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Reactive oxygen species-responsive miR-210 regulates proliferation and migration of adipose-derived stem cells via PTPN2

Hypoxia enhances the proliferation and migration of adipose-derived stem cells (ASCs) via the generation of reactive oxygen species (ROS). Therefore, this study primarily investigated whether or not ROS generation could regulate microRNA-210 (miR-210) expression, and increase proliferation/migration of ASCs. In addition, we tried to identify the signaling pathways involved in miR-210 upregulation and the direct target genes of miR-210 that mediate these functions. Various sources of ROS generation such as hypoxia, antimycin, rotenone, and platelet-derived growth factor (PDGF)-BB upregulated miR-210 expression, and increased the proliferation/migration of ASCs. There is a positive feed-forward loop between ROS generation and miR-210, and miR-210 itself increases ROS generation by downregulation of iron–sulfur cluster scaffold homolog 2 (ISCU2). Although hypoxia-inducible factor-1α was not involved in miR-210 expression, pharmacological or small interfering RNA (siRNA)-driven inhibition of Akt and ERK1/2 molecules reduced miR-210 expression. Transfection of siRNAs of NF-κB and Elk1 also reduced miR-210 expression, indicating that these signaling pathways mediate miR-210 upregulation. Protein tyrosine phosphatase, non-receptor type 2 (PTPN2) was selected for miR-210 target gene, and it was downregulated by ROS generators or miR-210 mimic treatment. PTPN2 was first proven to be a direct miR-210 target in luciferase activity assay, and pharmacological inhibition or overexpression of PTPN2 regulated the proliferation and migration of ASC. In conclusion, ROS generation from diverse sources induces miR-210 expression in ASCs via PDGFR-β, Akt and ERK pathways. Transcription of miR-210 expression is regulated by NF-κB and Elk1, and miR-210 increases the proliferation and migration of ASCs via ISCU2 and PTPN2 downregulation.


Subject Category: Experimental Medicine
Adipose-derived stem cells (ASCs) are isolated from subcutaneous fat and used for tissue repair and regeneration. Culturing ASCs in hypoxia (2% oxygen) increases their proliferation, migration and secretion of growth factors. [1][2][3] In addition, hypoxia preconditioning enhances regenerative potential of ASCs in vivo. [4][5][6] In a previous study, we investigated the key mediators and signal pathways involved in the stimulation of ASCs during hypoxia and found that reactive oxygen species (ROS) play a key role. Furthermore, exposure to a low concentration of antimycin or rotenone significantly increased the proliferation and migration of ASCs. 7 Hypoxia and ROS generation induced the phosphorylation of platelet-derived growth factor receptor-b (PDGFR-b), ERK1/2 and Akt. 8 Like this, acute responses of ASCs to hypoxia and ROS generation were investigated in our previous studies, but intermediate or chronic responses to hypoxia (i.e. microRNA regulation) have not been demonstrated.
Hypoxia influences cellular functions, including metabolism, survival, proliferation, migration and angiogenesis. All of these cellular functions are directly and indirectly controlled by a master transcription factor called hypoxia-inducible factor-1a (HIF-1a). 9,10 In response to hypoxia, HIF-1a levels increase a specific set of microRNA molecules called miR-210. This unique microRNA is highly expressed in hypoxic cells and tissues, and is reportedly regulated by HIF-1a stabilization. [11][12][13] Therefore, miR-210 is currently considered the 'master microRNA' of the hypoxic response, 14 and is known to influence physiological development as well as a number of hypoxia-dependent disease states. miR-210 mediates these functions by regulating a lot of target mRNAs. 15 For example, ephrin A3 and protein tyrosine phosphatase 1B (PTP1B) were validated as direct targets of miR-210, and downregulation of these proteins improves heart disease. 16 Similarly, iron-sulfur cluster scaffold homolog1/2 (ISCU1/2) facilitates the assembly of iron-sulfur clusters and regulates mitochondrial metabolism. 17 Furthermore, another direct target of miR-210, known as 'max-binding protein', controls the cell cycle and proliferation through reciprocal upregulation of c-myc activity. 18 Despite the knowledge that hypoxia enhances the proliferation and migration of ASCs via ROS generation, there has been no direct evidence that miR-210 is regulated by intracellular ROS generation and it mediates proliferation/ migration of ASCs. In addition, there are some evidences that expression of miR-210 is independent of HIF-1a pathway, but it was regulated by Akt-or TNF-a-related pathways. 19,20 Therefore, this study investigated: (1) Whether or not ROS generation regulates miR-210 expression, and increase proliferation/migration of ASCs? (2) Which signaling pathway(s) and transcription factor(s) are involved in the upregulation of miR-210? (3) What is (are) miR-210 target gene(s) involved in proliferation and migration of ASCs? We first found that ROS generation induces miR-210 expression, and identified the novel transcription factors and target genes of miR-210 in this study.

