Effects Of MiR-29b On The Proliferation And Apoptosis Of Pulmonary Artery Smooth Muscle Cells By Targeting CCND2 And Mcl-1

Although the research on the pathogenesis of PAH associated miRNAs has increased in recent years,there is little research on the association between miR-29 b and pathogenesis of PAH.We aim to study the effects of miR-29 b on CCND2 and Mcl-1 protein in pulmonary artery smooth muscle cells and analysis whether miR-29 b can inhibit pulmonary artery smooth muscle cells proliferation and promote apoptosis,try to explore the molecular mechanism of pulmonary arterial hypertension of the pathological process,provide a new theoretical basis of molecular pathological study of pulmonary arterial hypertension from the point of miR-29 b.Firstly monocrotaline induced and control groups of rats pulmonary tissue are under microarray analysis,screen meaningfull miRNAs in pulmonary artery hypertension formation process,further by qRT-PCR to validate miR-29 b expression in pulmonary tissues in monocrotaline induced and hypoxia-induced PAH model.To observe the effect of miR-29 b on the proliferation and apoptosis of pulmonary artery smooth muscle cells,PASMCs were processed respectively by miR-29 b mimics,inhibitoror,negative control and,OD value was detected.Cell cycle were detected by flow cytometry,using AnnexinV-PI flow cytometry experiments to detect apoptotic rate and compare each group’s rate.Use bioinformatics software to predict the target gene of miR-29 b.CCND2 and MCL-1 protein levels are measured by Western-blotting between the experimental groups.To compare the changes of right ventricular systolic pressure,right ventricular mass ratio and membrane ratio of pulmonary artery after MCT treatment and mimic miR-29 b treatment.Through the above methods,we observed that gene chip analysis showed miR-29 b expression in pulmonary artery tissue at different time points decreased continuously.miR-29 b expression was significantly reduced in PAH models.miR-29 b inhibited the proliferation of pulmonary artery smooth muscle cells,promoted apoptosis and thus affected the number of the cells.miR-29 b can inhibit the expression of Mcl-1 and CCND2 protein,and there was no significant difference in the proliferation and apoptosis of pulmonary artery smooth muscle cells after Mcl-1 and CCND2 were silenced.The increase of miR-29 b can make the right ventricular systolic pressure,right ventricular mass ratio and membrane ratio of pulmonary artery in the membrane decreased,the difference was statistically significant.Therefore,in this study we have demonstrated in vitro and in vivo: Gene chip analysis showed that miR-29 b expression in pulmonary artery tissue at different time points decreased continuously.Over expression of miR-29 b can significantly reduce the number of PASMCs,the expression of miR-29 b can significantly inhibit the PASMCs cell cycle and proliferation,and increase the apoptosis of PASMCs.Using bioinformatics software to predict the miR-29 b target genes and found that CCND2 and Mcl-1 were target genes of miR-29 b in PAH,over expression of miR-29 b could significantly inhibit the CCND2 and Mcl-1 protein expression by Western blot at the protein level and si-CCND2 and si-Mcl-1 silence CCND2 and Mcl-1 expression,transfection miR-29 b mimic or inhibitor almost has no influence on cell cycle and apoptosis of PASMCs,suggesting that in vitro miR-29 b in hibited the proliferation and promote apoptosis of PASMCs by inhibiting the expression of CCND2 and Mcl-1 respectively.miR-29 b mimic can reduce the increased right ventricular systolic pressure and right ventricular hypertrophy and improve pulmonary vascular remodeling and changes that in the MCT induced rat that have formed significantly,inhibit CCND2 and Mcl-1 protein expressionof MCT induced PAH in rat pulmonary vascular,suggesting that miR-29 b mimic in vivo can inhibit CCND2 and Mcl-1 protein expression and play a reversal role PAH pathology.