The Mechanism Of MIRNA98 In Mediating Vascular Smooth Muscle Cells Proliferation And Migration

BackgroundDiabetic lower extremity arterial disease(DLEAD)is one of the major vascular complications of diabetes mellitus,mainly due to atherosclerosis(AS),and its incidence is increasing year by year.It is a serious threat to the health and life of patients.Percutaneous transluminal angioplasty(PTA)is currently the preferred clinical treatment for DLEAD,but the incidence of postoperative restenosis(RS)remains high,which greatly limits the clinical efficacy and application of PTA.Effective prevention and treatment of postoperative restenosis is the focus of current efforts to improve the outcome of DLEAD.In our previous study,we found that unlike atherosclerotic lesions,vascular smooth muscle cells(VSMCs)in restenosis lesions exhibit unique biological characteristics:excessive proliferation and migration ability,in which Lin28a plays an important role.miR98 is usually highly expressed in differentiated cells with highly conserved sequences and functions,and is involved in the regulation of the vascular smooth muscle cells(VSMCs).sequences and functions that are involved in regulating cell proliferation,differentiation,death and other life processes.Previous studies have indicated that miRNA98 and Lin28a can be involved in regulating the proliferation and migration of cancer cells,but the regulatory mechanism between them has not been investigated for the proliferation and migration of vascular smooth muscle cells.In this study,we investigated the specific mechanisms of miR98/Lin28a regulation of proliferation and migration of VSMCs in restenotic lesions.This study provides new ideas and directions for the prevention and treatment of restenotic lesions after PTA in diabetic lower extremity vasculopathy.Objectives1.To clarify the involvement of miR98 and Lin28a in the development of restenotic lesions by constructing a rat iliac artery restenosis model with "double injury surgery".2.To investigate the specific molecular mechanisms by which miR98 and Lin28a regulate the proliferation and migration of VSMCs in restenotic lesions by regulating the expression of miR98 and Lin28a in vascular smooth muscle cells.Materials and Methods1.Construction of iliac artery AS and RS model in type 2 diabetic rats.(1)The rat iliac artery AS model was constructed by applying high-fat diet feeding+single injection of low-dose STZ+balloon dilatation strain.(2)The rat iliac artery RS model was constructed by high-fat diet feeding+single injection of low-dose STZ+balloon dilatation and straining+PTA surgery.2.AS and RS plaque detection and vascular specimen collection:4 weeks after balloon dilatation strain or PTA surgery,plaque formation was examined using Doppler ultrasound and perfusion sampling was performed in the modeled rats after anesthesia.3.Immunohistochemistry was performed to detect the localization and expression ofα-SMA in the vascular tissue of each group of rats.4.RT-qPCR was performed to detect the expression levels of miR98 and Lin28a in AS/RS vascular tissues.5.Extraction and culture of primary VSMCs:After obtaining primary VSMCs by stripping method,they were cultured in an incubator with an environment of 37℃ and 5%CO2.6.Transfection of VSMCs by up-/down-regulated Lin28a lentivirus and construction of stably transfected cell lines.(1)Lentivirus infection pre-experiment,virus reinfection index(MOI)assay:four orders of magnitude MOI values were taken and lentivirus was transfected to VSMCs,and the growth of cells when transfection efficiency reached about 80%,MOI values of this group and transfection conditions were recorded for formal experiments.(2)Formal transfection of Lin28a lentivirus:cells were transfected according to the optimal MOI and transfection conditions obtained from the pre-experiment,and the virus transfection in the cells was observed by fluorescence microscopy after 72 hours of transfection.(3)RT-qPCR and Western blot to detect the expression of Lin28a in VSMCs.7.Transfection of miR98 mimics or inhibitors into VSMCs with stably up-and down-regulated Lin28a or normal VSMCs.(1)Transfection of miR98 mimics or inhibitors and Lipofectamine 2000 mixture into stably upregulated Lin28a cell lines or normal VSMCs lines.(2)RT-qPCR assay of miR98 and Lin28a expression in VSMCs after transfection with miR98 mimics or inhibitors.