Results
Increased miR-210 expression by ROS generators. In a preliminary study, microRNA array was carried out to screen the altered microRNAs during hypoxia. The signal of hsa-miR-29a, -301a, -503 and -539 was slightly decreased (approximately 0.5-fold), but the signal of miR-210 was highly increased during hypoxia (4100-fold).
In the first place, we confirmed the miR-210 expression using quantitative real-time PCR (Q-PCR), and found that hypoxia (2%) significantly increased the miR-210 expression in a time-dependent manner (Figure 1a; Po0.01). miR-210 expression was not altered until 6 h under hypoxia. After 6 h, miR-210 expression started to increase, and it was highest at 24 h. Hypoxia-induced expression of miR-210 was attenuated by the ROS scavenger, such as N-acetyl-cysteine (NAC), indicating that hypoxia-increased miR-210 expression is mainly mediated by ROS generation (Figure 1b; Po0.01). Because NADPH oxidase 4 (Nox4) is involved in ROS generation by hypoxia, we investigated whether or not Nox4 knockdown attenuates miR-210 expression. However, Nox4 small interfering RNA (siRNA) partially reduced the hypoxiainduced miR-210 expression (Figure 1c; Po0.01), indicating that there are alterative ROS-generating systems in ASCs.
Therefore, we tested whether or not mitochondrial ROS generators could also induce miR-210 expression, and found that antimycin (10 nM) and rotenone (10 pM) significantly increased the miR-210 expression in a time-dependent manner (Figure 1d; Po0.01). miR-210 expression was increased after 6 h, and was highest at 24 h. Therefore, miR-210 expression was usually measured at 24 h after the treatment throughout the experiments.
Dose-dependent regulation of miR-210 by ROS generation was investigated. The results showed that low concentrations of antimycin (at 10 and 100 nM; Figure 1e; Po0.01) and rotenone (at 10 and 100 pM; Figure 1f; Po0.01) significantly increased the miR-210 expression, while high concentrations display no significant expression. This is consistent with our previous study that high concentrations of ROS generator did not increase the proliferation and migration of ASCs. 7 Increased ROS generation by miR-210 mimic treatment. We also investigated whether or not miR-210 upregulation could induce ROS generation in ASCs. 21,22 ROS generation was measured by flow cytometry (Figure 2a), and the florescence intensity of 2 0 ,7 0 -dichlorofluorescein diacetate (DCF-DA) was significantly increased by the transfection of miR-210 mimic (Figure 2b; Po0.01). In addition, fluorescence microscope images show that fluorescence signal of DCF-DA (green) was increased after transfection of miR-210 mimic compared with the negative/scramble control that did not contain miR-210 mimic (Figure 2c). These results indicate the positive feed-forward loop between ROS generation and miR-210 upregulation (ROS/miR210/ROS pathway) in ASCs (Supplementary Scheme 1).
ISCU2 involvement in ROS generation. It has been reported that PTP1B and ISCU1/2 are direct target of miR-210, and inhibition of these genes increases ROS generation. 16,21,22 Therefore, we further investigated whether or not these genes regulate ROS generation. However, downregulation of PTP1B by siRNA technique induced neither ROS generation nor proliferation/migration of ASCs (data not shown).
