Venkatachalam/aging_reg_dataset · Datasets at Fast360

MIR675

100033819

ncRNA

H9C2

--

Vascular disease

Prevent

EdU assay//SA--gal activity assay

The data showed that miR-675 mimic increased the expression of miR-675 and miR-675 inhibitor decreased the expression of miR-675.Importantly, the miR-675 mimic decreased ¦Â-gal staining and increased the numbers of proliferating cells, while the miR-675 inhibitor showed an opposite effect on ¦Â-gal staining and cell proliferation.

--

TGF¦Â1-p21

Downregulation

Luciferase reporter assay//qRT-PCR//Western blot

Dual-luciferase reporter assays showed that the miR-675 mimic decreased the luciferase activity compared to the control. To confirm that TGF-¦Â1 is a target of miR675 in H9C2 cells, we transfected miR-675 mimic or inhibitor into these cells. We found that miR-675 mimic and inhibitor did not alter the expression of TGF-¦Â1 mRNA, but miR-675 mimic decreased the expression level of TGF-¦Â1 protein and miR-675 inhibitor has the opposite effect. These findings verified TGF-¦Â1 as the target gene of miR-675. Moreover, p21 protein level was reduced by miR-675 mimic, but increased by miR-675 inhibitor.

Human

L

delay aging

30,889,706

Gene

0

1

0

1

0

LNCTAM34A

102724571

ncRNA

HCT116

Colon

Aging

Prevent

Cell viability assay//Knockdown//SA--gal activity assay

In addition, depletion of GUARDIN triggered cellular senescence.The effects of GUARDIN on cell viability were mirrored in the long-term survival of HCT116 cells in clonogenic assays and in their growth in nu/nu mice.

--

p53

--

Western blot//Cell viability assay//Knockdown

Although GUARDIN knockdown in the absence of p53 did not significantly affect cell viability, cell death was enhanced following induction of p53 . Conversely, overexpression of GUARDIN diminished the reduction in cell viability associated with p53 induction. Thus, although p53 controls the function of GUARDIN through regulating its expression, GUARDIN modulates the cytotoxic effect of p53.We extended our analysis to multiple lines, confirming that overexpression of wild-type p53 in U2OS osteosarcoma, A549 lung adenocarcinoma and HCT116 cells increased GUARDIN levels, whereas knockdown of p53 decreased GUARDIN expression.

Human

HL

delay aging

29,593,331

Gene

0

1

0

1

0

1

0

MIR21

406991

ncRNA

MCF-7

--

Breast cancer

Prevent

ELISA//Knockdown//MTT assay//SA--gal activity assay

Pre-miR-21 stimulated MCF-7 cell proliferation at the time points considered. In contrast, significant growth inhibition by ATRA was observed upon miR-21 silencing, indicating sensitization of MCF-7 cells. The effect of miR-21 silencing on ATRA-induced senescence was evaluated, using two molecular markers: -galactosidase and trimethyl K9 histone H3.

IL1B//ICAM-1//PLAT

Downregulation//--//Downregulation

Luciferase reporter assay//qRT-PCR//Western blot

We cloned the 3-UTR of the selected transcripts downstream of a luciferase reporter and evaluated the effect of miR-21 in 293T cells, which contain low levels of the miRNA (54) .MiR-21 inhibited the expression of the ICAM-1, PLAT, and IL1B constructs, indicating that they are direct targets.Forced expression of pre-miR-21 reduced ATRA-dependent induction of PLAT and IL1B mRNAs and proteins. By converse, the mRNA or protein expression pattern of ICAM-1 observed in ATRA-treated MDA-MB-231 cells was not affected by pre-miR-21 transfection. In contrast, pre-miR-21 enhanced the down-regulation of maspin mRNA and protein afforded by ATRA.

--

Human

HL

delay aging

21,131,358

Gene

0

1

0

1

0

1

0

1

0

ACSL5

51703

ncRNA

RT4,RT-112,Urotsa

Tumor tissue

Bladder cancer

Accelerate

SA--gal activity assay

Senescence-associated-¦Â-galactosidase activity was significantly associated with ACSL5 upregulation in tumours (p=0.03) and highest activity was also found in RT4 cells. RT112 and Urotsa showed no ¦Â-galactosidase activity, and in J82 enzyme activity was minimal.

--

Human

L

delay aging

23,348,389

Gene

0

1

0

MIR146A

406938

ncRNA

HTM636,HTM1073

--

Aging

Prevent

BrdU assay//Flow cytometry//SA--gal activity assay

Production of SA-¦Â-gal and iROS was significantly decreased, and BrdU incorporation was significantly increased by miR-146a in both HTM cell lines.

PAI-1//IRAK1

--//--

BrdU assay

We investigated whether the observed effects of miR-146a on SA-¦Â-gal activity, iROS, and cell proliferation in senescent HTM cells were mediated by the downregulation of either the direct target IRAK1 or the secondary target PAI-1.Overexpression of both PAI-1 and IRAK1, lacking the miR-146a target site, inhibited the effects of miR-146a on cell proliferation.

--

Human

HL

delay aging

20,053,980

Gene

0

1

0

1

0

1

0

MIR138-1

406929

ncRNA

HuH-7,HepG2,Hepatocyte

--

Hepatocellular carcinoma

Accelerate

BrdU assay//SA--gal activity assay

Telomeric repeat amplification protocol (TRAP) assay, senescence-associated ¦Â-galactosidase assay and BrdU incorporation assay were performed 24 and 48 h after transfection. The results showed that miR-138 functioned to decrease telomerase activity, which in turn induced cell senescence and suppressed cell proliferation.

TERT

Downregulation

Western blot//qRT-PCR//Luciferase reporter assay

The binding site of miR-138 in the 3¡¯UTR of TERT gene was predicted by miRNA target prediction tools. The results showed that wild-type miR-138, but not mutant miR-138 (mutated in the seed-complementary sequence),can significantly suppress the luciferase activities of Huh7 cells transfected with wild-type TERT 3¡¯UTR construct,Quantitative real-time PCR and Western blotting analysis showed that wild-type miR-138 mimic can suppress endogenous TERT mRNA and protein expression in Huh7 and HepG2 cells. These results indicated that TERT can be a direct target of miR-138 in HCC cells.

--

Human

L

delay aging

28,258,280

Gene

0

1

0

1

0

MIR101-1

406893

ncRNA

MCF-7

--

Aging

Accelerate

SA--gal activity assay

Further, continuous (upto 72 hrs) over-expression of miR-101 depicted an enhanced induction of senescence(2.3-Fold¡À0.25). miR-101 inhibition with anti-miR-101 oligonu-cleotides depicted a profile (0.9-Fold 60.25) which matched with the control cells.

UBE2N//SMARCA4

Downregulation//Downregulation

qRT-PCR

The validation of the two targets, UBE2N and SMARCA4, was established by measuring their endogenous cellular expression as well, which decreased significantly in over-expressing miR-101 cells.

--

Human

HL

delay aging

25,353,636

Gene

0

1

0

HOTAIR

100124700

ncRNA

WI-38,IDH4

--

Aging

Accelerate

Flow cytometry//SA--gal activity assay//qRT-PCR

The levels of senescent markers p53, p21 and p16 were elevated, and cells displayed the characteristic senescence- associated (SA) increase in G1 cell cycle compartment.That the increased HOTAIR was important for the implementation of senescence was evidenced by experiments in which silencing HOTAIR in IDH4 cells reversed the senescent phenotype, with a lower proportion of cells displaying the hallmark SA-¦Â-galactosidase activity along with reduced levels of senescence markers.

Ataxin-1//Snurportin-1

Downregulation//Downregulation

Western blot//Co-IP

Interestingly,the interaction of Dzip3 and Ataxin-1, as well as the interaction of Mex3b and Snurportin-1, as assessed by co-IP followed by western blot (WB) analysis, was facilitated by HOTAIR overexpression; furthermore, the levels of Ataxin-1 and Snurportin-1 decreased after HOTAIR overexpression.

--

Human

L

delay aging

24,326,307

Gene

0

1

0

1

0

1

0

MALAT1

378938

ncRNA

GBC-SD,SGC-996

--

Gallbladder cancer

Prevent

EdU assay//SA--gal activity assay//Transwell assay//Western blot

Our results indicated that when MALAT1 was silenced, the viability of the GBC-SD and SGC-996 cells was suppressed, and the expression of Ki67 and PCNA was reduced. Meanwhile, the capabilities of migration and invasion were inhibited, while the SA-¦Â-gal activity was promoted (p?<?0.05).

ABI3BP//EZH2

Downregulation//Binding

qRT-PCR//Pull-down assay

We carried out the correlation analysis and found that the expression of MALAT1 and ABI3BP was negatively correlated.The binding of MALAT1 to EZH2 was further verified by RNA pull down. The results demonstrated that Bio-MALAT1-WT pull-down more EZH2 relative to that of the Bio-probe and Bio-MALAT1-MUT, suggesting that MALAT1 promoted the enrichment of EZH2. Higher expression of ABI3BP was identified among cells treated with sh-MALAT1 than that in sh-NC.Taken together, a conclusion was drawn that MALAT1 inhibited the expression of ABI3BP by recruiting EZH2 to the promoter region of ABI3BP.

--

Human

HL

delay aging

31,174,563

Gene

0

1

0

1

0

1

0

1

0

SNHG6

641638

ncRNA

MGC803,MKN45

--

Gastric cancer

Prevent

Knockdown//SA--gal activity assay

The results showed that knockdown of SNHG6 in MGC-803 and MKN45 cells triggered an evident increase in the positive rate of senescent cells.

EZH2//p21

Upregulation//Downregulation

Western blot//Knockdown

Knockdown of SNHG6 reduced the expression level of EZH2 and overexpression of SNHG6 also increased EZH2 expression. The results showed that the expression level of p21 was strikingly augmented in SNHG6-knockdown GC cells;In contrast with SNHG6 knockdown, overexpression of SNHG6 decreased p21 levels and increased EZH2 levels

JNK

--

Western blot//Knockdown

--

Human

L

delay aging

30,031,062

Gene

0

1

0

1

0

MIR127

406914

ncRNA

WI-38,IMR-90

--

Aging

Accelerate

Cell counting//PI staining//SA--gal activity assay//Western blot

We observed that induced miR-127 expression caused a remarkable inhibition of cell proliferation and increased senescence-like phenotypes with positive staining of senescence-associated-¦Â-galactosidase (SA-¦Â-gal) in both WI-38 and IMR-90 cells.the senescence-like phenotype was associated with up-regulation of p53 and p21 and down-regulation of cyclin D1 (a pattern associated with senescence) in both WI-38 and IMR-90 cells.miR-127 overexpression induced cell cycle arrest at G0/G1phase.

BCL6

Downregulation

Western blot

Over-expression of miR-127 has markedly down-regulated BCL6 expression in both WI-38 and IMR-90 cells.

p53-p21

Upregulation

Western blot

The depletion of BCL6 inhibited cell proliferation was associated with increased p53 and p21 expression .

Human

L

delay aging

24,282,530

Gene

0

1

0

1

0

1

0

1

0

MIR449B

693123

ncRNA

SNU638

--

Gastric cancer

Accelerate

Flow cytometry//SA--gal activity assay

Compared to negative control microRNAs, re-introduction of miR-449b strongly affected the proliferation of SNU638 cells and visual inspection of the cells indicated induction of apoptosis and cellular senescence. Flow cytometric analysis of propidium iodide-stained cells transfected with miR-449b showed a G1accumulation 48 hours after transfection, followed at 72 hours post transfection by an accumulation of cells in the sub G1 fraction suggestive of cell death.The induction of cellular senescence was confirmed by acidic beta gal staining using miR-34a as a positive control microRNA.

--

p53

Activation

Western blot

Western blot analysis showing an increase of the p53 protein upon miR-449 and positive control miR-34a re-introduction into SNU638 cells compared to RNA scrambled control as well as an activation of the p53 downstream target p21 and apoptosis markers cleaved CASP3 and PARP.

Human

HL

apoptosis

21,418,558

Gene

0

1

0

1

0

MIR150

406942

ncRNA

Normal NK/T-cell Lymphoma cell line cell,HANK-1,MOTN-1,SNK-6

--

Malignant lymphoma

Accelerate

SA--gal activity assay//Southern blot analysis//TRAP assay

In addition, these cells stained positively for Ki-67 and senescence-associated beta-gal on day 21 after GFP selection and miR-150 transductants had a ¡®fried egg¡¯-like appearance, which is caused by senescence and is not seen in normal NK/T-cell lymphoma cells.Telomerase activity in miR-150 transductants (on day 21 after GFP selection) was markedly lower than in cells transduced with empty vector.We observed a shortening of telomeric DNA in the cells expressing miR-150 during continuous culture. Indeed, the telemetric restriction fragments of miR-150 transductants grew shorter with every successive passage.

--

PI3K-Akt

Downregulation

Luciferase reporter assay//Western blot

Upon insertion of the wild-type 30-UTR of DKC1, PIK3AP1, AKT3 or AKT2 into the reporter, we observed significant reductions in luciferase activity with DKC1, PIK3AP1 and AKT2, but not in AKT3 (data not shown), as compared with cells transduced with empty vector(control vector), suggesting that DKC1, PIK3AP1 (data not shown) and AKT2 have potential to function as direct targets of miR-150 .We therefore carried out a western analysis of pAKTser473/4expression and found it to be markedly reduced in the cells tested. We also detected upregulation of Bim (in MOTN-1, HANK-1 and SNK-6) and p53 (in SNK-6 and HANK-1).

