内含子源性27nt-microRNA对大鼠血管平滑肌细胞表型转换的影响及其机制研究  

Effect and mechanism of intron-derived 27-nt microRNA on phenotypic switch of rat vascular smooth muscle cells

作　　者：周炜潜 罗雪兰 王光耀 黄凤 欧和生 Zhou Wei-qian;Luo Xue-lan;Wang Guang-yao;Huang Feng;Ou He-sheng(Zhuang and Yao Medical Research Laboratory,International Zhuang Medical Hospital Affiliated to Guangxi University of Chinese Medicine,Nanning,Guangxi 530201,China;Department of Cardiovascular Medicine,International Zhuang Medical Hospital Affiliated to Guangxi University of Chinese Medicine,Nanning,Guangxi 530201,China)

机构地区：[1]广西中医药大学附属国际壮医医院壮瑶医药研究实验室,广西南宁530201 [2]广西中医药大学附属国际壮医医院心血管内科,广西南宁530201

出　　处：《中国现代医学杂志》2024年第14期36-45,共10页China Journal of Modern Medicine

基　　金：国家自然科学基金(No:82060655)。

摘　　要：目的 探讨内含子源性27nt-miRNA(27nt-miR)对血小板源性生长因子-BB(PDGF-BB)诱导大鼠血管平滑肌细胞(VSMCs)表型转换的影响及其分子机制。方法 用27nt-miR表达慢病毒转染培养的VSMCs,观察其对PDGF-BB诱导的VSMCs表型转换的影响;将细胞分为对照组、PDGF-BB诱导组、27ntmiR组、27nt-miR-sponge组及27nt-miR NC组(27nt-miR NC组)。雷帕霉素(100 nmol/L)与MHY-1485(10μmol/L)作用于27nt-miR组、27nt-miR-sponge组及27nt-miR NC组,以验证27nt-miR是否参与mTOR与p70S6k通路的调控。用生物信息学预测27nt-miR与mTOR的靶向结合位点,用CCK-8法与EdU法分别测定VSMCs活力与增殖情况,划痕试验测定VSMCs迁移能力,实时荧光聚合酶链反应检测α-SMA、SM22α及OPN mRNA表达,Western blotting检测α-SMA、SM22α、OPN、mTOR、p-mTOR、p70S6k与pp70S6k蛋白表达。结果 CCK-8法结果示,PDGF-BB诱导组细胞增殖率较正常值高(P <0.05),27ntmiR NC组与PDGF-BB诱导组比较,差异无统计学意义(P>0.05);27nt-miR组细胞增殖率较27nt-miR NC组低(P <0.05),27nt-miR-sponge组与27nt-miR NC组比较,差异无统计学意义(P>0.05)。PDGFBB诱导组细胞迁移率较对照组高(P <0.05),27nt-miR NC组与PDGF-BB诱导组比较,差异无统计学意义(P>0.05);27nt-miR组细胞迁移率较27nt-miR NC组低(P <0.05),27nt-miR-sponge组细胞迁移率与27nt-miR NC组比较,差异无统计学意义(P>0.05)。EdU法结果示,PDGF-BB诱导组细胞增殖率较对照组高(P <0.05),27nt-miR NC组与PDGF-BB诱导组细胞增殖率比较,差异无统计学意义(P>0.05);27nt-miR组细胞增殖率较27nt-miR NC组低(P <0.05),27nt-miR-sponge组与27nt-miR NC组比较,差异无统计学意义(P>0.05)。PDGF-BB诱导组α-SMA mRNA相对表达量较对照组低(P <0.05),两组SM22α与OPN mRNA相对表达量比较,差异无统计学意义(P>0.05)。27nt-miR NC组与PDGF-BB诱导组α-SMA、SM22α、OPN mRNA相对表达量比较,差异无统计学意义(P>0.05);27nt-miR组α-SMA、SM22α mRNA相对表达量较27nt-mObjective To investigate the effect and mechanism of intron-derived 27-nt microRNA(27ntmiR)on the phenotypic switch of rat vascular smooth muscle cells(VSMCs)induced by platelet-derived growth factor-BB(PDGF-BB).Methods VSMCs were transduced with 27nt-miR-expressing lentiviruses and their effects on phenotypic switch of VSMCs induced by PDGF-BB was observed.The cells were divided into control group,PDGF-BB induction group,27nt-miR group,27nt-miR-sponge group and 27nt-miR negative control(27nt-miR NC)group.The 27nt-miR group,27nt-miR-sponge group and 27nt-miR NC group were subjected to rapamycin(100 nmol/L)and MHY1485(10μmol/L)to verify the involvement of 27nt-miR in the regulation of mTOR and p70S6k pathways.The target binding sites of 27nt-miRNA and mTOR were predicted by bioinformatics.The viability and proliferation of VSMCs were determined by CCK-8 and EdU cell proliferation assays.The migration ability of VSMCs was determined by the scratch assay.The relative mRNA expressions ofα-SMA,SM22αand OPN were detected by qRT-PCR.The relative protein expressions ofα-SMA,SM22α,OPN,mTOR,p-mTOR,p70S6k and p-p70S6k were detected by Western blotting.Results The CCK-8 assay revealed that the cell proliferation rate in the PDGF-BB induction group was higher than normal(P<0.05),while there was no difference in the cell proliferation rate between the 27nt-miR NC group and the PDGF-BB induction group(P>0.05).The cell proliferation rate in the 27nt-miR group was lower than that in the 27nt-miR NC group(P<0.05),while there was no difference in the cell proliferation rate between the 27nt-miR-sponge group and the 27nt-miR NC group(P>0.05).The cell migration rate in the 27nt-miR group was lower than that in the 27nt-miR NC group(P<0.05),while there was no difference in the cell migration rate between the 27nt-miR-sponge group and the 27nt-miR NC group(P>0.05).The EdU cell proliferation assay demonstrated that the cell proliferation rate in the PDGF-BB induction group was higher than that in the control group(P<0.05),while the

关 键 词：27nt-microRNA 血管平滑肌细胞 mTOR/p70S6k信号通路 表型转换 

分 类 号：R543[医药卫生—心血管疾病]