Although mRNA expression of ISCU1/2 was not reduced, transfection of miR-210 mimic (20 and 50 nM) primarily reduced the protein level of ISCU2 in western blot analysis ( Figure 2d). Therefore, we investigated whether or not ISCU1/ 2 downregulation could affect ROS generation. Transfection of siRNA (5 nM) for ISCU1/2 reduced the ISCU1/2 protein level, but ISCU2 is primarily reduced by siRNA (Figure 2e). In addition, transfection of siRNA for ISCU1/2 increased the signal intensity of DCF-DA according to flow cytometry (Figure 2f     HIF-1a-independent miR-210 regulation. It is well known that miR-210 is primarily regulated by HIF-1a stabilization; 12,13,25 therefore, we investigated whether or not ROS-induced miR-210 expression is regulated by HIF-1a pathway. Although hypoxia (2%) increased the protein level of HIF-1a, a low dose of antimycin (10 nM) and rotenone (10 pM) treatment did not induce HIF-1a level in western blot analysis ( Figure 5a). In addition, hypoxia-induced HIF-1a On the other hand, we tested whether or not paracrine effects of ASCs could be regulated by miR-210. Although hypoxia-induced mRNA expression of vascular endothelial growth factor (VEGF) and hepatocyte growth factor were inhibited by Yc-1 treatment, it was not altered by the transfection of miR-210 mimic (Supplementary Figure 2). These results suggest that secretion of paracrine factors are not regulated by miR-210, but mainly by HIF-1a level under hypoxia (Supplementary Scheme 1).
Signal pathways involved in miR-210 upregulation. Because ASC stimulation by hypoxia was accompanied by the phosphorylation of signaling molecules such as PDGFR-b, ERK1/2 and Akt, 8 we further investigated whether or not these signaling pathways are involved in miR-210 upregulation.  NF-jB and Elk1 induce miR-210 transcription. Using Patch 1.0 and AliBaba 2.1 program, we searched for the possible transcription factors that promote the expression of miR-210 during ROS generation. The promoter region of miR-210 was analyzed to get some candidates. Because Akt and ERK pathways are involved in miR-210 expression and there are NF-kB and Elk1 binding sequence in the upstream region of the promoter sequence, we selected NF-kB and Elk1 as strong candidates for miR-210 transcription factors ( Figure 5g).
As expected, phosphorylation of NF-kB and Elk1 was significantly increased by low concentration of antimycin (10 nM; Figure 5h) and rotenone (10 pM; Figure 5i Chromatin immunoprecipitation (ChIP) assay was performed to identify the binding site of phosphorylated NF-kB and Elk1 in the upstream region of miR-210 promoter. As shown in Figure 5l, antimycin and rotenone treatment significantly increased the signal intensity of PCR products, which indicated that phosphorylated NF-kB and Elk1might bind to À 113 to À 123 and À 582 to À 595 regions, respectively, of the miR-210 promoter (Figure 5g).

PDGF-BB induces miR-210 through ROS generation.
Although PDGFR-a is highly expressed, 26 treatment of PDGF-AA is not as effective as PDGF-BB in the proliferation and migration of ASCs (our unpublished data). In addition, PDGF-AA (o10 ng/ml) treatment did not induce miR-210 expression and ROS generation ( Supplementary  Figures 3a and b). On the contrary, PDGF-BB treatment (5 or 10 ng/ml) induced ROS generation ( Figure 6a) and significantly increased the miR-210 expression in a time-dependent manner (Figure 6b; Po0.01). In addition, PDGF-BB increased the proliferation (Figure 6c Figure 4B). Analysis of gene ontology using the differentially expressed genes demonstrated that the genes related to signal transduction (20%), transcription (17%), cell proliferation (10%) and cell differentiation (9%) were primarily upregulated by hypoxia (Supplementary Figure 4C).
