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CircMEG3 inhibits telomerase activity by reducing Cbf5 in human liver cancer stem cells

Circular RNA (CircRNA) is a newly identified special class of non-coding RNA (ncRNA) that plays an important regulatory role in the progression of certain diseases. Herein, our results indicate that CircMEG3 is downregulated expression and negatively correlated with the expression of telomerase-related gene Cbf5 in human liver cancer. Moreover, CircMEG3 inhibits the growth of human liver cancer stem cells in vivo and in vitro. CircMEG3 inhibits the expression of m6A methyltransferase METTL3 dependent on HULC. Moreover, CircMEG3 inhibits the expression of Cbf5, a component of telomere synthetase H/ACA ribonucleoprotein (RNP; catalyst RNA pseudouracil modification) through METTL3 dependent on HULC. Thereby, CircMEG3 inhibits telomerase activity and shortens telomere lifespan dependent on HULC and Cbf5 in human liver cancer stem cell. Strikingly, increased Cbf5 abrogates the ability of CircMEG3 to inhibit malignant differentiation of human liver cancer stem cells. In summary, these observations provide important basic information for finding effective liver cancer therapeutic targets.


INTRODUCTION
It has been found that stem cells can be differentiated into malignant cells under unfavorable microenvironments. 1 Although most of the current support for malignant tumors stems from the malignant transformation of stem cells, the mechanism of stem cell deterioration is still controversial. 2,3 For example, METTL3-eIF3h promotes stem cell deterioration, 4 and farnesoid X receptor (FXR) regulates the proliferation of small-intestinal cancer stem cells (CSCs). 5-7 C-Myc is related to the malignant differentiation of leukemia stem cells. 8 Studies have found that JAK/STAT is highly activated in tumor stem cells. 9 Studies have confirmed that liver CSCs are closely related to the recurrence of liver cancer. 10 It is not clear what causes the accumulation of genetic errors of stem cells and changes in telomere function, which eventually evolve into malignant stem cells.
Circular RNA (CircRNA) is a newly identified special class of noncoding RNA (ncRNA) that plays an important regulatory role in the progression of certain diseases (such as tumors). 11,12 CircRNA can play the role of miRNA sponge. 13,14 Moreover, loop interactions between flanking introns can promote reverse splicing, thereby promoting the production of CircRNA. 15 For example, CircFOXP1 acts as a molecular switch that regulates Wnt and EGFR by acting as a sponge pad. 16 hsa_circ_0072387 suppresses glycolysis of oral squamous cell carcinoma. 17 Circ0031288/hsa-miR-139-3p/Bcl-6 influences the invasion of cervical cancer HeLa cells. 18 Circ_001653 silencing promotes the cell proliferation. 19 CircRNACCDC66 regulates cisplatin resistance in gastric cancer via the miR-618/BCL2 axis. 20 Furthermore, Circ0000790 is involved in pulmonary vascular remodeling, 21 and CircRNF20 promotes breast cancer tumorigenesis and Warburg effect through miR-487a/HIF-1a/HK2. 22 Our previous research found that long ncRNA MEG3 can form circular MEG3 (CircMEG3), and it is lowly expressed in human liver cancer. MEG3 participates in the regulation of various growth, 23 for example, MEG3 silencing can induce mouse pluripotent stem cells. 23 MEG3 inhibits the activation of liver satellite cells, 24 and MEG3 as ceRNA regulates liver fat metabolism. 25 Studies have shown that the expression patterns of various transcriptional variants of MEG3 are tissue cell specific. For example, fetal liver cells express 12 MEG3 transcriptional variants. 26 Furthermore, the downregulated expression of MEG3 is related to gene hypermethylation. 27,28 MEG3 inhibits malignant proliferation of tumor cells dependent on P53. 29 In addition, MEG3 can also inhibit tumor growth in a P53-independent manner. [30][31][32][33] It is well known that RNA m6A modification regulates RNA splicing, translocation, stability, and translation into protein. HULC is deregulated in cancer and acts as the potential biomarker and therapeutic target. Cbf5 is a component of telomere synthase H/ACA ribonucleoprotein (RNP). Our studies indicate that, first, CircMEG3 is downregulated expression and inversely correlated with the expression of telomerase-related gene Cbf5 in human liver cancer. Second, RNA sequencing indicates that CircleMEG3 inhibits HULC, METTL3, and Cbf5 (data not shown). Moreover, Protein chip indicates HULC enhances METTL3 and Cbf5 (data not shown). In particular, HULC overexpression abrogates the actions of CircleMEG3 that inhibits METTL3 and Cbf5 (data not shown). Moreover, we have clearly demonstrated that MEG3 inhibits METTL3 through blocking HULC in human liver cancer (data not shown). In this study, we identify that CircMEG3 inhibits the expression of METTL3 dependent on HULC and therefore inhibits the expression of Cbf5 in human liver CSCs. Given that there are multiple functions of METTL3 in human cancers, we also have reasons to investigate whether CircMEG3 inhibits the expression of Cbf5 dependent on METTL3 by reducing the methylation modification of Cbf5 mRNA. Thereafter, we will consider whether CircMEG3 affects the telomere function dependent on Cbf5 in human liver CSCs.
In conclusion, we have explored the effect of CircMEG3 on malignant differentiation of human stem cells in vivo and in vitro and focused on the important role played by CircMEG3 in regulating telomere re-modeling. These studies will play an important role in finding effective tumor therapeutic targets.
CircMEG3 inhibits the expression of m6A methyltransferase METTL3 RNA m6A modification regulates RNA splicing, translocation, stability, and translation into protein. m6A is catalyzed by the RNA meth-yltransferase METTL3. HULC is deregulated in cancer and acts as the potential biomarker and therapeutic target. Moreover, we have demonstrated that linear MEG3 inhibits METTL3 through blocking HULC in human liver cancer (data not shown). To further explore the effect of CircMEG3 on the expression of m6A methyltransferase METTL3 via HULC in human liver CSCs, METTL3 expression was first detected in the rLV-Tet-on-CircMEG3-hLCSCs, and CircMEG3 was significantly increased with increasing DOX concentration in DOX groups (0, 0.5, 1, 1.5, and 2 mg/mL) (Figure 2A). The ability of RNA polymerase II to bind to the METTL3 promoter was significantly decreased with increasing DOX concentration ( Figure 2B). The ability of RNA polymerase II to enter the METTL3 promoterenhancer loop was significantly decreased with increasing DOX concentration ( Figure 2C). However, excessive HULC abolished this function of CircMEG3 ( Figure 2D). The binding capacity of RNA polymerase II to the METTL3 promoter probe was significantly decreased with increasing DOX concentration ( Figure 2E); however, excessive HULC abolished this function of CircMEG3 ( Figure 2F). The pEZX-MT-METTL3 promoter-Luc luciferase activity was significantly decreased with the increase of DOX concentration ( Figure 2H). The expression of METTL3 was significantly decreased with increasing DOX concentration ( Figures 2I and 2J). However, excessive HULC abolished this function of CircMEG3 (Figures 2K and 2L). Collectively, these results suggest that CircMEG3 inhibits the expression of METTL3 dependent on HULC in human liver CSCs.

