Effect Of MiR-20b On The Proliferation, Differentiation,Apotosis, BMP Signaling Pathway And Mitochondrial Function Of Embryonic Carcinoma Cells

The heart is the first functional organ to form during embryogenesis. The coordination of time and space in heart development suggests it needs complex and precise procedure of genetic manipulation, which involves many related genes expression and multiple signaling pathways, such as BMP signaling pathway, Wnt signaling pathway, FGF signaling pathway, Notch signaling pathway, etc. The mutation and lack in any part will lead to cardiac malformations. Hence, the genetic and molecular ways provide a new direction for the prevention of congenital heart diseases (CHD).In recent years, many scientists have discovered that microRNAs (miRNAs) are very critical for CHD. MiRNAs are a set of non-coding small RNAs composed of19-23nucleotide. Many studies have indicated that miRNAs can degrade their target genes mRNA or inhibit their transcription level to reduce target protein expression by combining with3’UTR region of target gene mRNA. MiRNAs participate in and mediate a variety of physiological and disease process, such as the sequence development, cell apotosis, cell differentiation, hormone secretion, etc.Mir-20was differentially highly expressed in the embryonic heart tissues of ventricular septal defect fetals (VSDs) through Biochip technology. Mmu-miR-20b, a highly conserved miRNA, is a member of the miR-17microRNA precursor family and exists in embryonic heart of zebrafish, mice and many other creatures. There is no study about the effects of miR-20b on embryonic cardiac malformations. Therefore, our research aimed to observe the influence of miR-20b on cardiac development by mediating its expression. The study illustrated the effects of miR-20b on the proliferation, apotosis, differentiation, BMP signaling pathway and mitochondrial functions of P19cells, which is devided into three parts in total. The research discussed the mechanism of miR-20b affecting the differentiation of PI9cells into cardiomyocytes and analyzed the physiopathologic basis in congenital heart diseases. Part ⅠEffect of miR-20b Over-expression on the Proliferation,Differentiation, Apotosis and BMP Signaling Pathway of Embryonic Carcinoma Cells Objective:To explore the effect of miR-20b over-expression on the proliferation differentiation and apotosis of embryonic carcinoma cells in vitro. Methods:The over-expression vector of mmu-miR-20b and its corresponding empty vector were constructed and transfected into P19cells, respectively. A stably miR-20b over-expressed P19cell line was successfully selected by blasticidin screening for two weeks, which was confirmed by real-time quantitative polymerase chain reaction (RT-qPCR). A cell viability CCK-8assay was used to assess the proliferation of miR-20b over-expression P19cells and control cells. The distributions of cell cycle were analysed by flow cytometry. We performed RT-qPCR to evaluate the relative expression of some marker genes of cardiomyocytes in the stable sublines, such as cTnT, GATA4and ANP. The cells were induced to apotosis in FBS-free-a-MEM, which were examined by staining with Annexin V-FITC, Hoechst and measurement of caspase-3activity. BMP signaling pathway-related proteins were detected by Western blot. Results:We successfully established the stably miR-20b over-expressed P19cell line and the expression level miR-20b was up-regulated. Compared with empty vector, over-expressed miR-20b inhibited the proliferation and cell cycles indicated a decrease in the percentage of cells in S-phase. The apototsis of P19cell with miR-20b over-expression was significantly increased. Overexpression of miR-20b increased activation of the BMP signaling pathway in P19cells, which may regulate cardiomyocyte differentiation.Conclusion:MiR-20b over-expression might have the potential to prevent the proferlition and promote the differentiation and apotosis of embryonic carcinoma cells.Part ⅡEffect of miR-20b Over-expression on the Mitochondrial Function of Cardiac MyocytesObjective:To investigate the effects of miR-20b over-expression on the mitochondrial function of cardiac myocytes.Methods:The stably miR-20b over-expressed P19cells and the negative control (NC) were differentiated into cardiomyocytes with1%DMSO. The mitochondrial DNA (mtDNA) copy number was detected with the use of RT-qPCR. We evaluated the cellular ATP production by luciferase-based luminescence assay. The reactive oxygen species (ROS) was determined by DCFDA (2’,7’-Dichlorofluorescein diacetate, DCFDA) and the mitochondrial membrane potential by JC-1fluorescent probe with confocal laser microscopy and a FACScan flow cytometer using Cell Quest software.Results:Compared with the NC group, miR-20b over-expression had no obvious effect on mtDNA copy number. The study of luciferase-based luminescence assay demonstrated that miR-20b over-expression increased the production of cellular ATP. The mitochondrial membrane potential of over-expressed miR-20b cells was lowered and the ROS level was significantly increased.Conclusion:Up-regulated miR-20b might lead to a certain degree of damage in mitochondrial function.Part IIIEffect of miR-20b Silencing on the Differentiation, Apotosis,BMP Signaling Pathway and Mitochondrial Function of Embryonic Carcinoma CellsObjective:To explore the effect of miR-20b silencing on the differention, apotosis, and mitochondrial function of embryonic carcinoma cells.Methods:The silencing sponge vector of mmu-miR-20b and its corresponding empty vector were constructed and transfected into P19cells, respectively. A stably miR-20b silencing P19cell line was successfully established by puromycin screening for two weeks, which was confirmed by luciferase assay. We performed RT-qPCR to evaluate the relative expression of some marker genes of cardiomyocytes in the stable sublines, such as cTnT, GATA4and ANP. The cells were induced to apotosis in FBS-free-α-MEM, which were examined by staining with Annexin V-FITC, Hoechst and measurement of caspase-3activity. The stably miR-20b silencing P19cells and the negative control (NC) were differentiated by10days with1%DMSO. The mitochondrial DNA (mtDNA) copy number was detected with the use of RT-qPCR. We evaluated the cellular ATP production by luciferase-based luminescence assay. The reactive oxygen species (ROS) was determined by DCFDA (2’,7’-Dichlorofluorescein diacetate, DCFDA) and the mitochondrial membrane potential by JC-1fluorescent probe with confocal laser microscopy and a FACScan flow cytometer using Cell Quest software. BMP signaling pathway-related proteins were detected by Western blot.Results:1) We successfully established a miR-20b sponge vector to knockdown endogenous miR-20b expression. The expression level miR-20b was significantly lowered by luciferase assay.2) Compared with empty vector, miR-20b knockdown prevented the differentiation of P19cells into cardiac myocytes by inhibiting activation of the BMP signaling pathway.3) The apototsis of P19cell with miR-20b knockdown was significantly reduced.4) There was no significant difference in mtDNA copy number between the miR-20b silencing group and the NC one.5) miR-20b silencing reduced the production of cellular ATP.6) The mitochondrial membrane potential of over-expressed miR-20b cells was increased and the ROS level was significantly lowered.Conclusion:Mir-20b silencing can prevent P19to differentiate into cardiomyocytes and inhibit the apotosis. In addition, miR-20b knockdown might play some role in maintain normal function of mitochondrion.