LncRNA H19/miR-675-5p Regulate Proliferation And Apoptosis Of Vascular Smooth Muscle Cell By Targeting Mfn2

Objective: Vascular smooth muscle cell(VSMC),which is the main cell component of vascular wall tissue structure and can help maintain vascular tone,the proliferation and apoptosis disorder of VSMC is the cytopathological basis of vascular remodeling disease like atherosclerosis.Long non-coding RNA(lncRNA)is a class of RNA with more than 200 nucleotides in length and no protein-coding function,and it is widely involved in the regulation of VSMC function.Through transcriptome sequenceanalysis,we found that a large number of lncRNA express differential in the rat intimal hyperplasia vascular,in which H19 expression level abnormally increased.Whether H19 is involved in vascular intimal hyperplasia regulation? What is the molecular mechanism? It is unclear.So this study was to investigate the function and molecular mechanism of H19 in VSMC proliferation,VSMC apoptosis and intimal hyperplasia from the molecular,cellular and whole level.Methods:1 The model of vascular intimal injury was established by carotid artery balloon injury in rats.Samples were drawn from the intimal hyperplasia vascular and normal vascular,and then contrastively analyzed them via transcriptome sequence.Take GO and Pathway analysis of the differential expressed mRNA,and verified by qRT-PCR.2 VSMC were cultured in vitro and divided into 5 groups transfected with siRNA Control,siRNA H19,miR-675-5p inhibitor,siRNA H19 +mi R-675-5p mimic,mi R-675-5p mimic respectively.The cell viability was measured by CCK8 assay kit.Apoptosis and cell cycle were detected by Annexin V-FITC/PI staining assay kit with flow cytometry.The relate protein expressions were analyzed by Western Blot.3 To confirmed that Mfn2 is the target of miR-675-5p,we constructedpGL3-Mfn2-3’UTR(including wild-type and mutant)reporter gene recombinant vectors and tested by luciferase activity.4 H19 siRNA was used to intervene the rat model of intimal hyperplasia by carotid artery balloon injury,and the vascular samples were taken after 21 days.The intima/medial ratio was analyzed by frozen section with HE staining to analysis of intimal hyperplasia.Results:1 The transcriptome sequenceanalysis of rat intimal hyperplasia vascularThe transcriptome sequence results showed that compared with the normal vascular,there are 235 mRNA expression up-regulated,170 mRNA down-regulated,329 lnc RNA up-regulated and 473 lncRNA down-regulated(Log2 fold change≥1.0 or ≤-1,P<0.05).Compared with normal vascular,VSMC differentiation marker calponin(Cnn1),smooth muscle ?-actin(Acta2),smooth muscle 22 alpha(SM22 ?),Smooth muscle myosin heavy chain 11(Myh11),smoothelin(Smtn)expression levels were significantly decreased,indicating that the rat intimal hyperplasia vascular model was successfully established.The result of GO and Pathway analysis indicated that the biological processes related to the intimal hyperplasia after vascular injury were mainly manifested in skeletal muscle contraction,muscle filament sliding,and so on.And the high correlation pathways were mainly involved in hypertrophic cardiomyopathy,cardiac muscle contraction,tight junction,etc.The above-mentioned biological processes are closely related to the regulation of VSMC on vascular physiological function,which can be involved in the proliferation,and differentiation of VSMC from a variety of ways.2 H19/miR-675-5p promote VSMC proliferation and inhibit apoptosis by targeting Mfn2Further comparison of sequence analysis and q RT-PCR validation showed that the expression of H19 was significantly increased after intimal injury compared with control group.mi R-675-5p,as a micro RNA encoded by the first exon region of H19 gene,was also showed the higher expression levelin intimal injury vascular.It was suggested that H19/miR-675-5p might play an important role in the regulation of VSMC proliferation and apoptosis.Five groups of VSMC were cultured in vitro and make multiple testing after transfected with siRNA Control,siRNA H19,miR-675-5p inhibitor,siRNA H19+miR-675-5p mimic,miR-675-5p mimic respectively.The results demonstrated that compared with control,the cell viability of siRNA H19 group and miR-675-5p inhibitor group was significantly decreased,the ratio of apoptotic cells increased and the ratio of cells in G2/M phase decreased,the expression of apoptosis-related marker protein increased while proliferation-related marker protein expression decreased.Moreover,the results of miR-675-5p mimic group were completely opposite to siRNA H19 and mi R-675-5p inhibitor group.The results of co-transfected siRNA H19 and mi R-675-5p mimic showed that the addition of miR-675-5p mimic could effectively reverse the effect of siRNA H19 on VSMC(P<0.05).These results indicated that H19/miR-675-5p was involved in the regulation of VSMC cell activity,which could promote the proliferation and inhibit the apoptosis of VSMC.It was also found that there is a miR-675-5p binding site in the 3’UTR of Mfn2 through bioinformatics prediction analysis,suggesting that Mfn2 may be a downstream target of H19 acting through miR-675-5p.The luciferase reporter gene analysis result showed that miR-675-5p had a direct action with3’UTR of Mfn2 in rat.And compared with control,Western Blot analysis showed that Mfn2 expression was increased significantly in VSMC,which was transfected by miR-675-5p inhibitor(P <0.05).The results confirmed that Mfn2 is the target of miR-675-5p indeed.5 knockdown of H19 inhibite rat intimal hyperplasiaThe frozen section with HE staining showed that the intimal performe diffuse thickening and the lumen become smaller after balloon injury,but the degree of intimal thickening after knockdown of H19 was significantly less than that of the control group(P<0.05).Western Blot analysis showed that knockdown of H19 inhibited the expression of proliferation phenotype markergene PCNA and promoted the expression of contraction phenotype marker gene SM22?.Conclusions:1 In the process of intimal hyperplasia,a large number of lncRNA expression level have changed;2 Mfn2 is the regulatory target of miR-675-5p;3 H19 inhibit VSMC apoptosis,and promote VSMC proliferation and vascular intimal hyperplasia through miR-675-5p/Mfn2 regulatory axis.