The Role Of MicroRNA-4306 In The Regulation Of Autophagy In Phenotype Transformation Of Vascular Smooth Muscle Cells

Objective:Arteriosclerosis obliterans(ASO)is the most common peripheral vascular disease,which has seriously harmed the health of the elderly.Phenotype transformation of vascular smooth muscle cells is an important pathological process of ASO,but its specific molecular mechanism is not fully understood.Therefore,it is of great significance for the early diagnosis and treatment of ASO to further reveal and elucidate the role of phenotype transformation of vascular smooth muscle cells in the development of ASO and its molecular mechanism.MicroRNAs(miRNAs)are a kind of endogenous non coding single stranded microRNAs with a length of about 18~22 bp.They play a negative role in gene expression by binding the target gene 3 ’-non coding region(3’-UTR)at the post transcriptional level,thus regulating cell proliferation,differentiation,migration and apoptosis.Previous studies have confirmed that miRNAs play an important role in the pathophysiological process of ASO,which is expected to be the target of early diagnosis and treatment of ASO.In our previous experiments,we screened miR-4306 gene chip and found that the expression of miR-4306 decreased in the plasma of ASO patients.We preliminarily concluded that miR-4306 may be involved in the occurrence and development of ASO,but its specific mechanism is still unclear.Autophagy is a subcellular structuredegradation pathway of organelles and cytoplasm,which is highly conserved in eukaryotic cells.A large number of studies have shown that autophagy plays an important role in the genesis and development of ASO.At the same time,autophagy is involved in the phenotype transformation of vascular smooth muscle cells.However,it is not clear whether miR-4306 can regulate autophagy and participate in ASO.Therefore,the role of miR-4306 in regulating the phenotype transformation of vascular smooth muscle cells and its mechanism were discussed.Methods:Three pairs of plasma from ASO patients and normal control group were hybridized by miRNA gene chip,and the results of miRNAs differential expression in plasma of ASO patients were screened based on the gene chip.The expression of miR-4306 was verified by RT-qPCR in plasma and endovascular tissues of ASO patients and normal people,and the clinical Fontaine stage was compared and analyzed.Human aortic smooth muscle cells(HASMCs)were induced by oxidized low density lipoprotein(Ox-LDL)to simulate atherosclerosis.The cell model was established and the relative expression of miR-4306 was detected by RT-qPCR.Western blot was used to detect the relative expression of bone related protein(Runx-2,OPN)and autophagy related protein(Beclin-1,LC3-B).By transfection of miR-4306 and miR-4306 inhibitor,the expression of miR-4306 was up-regulated or down regulated,and the expression of bone related protein(Runx-2,OPN)was detected by WB.Finally,target scan analysis was used to predict the potential target genes of miR-4306 involved in the regulation ofphenotype transformation of vascular smooth muscle cells.To further explore the role of miR-4306 in regulating autophagy in Ox-LDL induced phenotype transformation of HASMCs.Results:The results of miRNA microarray showed that the expression of miR-4306 in the plasma of ASO patients was significantly lower than that of the control group(P<0.05).RT-qPCR further verified the expression of miR-4306 in ASO patients’ plasma and vascular intima.The results showed that the expression of miR-4306 in ASO patients’ plasma and vascular intima decreased significantly(P<0.05).The expression of miR-4306 was lower than that of normal control group(P<0.05),and the expression of osteogenic related protein(Runx-2,OPN),autophagy related protein(Beclin-1,LC3-B)was higher than that of normal control group(P<0.05).The expression of osteogenic related protein(Runx-2,OPN)was lower than that of normal control group(P<0.05),but the expression of autophagy related protein(Beclin-1,LC3-B)was higher than that of normal control group(P<0.05).After adding 3-MA,the expression of Runx-2 and OPN decreased significantly compared with the control group(P<0.05).Conclusion:1.The expression of miR-4306 in ASO patients’ plasma and vascular intima decreased significantly,and in Ox-LDL induced HASMCs decreased significantly.2.In the process of induction of HASMCs by Ox-LDL,the phenotype transformation was promoted and autophagy was involved.3.Up regulation of miR-4306 inhibited the phenotype transformation of HASMCsinduced by Ox-LDL.4.In the process of Ox-LDL induced phenotype transformation of HASMCs,miR-4306 was upregulated to promote autophagy of smooth muscle cells.