Human

L

apoptosis

21,502,955

Gene

0

1

0

1

0

1

0

MIR338

442906

ncRNA

U251

Brain

Malignant glioma

Accelerate

Flow cytometry//SA--gal activity assay

When compared with the blank group and the NC group, the cells were found relatively large in size with high degree of senescence in the miR-338-5p mimic group and the siRNA-FOXD1 group, while were small in size with low degree of senescence in the miR-338-5p inhibitor group.When compared with the blank group and the NC group, the G0/G1 phase lengthened (increased proportion of cells), but the S phase shortened (decreased proportion of cells) in the miR-338-5p mimic group and the miR-338-5p mimic group.

--

FOXD1//MAPK

Downregulation//Downregulation

qRT-PCR//Western blot

When compared with the control group, the miR338-5p expression and the mRNA and protein expression of Bax in cells of each group decreased significantly, and the protein and mRNA expression of FOXD1, MEK-2, ERK-1, Bcl-2, DAF, and PCNA increased obviously (p<0.05).

Human

HL

apoptosis

30,225,541

Gene

0

1

0

1

0

MIR195

406971

ncRNA

H1299,H1993,H358

--

Lung cancer

Accelerate

Flow cytometry//SA--gal activity assay

We observed significantly more cells in the G1 phase after miR-195 transfection, suggesting that miR195 causes arrest of NSCLC cells in the G1 phase.We found that miR-195 significantly increases the number of senescent cells, as measured by ¦Â-galactosidase staining assay .

CCND3//BIRC5

Downregulation//Downregulation

Luciferase reporter assay//Western blot//qRT-PCR

We confirmed that mRNA and protein levels of CCND3 and BIRC5 are decreased by miR-195 and increased by miR-195 inhibitors in NSCLC cells.BIRC5 is predicted to contain one miR-195 target site in its 3¡äUTR, while CCND3 is predicted to contain two. We confirmed direct and specific regulation of BIRC5 and CCND3 by miR-195 using a luciferase reporter assay.We demonstrated that miR-195 represses CCND3 and BIRC5 proteins in a pair of cell lines derived from the same patient, HBEC30-KT and HCC4017 .

Rb

Activation

Western blot

We also found decreased levels of p-Rb by miR-195, indicating both activation of the retinoblastoma (Rb) pathway and senescence .

Human

HL

apoptosis

29,416,000

Gene

0

1

0

1

0

MIR380

494329

ncRNA

Mammary epithelial cell

Mammary Gland

Neuroblastoma

Prevent

SA--gal activity assay

Tumor cells expressing activated HRAS plus control (empty vector or scrambled control) stained positive for senescence-associated ¦Â-galactosidase. In contrast, tumors expressing HRAS plus miR-380 or shRNA p53 did not express senescence markers .

p21

Downregulation

Immunohistochemical staining//qRT-PCR

p21waf1 was increased in wild-type mouse mammary epithelial cells and tumor cells expressing activated HRAS plus control. In contrast, p21waf1 expression was lower in tumor cells that co-express HRAS and miR-380 or shRNA p53.

p53

Downregulation

Western blot

Expression of miR-380-5p resulted in a significant ~40% decrease in basal p53 protein levels. UV irradiation led to a dose-dependent increase in p53 protein expression that was suppressed by the expression of miR-380-5p .

Human

L

apoptosis

20,871,609

Gene

0

1

0

MIR34A

407040

ncRNA

ECa-109

--

Endometrial cancer

Accelerate

Cell morphological analysis//SA--gal activity assay

After 4 days of incubation with miR-34a, we observed that the introduction of miR-34a in ECa-109 cells caused senescence-like phenotypes in most of the cells with positive staining for senescence associated ¦Â-galactosidase (SA-¦Â-Gal), and cell number decreased with enlarged cellular size and morphological changes compared to the control group .

SIRT1//p53//p21

Downregulation//Upregulation//Upregulation

Western blot

Western blot analysis revealed that the introduction of miR-34a (20 nM) decreased the expression of SIRT1 and increased the expression of p53 and p21 in ECa-109 cells;

--

Human

L

apoptosis

26,523,671

Gene

0

1

0

1

0

SNHG6

641638

ncRNA

SK-BR-3,MDA-MB-231

--

Breast cancer

Prevent

We found that the percentage of senescent cells was significantly increased in SNHG6 silenced SKBR-3 cells compared to that in scrambled siRNA transfected cells .SNHG6 silencing causes G1 cell cycle arrest in breast cancer cells and downregulation of Cyclin D1 and PCNA, two important factors in cell cycle progression, determined by RTqPCR following SNHG6 silencing.

--

Human

L

apoptosis

30,826,970

Gene

0

1

0

MIR125A

406910

ncRNA

HBMEC

--

Aging

Prevent

SA--gal activity assay

miR-125a-5p overexpression significantly decreased (by 50%) the percentage of senescence-associated ¦Â-galactosidase-positive HBMEC in both vehicle- and ox-LDL-treated groups.

--

Human

L

apoptosis

27,903,586

Gene

0

1

0

MIR30D

407033

ncRNA

OVCAR-5,OWA42,MDA-MB-231,T47D,HCT116

--

Cancer

Prevent

Flow cytometry//SA--gal activity assay

We found that mir-30d inhibitor transfection led to a significant cell-cycle arrest at the G0/G1 phase.Finally, we observed that transfection of a mir-30d inhibitor not only significantly blocked cancer cell proliferation but also remarkably increased the number of the cells with typical senescence morphology. Four to 6 days after transfection with a mir-30d inhibitor, the percentage of ¦Â-galactosidase¨Cpositive cancer cells was significantly increased compared with controls.

--

Human

HL

apoptosis

22,058,146

Gene

0

1

0

1

0

MIR1258

100302172

ncRNA

H1299

--

Lung cancer

Accelerate

SA--gal activity assay

H1299 cells transfected with miR-1258 showed a strong blue SA-¦Â-gal staining, while there were only scattered SA-¦Â-gal positive cells in miR©\1258 inhibitor group.

--

GRb2-Ras-ERK

Downregulation

Western blot//qRT-PCR

GRB2 relative expression levels were down©\regulated with miR©\1258©\down©\regulated expression of si©\GRB2. Western blotting results showed that the expression levels of p©\c©\Raf, p©\Mek1/2 and p©\Erk1/2 were significantly decreased when miR©\1258 was overexpressed. However, the expression levels of p©\c©\Raf, p©\Mek1/2 and p©\Erk1/2 were significantly increased in cells with low expression levels of miR©\1258. There was no significant difference in Ras, c©\Raf, Mek1/2 and Erk1/2 expression in either case. These results indicate that miR©\1258 may be involved in the regulation of the GRB2/Ras/Erk pathway.

Human

L

apoptosis

30,069,987

Gene

0

1

0

VIM-AS1

100507347

ncRNA

SW1116,SW480,HT29,SW48

--

Colorectal cancer

Prevent

DAPI staining//Flow cytometry//Knockdown//SA--gal activity assay

Flow cytometry data revealed that a significant increase in the proportion of cells in the G2/M phase and a decrease in the proportion of cells in the S phase in VIM- AS1 suppressed cells compared with the cells treated with scrambled siRNA, which implicated VIM- AS1 inhibition leads to G2/M arrest in colon cancer cells. The result displayed that the proportion of cells with increased ¦Â-galactosidase activity was significantly increased in VIM ¨CAS1 inhibited cells in comparison to cells treated with scrambled siRNA.(D) DAPI staining showed that the senescent cells were increased in VIM ¨CAS1 knocking down cells rather than the cells were treated with scrambled siRNA.

--

Human

L

apoptosis

29,656,793

Gene

0

1

0

1

0

1

0

1

0

MIR192

406967

ncRNA

HCT116,A549

--

Colorectal cancer

Accelerate

Flow cytometry//SA--gal activity assay

Whereas A549 cells (wt p53) were also retained in G1 by miR-192 and miR-215.HCT116 cells were transfected with control RNA or the microRNAs under study followed by SA-¦Â-Gal assay.MiR-192 and miR-215 were capable of inducing senescence to some extent but not as efficiently as miR-34a.

--

Human

HL

apoptosis

19,074,875

Gene

0

1

0

1

0

MIR215

406997

ncRNA

HCT116,A549

--

Colorectal cancer

Accelerate

Flow cytometry//SA--gal activity assay

Whereas A549 cells (wt p53) were also retained in G1 by miR-192 and miR-215.HCT116 cells were transfected with control RNA or the microRNAs under study followed by SA-¦Â-Gal assay. MiR-192 and miR-215 were capable of inducing senescence to some extent but not as efficiently as miR-34a.

--

Human

HL

apoptosis

19,074,875

Gene

0

1

0

1

0

MIR101-1

406893

ncRNA

SPAC-1-L,HEC-50

--

Endometrial cancer

Accelerate

SA--gal activity assay//Western blot

Introduction of miR-101 in SPAC-1-L and HEC-50 cells caused senescence-like phenotypes, such as positive staining for SA-¦Â-gal and enlarged, flattened cell morphology. Furthermore, immunoblot analysis revealed that miR-101 overexpression markedly enhanced the expression of pro-apoptotic gene Bax, apoptosis marker cleaved-PARP and senescence marker p21 in either cell line.

EZH2//MCL-1//FOS

Downregulation//Downregulation//Downregulation

Knockdown//qRT-PCR//Western blot

In agreement with this, a negative correlation between endogenous miR-101 levels and EZH2, MCL-1 and FOS mRNA expression was found in EC cells .Next, we confirmed that endogenous mRNA and protein levels of these genes were downregulated in 101-transfected SPAC-1-L cells . Knockdown of miR-101 by AS-101 conversely induced the mRNA and protein levels of EZH2, MCL-1 and FOS in HOUA-I cells.

--

Human

HL

apoptosis

25,153,722

Gene

0

1

0

1

0

MIR155

406947

ncRNA

HUVEC

--

Aging

Accelerate

SA--gal activity assay//Western blot//qPCR

Determination of cellular senescence of HUVECs was conducted using 2 indicators¨C anti-SMP-30 and SA ¦Â-Gal staining ¨C and TNF-¦Á significantly reduced SMP-30 protein expression by almost 50% and enhanced SA ¦Â-Gal staining by nearly 2-fold. Then, the role of miR-155 in senescence of HUVECs was assessed, and the results showed that miR-155 mimic transfection increased SA ¦Â-Gal staining and downregulated SMP-30 .Addition of the miR-155 inhibitor decreased SA ¦Â-Gal staining and increased SMP-30 protein levels.

--

SIRT1-FOXO1-p53-p21

--

SA-¦Â-gal activity assay//Western blot//Knockdown

Thus, the downstream signaling targets of SIRT1, including the protein levels of acetylated FoxO-1, acetylated p53, and p21, were also determined. Knockdown of miR-155 decreased p21, Ac-p53, and Ac-FoxO-1 protein expression, and SIRT1 silencing reversed this effect.Then, we assessed whether HUVEC proliferation and senescence were influenced by siSIRT1, and the results showed that siSIRT1 prevented the decrease in TNF-¦Á-induced cell proliferation triggered by miR-155. Similarly, the effect of miR-155 on the upregulation of senescence and SMP-30 expression were substantially decreased by SIRT1 knockdown.

Human

L

apoptosis

31,752,013

Gene

0

1

0

1

0

1

0

MIR34A

407040

ncRNA

A549

--

Idiopathic Pulmonary Fibrosis

Accelerate

SA--gal activity assay//qRT-PCR

It was found that cells overexpressing miR-34a,miR-34b, or miR-34c had a relative increase in the numbers of cells expressing SA-¦Âgal activity.In addition, levels of mRNA expression of p16 and p21 were increased between 1¨C1.9 fold in A549 cells expressing miR-34s.

--

Human

HL

telomere attrition

27,362,652

Gene

0

1

0

1

0

MIR34A

407040

ncRNA

HCT116,RKO

--

Colorectal cancer

Accelerate

Cell proliferation assay//SA--gal activity assay

We observed that introduction of miR-34a in HCT 116 cells caused senescence-like phenotypes with positive staining for senescence-associated ¦Â-galactosidase (SA-¦Â-gal) and enlarged cellular size.RKO cells also showed similar morphological changes with enlarged cellular size by miR-34a introduction,although few SA-¦Â-gal-positive cells were observed.The introduction of miR-34a caused a remarkable inhibition of cell proliferation in both HCT 116 and RKO cells compared with that of control miRNA.

--

E2F//p53

Downregulation//Upregulation

Western blot

We also observed that p53 and p21 started to accumulate at 2 and 4 h, respectively, and the accumulation continued until 48 h after treatment.As expected, LoVo and RKO cells increased expression of miR-34a similar to HCT 116 cells, but DLD1 and HT29 cells showed no change, like the HCT 116 p53/ cells. Accumulation of p53 and p21 was observed in HCT 116, LoVo, and RKO cells, whereas HCT 116 p53/ cells showed no accumulation, and DLD1 and HT29 cells expressing mutant p53 showed consistent levels of p53 and no accumulation of p21.As for the down-regulated genes, E2F1, E2F2, and some E2F-target genes, including DHFR, MCM3, and MCM10, were observed among the list.introduction of miR-34a decreased the accumulation of E2F-1 and -3. E2F-2 was not detectable in either case .

