The Mechanism Where By GATA4 Recruits PCG Proteins To Regulate The Expression Of MiR-29a In The Proliferation And Differentiation Of C2C12 Cells

Background and ObjectiveRhabdomyosarcoma(RMS) is a malignant tumor of skeletal muscle origin, also the most common soft tissue tumors among children. It is thought to arise as a consequence of a dysfunctional balance of the growth and terminal differentiation of muscle progenitor cells. But the molecular mechanism is poorly understood. Therefore, it is important to clarify the regulation mechanism of proliferation and differentiation of progenitor cells, and reveal the pathogenesis of RMS.GATA family is a transcription factor with zinc finger structure, and can combine with WGATAA sequence in the target gene promoter regions, so as to regulate the expression of genes. GATA4 plays an important role in the regulation of C2C12 proliferation and differentiation, but the mechanism of RMS occurrence, has not yet clear.Polycomb complex is a group of proteins to inhibit gene transcription. And the transcriptional activity of GATA4 can be repressed by EZH2, a member of the complex through methylation of the 299 lysine of GATA4 protein. But it is not yet clear the mechanisms in RMS occurrence.Studies have shown that, micro RNA dysregulation also has a close relationship with RMS. Micro RNA abnormalities such as muscle-specific micro RNA(miR-1, miR-133a/b, miR-206) and miR-29 family will induce RMS occurs, but the exact mechanism remains to be elucidated.The study has found that EZH2 and GATA4 not only regulate the proliferation and differentiation of C2C12 myoblast by regulating the expression of miR-29 a,, but also play a role in the occurrence of RMS. Therefore, the present study is to elucidate the molecular mechanisms of proliferation and differentiation of C2C12 and RMS occurrence, which can provide a theoretical basis for its treatment and prognosis.Research methods 1. Bioinformatics software forecast, cyclin D2 is the downstream target gene of miR-29 a.Plenty of methods were used to study miR-29 a targeting cyclin D2 in cell proliferation, cycle and apoptosis, including transient transfection, MTT assay, flow analysis, DAPI staining, luciferase reporter gene, QPCR and Westem Blot experiments2. In this study, we used stable transfection, QPCR, Westem Blotting, CO-IP and CHIP experiments and other methods to study the molecular mechanisms of GATA4 regulate miR-29 a.Results 1. MTT, flow cytometry and DAPI staining showed that, miR-29 a causeed inhibition of cell proliferation, cell cycle blockage and induction of apoptosis.2. Luciferase showed, miR-29 a was capable of specifically binding to the predicted occurrence cyclin D2 3’-UTR region, thereby reducing the luciferase activity.3. Western Blotting and QPCR confirmed the miR-29 a reduced cyclin D2 expression at the protein and m RNA levels, suggesting that miR-29 a can suppress cyclin D2 expression by post-transcriptional silencing, and cleared that cyclin D2 indeed is a target gene of miR-29 a.4. CHIP experiment showed that, GATA4 recruited PCG protein EZH2(PRC2), BMI1 and RING1B(PRC1), H3K27me3 to miR-29 a promoter regions which maked it be in the inhibitory state.5. CO-IP experiments further revealed the presence of interaction between EZH2and GATA4 in cells. These results elucidate the internal molecular mechanisms in GATA4 inhibiting miR-29 a transcriptional activity.6. Western Blotting and QPCR confirmed that GATA4 could suppress miR-29 a at the m RNA level, and promoted expression of cyclin D2 at the protein and m RNA levels.7. CHIP experiment showed that in C2C12 differentiation, GATA4 and PCG protein did not bind to the miR-29 a promoter region.Conclusion 1. miR-29 a causes C2C12 cell proliferation, cell cycle blockage and apoptosis by down-regulating the expression of its downstream target genes cyclin D2.2. In C2C12 cell proliferation, GATA4 recruits EZH2 and other Polycomb family protein binding to miR-29 a promoter regionto inhibit its expression and promote cyclin D2 expression, and promote C2C12 cell proliferation ultimately.3. During the process that C2C12 cells differentiate into skeletal muscle, GATA4 and EZH2 no longer bound to miR-29 a promoter region, so that the inhibition of miR-29 a expression is released, and promote skeletal muscle formation.