PTPN2 is a direct miR-210 target. Using 'microinspector' software (available at: http://bioinfo.uni-plovdiv.bg/microinspector/), we selected protein tyrosine phosphatase, nonreceptor type 2 (PTPN2) as miR-210 target from 15 genes downregulated by hypoxia (Supplementary Figure 4D). As expected, mRNA and protein expression of PTPN2 was downregulated at 24 h after hypoxia (Figure 7a). In addition, Ct value for PTPN2 was measured by Q-PCR, and PTPN2 mRNA was significantly decreased under hypoxia (Figure 7b; Po0.01). It is well known that microRNA negatively regulates protein expression by interacting with the 3 0untranslated regions of specific mRNA. However, 12 nucleotide sequences in miR-210 are complementary to the coding sequence of PTPN2, which might induce degradation of PTPN2 mRNA (Figure 7c). Similar to hypoxia, antimycin (10 nM) and rotenone (10 pM) treatment decreased the mRNA expression of PTPN2 (Figure 7d). Transfection of miR-210 mimic (20 nM) downregulated the PTPN2 mRNA and protein levels (Figure 7e). Using luciferase activity assay, we investigated whether or not PTPN2 is a direct target of miR-210 (Figure 7f). miR-210 mimic significantly reduced the PTPN2 luciferase activity in HEK293 cells (Figure 7g; Po0.01). Although miR-210 reduced wild-type PTPN2 in HEK293 cells (Po0.01), it did not reduce the luciferase activity of mutant PTPN2 (Figure 7h). Collectively, these results suggest that PTPN2 is a direct target of miR-210.

Discussion
MicroRNAs exert their actions primarily at the post-transcriptional level, either via translational repression and/or mRNA degradation. 29,30 This study attempted to identify a novel microRNA involved in the proliferation and migration of ASCs during hypoxia and ROS generation. In a microRNA array analysis and Q-PCR, miR-210 expression was significantly increased by various ROS generators (i.e. hypoxia, antimycin, rotenone and PDGF-BB). Transfection of miR-210 inhibitor significantly attenuated the ROS-mediated proliferation and migration of ASCs, while that of miR-210 mimic induced the (l) ChIP assay shows that antimycin and rotenone treatment significantly increased the signal intensity of PCR products, which indicated that phosphorylated NF-kB and Elk1 could bind to the upstream region of miR-210 promoter. **Po0.01. Con, control; Norm., normaxia proliferation and migration of ASCs. These results suggest that miR-210 is a key player in ROS-associated events in ASCs (Supplementary Scheme 1). miR-210 has been known as 'hypoxamiR' because it appears to control a network of multiple processes during the hypoxic responses. 31 However, since our study first identified 'ROS' as a regulatory mechanism of miR-210 expression, miR-210 could be renamed as 'ROSmiR'.
It has been reported that ROS generation or miR-210 expression is involved in adipocyte differentiation. [32][33][34][35] However, it is controversial whether ROS generation induces or reduces adiopocyte differentiation of ASCs. For example, antimycin (1-10 mM) and rotenone treatment induced adipocyte differentiation in ASCs in our preliminary study, but Nox4 silencing unexpectedly induced adipogenesis. This is consistent with the previous report that Nox4 knockout induced adipocyte differentiation in vivo. 36 Although Qin et al. 35 suggested that miR-210 could promote adipocyte differentiation by repressing WNT signaling through targeting Tcf7l2 in 3T3L1 cells, miR-210 was not highly induced during adipocyte differentiation of ASCs and treatment of miR-210 mimic did not induce the adipocyte differentiation in our preliminary study. Therefore, it is not clear whether or not ROS generation and miR-210 induce or reduce adipocyte differentiation in this stage, and further studies should be extended to define the role of ROS and miR-210 in ASC differentiation.