Human

HL

deregulated nutrient sensing

17,875,987

Gene

0

1

0

1

0

MIR29A

407021

ncRNA

MEF

Embryo

Aging

Accelerate

SA--gal activity assay

Increased abundance of miR-29 (represented miR-29a and miR-29c hereinafter) in early passaged cells (PD4) accelerated senescence whereas decreased amounts of miR-29 in late passaged cells (PD8) delayed senescent phenotypes.

H4K20me3

Downregulation

Western blot

Furthermore, forced expression or inhibition of miR-29, by individual transfection of miRNA mimics (M-miR-29) or inhibitors (I-miR-29a and I-miR-29c), resulted in decreased or increased H4K20me3 abundance, respectively. Gain of function of miR-29 via lentiviral infection led to reduced protein levels of Suv4-20h and H4K20me3.

--

Human

L

epigenetic alterations

29,967,491

Gene

0

1

0

TERC

7012

ncRNA

Squamous cell

Tumor tissue

Skin cancer

Accelerate

Cell proliferation assay//Telomere length assay

The percentage of apoptotic cells in Terc+/+ tumors averaged 1.1%. In contrast, the percentage of apoptotic cells in Terc-/- tumors was 5.5% (P < 0.04). Compared with Terc+/+ tumors.Average telomere length ratios were significantly lower in tumors compared with normal mucosa (2.1 vs. 0.78 for Terc+/+, 2.0 vs. 0.6 for G1 Terc-/-, and 1.22 vs. 0.27; P < 0.001).

--

Human

HL

genomic instability

21,593,138

Gene

0

1

0

1

0

MIR22

407004

ncRNA

MCF-7,MDA-MB-231,SiHa

--

Aging

Accelerate

SA--gal activity assay//Western blot//qPCR

Introduction of miR-22 into cancer cells caused a senescence-like phenotype, as observed by the increased senescence-associated ¦Â-galactosidase (SA-¦Â-gal) activity.Thus, miR-22¨Cmediated downregulation of MDC1 expression is a common mechanism in aging cells and is utilized indiverse and widespread tissue types in mammals.

MDC1//Akt1

Downregulation//--

qPCR//Western blot//Luciferase repoter assay

Using real-time quantitative PCR (qPCR), we found that MDC1 mRNA was reduced more than 50% when miR-22 was overexpressed,indicating that miR-22 posttranscriptionally downregulates MDC1, probably by promoting both mRNA decay and inhibiting translation.We found that inhibition of endogenous miR-22 led to an increase in MDC1 protein and mRNA in CA-Akt1¨Coverexpressing cells. Moreover, CA-Akt1 significantly inhibited MDC1 30-UTR luciferase activity relative to control.Under these experimental conditions, CA-Akt1¨Cinduced decrease of luciferase activity was significantly attenuated when anti¨CmiR-22 was introduced, suggesting that CA-Akt1 negatively regulates MDC1 via miR-22.

--

Human

HL

genomic instability

25,627,978

Gene

0

1

0

1

0

1

0

BMP2

650

protein coding

RasV12

--

Aging

Accelerate

Knockdown//SA--gal activity assay

Bmp2-knocked-down RasV12 cells escaped from senescence with decreased number of SA-¦Âgal(+) cells compared to RasV12 cells.The cells showed increased number of SA-¦Âgal(+) cells in dose-dependent manner,even to the level of RasV12 cells when rBMP2 was at 200 ng/mL.

Smad1//Smad6

Upregulation//Upregulation

Knockdown//Western blot//CHIP-seq

Smad1 target genes upregulated most by rBMP exposure included Smad6, which was upregulated by 4.5-fold in MEF.

--

Human

HL

cellular senescence

22,072,987

Gene

0

1

0

1

0

MIR1827

100302217

ncRNA

HCT116,RKO

--

Colorectal cancer

Accelerate

Knockdown//SA--gal activity assay

MiR-1827 enhanced p53-mediated senescence in HCT116 cells treated with Doxorubicin.MiR-1827 enhanced p53-mediated senescence in RKO cells treated with Doxorubicin. miR-1827 inhibitor reduced p53-mediated senescence in HCT116 cells treated with Doxorubicin.Compared with control inhibitor, miR-1827 inhibitor significantly inhibited Doxorubicin-induced senescence in HCT116 p53+/+ cells but not in HCT116 p53¨C/¨Ccells.

MDM2//p53

Downregulation//Upregulation

Western blot

miR-1827 reduced MDM2 protein levels and increased p53 protein levels in HCT116 cells treated with Doxorubicin (Dox).

--

Human

HL

apoptosis

26,840,028

Gene

0

1

0

1

0

MIR23A

407010

ncRNA

PBMC

Blood

Coronary artery disease

Accelerate

FISH

Levels of miR-23a were negatively correlated with levels of RTL in all subjects ( miR-23a vs. RTL, r = -0.58, P < 0.01).

TRF2

Downregulation

Flow cytometry

The miR-23a mimic transfection resulted in a decrease in levels of TRF2 compared with the mock transfection.The miR-23a inhibitor transfection also resulted in an increase in levels of TRF2 compared with the mock transfection.

--

Human

HL

telomere attrition

28,646,123

Gene

0

1

0

MIR494

574452

ncRNA

AsPC-1,PANC-1

--

Pancreatic cancer

Accelerate

Flow cytometry//PI staining//SA--gal activity assay

MiR-494 induced the G1-phase arrest of AsPC-1 and PANC-1 cells (measured by propidium iodide (PI) staining and flow cytometry). (f) MiR-494 induced senescence of AsPC-1 and PANC-1 cells (measured by ¦Â-Gal staining).As well as increased the proportion of cells in the G1 phase in AsPC-1 (63.3¡À3.1% vs 73.7¡À2.5%, P<0.05) and PANC-1 cells (40.5.6¡À4.2% vs 58.6¡À3.7%, P<0.01). Meanwhile, the senescence was also increased in AsPC-1 (17.5¡À2.1% vs 40.7¡À1.7%, P<0.01) and PANC-1 cells (14.6¡À3.3% vs 60.9¡À3.8%, P<0.01).

c-Myc//SIRT1

Downregulation//Downregulation

qRT-PCR//Western blot

The qRT-PCR and western blot results confirmed that miR-494 restoration led to the downregulation of c-Myc and SIRT1 expression, whereas the inhibition of miR-494 expression increased c-Myc and SIRT1 expression.

--

Human

L

apoptosis

25,965,392

Gene

0

1

0

1

0

1

0

MIR19B1

406980

ncRNA

Hela,MCF-7

--

Cancer

Prevent

Flow cytometry//SA--gal activity assay

Further investigation of miR-19b's effect on cell cycle arrest revealed that overexpression of exogenous miR-19b caused exiting from the G1 phase and entry into S or G2/M phase in both Hela and MCF7 cells .¦Â-galactosidase staining showed that cellular senescence was reduced when miR-19b was overexpressed in both Hela cells and MCF7 cells, but increased 50% when miR-19b was silenced or p53 was overexpressed simultaneously.

p53

Downregulation

Western blot//qRT-PCR

miR-19b was transfected into Hela cells and p53 levels of both transcriptional and translational were examined after 48 h. Western blot and qRT-PCR showed that p53 protein levels decreased by 50%, while mRNA levels were not altered. Similarly, the decrease of endogenous p53 protein was observed in both MCF7 and Huh7 cell lines when miR-19b was overexpressed, while mRNA level remained unchanged.

--

Human

L

apoptosis

24,742,936

Gene

0

1

0

1

0

MIR30D

407033

ncRNA

293T,H1299,HCT116

--

Cancer

Prevent

Flow cytometry//SA--gal activity assay

In our experiment, Gadd45¦Á was down-regulated along with p53 in 293T cells transfected with both miR-25 and miR-30d and a cell cycle analysis showed that fewer cells were arrested at the G2-M phase than in the control. We then performed a cell cycle analysis and found that fewer cells were arrested at the G1-S phase (G1 arrest) when miR-25 or miR-30d was co-overexpressed with the p53 gene with a wt 3¡¯UTR but not with the one with a respective mutant .Senescence was reduced in HCT116 cells with miRNA overexpression and lower levels of p53 and p21.

p53//p21

Downregulation//Downregulation

Western blot

We found that both miR-25 and miR-30d down-regulated the expression of the ectopic p53 gene carrying a wt 3¡¯UTR but not the one with a mutant. The expression of both p21 and Bax was either reduced or minimally changed, following p53 expression. These results suggest that miR-25 and miR-30d regulate cellular senescence through down-regulating p53 expression and subsequently the expression of p21 and that such regulation depends on miRNA binding sites of TP53 3¡¯UTR.

--

Human

L

apoptosis

20,935,678

Gene

0

1

0

1

0

MIR25

407014

ncRNA

293T,H1299,HCT116

--

Cancer

Prevent

Flow cytometry//SA--gal activity assay

In our experiment, Gadd45¦Á was down-regulated along with p53 in 293T cells transfected with both miR-25 and miR-30d and a cell cycle analysis showed that fewer cells were arrested at the G2-M phase than in the control. We then performed a cell cycle analysis and found that fewer cells were arrested at the G1-S phase (G1 arrest) when miR-25 or miR-30d was co-overexpressed with the p53 gene with a wt 3¡¯UTR but not with the one with a respective mutant.Senescence was reduced in HCT116 cells with miRNA overexpression and lower levels of p53 and p21.

p53//p21

Downregulation//Downregulation

Western blot

We found that both miR-25 and miR-30d down-regulated the expression of the ectopic p53 gene carrying a wt 3¡¯UTR but not the one with a mutant. The expression of both p21 and Bax was either reduced or minimally changed, following p53 expression. These results suggest that miR-25 and miR-30d regulate cellular senescence through down-regulating p53 expression and subsequently the expression of p21 and that such regulation depends on miRNA binding sites of TP53 3¡¯UTR.

--

Human

L

apoptosis

20,935,678

Gene

0

1

0

1

0

MIR339

442907

ncRNA

HCT116

--

Colorectal cancer

Accelerate

SA--gal activity assay

MiR-339-5p enhanced p53-mediated senescence in HCT116 cells treated with Doxorubicin.The miR-339-5p inhibitor reduced p53-mediated senescence in HCT116 cells treated with Doxorubicin.

MDM2//p21//p53

Downregulation//Upregulation//Upregulation

Western blot

MiR-339-5p decreased MDM2 protein levels and increased the p53 protein accumulation and transcriptional activity toward p21 in response to stress in HCT116 cells. (C) MiR-339-5p decreased MDM2 protein levels and increased p53, p21 protein levels in response to stress in RKO cells.

--

Human

L

apoptosis

25,193,859

Gene

0

1

0

MIR200C

406985

ncRNA

HUVEC

--

Aging

Accelerate

SA--gal activity assay

In HUVEC transduced with miR-200c the percentage of SA-¦Â-gal-positive cells was strongly increased compared with control.

ZEB1

Downregulation

Western blot

As expected, ZEB1 protein was downmodulated by miR-200c overexpression.

--

Human

L

apoptosis

21,527,937

Gene

0

1

0

MIR34A

407040

ncRNA

A549,BEAS-2B

--

Idiopathic pulmonary fibrosis

Accelerate

BrdU assay//Flow cytometry//SA--gal activity assay//Western blot//qRT-PCR

MiR-34a was potent in inducing senescence of human lung epithelial A549 cells, as indicated by elevated senescence-associated ¦Â-galactosidase activity and increased expression of senescence markers p21 and PAI-1 in miR-34a transfected lung epithelial cells. Consistent with this senescent phenotype, miR-34a transfected lung epithelial cells also demonstrated decreased proliferation. The prosenescence activity of miR-34a was also confirmed in human lung epithelial cell line BEAS-2B.

SIRT1

Downregulation

qRT-PCR

We confirmed that Sirt1 expression was decreased in lung epithelial cells that were transfected with miR-34a.

--

Human

L

apoptosis

27,979,858

Gene

0

1

0

1

0

1

0

1

0

1

0

MIR34A

407040

ncRNA

Chondrocyte

Bone

Osteoarthritis

Accelerate

CCK-8 assay//Flow cytometry//SA--gal activity assay

Cells characterized by positive SA-¦Â-gal were enhanced in chondrocytes that underwent transfection with the miR-34a mimic.Compared to the TRAIL alone treatment group, the inhibitor of miR-34a suppressed the number of apoptotic cells.The miR-34a mimic transfection remarkably suppressed the growth of these cells.

DLL1

Downregulation

Luciferase reporter assay//Western blot

In order to explore the interaction between miR-34a and the DLL1 3¡ä-UTR, the miR-34a binding region was mutated in the DLL1 3¡ä-UTR. Subsequent to luciferase vector co-transfection with DLL1 3¡ä-UTR as well as the miR-34a mimic, the miR-34a mimic was discovered to be unable to inhibit expression of luciferase. The findings indicated that transcription, secretion, and translation of DLL1 were reversely modulated through miR-34a.

PI3K-Akt

Downregulation

Western blot

He findings revealed that the level of total AKT and p-AKT declined in the groups that underwent miR-34a transfection.

