A Preliminary Study On The Function Of Long Noncoding RNA Uc40 In Myocardial Cells

Objective:To observe the effect of stable uc40-siRNA-44 silenced P19 cell on the differentiation,proliferation,apoptosis and mitochondrial function of them and the changes of myocardial markers and key gene PBX1 during the differentiation of uc40-siRNA-44 silenced P19 cells.Methods:Construct uc40-siRNA-44 plasmid vector and transfect into P19 cells.The transfection efficiency was observed by fluorescence microscope,and the cells were screened with neomycin for 5 days and cloning cultured to obtain stable expression cell lines.Real-time quantitative PCR(qRT-PCR)was used to verify the uc40 silencing expression.The effect of uc40-siRNA-44 on the cell cycle was detected by PI staining and CCK-8 assay,and the effect of uc40-siRNA-44 on apoptosis was analyzed by Annexin V PE/7-AAD and caspase 3 activity assay.Culture uc40-siRNA-44 silenced stable P19 cell lines in vitro and induce it with 1%DMSO to differentiate into myocardial like cells and morphological changes of cell differentiation were observed by inverted microscope.The mitochondrial DNA(mtDNA)copy number was detected by PCR.The cellular ATP production was detected by luciferase-based luminescence assay.The reactive oxygen species(ROS)was determined by 2’,7’-dichlorofluorescein diacetate(DCFDA)fluorescent probe and the mitochondrial membrane potential(MMP)was determined by JC-1 fluorescent probe with fluorescence microscope and FCM.The changes of key gene PBX1 mRNA and mRNA level of GATA4,Nkx2.5,cTnI,ANP in the differentiation of P19 cells were detected by PCR.The protein expression levels of cTnI and ANP was detected by western blot.Results:The stable cell line of uc40-siRNA-44 silencing was successfully constructed and verified.The increased level of gene GATA4 mRNA and Nkx2.5 mRNA indicated that the pulsatile like cells were cardiac like cells.Uc4O-siRNA-44 can significantly promote the differentiation of P19 cells either in size or amount of the embryonic like bodies,but there were no significantly difference in the morphology or time taken for the appearance of beating-like myocardial cells between two groups.Uc40-siRNA-44 can promote P19 cells into the S phase,and induce the proliferation of P19 cells and inhibit the apoptosis of P19 cells.There was no significant difference in mtDNA copy number and cellular ATP level between two groups.The MMP of uc40-siRNA-44 cells was increased slightly and the ROS level was significantly lowered in uc40-siRNA-44 group.During the differentiation of the cells,the myocardial differentiation associated gene PBX1 in uc40-siRNA-44 group increased with the differentiation and significantly higher than that in siNC control group,while the increasing tendency of myocardial markers such as ANP and cTnI in uc40-siRNA-44 group is much lower than those in siNC group.Conclusion:Uc40-siRNA-44 has the effect of promoting cell proliferation and inhibiting cell apoptosis.Uc40-siRNA-44 can significantly promote the differentiation of P19 cells either in size or amount of the embryonic like bodies,but there were no significantly difference in the morphology or time taken for the appearance of beating-like myocardial cells.Uc40-siRNA-44 may play some role in maintaining normal function of mitochondrion.The mechanism of uc40-siRNA-44 silencing may undo the inhibition of IncRNA on its target gene PBX1 and activate its signaling pathway,thus break the balance development and cause the cell to develop into abnormal myocardial cells.