DISCUSSION
CSCs in hepatocellular carcinoma are able to exclusively initiate tumorigenesis. [34][35][36] For example, LCSC-related mitochondrial metabolism contributes to the liver CSC features. 37 The Wnt/beta-catenin is believed to play an important role in the pathogenesis of CSC formation. 38 To date, the functions and regulatory mechanisms of CircMEG3 have not fully been elucidated in liver CSCs. We first demonstrate that CircMEG3 inhibits the growth of liver CSCs by inhibiting telomerase activity dependent on HULC and Cbf5 in human liver CSCs (Figure 7).  It is worth mentioning that our findings in this study provide novel evidence for a suppressor role of CircMEG3 in human liver cancer. This assertion is based on several observations: (1) CircMEG3 is downregulated expression and negatively correlated with the expression of telomerase-related gene Cbf5 in human liver cancer; and (2) CircMEG3 inhibits the growth ability of human liver CSCs in vitro and in vivo. A MEG3 acts as an antitumor component in different cancer cells, such as breast and liver cancer cells. 39 MEG3 activated by vitamin D suppresses glycolysis in cancer, 40 and MEG3 induces invasion of glioma cells via autophagy. 41 Moreover, MEG3 promotes differentiation of porcine satellite cells by sponging miR-423-5p 42 and is involved in pituitary tumor invasiveness. 43 In addition, MEG3 inhibits the inflammatory response of ankylosing spondylitis, 44 and MEG3 inhibits HMEC-1 cell growth and migration. 45 Also, MEG3 inhibits the progression of prostate cancer by facilitating H3K27 trimethylation, 46 and MEG3 knockdown attenuates endoplasmic reticulum stress-mediated apoptosis. 47 Furthermore, uric acid enhances autophagy through the MEG3/miR-7-5p/EGFR axis. 48 Interestingly, MEG3 interacts with miR-494 to repress bladder cancer progression through targeting PTEN, 49 and MEG3 binds with miR-27a to promote PHLPP2 protein translation and impairs bladder cancer invasion. 50 In addition, MEG3 inhibits breast cancer growth via upregulating endoplasmic reticulum stress. 51 Our present results are consistent with these reports and provide novel evidence for an active role of CircMEG3 in inhibiting the growth of liver CSCs. Importantly, our results suggest that CircMEG3 inhibits the expression of m6A methyltransferase METTL3 dependent on HULC in human liver CSCs. METTL3 is implicated in many aspects of tumor progression, including tumorigenesis, proliferation, and invasion, 52 and promotes the progression of prostate carcinoma via mediating MYC methylation 53 and enhances cell adhesion through stabilizing integrin b1. 54 Moreover, m6A-dependent glycolysis enhances colorectal cancer progression. 55 Evidentially, our findings provide novel evidence that CircMEG3 inhibits the expression of Cbf5 through METTL3 dependent on HULC in human liver CSCs. A single H/ACA small nucleolar RNA mediates tumor suppression downstream of oncogenic RAS, [56][57][58] and H/ACA box small nucleolar RNA 7B acts as an oncogene and a potential prognostic biomarker in breast cancer. 59 Our present results are consistent with these reports and provide novel evidence for an active role of CircMEG3 liver CSCs.
Notably, our results suggest that CircMEG3 inhibits telomerase activity dependent on HULC and Cbf5 in human liver CSCs. Studies in telomere-related protein complexes include TRF1, TRF2, Rap1, POT1, TIN2, etc. [60][61][62] The formation of the T loop of the granules inhibits the ATM-mediated DNA damage response. 63 Telomerase core components include telomerase reverse transcriptase (TERT) and TERC. 64,65 Moreover, telomerase is involved in stem cell selfrenewal. 66,67 Studies have shown that mammalian cell telomeres exhibit high levels of histone H3K9me3 and H4K20me3 modifications, 68 and telomeres can rely on RNA polymerase II to generate long-chain ncRNA TERRA. 69,70 It was found that TERRA deletion would result in a reduction in the apparent modification of H3K9me3 at the telomeres. 71 In particular, the 5 0 -UUAGGG-3 0 repeat sequence of TERRA can bind to TERC through base-pairing, which competitively inhibits the activity of telomerase. 72,73 In recent years, new phenomena that regulate cell telomere function have been discovered, such as selective extension of telomere through DNA break-induced replication mechanism. 74,75 Telomere shieldin complex mediates P53BP1-dependent DNA repair, 76 and telomere necrosis activates autophagic death. 77,78 What kind of factor can dynamically control telomere remodeling at the spatiotemporal level and control the fate of cells needs to be further investigated.