Many reports have shown evidence of the involvement of HIF-1a pathway in miR-210 upregulation and of a feedback response between miR-210 and HIF-1a. 11,13,37,38 For example, transfection of HIF-1a in HT-29 cells significantly increased miR-210 expression. 38 Similarly, green tea polyphenol, EGCG, suppressed lung cancer cell viability by upregulating both HIF-1a and miR-210. 11 Kelly et al. 37 also demonstrated that hypoxia promotes HIF-1a stability through suppression of the expression of glycerol-3-phosphate dehydrogenase 1-like protein by miR-210. However, this study demonstrated HIF-1a-independent miR-210 induction by ROS generation. For example, low concentration of antimycin and rotenone did not induce HIF-1a levels (Figure 5a), and Yc-1 (an HIF-1a inhibitor) did not reduce miR-210 expression under hypoxia (Figure 5c). Therefore, it is reasonable that miR-210 upregulation by low concentrations of ROS generators is independent of HIF-1a pathway.
Until now, little is known about HIF-independent miR-210 regulation. Mutharasan et al. 39 first reported that miR-210 was upregulated in hypoxic cardiomyocytes through Akt-and p53-dependent pathways, and exerted cytoprotective effects through both HIF-dependent and -independent regulation. 39 This study focused on the HIF-independent miR-210 regulation by ROS generation. Specifically, phosphorylation of PDGFR-b is followed by phosphorylation of Akt and ERK1/2, which induces the phosphorylation of miR-210 transcription factors such as NF-kB and Elk1. In addition to Akt and NF-kB, we first found the novel signal molecules and transcription factors that PDGFR-b, ERK1/2 and Elk1 are involved in miR-210 induction. Protein tyrosine phosphatases are natural receptor tyrosine kinase antagonists and serve as regulators of both non-receptor and receptor tyrosine kinases. 40,41 Of them, PTPN2 is a ubiquitously expressed phosphatase and is implicated in the regulation of growth factor receptor signaling.
PTPN2 directly controlled the PDGFR-b phosphorylation in a site-selective manner, and also negatively regulated the migratory response to PDGF-BB. 27 In addition, loss of PTPN2 specifically redirected the PDGFR-b toward rapid recycling and induced PDGFR-b-related signal pathways,  (f-h) Using luciferase activity assay in HEK293 cells, we investigated whether or not PTPN2 is a direct target of miR-210. Scheme of luciferase activity assay (f). miR-210 mimic significantly reduced the PTPN2 luciferase activity in HEK293 cells (g). Although miR-210 reduced the luciferase activity of wildtype PTPN2, it did not reduce the mutant PTPN2 in HEK293 cells (h). (i, j) Ethyl-3,4-dephosphostatin, a PTPN2 inhibitor, increased the proliferation (i) and migration (j) of ASC. (k, l) However, transfection of PTPN2 cDNA significantly reduced the hypoxia-induced proliferation (k) and migration (l) of ASCs **Po0.01. Cont, control; CMV, cytomegalovirus; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NC, negative control; Nor., normaxia; Hyp., hypoxia but not PDGFR-a. 28 Furthermore, we first found in this study that there is a positive feed-forward loop between PDGFR-b activation and PTPN2 downregulation, and it is mediated by Akt or ERK pathway, and miR-210 upregulation (Supplementary Scheme 1).
Growth factors are added to the culture medium to maintain the self-renewal capability and to increase the proliferation of stem cells. [42][43][44][45] In a previous study, we tested and compared the proliferating effects of six growth factors (i.e. PDGF-AA, PDGF-BB, VEGF, insulin-like growth factor, basic fibroblast growth factor and epidermal growth factor) in ASCs. 46 Among them, PDGF-BB showed the best stimulatory effect on ASC proliferation at a physiologically relevant level (at 10 ng/ml). Compared with PDGF-AA and other growth factors, PDGF-BB treatment highly induces ROS generation and miR-210 level, and it might be responsible for the highest proliferative effect of PDGF-BB in ASCs.
In addition to hypoxia, ROS generation from diverse sources (mitochondrial ROS donors and PDGF-BB) induces miR-210 expression; therefore, it can be regarded as ROSresponsive microRNA. PDGFR-b, Akt and ERK1/2 molecules are involved in ROS-induced signaling cascades, and NF-kb and Elk1 induce the transcription of miR-210. PTPN2 and ISCU2 are found to be a direct target of miR-210, which collectively mediates miR-210-induced ROS generation and proliferation/migration of ASCs.
