| The heart as the first functional organ formation during embryonic development, involves many related genes with differential expression of temporal and steric order, and also multiple signaling pathways(such as Notch signaling pathway, BMP signaling pathway, Wnt signaling pathway,etc.). Such precise control ensures the correct migration, proliferation and differentiation of myocardial cells. The mutation or lack in any part will result in the occurrence of congenital heart disease. Congenital heart disease(CHD) is one of the most common congenital anomalies during neonatal period; the incidence rate is as high as 0.8%~1%. It is one of the main causes of miscarriage, stillbirth, neonatal developmental defects, or even death, and seriously affect the quality of human life, bring extreme burden to the family and society. Therefore, to explore the mechanism of formation and development of embryo heart has become one of the new directions of congenital heart disease from gene and molecular level.MicroRNA(miRNA) is a class of about 22 nt of non-coding RNA. By combining the 3 ’UTR region of the target gene mRNA, it mediates mRNA degradation or inhibition through the post-transcriptional level, resulting in the target labeled protein decreased to participate and regulate heart development, apoptosis, cell differentiation, secretion of hormones and other physiological processes and a variety of disease processes. The change of miRNA in the level of expression is closely related with the occurrence of many diseases. Studies have found that miRNA plays an important role no matter in cell proliferation, differatiation, or in the biological development and disease development, which causes more and more attention of researchers. Relative researches indicated that microRNA is not only involved in heart development and cardiac remodeling, and plays a role in the development of heart disease.Previously, our research team found that the expression of miR-375 was significantly up-regulated in the fetal myocardial tissues than that in normal abortion embryonic myocardium. We verified this result by using quantitative real-time PCR afterwards. Mealwhile, our further studies revealed that miR-375 was highly expressed in the maternal blood with fetal congenital heart disease, indicating that miR-375 might play an important role in embryonic heart development. However literatures about mechanism of miR-375 in the heart development have not been reported yet. Considering that P19 cells and zebrafish are both classsic models in the molecular mechnism of cardiac development, we altered the expression level of miR-375 in these two models,explored the effect of miR-375 overexpression on P19 cell proliferation, apoptosis and differentiation, and evaluated its functions on the zebrafish embryonic heart development. We also analyzed the downstream targeted genes of miR-375, providing new clues for basic research on the pathogenesis of congenital heart disease.Part I Effect of miR-375 overexpression on the Proliferation, Apoptosis of P19 CellsObjective: Generate miR-375 overexpression plasmids and select a stably miR-375 overexpressed P19 cell line to investigate the role of miR-375 on P19 cell proliferation, apoptosis, and obtain the overexpression of miR-375 zebrafish embryonic heart development phenotype.Methods: The mmu-miR-375 overexpression and mmu-miR with GFP control plasmids were transfected into P19 cells and selected by puromycin for two weeks. Stable cells were imaged using fluorescence microscopy and screened using quantitative real-time PCR(qRT-PCR). CCK-8 assay was used to assess the proliferation, flow cytometry was used to analyse the cell cycle and the apoptosis was detected by Hoechst stain, flow cytometry and BAX, BCL-2 expression level was measured by qRT-PCR and Western blot in miR-375-overexpressing P19 cells. The miR-375 minic was microinjected in the zebrafish single-cell stage to observe the morphological change.Results: miR-375 overexpression inhibited P19 cells proliferation and cell cycles by decreasing the percentage of cells in S-phase. The ratio of BAX/BCL-2 were significantly increased in miR-375-overexpressing cells, which was related to cell apototsis. The morphological observations showed that overexpression of miR-375 in zebrafish embryos lead to significant pericardial edema.Conclusion: miR-375 overexpression affects the proliferation and apoptosis of P19 cells and leads pericardial edema in the morphological of zebrafish embryonic heart development.Part 2 Effect of miR-375 overexpression on the Differentiation and Notch signaling pathway of P19 CellsObjective: To explore the effect of miR-375 overexpression on P19 cell differentiation and Notch signaling pathway.Methods: Induce the stably miR-375 over-expressed P19 cells differentiate into cardiomyocytes. QRT-PCR was used to detect the expression of GATA binding protein 4(GATA4), cardiac troponin T(cTnT), myocyte enhancer factor 2c(Mef2c) and Notch signaling pathway related genes during differentiation. Related proteins were detected by Western blot. Luciferase assay was used to validate whether Notch is miR-375 target.Results: Compared to the controls, miR-375-overexpressing inhibit the differentiation of P19 cells. Luciferase assay confirmed that Notch2 was a downstream target of miR-375. MiR-375 overexpression possibly affects the P19 cells differentiation by suppressing Notch signaling pathway.Conclusion: miR-375 overexpression may affect the differentiation of P19 cells towards cardiomyocytes by the Notch signaling pathway. |
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