Human

L

apoptosis

30,048,987

Gene

0

1

0

1

0

1

0

MIR34C

407042

ncRNA

KG-1a

--

Leukemia

Accelerate

EdU assay//Flow cytometry//SA--gal activity assay//Western blot//qRT-PCR

Forced expression of miR-34c-5p resulted in a decreased cell number, proliferation index, and percentage of EdU+ cells.. Increased miR-34c5p induced an increase in the number of G0/G1 phase cells and decreased cell cycle-associated protein levels.Up-regulation of miR-34c-5p failed to induce apoptosis but resulted in significantly increased senescence in KG-1a cells . Furthermore , two typical senescence-associated secretory factors , IL-6 and plasminogen activator inhibitor-1 (PAI-1), were elevated under treatment with miR-34c-5p mimic at 48 h postelectrotransfection .

RAB27B

Binding

qPCR//Western blot//Luciferase reporter assay

Using qPCR and Western blotting, we further confirmed that RAB27B mRNA and protein levels were decreased in 34cOE-KG-1a cells.We selected one of the most likely binding sites according to the folding energy and P- value parameters provided by rna22 and performed a luciferase reporter assay. We found that co-transfection of the luciferase reporter and miR-34c-5p mimic into KG-1a cells produced lower luciferase activity than cells co-transfected with miR-NC, but miR-34c-5p mimic did not reduce luciferase activity with the mutated RAB27B 3¡ä-UTR, indicating that miR-34c-5p is a specific regulator of RAB27B.

--

Human

HL

delay aging

29,479,064

Gene

0

1

0

1

0

1

0

1

0

1

0

MIR106B

406900

ncRNA

MTMEC

--

Breast cancer

Prevent

EdU assay//SA--gal activity assay

Overexpression of the miR-106bB25 cluster allowed cells to bypass doxorubicin-induced senescence and increased the capacity of MTMEC cells to generate doxorubicin-resistant cells.

EP300

Binding

Luciferase reporter assay

To experimentally confirm that EP300 mRNA was a target of the miR-106bB25 cluster, we used a luciferase reporter with the 30 -UTR of EP300 and transiently transfected either control or cells overexpressing the cluster or individual miRs. In all cases, luciferase expression from the plasmid containing the EP300 30 -UTR was lower in cells overexpressing both the miR-106bB25 cluster and its individual miRs.which overexpress the miR-106bB25 cluster,showed reduced EP300 and CDH1, but increased VIM(encoding vimentin, a mesenchymal intermediate filament protein activated during EMT4) mRNA levels.

--

Human

HL

delay aging

24,270,410

Gene

0

1

0

1

0

MIR31

407035

ncRNA

MDA-MB-231,MCF-7,MRC-5

--

Aging

Accelerate

EdU assay//Immunostaining//SA--gal activity assay

The data showed that indeed, miR-31 overexpression increased SA-¦Â-gal positive cells and decreased EdU positive cells indicating that miR-31 is a potent inducer of cell senescence. The role of miR-31 in senescence was further confirmed by examining ¦ÃH2AX foci formation, which is another marker of cell senescence. The data indicated that miR-31 overexpression leads to an increase in ¦ÃH2AX foci formation in MRC5 cells.Induction of senescence by miR-31 overexpression was also confirmed in MDA-MB-231 and MCF7 cells.

BMI1

Downregulation

Western blot

We found that miR-31 overexpression leads to repression of BMI1 and its inhibitor up-regulates BMI1.miR-31 overexpression also led to significant down-regulation of BMI1.

--

Human

HL

cellular senescence

25,737,447

Gene

0

1

0

1

0

1

0

DPP4

1803

protein coding

HUVECs

--

Aging

Accelerate

Immunochemical staining//SA--gal activity assay//Western blot

The fraction of senescence-associated ¦Â-galactosidase (SA-¦Â-gal) positive cells was increased with aging, whereas DPP4 inhibition mitigated this enhancement. In addition, immunohistochemical staining and western blot analysis revealed that the aging markers, including p53 and p21, were increased with advancing age, whereas DPP4 inhibition reversed these changes. Taken together, these data demonstrated that inhibiting DPP4 activity partly prevented vascular aging. Small interfering RNA (SiRNA) for DPP4 silencing was used. After exposure to H2O2, the fraction of cells that stained positive for SA-¦Â-gal was markedly increased, accompanied with augmented p53 and p21 expression. DPP4 knockdown significantly reversed H2O2-induced increase in SA-¦Â-gal + cell numbers, and p53, p21 expression. Inhibiting DPP4 with saxagliptin repressed both the activity and expression of DPP4 in endothelial cells, and inhibiting DPP4 reversed H2O2-induced augmentation of SA-¦Â-gal + cell numbers, as well as p53 and p21 expression.

--

AMPK-SIRT1-Nrf2

Activation

Immunochemical staining//Western blot//Immunofluorescence

Immunohistochemical staining and western blot analysis revealed that SIRT1 and AMPK¦Á expression, as well as phosphorylation level of AMPK¦Á, was decreased with aging, and that DPP4 inhibition was able to restore these changes in aging aortas. As observed in in vivo studies, SIRT1 and AMPK¦Á expression, as well as - phosphorylation level of AMPK¦Á, was also decreased in H2O2-induced senescent endothelial cells, whereas DPP4 knockdown, or inhibition with saxagliptin, significantly reversed these changes. Our data from immunofluorescence staining revealed that SIRT1 activation and DPP4 silencing promoted the translocation of Nrf2 into the nucleus. However, inhibiting SIRT1 reversed the effect of DPP4 knockdown on Nrf2 translocation. Moreover, Nrf2 expression was decreased with cell senescence, whereas DPP4 silencing or SIRT1 activation reversed Nrf2 expression. The results from western blot analysis demonstrated that Nrf2 expression was decreased with vascular aging, while DPP4 inhibition markedly abolished this change. Similarly, Nrf2 expression was reduced in senescent endothelial cells, while DPP4 silencing reversed this change.

Human

L

cellular senescence

32,251,672

Gene

0

1

0

1

0

1

0

PIM1

5292

protein coding

Cardiomyocytes

--

Aging

Prevent

Fluorescence quenching assay//RT-PCR

PIM1 cells contained 1.55- fold longer telomeres than GFP cells after three passages as revealed by quantitative RT-PCR.Telomeres were 27% longer in PIM1 myocardium and 31% shorter in Pim1-KO hearts compared to NTg as revealed by qRT¨CPCR.Telomere fluorescence intensity was 28% greater in PIM1 cardiomyocytes and reduced by 30% in Pim1-KO cardiomyocytes compared to NTg controls.

--

TGF¦Â

Downregulation

Western blot//qRT-PCR//Immunoblotting(ÃÏÀö¼ÓµÄ)

In the presence of TGFb1 agonist, PIM1 exhibited a 41% reduction in pSMAD2 levels compared to GFP as evaluated by immunoblotting. Inhibitory effects of PIM1 on TGFb signalling were confirmed by expression of canonical downstream target SERPINE1 and non-canonical target N-CADHERIN as measured via qRT-PCR. PIM1 displayed a 64% and 42% reduction in mRNA expression of SERPINE1 and N-CADHERIN, respectively, compared to GFP.Cardiac-specific overexpression of PIM1 resulted in a 39% reduction in pSmad2 levels compared to NTg.Pim1-KO cardiomyocytes also displayed an up-regulation of pSmad2 compared to PIM1 Immunoblot analysis of pSmad3 also revealed a reduction of TGF¦Â signalling in PIM1 compared to NTg and Pim1-KO.

Human

L

cellular senescence

32,176,281

Gene

0

1

0

1

0

IRX5

10265

protein coding

SW480

--

Aging

Accelerate

SA--gal activity assay

Indeed approximately 60% to 70% of IRX5 overexpression cells exhibited positivity for the se- nescence hallmark, SA ¦Â gal compared to control ones.

--

ATM

Activation

Immunofluorescence//RT-PCR//Western blot

As we expected the immunofluorescence staining of ATM increased in IRX5 overexpression cells compared with that in controls. To confirm the inactivation of the ATM pathway by siRNA, real time PCR was performed. According to the efficiency of siRNA, we chose si1 in SW480 and si4 in DLD 1. Meanwhile, the inhibition of ATM significantly reduced the RH2A protein level.

Human

L

cellular senescence

32,162,364

Gene

0

1

0

MTOR

2475

protein coding

Fibroblasts

--

Aging

Accelerate

SA--gal activity assay//Western blot

Serum-starved fibroblasts exposed to R showed lowered CDNK1A and CDKN2A protein levels and decreased SA-GLB1/¦Â-gal activity.RICTOR silencing suppressed AKT1 phosphorylation at Ser473 along with repression of myofibroblast differentiation inhibition of CDKN1A and CDKN2A expression, and inhibition of SA-GLB1/¦Â-gal activity RPTOR silencing led, however, to inhibition of SA-GLB1/¦Â-gal activity .We silenced RICTOR and RPTOR concomitantly in serum-starved fibroblasts.

AKT1

Upregulation

Western blot//SA-¦Â-gal activity assay

RICTOR silencing suppressed AKT1 phosphorylation at Ser473 along with repression of myofibroblast differentiation inhibition of CDKN1A and CDKN2A expression, and inhibition of SA-GLB1/¦Â-gal activity.

--

Human

L

cellular senescence

31,931,659

Gene

0

1

0

1

0

HNRNPF

3185

protein coding

hMSCs

--

Aging

Prevent

CCK-8 assay//Cell cycle analysis//Colony formation assay//Immunofluorescence//SA--gal activity assay

Knockdown of hnRNP F in 5PDs of hMSCs largely accelerated cell senes- cence, which indicated by the dramatic increase of SA-¦Â-gal activity.More severe retardation of cell proliferation was observed after knockdown of hnRNP H1 in hnRNP F KD HeLa cells. Furthermore, knockdown of both hnRNP H1 and H2 in hnRNP F KD HeLa cells further reduced cell proliferation, indicating an additive effect of hnRNP F, H1, and H2 on regulating cancer cell proliferation. We also found that hnRNP F depletion caused significant G2/M accumulation and triple knockdown of hnRNP F, H1, and H2 triggered more obvious G2/M arrest.Similar to HeLa cells, hnRNP F deletion significantly reduced hMSCs telomerase activity ,drastically inhibited hMSCs proliferation and Ki67-positive cell numbers , and caused significant hMSCs G2/M cell cycle arrest.HnRNP F or H1 overexpression in 12PDs of hMSCs remarkably retarded cell senescence . HnRNP F or H1 overexpression also significantly elevated Ki67-positive cell numbers , indicating that enforced expression of hnRNP F/H preserve hMSCs proliferation capacity.

TCAB1//hTERC

Binding//Binding

Immunofluorescence//Biotinylated RNA pull down assay

The immunofluorescent signals of FLAG-hTERT overlapped with a specific Cajal bodies marker Coilin signals in the nuclei in both HeLa and U2OS control cells.The results showed that hnRNP F/H may form stable ribonucleoprotein complex with hTERC through direct binding.

--

Human

L

cellular senescence

31,863,069

Gene

0

1

0

1

0

1

0

1

0

1

0

CGRP

796

protein coding

NPCs

--

Intervertebral disc degeneration

Accelerate

CCK-8 assay//Immunochemical staining//RT-PCR//Safranin O-fast green staining

To further explore the relationship between CGRP and its receptors, CALCRL and RAMP1, and age, mice with different age of months were further used. As shown in safranin O-fast green staining, the shape of IVD tissue was collapsed in aged mice at 12 months and 18 months compared with 6 months, suggesting the intervertebral disc gradually experienced degeneration with aging. In addition, immunohistochemical analysis revealed the gradually elevated protein expression of CGRP and its receptors, CALCRL and RAMP1, with increasing age in mice. The cell viability of NP cells decreased after being treated by CGRP in a concentration-dependent manner.The mRNA expression of PCNA in human NP cells dose-dependently decreased after being treated with CGRP.

--

MAPK//NF-¦ÊB

Activation//Activation

Western blot//Immunofluorescence

Western blot demonstrated that CGRP could dose-dependently enhance the phosphory- lation of MAPK. Besides, NF-¦ÊB signaling pathway was also concentration-dependently activated by CGRP, and this activated effect was achieved through the phosphorylation of I¦ÊB-¦Á. Immunofluorescence results suggested that the nucleus translocation of p65 was increased when human NP cells were treated with CGRP.

Human

L

loss of proteostasis

34,987,701

Gene

0

1

0

1

0

1

0

1

0

PRKG1-AS1

100506939

ncRNA

Myoblasts

Skeletal muscle

Aging

Accelerate

CCK-8 assay//Cell viability assay//Flow cytometry//Western blot//qRT-PCR

CCK-8 assay showed that knock-down of PRKG1-AS1 could significantly increase cell viability in a time-dependent manner. Besides, flow cytometry demonstrated that cell apoptosis was significantly decreased by knocking-down of PRKG1-AS1 compared with the model group. In order to validate the effect of PKG1-AS1 at molecular level, we detected the mRNA and protein expression of muscle regulatory factors, including MyoD, MyoG, Myf2c and Myf5. These four factors were significantly decreased in the dexamethasone-induced muscle atrophy cell model (P < 0.05). After transfection with si-PRKG1-AS1, their expression was significantly upregulated both at mRNA level (P < 0.05). Western blot analysis showed consistent results with qRT-PCR.