Furthermore, our study found that CircMEG3 inhibited the expression and function of the telomerase component Cbf5 in human liver CSCs, thereby inhibiting the lifespan of telomeres in liver CSCs. It is worth noting that mutations in the Cbf5 protein in H/ACA RNP that catalyzes the modification of RNA pseudouracil and the synthesis of telomeres will cause shortening of telomeres. 79 Studies have shown that TCAB1 is a component of telomerase, and it plays a role in the nuclear processing of the Cajal body. 80 It has also been found that the TRFH domain of TRF2 regulates the formation of telomere T loops while inhibiting ATM activity. 81 An activity switch in human telomerase was based on RNA conformation and shaped by TCAB1. 82 Minimized human telomerase maintains telomeres and resolves endogenous roles of H/ACA proteins, TCAB1, and Cajal bodies. 83 WRAP53b mediates site-specific interactions between Cajal Figure 7. Schematic diagram about molecular mechanism by which CircMEG3 blocks telomere function dependent on Cbf5 and inhibits the malignant differentiation of human liver cancer stem cells body factors and DNA repair proteins. 84 Reptin drives tumor progression, 85 and Pontin/Tip49 negatively regulates JNK-mediated cell death. 86 Strikingly, our studies have found that CircMEG3 can promote DNA damage repair and inhibit DNA instability. CircMEG3 is involved in DNA damage repair and DNA microsatellite instability. Studies have shown that when the genome is damaged by DNA stimulation inside and outside the cell, it may lead to genome instability. 87 Therefore, DNA repair in nucleosomes is essential for gene regulation, 88 and various DNA repair pathways maintain the genome stability. 89 DNA breaks and the activation of the DNA damage response arise from endogenous replication stress. 90 Octamerbinding transcription factor 4 (OCT4) is essential in embryogenesis and pluripotency. 91 SOX2 protein may serve as a novel prognostic factor for colorectal cancer. 92 KLF4 regulates gene expression through transcriptional activation or repression. 93 NANOG is a novel therapeutic target for ovarian cancer (OC), 94 and Kinesin family member 2C aggravates the progression of hepatocellular carcinoma. 95 In addition, Plk1 regulates the kinesin-13 protein Kif2b to promote chromosome segregation, 96 and PCAT-1 plays an oncogenic role in epithelial OC by modulating cyclinD1/CDK4. 97 It was confirmed that our present results are consistent with these reports and provide novel evidence for a suppressor role of CircMEG3 in inhibiting malignant growth of liver cancer.
Another significant finding is that long ncRNA HULC plays an important role for regulating CircMEG3. Our present results are consistent with these reports and provide novel evidence for a suppressor role of CircMEG3 in inhibiting malignant growth of liver cancer via altering HULC. HULC is highly upregulated in hepatocellular carcinoma and in several other cancers. 98 Also, HULC induces the progression of osteosarcoma by regulating the miR-372-3p/HMGB1 signaling axis, 99 and HULC accelerates the growth of human liver CSCs via autophagy. 100 In particular, miR24-2 promotes malignant progression of human liver CSCs dependent on HULC. 101 In addition, H-Ras is a unique isoform of the Ras GTPase family. 102 Moreover, inhibiting the cell-cycle kinases CDK4 and CDK6 results in a significant therapeutic effect in several cancers. 103 In conclusion, our results suggest that increased Cbf5-telomerase activity abrogates the ability of CircMEG3 to inhibit malignant www.moleculartherapy.org differentiation of human liver CSCs. These observations provide important basic information for finding effective liver cancer therapeutic targets. We will further study the exact mechanism of Circ-MEG3 in the development of liver cancer and its clinical application.

