The Mechanism Research Of MiR-4463 On Vascular Smooth Muscle Cells Through JNK Pathway In Arteriosclerosis Obliterans

Objective: Arteriosclerosis obliterans(ASO)has become the primary disease of peripheral vascular diseases,which seriously harm to people’s physical and mental health.And vascular smooth muscle cells are involved in the important process of the pathological formation of ASO,and its molecular mechanism is not clear,so it is particularly urgent and necessary for the early diagnosis and treatment of ASO.In recent years,with the research of non-protein mechanism and posttranscriptional mechanism becoming clearer,some researches focus on exploring the relationship between microRNAs(miRNAs)and ASO.In the previous experiments,we screened the differential expression of miRNA in the plasma of ASO patients and the control population by using the miRNA chip method,and the expression of mi R-4463 is different.Next,we used RT-qPCR technology to verify.Therefore,in this article,we mainly discuss the regulation of miR-4463 on vascular smooth muscle in ASO disease.Methods: We used the experimental conditions of ox-LDL intervention to simulate the conditions of the occurrence of atherosclerotic diseases.We detected the expression of miR-4463 in the plasma of the ASO patients and the control group by PT-qPCR,and the expression of the vascular smooth muscle with ox-LDL intervention or not by PT-qPCR.The expression of miR-4463 was regulated by transfection of miR-4463 mimic or miR-4463 inhibitor,and the effects of mi R-4463 on the vascular smooth muscle cells were further examined by cycle experiments,apoptosis experiments,migration experiments and so on.According to the results of functional tests,we predicted the target genes and related downstream pathways of miR-4463 through TargetScan,and we chose JNK pathway as a further research object,which is closely related to cell migration and smooth muscle cells phenotype transformation.Results: First,we found that the expression of mi R-4463 in the plasma of ASO patients decreased 53%(P=0.0145)than normal control group,and the expression of miR-4463 in vascular smooth muscle cell under the condition of ox-LDL intervention was 75%(P=0.0024)lower than normal cell group.According to the experimental results of cycle experiments,apoptosis experiments and migration experiments,we found that miR-4463 mimic inhibits the migration of vascular smooth muscle cells,and mi R-4463 inhibitor promotes the migration of vascular smooth muscle cells.However,from the results of cell cycle and apoptosis experiments,there was no significant difference between miR-4463 mimic and miR-4463 inhibitor group and NC group.The changes in the expression of migration related marker protein N-Cadherin and E-Cadherin further confirm that miR-4463 does have an effect on the migration of vascular smooth muscle by Western Blot test.From the results of cytoskeleton experiments,we can see that miR-4463 mimic keeps the vascular smooth muscle cells being contractile type,while miR-4463 inhibitor promotes the transformation of vascular smooth muscle cells to secretory type.Then,we detected the contractile marker protein SMA and the secretory marker protein OPN of vascular smooth muscle cell by Western Blot experiment.The experimental results also suggested that the expression of OPN was enhanced in both femoral artery tissues in ASO group than normal control group,and the expression of SMA was weakened in ASO group.From the cell level,the expression of OPN was weakened while the cell with mi R-4463 mimic transfection and the expression of SMA was enhanced.Compared with the NC group,the expression of OPN in the transfected miR-4463 inhibitor group was enhanced and the expression of SMA protein was weakened.After transfecting smooth muscle cells with NC,miR-4463 mimic and miR-4463 inhibitor,and dividing into three groups,the expression of P-JNK/JNK protein was detected by Western Blot test.We found that the expression of P-JNK/JNK protein transfected with miR-4463 mimic was stronger than that of NC group,and the expression of the protein was weaker with miR-4463 inhibitor transfected group than that of the NC group,butthere was no significant difference in the difference between them.Interestingly,the smooth muscle cells intervention with ox-LDL also got the same experimental results,and the results showed significant statistical differences.SP600125,an inhibitor of JNK pathway,interfered vascular smooth muscle cells after it transfected with miR-4463 inhibitor.We found that the effects of miR-4463 inhibitor on the migration and cytoskeleton change could be reversed after the SP600125 intervention was added.Conclusion: 1.The expression of miR-4463 was decreased in the plasma of the ASO patients indicates that miR-4463 may be involved in the pathophysiological process of the ASO disease.2.The expression of miR-4463 in vascular smooth muscle cells under ox-LDL intervention is lower than that in normal control group,suggesting that miR-4463 may participate in the pathological process of ASO disease.3.Cell function tests showed that miR-4463 could affect the migration and cytoskeleton of vascular smooth muscle cells.4.MiR-4463 may affect the migration and cytoskeleton of vascular smooth muscle cells through JNK pathway.