Study On The Effect Of LncRNA MALAT1 In Phenotypic Switching Of Vascular Smooth Muscle Cells

Phenotypic switching of vascular smooth muscle cells is a common pathological process of multiple cardiovascular disease and is a complex biological process.In recent years,numerous studies confirm that lncRNAs are involved in the development of atherosclerosis and phenotypic switching process of VSMCs,including the regulation of proliferation and migration of VSMCs.The effect of lncRNA MALAT1 in phenotypic switching of VSMCs is unknown.Therefore,the aim of this study is to investigate the role of MALAT1 on phenotypic switching process of VSMCs and its influence on several key transcription factors.First,two models were established.one was VSMCs differentiation model induced by BMP-7,and the other was VSMCs proliferation model induced by PDGF-BB.The two models were successfully established,confirmed by cellular morphology,differentiated and proliferative marker gene expression.Second,the expression of key transcription factor Myocardin,SRF and MKL1 were tested the in two models.The results showed that Myocardin and SRF are key transcription factors of differentiated phenotype,and MKL1 is one key transcription factor of proliferative phenotype.Third,we examined the expression of MALAT1 in two models.The results showed that the expression of MALAT1 was decreased in BMP-7-induced differentiation model,whereas,the expression of MALAT1 was increased in PDGF-BB-induced proliferation model.Therefore,these data suggested that MALAT1 could be involved in smooth muscle cell phenotype transformation.Next,the effect of MALAT1 on VSMCs differentiation,proliferation and migration were detected,when the level of MALAT1 was decreased using siRNA of MALAT1.First,HA-VSMCs treated with siRNA of MALAT1 became elongated fusiform,had rich filaments in cytoplasm and exhibited differentiated phenotype forms.Treatment with MALAT1 siRNA increased the expression of differentiation marker genes ACTA2 and SM-22 by immunofluorescence,real-time PCR and western blot.Second,low level of MALAT1 inhibited HA-VSMCs cell proliferation,confirmed by MTT assay and EdU assay.And the expression of proliferation marker gene PCNA,CyclinDl and OPN was decreased in MALAT1 siRNA-transfected HA-VSMCs.Third,The migration of HA-VSMCs was inhibited,confirmed by wound scratch assay,when HA-VSMCs were transfected with MALAT1 siRNA.Knockdown of MALAT1 also reduced the expression of cell migration markers CYR61 MYL9.The results determined that the knockdown of MALAT1 could inhibit proliferation of smooth muscle and promote VSMCs transfer to differentiated phenotype.Finally,the expression of myocardin,SRF and MKL1 were detected by Western blotting and real-time PCR,when HA-SMCs were transfected with siRNA for MALAT1.The results showed that the expression of transcription factor myocardin and SRF were increased in HA-SMCs transfected with MALAT1 siRNA,while the expression of transcription factor MKL1 was decreased.These results preliminarily determined that the role of MALAT1 in phenotypic switching of VSMCs might be related to transcription factors myocardin,SRF and MKL1.