27nt-miRNA通过靶向哺乳动物雷帕霉素靶蛋白调控血管平滑肌细胞凋亡及其分子机制研究  

Impact of 27nt-miRNA on Apoptosis of Vascular Smooth Muscle Cells and Related Mechanism

作　　者：李丹 杨鹏 滕红丽[2] 颜渊鸳 罗雪兰 欧和生[1] LI Dan;YANG Peng;TENG Hongli;YAN Yuanyuan;LUO Xuelan;OU Hesheng(College of Pharmacy,Guangxi Medical University,Nanning(530021),Guangxi Zhuang Autonomous Region,China)

机构地区：[1]广西医科大学药学院,广西壮族自治区南宁市530021 [2]广西中医药大学附属广西国际壮医医院科技部

出　　处：《中国循环杂志》2020年第4期401-407,共7页Chinese Circulation Journal

基　　金：国家自然科学基金资助项目(81373403);广西自治区教育厅中青年教师基础能力提升项目(2019KY0344);南宁市良庆区科学研究与技术开发计划项目(201911);广西中医药大学博士科研启动基金项目(2018BS059)。

摘　　要：目的:探讨来源于一氧化氮合酶基因第四内含子的27碱基重复序列微小RNA(27nt-miRNA)对哺乳动物雷帕霉素靶蛋白(mTOR)介导的主动脉血管平滑肌细胞(VSMC)凋亡调控作用及潜在分子机制。方法:将大鼠主动脉VSMC分为空白对照组、雷帕霉素组、27nt-miRNA组、27nt-miRNA反义序列(anti-27ntmiRNA)组、阴性对照组。27nt-miRNA组:转染27nt-miRNA正义链慢病毒载体;anti-27nt-miRNA组:转染27ntmiRNA反义链慢病毒载体;阴性对照组:转染随机阴性序列慢病毒载体。除空白对照组外,雷帕霉素组与各转染组细胞给予100 ng/ml雷帕霉素诱导培养2 h诱导凋亡。荧光定量逆转录PCR(qRT-PCR)验证27nt-miRNA慢病毒稳转细胞株构建情况;CCK-8法、流式细胞术、qRT-PCR法与蛋白免疫印迹(Western blot)法分别检测各组细胞增殖能力、细胞凋亡率与周期分布以及mTOR、磷酸化-mTOR(p-mTOR)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)和半胱氨酸天冬氨酸蛋白酶-3(caspase-3)的信使RNA(mRNA)和(或)蛋白表达水平。结果:慢病毒转染VSMC后,27nt-miRNA稳转细胞株构建成功,雷帕霉素可抑制VSMC的mTOR mRNA与p-mTOR蛋白表达水平。与阴性对照组相比,27nt-miRNA组细胞的活力与增殖能力显著增强,且细胞凋亡率显著降低(P均<0.05),细胞周期G1期向S期分裂进程阻滞减弱(P<0.05);Bax和caspase-3蛋白表达量显著减少,Bcl-2蛋白表达量显著增加(P均<0.05);mTOR基因转录水平与p-mTOR蛋白表达水平均下降(P<0.05)。结论:27nt-miRNA通过负调控m TOR基因转录水平与蛋白磷酸化水平抑制VSMC凋亡,其机制可能与上调Bcl-2蛋白表达,下调Bax和caspase-3蛋白表达有关。Objectives: This study investigated the regulation of 27 nt-miRNA on apoptosis of aortic vascular smooth muscle cells(VSMC) and related mechanisms focusing on mammalian target of rapamycin protein(mTOR) pathway. Methods: Rat aortic VSMC were divided into control group, rapamycin group, 27 nt-miRNA group, anti-27 nt-miRNA group and negative control group. The rapamycin group, 27 nt-miRNA group, anti-27 nt-miRNA group and the negative control group were treated with 100 ng/ml rapamycin for 2 hours. The construction of 27 nt-miRNA lentivirus stable cell line was verified by qRT-PCR. CCK-8 assay and flow cytometry were used to detect the proliferative capacity, apoptotic rate and cell cycle distribution;meanwhile, q RT-PCR and Western blot were used to determine the mRNA or protein expression level of mTOR, phospho-mTOR(p-mTOR), Bcl-2, Bax and caspase-3.Results: The 27 nt-miRNA stable cell line was successfully constructed after transfection. Compared with the negative control group, the proliferative capacity was enhanced, while apoptotic rate was significantly decreased in 27 nt-miRNA group(P<0.05). Additionally, the proportion of cells at G1-phase was significantly decreased, while the proportion of cells at S-phase was significantly increased in 27 nt-miRNA group(P<0.05). Moreover, Bax and caspase-3 protein expression was significantly downregulated but the Bcl-2 protein expression was significantly upregulated in 27 nt-miRNA group(P<0.05). The mRNA expression of mTOR and the protein expression of p-mTOR were significantly decreased in 27 nt-miRNA group(P<0.05).Conclusions: 27 nt-miRNA could inhibit the apoptosis of VSMC by negatively regulating the mTOR at mRNA and protein phosphorylation level, and by the up-regulation of Bcl-2 protein expression and down-regulation of Bax and caspase-3 protein expression.

关 键 词：27nt-miRNA 血管平滑肌细胞 凋亡 哺乳动物雷帕霉素靶蛋白 磷酸化 

分 类 号：R54[医药卫生—心血管疾病]