MyoD//MyoG//Mef2c

Downregulation

qRT-PCR//Western blot

In order to validate the effect of PKG1-AS1 at molecular level, we detected the mRNA and protein expression of muscle regulatory factors, including MyoD, MyoG, Myf2c and Myf5. These four factors were significantly decreased in the dexamethasone-induced muscle atrophy cell model (P < 0.05). After transfection with si-PRKG1-AS1, their expression was significantly upregulated both at mRNA level (P < 0.05). Western blot analysis showed consistent results with qRT-PCR.

--

Human

HL

apoptosis

34,051,073

Gene

0

1

0

1

0

1

0

1

0

1

0

MIR99B

407056

ncRNA

Chondrocyte

--

Osteoarthritis

Accelerate

Western blot

We then explored the role of miR-99b-5p in OA development. Increased MMP13 and decreased COL2 were detected in mimics-treated chondrocytes, accompanied by enhanced senescence-related hallmarks P16, P21, and P53.

MFG-E8

Downregulation

Western blot

We found inhibited expression of MFG-E8 in cells treated with miR-99b-5p mimics and upregulated expression in cells treated with miR-99b-5p inhibitor.

p65 NF-¦ªb

Activation

Immunofluorescence//Western blot

We found that MFG-E8 treatment rescued the phosphorylation of p65 NF-¦ÊB signaling, while neutralizing MFG-E8 significantly enhanced p65 phosphorylation both in cartilage and in synovium post OA surgery. In vitro studies in murine primary chondrocytes and RAW264.7 cells con- firmed the suppressive effect of MFG-E8 in p65 NF-¦ÊB signaling.

Human

L

apoptosis

34,031,369

Gene

0

1

0

MFGE8

4240

protein coding

Chondrocyte

--

Osteoarthritis

Prevent

Immunofluorescence//SA--gal activity assay//Western blot

¦Â-galactosidase staining revealed that rmMFG-E8 markedly rescued the senescence phenotype induced by IL-1¦Â in primary murine chondrocytes. Senescence-related hallmarks P16, P21 and P53 were significantly downregulated after ectopic application of MFG-E8 in IL-1¦Â-pretreated primary chondrocytes, while MFG-E8 loss strongly promoted the expression of these senescence factors. Intraarticular injection of MFG-E8 neutralizing antibody obviously upregulated senescence markers, which were suppressed by rmMFG-E8, indicating the protective effect of MFG-E8 against chondrocyte senescence.

--

p65 NF-¦ÊB

Downregulation

Immunofluorescence//Immunoblotting

We found that MFG-E8 treatment rescued the phosphorylation of p65 NF-¦ÊB signaling, while neutralizing MFG-E8 significantly enhanced p65 phosphorylation both in cartilage and in synovium post OA surgery. We found that the NF-¦ÊB pathway inhibitor JSH-23 partially reduced MFG-E8 loss-induced expression of P53, P21 and P16 in murine primary chondrocytes, and reduced the enhancing tendency of M1 macrophages caused by MFG-E8 deficiency. While NF-¦ÊB pathway activator phorbol 12-myristate 13-acetate (PMA) dismissed the inhibition of catabolism and senescence in chondrocytes, and decreased the M2 macrophage polarization induced by rmMFG-E8 .

Human

L

apoptosis

34,031,369

Gene

0

1

0

1

0

1

0

TBK1

29110

protein coding

NPCs

--

Intervertebral disc degeneration

Prevent

SA--gal activity assay//TUNEL assay//Western blot

We markedly found that cells transfected with lentivirus-TBK1 (Lv- TBK1) remarkably exhibited less p16INK4a (p16) activity than cell transfected with lentivirus-Control (Lv-Ctrl) at passage 15, show- ing that TBK1 exerted an enormous function on NPCs senescence. The p16 and cleaved caspase-3 (CC3) expression levels were assessed to evaluate the TBK1-mediated anti-apoptotic and anti- senescence. Therefore, the substantial results in our study showed that TNF-a significantly increased in the NPCs and the expressions level of p16 and CC3, suggesting that TNF-a induced apoptosis and premature senescence, which were inhibited by Lv-TBK1. From the TUNEL staining and SA-b-gal staining, we could observe that TNF-a stimulation markedly increased the positive points of TUNEL and SA-b gal. Conversely, administration of Lv- TBK1 significantly reversed the TNF-1a induced excessive senes- cence and apoptosis.

--

Human

L

apoptosis

34,026,282

Gene

0

1

0

1

0

1

0

CDK4

1019

protein coding

BxPC-3,Panc-1,MiaPaCa-2

--

Aging

Prevent

SA--gal activity assay

The ¦Â-galactosidase assay was used to determine senescence following treatment with LEE011 and MEK162. Combined inhibition resulted in a significant increase in ¦Â-galactosidase staining when compared to monotherapy in all cell lines, suggesting that combined inhibition of CDK4/6 and MEK with LEE011 and MEK162, respectively, reduces proliferation through the induction of cellular senescence rather than through pro-apoptotic mechanisms in PDAC cell lines.

pERK

Upregulation

Western blot

We observed reduced phosphorylated ERK (pERK) levels at all doses and in a time-dependent manner. Finally, ctreatment with MEK162 and LEE011 alone and in combination resulted in suppressing both pERK and pRb levels in all cell lines.

--

Human

L

apoptosis

34,001,634

Gene

0

1

0

CDK6

1021

protein coding

BxPC-3,Panc-1,MiaPaCa-2

--

Aging

Prevent

EdU cell proliferation assay//Flow cytometry//SA--gal activity assay

The ¦Â-galactosidase assay was used to determine senescence following treatment with LEE011 and MEK162. Combined inhibition resulted in a significant increase in ¦Â-galactosidase staining when compared to monotherapy in all cell lines, suggesting that combined inhibition of CDK4/6 and MEK with LEE011 and MEK162, respectively, reduces proliferation through the induction of cellular senescence rather than through pro-apoptotic mechanisms in PDAC cell lines. In BxPC-3 cells, a significant decrease in proliferation was seen with individual treatment with LEE011 and MEK162 as well as the combination treatment. Compared to single-agent treatment, the combination of LEE011 and MEK162 resulted in a significant decrease in proliferation in Panc-1 and MiaPaCa-2 cells.In cell cycle analysis, BxPC-3, Panc-1 and MiaPaCa-2 cell lines displayed reduced S phase fraction and increased G0/G1 phase arrest, confirming inhibition of cell proliferation.

pERK

Upregulation

Western blot

We observed reduced phosphorylated ERK (pERK) levels at all doses and in a time-dependent manner. Finally, ctreatment with MEK162 and LEE011 alone and in combination resulted in suppressing both pERK and pRb levels in all cell lines.

--

Human

L

apoptosis

34,001,634

Gene

0

1

0

1

0

1

0

BRAF

673

protein coding

HPCs,LCH cells

--

Langerhans cell histiocytosis

Accelerate

ELISA//Immunofluorescence//RNA-seq//RT-PCR//SA--gal activity assay

HPCs isolated from BRAF-V600EScl+ mice were positive for SA¦ÂGal; and pro- duced SASP-associated proteins, all features consistent with a senescent state. LCH cells isolated from peripheral tissues of BRAF-V600EScl+ mice were also in a senescent state, as shown by reduced Ki-67 expression, expression of Cdkn2a, SA¦ÂGal activity and the production of SASP-associated proteins.Human BRAFV600E-transduced HPCs exhibited canonical markers of cellular senescence, including enlarged cellular size, positivity for SA¦ÂGal staining, senescence-associated heterochromatin foci and increased production of SASP cytokines. Gene expression profiling of transduced human BRAF-V600E+GFP+ HPCs confirmed the senescence signature16 and LCH lesions that formed in humanized mice reconstituted with human BRAFV600E CD34+ cells also had increased SA¦ÂGal activity and high p16INK4a levels.CD34+ BM cells isolated from patients with LCH also expressed a senescence signature, including increased expression of CDKN2A, CDKN2C, CDKN2D, CD9, MDM2 and genes encoding matrix metalloproteinases.

--

mTOR

Activation

ELISA//Flow cytometry

We first confirmed that inhibition of the mTOR pathway was sufficient to reduce the production of inflammatory cytokines by BRAF-V600E+ senescent HPCs in vitro. And in line with our hypothesis, SASP inhibition significantly reduced BRAF-V600E+ HPC skewing into MNPs in vitro. Rapamycin treatment also partially reduced excess MNP differen- tiation from BRAF-V600E GFP HPCs co-cultured with senescent BRAF-V600E+GFP+ HPCs, further establishing that mTOR inhi- bition reduced the release of SASP-associated molecules driving HPC-sustained MNP differentiation potential.

Human

HL

apoptosis

33,958,797

Gene

0

1

0

1

0

1

0

1

0

1

0

TNF

7124

protein coding

A375,HT29

--

Melanoma

Accelerate

SA--gal activity assay

Characteristic enlarged senescent cells containing blue-dyed precipitates indicating increased senescence-associated ¦Â- galactosidase activity appeared in both melanoma lines A375hTNFa and M4BeuhTNFa. Senescence induction was observed also in parental melanoma cells treated with recombinant human TNF¦Á protein. In colorectal carcinoma cells HT29hTNFa and HCT116h TNFa, no differences in the incidence of senescent cells were observed. HCT116 parental cells entered senescence spontaneously and more frequently in comparison to the HT29 cells.

TRAIL//IL6

Upregulation//Upregulation

qRT-PCR

CSCs- like markers remained unchanged In engineered melanoma cells A375hTNFa, significant overexpression of inflammatory cytokine IL6 was in- duced. Overexpression of TNF¦Á in cells HT29hTNFa significantly stimulated expression of apoptosis-inducing ligand TRAIL.

--

Human

L

apoptosis

33,957,885

Gene

0

1

0

ATG5

9474

protein coding

NPCs

--

Intervertebral disc degeneration

Prevent

SA--gal activity assay//Western blot

IL-1¦Â-increased percentage of senescence-associated beta-galactosidase (SA-¦Â-gal)-positive cells. Increased p16/INK4A, p21/WAF1/CIP1, and p53 expression, indicating senescence induction.

--

Human

L

apoptosis

33,921,398

Gene

0

1

0

1

0

DKC1

1736

protein coding

A549 cells,PC-9 cells

--

Aging

Prevent

SA--gal activity assay//Western blot

There was more senescence associated ¦Â-galactosidase positive cells in DKC1 silenced A549 and PC-9 cells, and the expression of cell senescence and apoptosis marker P21 and DNA damage marker ¦ÃH2A. X are higher in A549 and PC-9 cells with DKC1 downregulation compared to control cells.

TERC

Upregulation

SA-¦Â-gal activity assay//Western blot//qPCR//Spearman's rank correlation analysis

Both the level of TERC and telom- erase activity were significantly decreased in DKC1 silenced A549 and PC-9 cells. In accordance with this phenomenon, DKC1 downregulated A549 and PC-9 cells showed reduced length of tel- omere. There was more senescence associated ¦Â-galactosidase positive cells in DKC1 silenced A549 and PC-9 cells, and the expression of cell senescence and apoptosis marker P21 and DNA damage marker ¦ÃH2A. X are higher in A549 and PC-9 cells with DKC1 downregulation compared to control cells. Both telomerase RNA component (TERC) and telomerase reverse transcriptase (TERT) expression were positively correlated with DKC1 expression in TCGA datasets.

--

Human

HL

cellular senescence

33,879,171

Gene

0

1

0

1

0

CXCR2

3579

protein coding

LL2 cells,H460 cells

--

Lung cancer

Prevent

CCK-8 assay//SA--gal activity assay//Western blot//qRT-PCR

Selective inhibitor of CXCR2, SB225002, was confirmed to inhibit the proliferation of lung cancer cells in a both time-dependent and dose-dependent manner. To investigate the role of CXCR2 in tumor cell senescence, the expres- sion of SA-¦Â-gal was detected while using SB225002 on LL2 and H460 cells. After 24 h, the result showed that the percentage of ¦Â-gal-positive cells in SB225002 group was higher than that of control group. The senescence-associated markers p16 and p21 were up- regulated after using SB225002 and down-regulated after using CXCL2.

--

p38-ERK MAPK

Activation

Western blot

After using SB225002 and CXCL2 on LL2 cells, p38/ERK MAPK signaling pathway was found involved in CXCR2-associated signaling pathway. The levels of p38 and ERK proteins phosphorylation were obviously al- tered, whereas p-JNK almost remained unchanged during activation or inhibition of CXCR2.

Human

HL

apoptosis

33,814,009

Gene

0

1

0

1

0

1

0

1

0

BMP5

653

protein coding

Chondrocytes

--

Osteoarthritis

Accelerate

SA--gal activity assay//Western blot//qRT-PCR

We then performed SA-¦Â-gal staining assay to determine the role of BMP5 in OA-related chondrocyte senescence. IL-1¦Â-treated BMP5 knockdown chondro- cytes showed significantly lower SA-¦Â-gal activity than the corresponding controls. Western blotting analysis showed that p16, p21, HMGB1, and p53 protein levels were significantly reduced in the IL-1¦Â-treated BMP5- silenced chondrocytes compared to the corresponding controls. Next, we analyzed ¦ÃH2AX staining to determine if BMP5 knockdown reduced premature senescence of the chondrocytes. We observed that the relative area of ¦ÃH2AX staining was significantly lower in the IL-1¦Â- treated BMP5-depleted chondrocytes compared to the IL-1¦Â-treated control chondrocytes. We also demonstrated that p16 and p21 protein levels were significantly lower in the knee joint cartilage tissues from the DMM+LV-siBMP5 group mice compared to those from the DDM+LV-siNC group mice.