(3)Western blot to detect the expression of Lin28a in VSMCs.8.EdU cell proliferation assay:The EdU cell proliferation assay was performed on VSMCs in different treatment groups,and the EdU staining of VSMCs in 96-well plates was observed by fluorescence microscopy and photographed for counting.9.Transwell cell migration assay was performed to detect the migration ability of VSMCs in different treatment groups.10.Statistical analysis:All statistical analyses were performed using SPSS 22.0.Comparisons between two groups of data were performed using t-test,and comparisons between multiple groups of data were performed using one-way ANOVA.All data were expressed as mean 士 standard deviation(mean±SD),and the statistical significance of differences between groups was set at P<0.05(two-sided).Statistical analysis was performed after all experiments were repeated more than three times.Results1.Restenotic plaques exhibit pathophysiological features that are different from those of atherosclerotic plaques.The excessive migration and proliferation of VSMCs were the main cause of RS lesion development after PTA.Compared with atherosclerotic plaques,restenotic lesions exhibited unique pathophysiological features,and immunohistochemical results showed that the number of vascular smooth muscle cells was significantly elevated in restenotic plaques.Meanwhile,RT-qPCR results showed that the expression of Lin28a was significantly increased in restenotic plaque tissues,while the content of miR98 was decreased compared with that in atherosclerotic plaque tissues(P<0.05),and the results indicated that miR98 and Lin28a were involved in the formation of restenotic lesions.2.In restenotic plaques,Lin28a was able to promote the proliferation and migration of vascular smooth muscle cells.Lentiviruses with up-and down-regulated Lin28a and corresponding negative control lentiviruses were transfected in VSMCs,and Western blot verified that Lin28a was successfully regulated and stable in VSMCs after transfection.edU and Transwell assayed the changes of proliferation and migration ability in VSMCs after regulating Lin28a expression,and the results showed that up-regulated The results showed that upregulation of Lin28a promoted the proliferation and migration of vascular smooth muscle cells,while inhibition of Lin28a inhibited the proliferation and migration of vascular smooth muscle cells,i.e.Lin28a had a regulatory effect on the proliferation and migration of VSMCs(P<0.05).3.In vascular smooth muscle cells,miR98 was able to inhibit the migration and proliferation of VSMCs.VSMCs were transfected with miR98 mimics and inhibitors,and qRT-PCR confirmed that miR98 expression was successfully regulated in VSMCs(P<0.05).Subsequently,EDU and Transwell examined the proliferation and migration ability of VSMCs after miR98 mimic or inhibitor transfection,respectively,and the results confirmed that miR98 was able to inhibit the proliferation and migration ability of VSMCs compared with the control group(P<0.05).4.In vascular smooth muscle cells of restenotic plaques,Lin28a was a downstream target of miR98.As overexpression of miR98 inhibits the proliferation and migration of VSMCs.Transfection of miR98 mimics and inhibitors in VSMCs.qRT-PCR verified that miR98 was successfully regulated and stably functioned in VSMCs after transfection.Subsequently,we used RT-qPCR to detect Lin28a expression in VSMCs after regulation of miR98 expression,and the results showed that upregulation of miR98 could inhibit Lin28a expression,while inhibition of miR98 could promote Lin28a expression.Combined with the in vitro and out vivo results,we concluded that Lin28a was a downstream target regulated by miR98 inhibition(P<0.05).5.miR98 was able to regulate VSMCs migration and proliferation by inhibiting Lin28a expression.Secondary transfection of miR98 mimics or inhibitors in VSMCs transfected with Lin28a lentivirus and subsequent assay of proliferation and migration ability of regulated vascular smooth muscle cells using EDU and Transwell showed that simultaneous modulation of Lin28a expression "rescued" VSMCs alone.The results showed that simultaneous regulation of Lin28a expression could "rescue" the changes in proliferation and migration ability of VSMCs induced by miR98 alone(P<0.05),confirming that miR98 could regulate VSMCs migration and proliferation by inhibiting downstream Lin28a.Conclusion1.Atherosclerotic and restenotic lesions show different pathophysiological features,miR98 and Lin28a are involved in the development of restenotic lesions.2.In VSMCs,Lin28a is a downstream target regulated by miR98 inhibition.miR98 is able to exert inhibitory effects on the proliferation and migration of VSMCs by suppressing Lin28a expression.