hLCSC sorting
The hLCSCs were isolated from human liver cancer line Huh7 using CD133/CD44/CD24/EpCAM MicroBead Kits (MACS Technology, Miltenyi Biotech, Boston, MA, USA) and MACS Technology operation according to the manufacturer.

RT-PCR
cDNA was prepared by using oligonucleotide (dT), random primers, and First-Strand Synthesis System (Invitrogen). PCR analysis was performed according to the manufacturer. b-Actin was used as an internal control.

Western blotting
Proteins were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membranes (Invitrogen). The blots were incubated with antibody overnight at 4 C. Signals were visualized by enhanced chemiluminescence plus kit (GE Healthcare).

Super-RNA-EMSA
Cells were washed and scraped in ice-cold phosphate-buffered saline (PBS) to prepare nuclei for electrophoretic gel mobility shift assay with the use of the gel shift assay system (Promega) modified according to the manufacturer's instructions.

Chromatin immunoprecipitation (ChIP) assay
Crossed-linked cells were washed with PBS, resuspended in lysis buffer, and sonicated for 10 min in a SONICS VibraCell to generate DNA fragments. Chromatin extracts were pre-cleared with protein A/G-Sepharose beads and immunoprecipitated with specific antibody on protein A/G-Sepharose beads. After washing, elution, and decross-linking, the ChIP DNA was measured by PCR.

Telomere length assay
ScienCell's Relative Human Telomere Length Quantification qPCR Assay Kit (RHTLQ) is designed to directly compare the average telomere length of the samples.

Cell colony formation efficiency assay
Cells were incubated in a humidified atmosphere of 5% CO 2 incubator at 37 C for 10 days. For visualization, colonies were stained with 0.5% crystal violet (sigma) in 50% methanol and 10% glacial acetic acid.

Tumorigenesis test in vivo
Four-week-old male athymic BALB/c mice were maintained in the Tongji university animal facilities approved by the China Association for accreditation of laboratory animal care. Athymic BALB/c mice were injected with LCSC cells at the armpit area subcutaneously. The mice were then sacrificed and the tumors recovered. A portion of each tumor was fixed in 4% paraformaldehyde and embedded in paraffin for histological examination and immunohistochemical staining.