--

p38-ERK

Activation

Western blot

We analyzed the status of the p38/ERK signaling pathway in control and BMP5-silenced chondrocytes subjected to IL-1¦Â treatment by western blotting. The levels of total and phosphorylated p38 and ERK1/2 proteins were significantly higher in the IL-1¦Â- treated control chondrocytes compared to the IL-1¦Â treated BMP5-silenced chondrocytes. Moreover, the levels of p38 and ERK1/2 proteins were significantly increased in a dose-dependent manner by incubating chondrocytes with recombinant BMP5.

Human

L

apoptosis

33,744,859

Gene

0

1

0

1

0

1

0

CRY1

1407

protein coding

UMUC3 Res cells,EJ Res cells

--

Bladder cancer

Prevent

SA--gal activity assay//Western blot//qPCR

After CRY1 knockdown, p21 mRNA expression was significantly downregulated, but its protein expression was not significantly changed in both Res cells. The p53 mRNA expression did not change significantly in EJ Res cells, but was significantly increased in UMUC3 Res cells. Moreover, its protein expres- sion was significantly enhanced after CRY1 knockdown in both cell lines. The si-CRY1-1 demonstrated a higher efficiency compared with the si CRY1 2 to inhibit CRY1 expression. Therefore, the si-CRY1-1 was used in subsequent experiments. It was identified that CRY1 knock- down induced apparent senescence in the PTX-treated Res cells.

P53

Downregulation

Western blot//qPCR

The p53 mRNA expression did not change significantly in EJ Res cells, but was significantly increased in UMUC3 Res cells. Moreover, its protein expres- sion was significantly enhanced after CRY1 knockdown in both cell lines.

--

Human

L

apoptosis

33,650,658

Gene

0

1

0

1

0

1

0

MIR340

442908

ncRNA

HT22

--

Alzheimer's disease

Prevent

MTT assay//SA--gal activity assay

Compared with untreated HT22 cells, the viability of A¦Â42 induced cell viability was decreased, the apoptotic rate was increased, and intracellular ¦Â galactose deposition was increased, which suggested accelerated cellular senescence, but these signs were alle- viated after POT1 downregulation.£¨É¾³ý£¿£©Compared with cells in the A¦Â42 group, cell viability was increased, apoptosis rate was decreased, and intracellular ¦Â galactose deposition was reduced after overexpression of miR-340-5p.

POT1

Downregulation

Dual-Luciferase reporter assay//qRT©\PCR//SA-¦Â-gal activity assay

A dual luciferase reporter vector was con- structed to validate the targeted binding relationship between miR 340 5p and POT1. MiR-340-5p was overexpressed using agomiRNA in A¦Â42 induced HT22 cell, and intracellular POT1 level was decreased after overexpression of miR-340-5p. Compared with cells in the A¦Â42 group, cell viability was increased, apoptosis rate was decreased, and intracellular ¦Â galactose deposition was reduced after overexpression of miR-340-5p. TRF analysis showed that cel- lular telomere length was significantly increased.

--

Human

L

cellular senescence

33,624,913

Gene

0

1

0

1

0

SIRT6

51548

protein coding

LFH cells

--

Lumbar spinal stenosis

Prevent

SA--gal activity assay//qRT-PCR

SIRT6 overexpression significantly reduced the frequency of SA-¦Â-gal-positive LFH cells to 11.8%. Trends in the expression of senescence- related genes including p16INK4a, MMP3, and long interspersed nuclear element 1 were consistent with frequencies of SA-¦Â-gal-positive cells, with the exception of L1, which was inhibited in pSIRT6 and sh- hTERT co-transduced cells, while the same was not true for p16 or MMP3.

hTERT

Upregulation

SA-¦Â-gal activity assay//qRT-PCR

SIRT6 overexpression significantly reduced the frequency of SA-¦Â-gal-positive LFH cells to 11.8%, whereas hTERT knockdown increased this frequency to 28.4%. Importantly, hTERT knockdown in SIRT6-overexpressing cells reversed the beneficial impact of SIRT6 overexpression and increased the frequency of SA-¦Â-Gal positive LFH cells to 26.2%. Trends in the expression of senescence- related genes including p16INK4a, MMP3, and long interspersed nuclear element 1 were consistent with frequencies of SA-¦Â-gal-positive cells, with the exception of L1, which was inhibited in pSIRT6 and sh- hTERT co-transduced cells, while the same was not true for p16 or MMP3.

--

Human

HL

cellular senescence

33,568,575

Gene

0

1

0

1

0

MIR130A

406919

ncRNA

SH-SY5Y

--

Aging

Prevent

Western blot

Overexpression of miR-130a in D-galactose (D-gal)-induced SH-SY5Y cell senescence model attenuated D-gal-induced impaired autophagy and cell senescence, demonstrated by decreased levels of LC3, Ac-p53, p21 and increased p62

--

Human

L

cellular senescence

33,964,346

Gene

0

1

0

AHNAK

79026

protein coding

BJ cells

--

Aging

Prevent

SA--gal activity assay

To induce senescence by Nutlin-3, BJ cells were seeded at low density, and then transfected with indicated siRNA. Subsequently, cells were left treated with DMSO or Nutlin-3 (20 mM) for indicated days, and SA-b galactosidase assay is performed as described previously.

53BP1

Binding

IP//Western blot

Western blot (WB) using anti-mCherry and anti-AHNAK antibodies after immunoprecipitation (IP) with IgG or Anti-AHNAK beads as indicated.

p53

Downregulation

Western blot

Immunoblot analysis of BJ fibroblast following transfection with control or indicated siRNA. Cell lysates prepared from untreated and NCS-treated cells were analyzed using immunoblot using the specified antibodies.

Human

HL

apoptosis

33,961,796

Gene

0

1

0

CD276

80381

protein coding

HCT116,RKO

--

Colorectal cancer

Prevent

CCK-8 assay//Colony formation assay//Flow cytometry//SA--gal activity assay//Western blot

Western blot analysis of p21 and p53 in sh-NC cells or shB7-H3 CRC cells treated with DOX. SA-¦Â-Gal activity of sh-NC cells or shB7-H3 CRC cells treated with DOX was examined. Cell viability in sh-NC cells or shB7-H3 CRC cells treated with DOX after 24, 36, 48, and 96 h was examined by CCK-8 assays. The colony formation of shNC cells or shB7-H3 CRC cells treated with DOX was examined. Cell cycle analysis by PI staining in sh-NC cells or shB7-H3 CRC cells with DOX treatment was examined through flow cytometry.

TM4SF1

Upregulation

RT-qPCR//Western blot

The mRNA expression of TM4SF1 in EV cells or B7-H3 CRC cells treated with DOX. Western blot analysis of TM4SF1 and p21 in sh-NC cells or shB7-H3 CRC cells treated with DOX. Western blot analysis of TM4SF1 and p21 in EV cells or B7-H3 CRC cells treated with DOX.

AKT-TM4SF1-SIRT1

Upregulation

Western blot

The western blot results showed that B7-H3 knockdown decreased SIRT1 protein expression, while TM4SF1 overexpression reversed the effect of B7-H3 knockdown on SIRT1 expression in low-dose DOX induced senescent HCT116 and RKO cells. Moreover, B7-H3 overexpression increased SIRT1 protein expression, while TM4SF1 knockdown abolished the effect of B7-H3 overexpression on SIRT1 expression in low-dose DOX-induced senescent CRC cells.

Human

HL

delay aging

33,958,586

Gene

0

1

0

1

0

1

0

1

0

1

0

MIR27B

407019

ncRNA

B-MSC

--

Osteoarthritis

Accelerate

SA--gal activity assay

Western blot analysis, enzyme-linked immunosorbent assay, and¦Â-galactosidase activity assay were conducted with the results showing that knockdown of miR-27b-3p promoted expression of the osteogenic differentiation markers while inhibiting expression of the adipogenic differentiation markers, inflammatory factors, and cellular senescence of bone marrow mesenchymal stem cells (BMSCs).

KDM4B

Downregulation

Luciferase reporter assay//CHIP

After that, the interactions between miR-27b-3p, lysine Demethylase 4B (KDM4B), and Distal-Less Homeobox 5 (DLX5) identified using dual-luciferase reporter gene assay and ChIP assay revealed that miR27b-3p inhibited KDM4B and further reduced expression of DLX5.

--

Human

HL

cellular senescence

32,856,377

Gene

0

1

0

MTX2

10651

protein coding

Primary Fibroblast

--

Mandibuloacral dysplasia

Prevent

SA--gal activity assay

Senescence was examined in cells by a senescence associated ¦Â-galactosidase assay following the manufacturer¡¯s protocol (Cell Signaling #9860).

MTX1

Upregulation

Western blot

Immunoblot analysis of MTX2 and other outer and inner mitochondrial membrane proteins of whole-cell extracts from healthy control (WT) and patients¡¯ (MADM2, MADM3)fibroblasts. Actin or REVERT total proteins were used as loading controls. n = 3 independent experiments for MTX2, TOMM22, SAMM50, CHCHD3 and n = 2 independent experiments for MTX1, VDAC2, TOMM40.

--

Human

HL

apoptosis

32,917,887

Gene

0

1

0

MIR29A

407021

ncRNA

HMSC

--

Aging

Prevent

SA--gal activity assay//Western blot

Senescence-associated-¦Â-galactosidase (SA-¦Â-gal) staining. Compared with the siGFP-transfected group, siDGCR8-transfected hMSCs showed increased numbers of The percentage of SA-¦Â-gal-positive cells was significantly higher in siDGCR8-transfected hMSCs. Western blot showing the protein levels of tumour suppressors and senescence markers, including p21, p53, and p-p53.

DNMT3A

Downregulation

Luciferase reporter assay//RT-qPCR//Western blot

293T cells were co-transfected with luciferase reporters carrying either the wild-type DNMT3A 3¡ä UTR (Dnmt3a) or the mutagenised DNMT3A 3¡ä UTR (mDnmt3a), as well as 50 nM negative control mimic (miR-Control) or miR-29a-3p or/and miR-30c-5p mimics. The mRNA levels of DNMTs were measured by quantitative real-time PCR. Immunoblot showing DNMT3A protein levels in DGCR8 knockdown cells with or without miR-29a-3p or/and miR-30c-5p forced expression (bottom).

--

Human

HL

cellular senescence

32,961,441

Gene

0

1

0

1

0

MIR30C1

407031

ncRNA

HMSC

--

Aging

Prevent

SA--gal activity assay//Western blot

Senescence-associated-¦Â-galactosidase (SA-¦Â-gal) staining. Compared with the siGFP-transfected group, siDGCR8-transfected hMSCs showed increased numbers of The percentage of SA-¦Â-gal-positive cells was significantly higher in siDGCR8-transfected hMSCs. Western blot showing the protein levels of tumour suppressors and senescence markers, including p21, p53, and p-p53.

DNMT3A

Downregulation

Luciferase reporter assay//RT-qPCR//Western blot

293T cells were co-transfected with luciferase reporters carrying either the wild-type DNMT3A 3¡ä UTR (Dnmt3a) or the mutagenised DNMT3A 3¡ä UTR (mDnmt3a), as well as 50 nM negative control mimic (miR-Control) or miR-29a-3p or/and miR-30c-5p mimics. The mRNA levels of DNMTs were measured by quantitative real-time PCR. Immunoblot showing DNMT3A protein levels in DGCR8 knockdown cells with or without miR-29a-3p or/and miR-30c-5p forced expression (bottom).

--

Human

HL

cellular senescence

32,961,441

Gene

0

1

0

1

0

PIK3R3

8503

protein coding

LoVo,SW48

--

Colorectal cancer

Prevent

CCK-8 assay//Flow cytometry//SA--gal activity assay//Western blot

WB of PIK3R3 and p21 both in LoVo and SW48 cells transfected with siNC, siPIK3R3#1, and siPIK3R3#2. CCK8 assays of LoVo and SW48 cells transfected with PIK3R3-specific siRNA. Cell cycle distribution of LoVo and SW48 cells transfected with siNC, siPIK3R3#1, and siPIK3R3#2 was measured byflow cytometry. SA-¦Â-gal staining of PIK3R3-knockdown LoVo or SW48 cells after treatment of Dox (0.25¦ÌM, 48 h).

P53

Binding

IP

Immunoprecipitation assays were performed for LoVo cells with antiPIK3R3 or anti-p53 antibodies.

p53-p21

Downregulation

CHIP//Western blot

ChIP assays of p53 binding to the CDKN1A promoter region in LoVo transfected with PIK3R3-expressing plasmid. WB of PIK3R3, p53, and p21 in LoVo and SW48 cells transfected with PIK3R3 plasmid or/and His-p53 plamid.

Human

L

cellular senescence

32,973,127

Gene

0

1

0

1

0

1

0

1

0

POLR3A

11128

protein coding

fibroblast

--

Wiedemann-Rautenstrauch syndrome (WRS)

Prevent

Immunostaining//SA--gal activity assay//Telomere length assay//Western blot

Beta-galactosidase staining (pH 6) was evident at PD 45 in controls, at PD 27 in HGPS, and at PD 36 in WRS fibroblasts (A). Nuclear morphology analyzed by Lamin A immunostaining is shown for HGPS cells (C), control cells (D), and WRS (E) cells. Relative telomere length is shown in (G).

