| Objective:Atherosclerosis(AS)has become a serious harm to human life and health of the frequently occurring and common diseases,is the main cause of death in developed countries,the mortality rate in China is also increasing year by year trend.Atherosclerosis is caused by various stimuli in the artery wall,is a major cause of cardiovascular and cerebrovascular disease.Because of its pathogenesis has not been fully elucidated,so to explore the pathogenesis of cardiovascular disease prevention and treatment of great significance.The development of atherosclerosis related VSMCs function changes,while VSMCs is regulated by mediators such as platelet-derived growth factor(Platelet Derived Growth Factor,PDGF)stimulation from media to intima migration is a key step in the formation of atherosclerotic plaque.Rho-associated coiled-coil containing protein kinase(ROCK)is a serine/threonine kinase that is downstream target of the small GTPases RhoA.Its main function is to regulate the activity of the actin cytoskeleton.ROCK plays an important role in VSMC migration and migration by regulating microfilm assembly and local attachment.However,the regulatory mechanism of ROCK on vascular remodeling in the process of AS is not yet fully defined.This study aims to explore the miRNAs related to the ROCK-incuced proliferation and migration of vascular smooth muscle cells and its mechanism by culturing VSMCs.Methods:(1)The expression level of miR-92 a were measured by qPCR in A7r5,after treatment with ROCK inhibitor Y27632.(2)Using siRNA interference technology,ROCK gene was downregulated by liposome transfection of ROCK siRNA(siROCK),qPCR detection of ROCK mRNA expression was successfully downregulated.(3)Transient transfection technique was used to transfect Pre-mi R-92 a and Anti-miR-92 a to inhibit or overexpression in VSMCs.qPCR was used to detect whether miR-92 a was up-regulated or down-regulated.(4)The expression of ROCK mRNA was detected by qPCR after up-regulation and down-regulation of miR-92 a by using siRNA interference technique.(5)The effects of miR-92 a and ROCK on the migration of VSMCs were detected by Boyden Chamber microporous membrane double-cell method.The effects of miR-92 a and ROCK on the proliferation of VSMCs were detected by CCK-8.(6)The effects of miR-92 a target gene KLF4 on the proliferation and migration of VSMCs were detected by CCK-8 and Boyden Chamber methods.(7)Western blot was used to detect the expression of KLF4 in VSMCs after treat with Y27632.(8)The effect of miR-92 a and ROCK on the pseudopodal extension of VSMCs and the expression of KLF4 were analyzed by immunofluorescence.Results:(1)ROCK-specific inhibitor Y27632 inhibited the expression of miR-92 a.(2)Anti-mi R-92 a and Pre-miR-92 a had no significant effect on the expression of ROCK.(3)Anti-miR-92 a reduced the proliferation and migration of VSMCs under the stimulus of PDGF-BB.However,pre-miR-92 a promoted the proliferation and migration of VSMCs.(4)ROCK specific inhibitor Y27632 can inhibit the proliferation and migration of A7r5 cells induced by PDGF.(5)Pre-miR-92 a could restore the inhibitory effect of Y27632 on proliferation and migration of VSMCs.(6)Inhibition of VSMCs proliferation and migration by KLF4.(7)ROCK inhibitor Y27632 upregulates the expression of KLF4.(8)Anti-miR-92 a increased KLF4 expression and inhibited filopodia formation.siROCK increased the expression of KLF4 and inhibited the formation of filopodia,while the pre-mi R-92 a could partially recover this phenomenon.Conclusion:(1)Both ROCK and miR-92 a can promote the proliferation and migration of VSMCs.(2)ROCK promotes VSMCs proliferation and migration by regulating miR-92 a.(3)ROCK acts on KLF4 through miR-92 a to regulate VSMC proliferation and migration mediated by PDGF-BB. |
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