| MicroRNA(miRNA)is a kind of small noncoding single-stranded RNA,with 22 nucleotides length,which exists widely in plant and nematodes and human cells.Mi RNA is known to combine with target gene m RNA3 ’UTR region in order to mediate the degradation of the target gene m RNA or inhibit the expression of the target gene in transcription level,so as to participate in sequence development,cell apoptosis,cell differentiation,hormone secretion and various physiological process.Many of scientists found that micro RNAs play an important role in congenital heart disease,meanwhile the heart is regarded as the earliest formed organ in the process of embryonic development and is functional among important organs,which involves a variety of complex genes to manipulate in time and in correct space.It may related to certain gene expression and signaling pathway.Therefore,we can explore the the possible mechanisms of congenital heart disease through detecting the level of gene and molecular expression to understand.We found that miR-30c expression significantly raised in ventricular septal defect fetal ventricular muscle tissue through miRNAs biochip screening.It shows that miR-30c may play an important role in the process of heart development.mmu-miR-30c belongs to miR-30 family,it is highly conserved in evolution,and studies revealed that miR-30c can participate in myocardial matrix reconstruction.It provides us a new way to learn about heart development.By changing the miR-30c expression level,we observe its effect on the heart development in P19 cells.Thewhole research is divided into two parts respectively,we explore the function of proliferation,apoptosis,cell differentiation via shh signaling pathways of overexpression and knockdown of mmu-miR-30c in P19 cells,and trying to explain possible mechanisms of congenital heart disease,so as to analyzing the possible pathophysiological mechanisms of congenital heart disease.Part I Effect of miR-30c Over-expression on the Proliferation,Differentiation,Apotosis of Embryonic Carcinoma CellsObjective: To explore the effect of miR-30c over-expression and knockdown on the proliferation and apotosis of embryonic carcinoma cells in vitro.Methods: Transfect the overexpression/knockdown vector of mmu-miR-30c and its corresponding empty vector into P19 cells,respectively.A stable miR-30c overexpression/knockdown P19 cell line was selected successfully by Blasticidin for two weeks.Mi R-30c overexpression cell line was confirmed by real-time quantitative polymerase chain reaction(RT-q PCR)and Mi R-30c knockdown cell line was confirmed by dual luciferase report system.CCK-8 assay was used to assess the proliferation of miR-30c P19 cells.The distributions of cell cycle were analysed by flow cytometry.The cells were induced to apotosis in FBS-free-α-MEM,which were examined by staining with Annexin V-FITC and Hoechst.Results: We successfully established the stably miR-30c overexpression/knockdown P19 cell line.Compared with control group,overexpression of miR-30c promoted the proliferation and showed an increasing in the percentage of cells in S-phasecell of cycles.Knockdown of miR-30c inhibited the proliferation and decreased the percentage S-phase of cells cycle.The apoptosis of P19 cell with miR-30c knockdown was significantly increased.Conclusion: Overexpression of miR-30c increased proliferation and knockdown of miR-30c decreased proliferation in P19 cells.Knockdown of miR-30c inhibited apoptosis in P19 cells.Part II Effect of miR-30c overexpression/knockdown on the Differentiation via shh Signaling Pathway in Embryonic Carcinoma CellsObjective: To explore the effect of miR-30c overexpression/knockdown on the differentiation via shh signaling pathway in embryonic carcinoma cells.Methods: The stably P19 cell lines of miR-30c overexpression/knockdown were induced to differentiate with 1%DMSO for 10 days.During cell differentiation of d0,d2,d4,d6,d8,d10,we performed RT-q PCR to evaluate the relative expression of some cardiomyocytes differentiation marker genes including c Tn T,GATA4,ANP and important genes of shh signaling pathway including Gli2,shh,Smo,ptc.All related proteins were detected by Western blot.Results: Compared with control group,both of miR-30c overexpression and knockdown prevented the differentiation of P19 cells into cardiac myocytes.Compared with control group,the level of shh and Smo increased in miR-30c overexpression group,which decrease in miR-30c knockdown group.Conclusion: Mi R-30c overexpression/knockdown can prevent P19 to differentiate into cardiomyocytes.In addition,miR-30c overexpression inhibit shh signaling pathway and miR-30c knockdown may active shh signaling pathway... |
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