--

Human

L

cellular senescence

32,976,914

Gene

0

1

0

1

0

1

0

1

0

CDKN2B-AS1

100048912

ncRNA

HEK293T,HTM

--

POAG

Prevent

PCR//SA--gal activity assay

The human cellular senescence and extracellular matrix and adhesion molecules RT2 Profiler PCR arrays showed differential gene expression profile in TM cells after 10 nM CDKN2B-AS1 siRNA and control siRNA treatment for 72 h. Senescence-related¦Â-galactosidase staining of TM cells after 10 nM CDKN2B-AS1 siRNA and control siRNA treatment.

CDKN2B

Downregulation

immunocytochemistry//RT-PCR

A marked increase in CDKN2B expression was also observed in TM cells after 10 nM CDKN2B-AS1 siRNA treatment as assessed by immunocytochemistry and RT-PCR.

--

Human

HL

cellular senescence

32,825,664

Gene

0

1

0

1

0

FOXO4

4303

protein coding

UC-MSC

--

Aging

Accelerate

CCK-8 assay//SA--gal activity assay

Senescence of hUC-MSCs detected by ¦Â-galactosidase staining. Cell viability assayed by CCK-8.

--

Human

L

apoptosis

32,820,304

Gene

0

1

0

1

0

LMNA

4000

protein coding

HSF,MEF

--

HGPS

Prevent

SA--gal activity assay//Western blot

Representative images of SA-¦ÂGal staining in HSFs (P17) treated with the indicated siRNAs. Western blot analysis of METTL14, p21, and p16 levels in MEFs (P3) treated with the indicated siRNAs.

METTL3/14

Binding

Co-IP//Western blot

Co-IP and Western blot analysis of the endogenous interaction between Lamin A and METTL14 and Lamin A and METTL3 in MEFs.

--

Human

L

delay aging

32,813,328

Gene

0

1

0

1

0

MIR483

619552

ncRNA

ADSC

--

Aging

Accelerate

SA--gal activity assay

SA-¦Â-gal staining of hADSCs (P14) transfected with 483-3p-M and corresponding control.

IGF1

Downregulation

RT-qPCR//ELISA

Efficiency of IGF1 inhibition was assayed by RT-qPCR and ELISA.

--

Human

HL

cellular senescence

32,805,717

Gene

0

1

0

CLOCK

9575

protein coding

HMSC

--

Aging

Prevent

Clonal expansion assay//RT-qPCR//SA--gal activity assay

Increased SA-¦Â-gal activity, decreased proliferative potential, and decreased percentage of S-phase cells were detected in CLOCK¨C/¨C hMSCs. CLOCK deficiency resulted in the enhancement of DNA damage response and SASP. we observed downregulated transcription levels of LMNB1 and TMPO and upregulated mRNA expression of the aging markers CDKN1A (P21) and CDKN2A (P16) in CLOCK¨C/¨C hMSCs.

Lamin B1,Emerin,KAP1

Binding

Co-IP

Co-immunoprecipitation (Co-IP) assays verified these new interactions between CLOCK and nuclear lamina/heterochromatin-associated proteins in HEK293T cells and hMSCs.

--

Human

HL

cellular senescence

32,737,416

Gene

0

1

0

1

0

1

0

SIRT1

23411

protein coding

NP

--

IVDD

Prevent

JC-1 probe staining//ROS staining//SA--gal activity assay//Western blot

The degree of senescence of the NP cells subjected to 0% or 20% (high-magnitude) compressive deformation was analyzed using SA-¦Â-Gal staining (200¡Á). The SIRT1, PGC1¦Á and senescence-related protein (AMPK, P53, P21 and P16) levels were analyzed by Western blotting. After high-compression or high-compression plus Ad-SIRT1 treatment, the ROS level in NP cells was observed and measured by fluorescence microscopy (200¡Á) and flow cytometry following DCFH-DA fluorescent probe staining. JC-1 probe staining (Beyotime, China) was utilized to detect the mitochondrial membrane potential of NP cells.

--

Human

L

cellular senescence

32,687,063

Gene

0

1

0

1

0

1

0

1

0

MIR185

406961

ncRNA

HTC75,HFF,MRC5

--

Aging

Accelerate

Immunofluorescence//SA--gal activity assay//TRF assay//qRT-PCR

Immunofluorescence and fluorescence in situ hybridization (IF-FISH) were performed in POT1 knockdown, miR-185 overexpression and POT1 rescue HTC75 cells with the indicated¦Ã-H2AX antibody and the TTAGGG telomere probe. Telomere restriction fragment (TRF) analysis showed the telomere length of HTC75 cells stably infected with the indicated viruses at the indicated population doublings. Either POT1 knockdown or miR-185 overexpression in HFF cells significantly increased CDKN1A (P21) and CDKN2A (P16) expression levels. Senescence-associated ¦Â-galactosidase (SA-¦Â-gal) staining was performed to detect senescent cells.

POT1

Upregulation

Dual-luciferase reporter assay//qRT-PCR//Western blot

By using dual luciferase reporter assay, we compared the effects of miR-185 overexpression on wild-type POT1 (wPOT13¡¯-UTR) or mutant POT1 (mPOT1-3¡¯-UTR) luciferase activity. We stably transfected miR-185 into HTC75 cells and observed a decrease in POT1 mRNA levels. Consistent with the change in mRNA levels, POT1 protein levels were also decreased after miR-185 overexpression.

ATR

Downregulation

Western blot

The total ATR protein level reduced upon miR-185 overexpression.

Human

L

telomere attrition

32,687,062

Gene

0

1

0

1

0

1

0

1

0

ERG

2078

protein coding

LNCaP

--

Aging

Prevent

SA--gal activity assay

Analysis of cellular senescence by senescence-associated (SA)-b-galactosidase (Gal) activity measurement in LNCaP cells after treatment with 0.5 mM daunorubicin or DMSO as control.

p53

Upregulation

Western blot//Immunofluorescence microscopy

Protein levels of p53 and ERG in control vector and DN-ERG transfected normal human prostatic epithelial cells (hPREC) and LNCaP cells were analyzed by immunoblotting. ERG and p53 expression were evaluated by immunofluorescence microscopy in LNCaP cells transfected with control vector or DN-ERG for 48 h.

--

Human

HL

apoptosis

32,674,955

Gene

0

1

0

MIB1

57534

protein coding

HCT116,HEK293T

--

Aging

Accelerate

SA--gal activity assay

Cellular senescence was detected by senescence-associated ¦Â-galactosidase (SA-¦Â-Gal) assay.

WRN

Binding

Co-IP

The protein was extracted for co-immunoprecipitation (co-IP) to detect the interaction between WRN and MIB1.

--

Human

L

cellular senescence

32,652,764

Gene

0

1

0

MIR34A

407040

ncRNA

HADMSC

--

Aging

Accelerate

RT-PCR//SA--gal activity assay//Western blot

The activity of senescence associated¦Â¨C galactosidase (SA-¦Âgal) was assessed by histochemical staining. Moreover, the senescence associated molecular alterations including that of pro-senescence (P53, P21 and P16) and anti-senescence (SIRT1, HTERT and CD44) genes were examined by quantitative RT-PCR and western blot assays.

--

Human

L

delay aging

32,634,429

Gene

0

1

0

1

0

1

0

IQGAP1

8826

protein coding

VMSC

--

Aging

Prevent

SA--gal activity assay

Indeed, we observed an increased number of cells with elevated activity of senescence-associated ¦Â-galactosidase (SA-¦Â-gal)¨C a most commonly used senescence marker.

--

Human

L

altered intercellular communication

32,592,713

Gene

0

1

0

NSD2

7468

protein coding

IMR-90

--

Aging

Prevent

Flow cytometry//SA--gal activity assay

Cell cycle analysis by propidium iodide staining revealed that the population of cells in G2/M phase was slightly increased on day 6 in NSD2-KD cells. Furthermore, NSD2-depleted cells exhibited SA-¦Â-Gal staining starting on day 3 after siRNA transfection.

--

RB

--

Knockdown

Knockdown of RB completely abolished the reduced expression of RB-associated genes such as MCM3, LMNB1, and EZH2 in NSD2-KD cells.

Human

HL

delay aging

32,573,059

Gene

0

1

0

1

0

RTEL1

51750

protein coding

fibroblast

--

HHS

Prevent

Southern blot

Southern blot and in-gel hybridization analyses were done as described (23) except for the telomeric oligonucleotide probe, which was (AACCCT)3.

--

Human

L

cellular senescence

32,542,379

Gene

0

1

0

SIRT7

51547

protein coding

HMSC

--

Aging

Prevent

Clonal expansion assay//RT-qPCR//SA--gal activity assay//Western blot

We also observed increased percentages of senescence-associated ¦Â-galactosidase (SA-¦Âgal)-positive cells, compromised clonal expansion ability, decreased ratios of Ki67-positive cells and reduced percentages of S-phase cells in SIRT7?/? relative to SIRT7+/+ hMSCs. Consistent with the accelerated senescence phenotypes, we observed the upregulation of cyclin-dependent kinase inhibitors and aging markers CDKN1A (P21) and CDKN2A (P16) at both mRNA and protein levels, along with transcriptional downregulation of LMNB1 and TMPO in SIRT7?/? hMSCs.

LINE1

Binding

ChIP-qPCR

Using ChIP-qPCR assay, we detected that SIRT7 bound specific LINE1 promoter regions in SIRT7+/+ but not in SIRT7?/? hMSCs.

cGAS-STING

Downregulation

Western blot

Activation of the cGAS-STING pathway was evident in SIRT7deficient hMSCs, as detected by increased levels of phosphorylated forms of NF-¦ÊB P65, TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3).

Human

L

cellular senescence

32,504,224

Gene

0

1

0

1

0

1

0

1

0

ZKSCAN3

80317

protein coding

HMSC

Muscles of nude mice

Aging

Prevent

Clonal expansion assay//Ki67 staining//SA--gal activity assay//Western blot//qPCR

SA-¦Â-gal staining of ZKSCAN3+/+ and ZKSCAN3-/-hMSCs (P10). Clonal expansion ability analysis of ZKSCAN3+/+ and ZKSCAN3-/- hMSCs (P10). Telomere length analysis of ZKSCAN3+/+ and ZKSCAN3-/- hMSCs (P10) by qPCR. Western blot analysis of P16, P21 in ZKSCAN3+/+ and ZKSCAN3-/- hMSCs (P10). Immunostaining of Ki67 in ZKSCAN3-/- hMSCs (P8) transduced with lentiviruses expressing luciferase (Luc) or ZKSCAN3.

Lamin B1,LBR,KAP1,HP1¦Á

Binding

Co-IP//Western blot

Co-IP analysis showing that exogenous ZKSCAN3 interacts with Lamin B1, LBR, KAP1 and HP1? proteins in HEK293T cells. Western blot analysis of the expression levels of Lamin B1, LBR, KAP1 and HP1? proteins in ZKSCAN3+/+ and ZKSCAN3-/- hMSCs (P10).

--

Human

HL

abnormal gene expression

32,427,330

Gene

0

1

0

1

0

1

0

1

0

1

0

MFN2

9927

protein coding

Chondrocyte

--

Osteoarthritis

Accelerate

Flow cytometry//SA--gal activity assay

Representative microscopy images of senescence-associated b-galactosidase activity. Flow cytometry analysis of mitochondrial ROS level of NC and siMfn2#3 chondrocytes.

PARKIN

Binding

Western blot//Co-IP

The protein expression of PARKIN, MFN2 in NC siRNA, PARKIN siRNA#1, #2, #3-treated chondrocytes. Chondrocytes were immunoprecipitated with anti-MFN2 or control IgG, the immunoprecipitated complex was detected by Western blotting with anti-PARKIN.

--

Human

L

mitochondrial dysfunction

32,416,221

Gene

0

1

0

1

0

FBP1

2203

protein coding

HSC

--

Aging

Accelerate

IHC staining//SA--gal activity assay

We detected significant numbers of SA-¦Â-Gal+ cells in DEN/Cre livers through senescence-associated¦Â-galactosidase (SA-¦Â-Gal) staining. Representative SA-¦Â-Gal and CD140B IHC staining of cryosections from mouse liver sections at 36 weeks. Representative p21 and FOXO4 IHC staining of mouse liver sections at 36 weeks.

HMGB1

Upregulation

IF//Immunoblotting

We therefore treated DEN/GFP or DEN/Cre animals with inflachromene (ICM), a small molecule shown to block HMGB1 release45. ICM treatment greatly reduced not only surface tumours, but also microscopic lesions in DEN/Cre mice. ICM did not affect hepatic steatosis, based on comparable TG levels. As expected, cytosolic HMGB1 levels decreased while nuclear HMGB1 levels increased in ICM-treated livers. ICM also substantially reduced numbers of HSCs expressing SASP components.