For Measurement of miR-210 expression level. Total cellular RNA was extracted with TRIzol reagent (Invitrogen), followed by a reverse transcription using miRNA First-Strand Synthesis Kit (Clonetech, Mountain View, CA, USA). cDNA was synthesized from 500 ng of total RNA using mRQ enzyme. The miR-210 expression level was measured by Q-PCRs, using 10 mM of the miR-210 primer (Genolution, Seoul, Korea) and quantitect SYBR green (Qiagen, Hilden, Germany). The Q-PCR was performed using StepOne real-time PCR system (Applied Biosystems, Carlsbad, CA, USA) under the following steps: initial denaturation step at 94 1C for 5 min, 40 thermal cycling step consisted of 30 s at 94 1C, 30 s at 56 1C and 30 s at 72 1C, and a final extension step at 72 1C for 5 min for termination. For miR-210 quantification, analysis of fold change was calculated by using DCt value, relative to the level of U6 snRNA. miR-210 mimic or inhibitor transfection. ASCs were seeded with complete medium without antibiotics. On the following day, confluent ASCs were siRNA or PTPN2 transfection. ASCs were seeded with complete medium without antibiotics. On the following day, confluent ASCs were kept in serum-free medium without antibiotics for 2 h. Then, siRNA for PDGFR-b, Akt, ERK and ISCU1/2 (20 nM; Santa Cruz Biotechnology), or PTPN2 cDNA clone (0.02, 0.05 and 0.1 mg; Origene, Rockville, MD, USA) were transfected to ASCs using Lipofectamine 2000 (Invitrogen). Cells were incubated for 24 or 48 h after siRNA or cDNA transfection.
ROS generation assay. Intracellular ROS production was measured using DCF-DA (Molecular Probes, Eugene, OR, USA). ASCs were seeded in 60-mm culture dish. Cells were transfected with miR-210 mimic, inhibitor or siRNA for 24 h and were incubated with DCF-DA (20mM) for 20 min at 37 1C in the dark place. After incubation, cells were harvested by trypsin-EDTA solution. Then, cell suspensions were collected in a centrifuge and the supernatant was removed. Fluorescence images were taken after DCF-DF staining by fluorescence microscopy (ECLIPSE E600; Nikon, Tokyo, Japan). Otherwise, fluorescence intensity of DCF-DA was measured and calculated using flow cytometry (Becon Dickinson, Franklin Lakes, NJ, USA).
Proliferation assay. ASCs were seeded in 24-well plates and transfected with miR-210 mimic, inhibitor or siRNA. After 48 h incubation, the medium was removed and cell number was measured using CCK-8 assay kit (Dojindo, Gaithersburg, MD, USA). CCK-8 solution (150 ml) was added to each well and incubated for 2 h. After the incubation, the absorbance was measured at 450 nm using a microplate reader (Tecan, Grodig, Austria).
Migration assay. To determine the migration rate of ASCs, ASCs were seeded in six-well plates with complete medium. On the following day, confluent ASCs were kept in serum-free medium for 12 or 24 h. Serum-free/starved ASCs were wounded using migration micropipette tip, and were transfected with miR-210 mimic, inhibitor or siRNA. At 24 h after the wounding, cell migration was determined by bright-field microscopic examination. For the evaluation of ASC migration, five randomly selected points along each wound were marked, and the horizontal distance of migrating cells from the initial wound was measured.
Western blotting. Total proteins of ASCs were isolated using SDS sample buffer. Lysates were loaded by 10 or 8% SDS-PAGE and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% skim milk for 1 h at room temperature and then incubated with primary antibody at 4 1C for 24 h. On the following day, the membrane was washed with TBS-T (0.1% Tween-20 in Tris-buffered saline) and then incubated with HRP-conjugated secondary antibody for 1 h. The membrane was also reacted with the ECL solution (Millipore) and developed in an X-ray film.
Statistical analysis. All data were representative of triplicate independent experiments. The statistical significance of the differences among groups was tested using the ANOVA or Student's t-test. Po0.01 is considered to be significant.