Forty-eight hours post-transfection, the cells on the glass coverslips were rinsed with PBS and fixed with 95% ethanol for 30 min at room temperature. regulation, cell growth, cell differentiation Introduction GATA-binding protein 4 (GATA-4), a zinc finger transcription factor, is a master regulator of developmental processes of the heart, such as cardiac myocyte proliferation, differentiation and survival.1-6 Recent studies indicate that it is also involved in a number of other processes such as female fertility and carcinogenesis.7-9 As a regulator of several target genes, GATA-4 plays many important roles.4,9-12 However, the precise mechanisms by which GATA-4 itself is regulated are not yet fully understood. The expression of GATA-4 could be regulated at the post-translational or post-transcriptional level. Mechanisms of post-translational regulation include protein phosphorylation, acetylation, sumoylation and methylation, whereas post-transcriptional modification mechanisms include the stabilization of mRNA prior to protein synthesis. Although it has been established that the activity of GATA-4 can be modulated through post-translational modifications, including protein phosphorylation, acetylation, sumoylation and methylation,13,14 the mechanisms underlying the post-transcriptional regulation of GATA-4 remain unclear. MicroRNAs (miRNAs) are short, highly conserved noncoding RNA molecules that play a role in post-transcriptional regulation by targeting the 3 untranslated region (3-UTR) of target gene mRNAs, leading to mRNA degradation and translational repression. Recent studies have shown that miR-26b binds the GATA-4 3-UTR c-Fms-IN-1 to repress its translation.15 Interestingly, bioinformatic analysis predicted that the 3- UTR of GATA-4 also contains a miR-200b target site, raising the possibility that miR-200b targets GATA-4. The miR-200 family consists of five members, miR-200a, miR-200b, miR-200c, miR-429 and miR141, which regulate the transcription factors Zeb1 and Ets-1 as well as Suz12, a subunit of the polycomb repressor complexes.16-18 Previous studies have shown that miR-200b is involved in epithelial to mesenchymal transition, formation and maintenance of cancer stem cells, invasion of prostate cancer cells and gastric carcinoma.16-24 Recently, miR-200b was found to be involved in the angiogenic response of endothelial cells.18 miR-200b exerts these effects through targeting specific genes, such as ZEB1 and SIP1, Suz12 and Ets-1.16-18 However, it remains unclear c-Fms-IN-1 whether miR-200b targets the transcription factor GATA-4. Bioinformatics analyses suggest that the mouse GATA-4 3-UTR contains binding sites for miR-26ab/1297/4465, miR-200bc/429/548a, miR-122/122a/1352 and miR-208ab. Among these miRNAs, miR-26b has been demonstrated to target GATA-4 during cardiac hypertrophy,15 so it would be interesting to determine whether miR-200b targets GATA-4, which contributes to the establishment of the post-transcriptional mechanisms in regulating GATA-4. In this study, we have identified GATA-4 as a novel direct target of miR-200b. We demonstrate for the first time that miR-200b-mediated downregulation of GATA-4 leads to subsequent downregulation of cyclin D1 and myosin heavy chain (MHC) expression, resulting in inhibition of cell growth and differentiation. Results miR-200b inhibits cell proliferation by inducing cell cycle arrest and apoptosis To elucidate the specific role of miR-200b in cell growth, C2C12 and P19CL6 cells were stably transfected with pri-miR-200b to upregulate endogenous miR-200b and subsequently plated in 96-well plates to measure c-Fms-IN-1 cell viability. Mouse monoclonal to PRAK The miR-200b level in each stable c-Fms-IN-1 cell line was determined by quantitative real-time PCR (qPCR) (Fig.?1A, upper right panel), and cell viability was measured by the MTT assay (Fig.?1A, upper left panel). Interestingly, C2C12 and P19CL6 cells stably expressing miR-200b demonstrated a 44% and 41% reduction in cell number and a 4.3- and 6.9-fold increase in miR-200b levels, respectively (Fig.?1). These data suggested that miR-200b has an anti-proliferative effect on C2C12 and P19CL6 cells. To further determine whether C2C12 cells stably transfected with pri-miR-200b were reserved in an undifferentiated state, the expression of myogenin, MyoD and -MHC, three muscle-specific genes, was analyzed by real-time PCR. As shown in Figure?1A (lower), when compared with C2C12 cells on differentiation day 3 and day 6, myogenin, MyoD and -MHC mRNA levels were significantly decreased, suggesting that miR-200b maintains C2C12 cells in an undifferentiated state. Open in a separate window Figure?1. miR-200b inhibits cell growth. (A) The effect of miR-200b on cell proliferation. C2C12 and.