--

Human

HL

cellular senescence

32,367,049

Gene

0

1

0

1

0

MIR21

406991

ncRNA

CD4+T,HEK293T

--

Aging

Prevent

Western blot//miRNA qRT-PCR//qRT-PCR

In the CD4+ T cells of D-gal treated mice, the level of miR-21was significantly decreased in the hPMSC-Exo miR-21 inhibitor treatment group compared with hPMSC-Exo group. Furthermore, the miRNA level of PTEN was markedly increased, while the mRNA levels of HO-1 and NQO1 were significantly decreased after hPMSC-Exo miR-21 inhibitor treatment. In addition, the aging-related protein expression of p53 and ¦Ã-H2AX were also improved significantly in the hPMSC-Exo miR-21 inhibitor treatment group compared with hPMSC-Exo group.

PTEN

Downregulation

qRT-PCR

The exosomes treated with different plasmids were co-cultured with CD4+?T cells, and the expression of miR-21 and PTEN was detected. The expression of miR-21 was markedly improved in CD4+?T cells after co-cultured with hPMSC-Exo compared PBS treatment group. Conversely, hPMSC-Exo treatment greatly decreased the expression of PTEN in CD4+?T cells compared with the PBS group. Moreover, hPMSC-Exo treated with miR-21 mimics further increasing the expression of miR-21 and decreasing the expression of PTEN in CD4+?T cells compared hPMSC-Exo treatment group, while the opposite trend appeared in the hPMSC-Exo treatment miR-21inhibitor group.

PTEN/PI3K-Nrf2

Activation

Western blot

The expression of PTEN significantly decreased in activated CD4+ T cells after cultured for 72 h, while hPMSC-Exo intervention further decreased the expression of PTEN in activated CD4+ T cells. Furthermore, the phosphorylation of PI3K and Akt were significantly increased in activated CD4+ T cells, and the hPMSC-Exo intervention further improved the phosphorylation of PI3K and Akt. In addition, we found that the expression of nuclear Nrf2 and downstream target antioxidant genes NQO1 and HO-1 was markedly improved in activated CD4+ T cells, and the hPMSC-Exo treatment further improved the expression of these proteins. Moreover, significantly improve nuclear Nrf2, NQO1, and HO-1 expression were also found in hPMSC-Exo treated CD4+ T cells after bpV(HOpic) supplementation.

Human

HL

cellular senescence

34,887,868

Gene

0

1

0

1

0

1

0

MIF

4282

protein coding

Primary Human NP Cells

Nucleus pulposus

Intervertebral disc degeneration

Prevent

Flow cytometry//SA--gal activity assay//Western blot

The SA-¦Â-Gal staining result indicated that MIF-KO did not affect the senescence rate of the NP cells, but HC loading significantly aggravated the senescence of NP cells. However, MIF-KO further exacerbated the HC loading-induced senescence of NP cells. Flow cytometry was then used to analyze the content of intracellular ROS in NP cells marked by DCFH-DA molecular probes, and the result revealed that knockout of MIF alone did not induce the accumulation of ROS. However, HC loading did increase the content of ROS in NP cells, and MIF-KO markedly exacerbated the ROS content of the HC loading-treated NP cells. In addition, the western blot analysis revealed that the expression of MIF was markedly eliminated by MIF-KO, but the expression of senescence-associated markers and oxidative stress-related marker (NT) was not affected by MIF-KO. HC loading depressed the expression of MIF compared with the control group and markedly enhanced the P53, P21, P16, and NT expression. Similarly, knockout of MIF in HC loading-treated NP cells further aggravated the expression of senescence-related and oxidative stress-related markers.

--

Human

L

mitochondrial dysfunction

34,306,312

Gene

0

1

0

1

0

1

0

UNC5B

219699

protein coding

HUVEC

--

Aging

Accelerate

CCK-8 assay//EdU assay//SA--gal activity assay//qRT-PCR

The proportion of senescent cells was lower among UNC5B-silenced cells than among the control. The mRNA expression of the SASP genes, i.e., IL-1¦Á, IL-1¦Â, IL-8, MCP-1, and ICAM-1, was significantly attenuated by inhibition of UNC5B. Further, the proliferation of control and UNC5B-silenced cells was assessed. As a result, UNC5B-silenced cells proliferated at a faster rate than the control cells based on the growth curve analysis and Edu incorporation assay. These results reveal that the downregulation of UNC5B ameliorated the senescence-associated phenotype. UNC5B-overexpressing cells displayed an increase in the proportion of SA-¦Â-gal-positive cells than control cells. The mRNA expression of the SASP genes, i.e., IL-1¦Á, IL-1¦Â, IL-8, MCP-1, and ICAM-1, was also significantly upregulated in UNC5B-overexpressing cells compared to that of the controls. Moreover, overexpression of UNC5B reduced total cell numbers according to growth curve analysis and inhibited cell proliferation potential as indicated by fewer Edu-positive cells.

--

P53

Activation

Western blot//EdU assay//CCK-8 assay

UNC5B-silencing cells showed downregulated protein expression levels of P53 and P21. For further clarification on the relationship between the P53 pathway and senescence induced by UNC5B, this work used PFT¦Á, a P53-specific inhibitor. The presence of PFT¦Á inhibited the upregulation of P53 induced by UNC5B overexpression and partially suppressed the expression of P21. Additionally, the presence of PFT¦Á reversed the decrease in the percentage of Edu-positive cells induced by UNC5B overexpression.

Human

L

cellular senescence

34,239,689

Gene

0

1

0

1

0

1

0

1

0

CARTPT

9607

protein coding

Primary cortical neurons

Cortex

AD

Prevent

Western blot//qRT-PCR

The western blotting results indicated that p16, TNF¦Á and IL-1¦Â protein expression levels were significantly increased in A¦Â1¨C42-treated primary cortical neurons, but were significantly decreased following pretreatment with CART. Furthermore, RT-qPCR results demonstrated that p16, p53, IL-6 and IL-1¦Â mRNA expression levels were significantly increased in A¦Â1¨C42-treated primary cortical neurons, but were significantly decreased following pretreatment with CART. SOD1 protein and SOD2 mRNA expression levels were significantly decreased in the A¦Â1¨C42-treated primary cortical neurons, but were significantly increased following pretreatment with CART.

--

Human

L

cellular senescence

33,610,183

Gene

0

1

0

1

0

SIRT3

23410

protein coding

NP Cells

Nucleus pulposus

Intervertebral disc degeneration

Prevent

SA--gal activity assay//Western blot

The proportion of SA©\¦Â©\Gal©\positive cells and p16INK4¦Á expression in the LV©\shSIRT3 + H2O2 group was significantly higher than those in the LV©\Ctrl and LV©\Ctrl + H2O2 groups, whereas overexpression of SIRT3 distinctly decreased the percentage of SA©\¦Â©\Gal©\positive cells and p16INK4¦Á expression. In addition, whether at the protein or messenger RNA (mRNA) level, p16INK4¦Á and SASP (such as IL©\1¦Â,IL©\6, MMP3, and MMP9) among groups of NPC exhibited a very similar trend, which was consistent with the results of SA©\¦Â©\Gal staining.

--

AMPK-PGC©\1¦Á

Activation

ROS Assay//Immunofluorescence//Western blot//SA-¦Â-gal activity assay//qRT-PCR

Furthermore, activation of AMPK in NPC noticeably reduced high the iNOS expression and ROS production caused by downregulation of SIRT3; conversely, the inhibition of AMPK in NPC counteracted the decline in iNOS expression and ROS production after SIRT3 overexpression. Additionally, western blot analysis showed that the expression of iNOS and the ratios of Ac©\SOD2/SOD2 in each group were similar to the results of iNOS immunofluorescence staining. In Figure S4F, we found that the SOD2 activity in the LV©\shSIRT3 + AICAR + H2O2 group was significantly higher than that in the LV©\shSIRT3 + H2O2 group, whereas, in the LV©\SIRT3 + Compound C + H2O2 group, its activity was obviously lower than that in the LV©\SIRT3 + H2O2 group. Subsequently, we observed that the activation of AMPK activity in NPC significantly reduced the number of SA©\¦Â©\Gal©\positive cells induced by LV©\shSIRT3; in contrast, the inhibition of AMPK activity in NPC evidently increased the number of SA©\¦Â©\Gal©\positive cells reduced by LV©\SIRT3. In addition, the results of immunofluorescence staining also illustrated that the activation of AMPK declined the expression of p16INK4¦Á, which is highly expressed in NPC by silencing SIRT3; in contrast, the inhibition of AMPK activity in NPC obviously increased the expression of p16INK4¦Á after LV©\SIRT3 intervention. Moreover, RT©\PCR and western blot results demonstrated that the expression changes of p16INK4¦Á and SASP (including IL©\1¦Â,IL©\6, MMP3, and MMP9) among the different groups showed consistency at the mRNA and protein levels.

Human

L

cellular senescence

33,565,085

Gene

0

1

0

1

0

EGR2

1959

protein coding

DS HMFs

--

Aging

Accelerate

ELISA//Immunofluorescence//qRT-PCR

Using multi©\parameter phenotypic analysis to control for off©\target effects, we identified ¡®EGR2 3¡¯ siRNA as the most potent siRNA transfected individually which significantly increased cell number and significantly reversed cell area, nuclear area and cell elongation. ¡®EGR2 2¡¯, the second most potent siRNA transfected individually, produced a modest increase in cell number and significantly reversed nuclear area and cell elongation. Finally, the least potent siRNA, ¡®EGR2 1¡¯, only significantly reversed cell elongation compared to the DS?+?siGLO control. Further characterisation of the changes to the senescence phenotype following ablation of the?EGR2?in DS HMFs revealed a significant down©\regulation of the SASP factor,?IL©\6, at the transcript level and at the secreted protein level.

ARF//p16

Upregulation

CHIP//Western blot

Cells transfected with the pGL3 ARF 800 or with the complete ARF promoter, pGL3 ARF 3.4, displayed a significant increase in luciferase activity following transfection with the EGR2 expression vector or E2F1 positive control, but not EGR1, EGR3 or EGR4 expression vectors, thus confirming EGR2 as a direct activator of the ARF promoter.Validation of the interaction between EGR2 and the ARF promoter was performed using chromatin immunoprecipitation (ChIP) on cross©\linked DNA from quiescent interleukin©\2 (IL©\2)©\dependent Kit225 human T©\lymphocytes, with low levels of ARF expression and Kit225 cells following IL©\2 activation which results in increased ARF expression.Subsequently, ChIP was performed with polyclonal antibodies against EGR2 or E2F1, which acted as a positive control. Addition of IL©\2 to Kit225 cells resulted in significantly increased binding of E2F1 and EGR2 to the ARF promoter, demonstrating that EGR2 can be detected at the endogenous ARF promoter.Furthermore, ChIP performed in EP and DS HMFs revealed significantly increased binding of EGR2 to the p16 promoter in DS HMFs, thus confirming that EGR2 can bind to the endogenous p16 promoter

p16-pRB//ARF-p53-p21

Activation

Immunofluorescence

Using immunofluorescence staining and high©\content analysis, we further examined p16 and p21 on a cellular level and found a significant increase in the proportion of double negative (p16? p21?) ¡®reversed¡¯ cells in DS HMFs following EGR2 knockdown in combination with?p21?siRNA (¡®DS?+?EGR2?+?p21?siRNA) compared to the DS?+?p21 siRNA HMFs, an increase similar to that seen in the reversed DS?+?p16?+?p21 HMFs.

Human

HL

cellular senescence

33,547,862

Gene

0

1

0

1

0

1

0

ITPR2

3709

protein coding

MRC5

--

Aging

Accelerate

EdU assay//RNA-seq//SA--gal activity assay//Western blot//qRT-PCR

To further confirm a link between the loss of Itpr2 and decreased levels of senescence, beyond the liver, we explored the replicative potential of Itpr2 KO- and WT-derived MEFs. Late passage Itpr2 KO MEFs showed a decreased level of cellular senescence, as illustrated by a decrease in the level of several cellular senescence markers, namely the senescence associated-¦Â-galactosidase activity (SA-¦Â-Gal) (Fig. 2g and Supplementary Fig. 2c), cell proliferation (Supplementary Fig. 2d), p16ink4a mRNA (Fig. 2h) and protein (Fig. 2i) levels and finally a reversion of the enhanced inflammatory response as evidenced by transcriptomic data

--

Human

HL

cellular senescence

33,526,781

Gene

0

1

0

1

0

1

0

1

0

1

0

DKC1

1736

protein coding

CD34+ cells

--

Dyskeratosis congenita

Prevent

TRAP assay//Western blot

These results showed marked decreases in the telomerase activity of CD34+ cells that had been transduced with iDKC1-, iDKC4-, or iDKC7-LVs, which showed values of 42.8%?¡À?19%, 61.5%?¡À?3.6%, and 43.7%?¡À?15.6%, respectively, of values determined in the control group. Analyses of ¦ÃH2AX foci in the nucleus of cells transduced with iDKC1-, iDKC4-, or iDKC7-LVs revealed that only 19% of cells transduced with the scrambled shRNA LV showed more than 10 ¦ÃH2AX foci per cell. However, an important increase in the proportion of CD34+ cells with ¦ÃH2AX foci was observed in cells transduced with either the iDKC1- (76%), iDKC4- (42%), or the iDKC7- (61%) LVs. As shown in Fig. ?Fig.2b,2b, phosphorylated p53 expression was higher in CD34+ cells transduced with the DKC1-shRNA LVs.

--

Human

L

cellular senescence

33,514,435

Gene

0

1

0

1

0