miRNA-652在血管平滑肌细胞增殖中的作用及可能机制

Effect and Mechanism of miRNA-652 on the Proliferation of Vascular Smooth Muscle Cells

目的 探讨miRNA-652在血管平滑肌细胞(VSMCs)增殖中的作用及可能机制。方法 本实验时间为2019年1月至2020年7月。采用不同浓度(0、1、10、100 nmol/L)的血管紧张素Ⅱ(AngⅡ)刺激VSMCs 24 h后,检测miRNA-652表达情况,确定10 nmol/L AngⅡ为最佳刺激浓度。采用10 nmol/L AngⅡ分别刺激VSMCs 0、6、12、24 h后,检测miRNA-652表达情况,确定AngⅡ最佳刺激时间为24 h。将VSMCs随机分为miRNA-652模拟物组、模拟物对照组、miRNA-652抑制剂组、抑制剂对照组,分别转染miRNA-652 agomir、agomir NC、miRNA-652 antagomir及antagomir NC 24 h/48 h。采用qRT-PCR检测VSMCs中miRNA-652、NPTN mRNA相对表达量,采用CCK8试剂盒检测细胞增殖能力(相对吸光度)。通过miRNA靶基因预测数据库miRDB、TargetScan、miRTarBase在线进行生物信息学分析。为了验证NPTN是miRNA-652的靶基因,将含有miRNA-652结合位点的NPTN 3’UTR克隆到报告基因载体pmirGLO中,构建野生型pmirGLO-NPTN 3’UTR-WT和突变型pmirGLO-NPTN 3’UTR-MUT,并分别与寡核苷酸miRNA-652 agomir、miRNA-652 antagomir、agomir NC及antagomir NC共转染293 T细胞,转染48 h后测定荧光素酶相对活性。结果 转染24 h后,miRNA-652模拟物组miRNA-652相对表达量高于模拟物对照组,相对吸光度低于模拟物对照组(P<0.05);miRNA-652抑制剂组miRNA-652相对表达量低于抑制剂对照组,相对吸光度高于抑制剂对照组(P<0.05)。生物信息学分析结果显示,3个数据库均检测到NPTN是miRNA-652的靶基因。双荧光素酶报告基因检测结果显示,转染48 h后,miRNA-652模拟物组pmirGLO-NPTN 3’UTR-WT荧光素酶相对活性低于模拟物对照组(P<0.05)。转染48 h后,miRNA-652模拟物组NPTN mRNA相对表达量低于模拟物对照组,miRNA-652抑制剂组NPTN mRNA相对表达量高于抑制剂对照组(P<0.05)。结论 过表达miRNA-652可以抑制VSMCs增殖,而抑制miRNA-652可以促进VSMCs增殖;miRNA-652对NPTN具有负调控作用,其可能通过调控靶基因NPTN而影响VSMCs增殖。

Objective To investigates the effect and mechanism of miRNA-652 on the proliferation of vascular smooth muscle cells(VSMCs).Methods The time of this experiment was from January 2019 to July 2020.The relative expression of miRNA-652 was detected after VSMCs were stimulated with different concentrations(0,1,10,100 nmol/L)of AngⅡfor 24 h.Ten nmol/L angiotensinⅡ(AngⅡ)was determined as the optimal stimulating concentration.And then,the relative expression of miRNA-652 was detected after VSMCs were stimulated with 10 nmol/L AngⅡfor different time(0,6,12,24 h),and 24 h was determined as the optimal stimulating time.VSMCs were randomly divided into miRNA-652 agomir group,agomir negative control(NC)group,miRNA-652 antagomir group and antagomir NC group,transfected with miRNA-652 agomir,agomir NC,miRNA-652 antagomir and antagomir NC for 24 h/48 h,respectively.The relative expression of miRNA-652 and NPTN mRNA in VSMCs was detected by qRT-PCR,and the cell reproductive capacity(relative absorbance)was detected by CCK8 kit.Bioinformatics analysis was carried out online through miRDB,TargetScan and miRTarBase.In order to verify that NPTN may be the target gene of miRNA-652,the NPTN 3'UTR containing the miRNA-652 binding site was cloned into the reporter gene vector pmirGLO to construct the wild-type recombinant plasmid pmirGLO-NPTN 3'UTR-WT,mutant recombinant plasmid pmirGLO-NPTN 3'UTR-MUT,which were co-transfected with oligonucleotides miRNA-652 agomir,miRNA-652 antagomir,agomir NC and antagomir NC into 293T cells respectively,and the luciferase activity was measured 48 h after transfection.Results At 24 h after transfection,the relative expression of miRNA-652 in the miRNA-652 agomir group was higher than that in the agomir NC group,and the relative absorbance was lower than that in the agomir NC group(P<0.05).The relative expression of miRNA-652 in the miRNA-652 antagomir group was lower than that in the antagomir NC group,and the relative absorbance was higher than that in the antagomir NC group(P<0.05).The results of bioinformatics analysis showed that NPTN was detected as the target gene of miRNA-652 in all three databases.The result of detection of double luciferase reporter gene showed that the relative luciferase activity of pmirGLO-NPTN 3'UTR-WT in miRNA-652 agomir group was lower than that in agomir NC group at 48 hours after transfection(P<0.05).At 48 h after transfection,the relative expression of NPTN mRNA in miRNA-652 agomir group was lower than that in agomir NC control group,and the relative expression of NPTN mRNA in miRNA-652 antagomir group was higher than that in antagomir NC group(P<0.05).Conclusion Overexpression of miRNA-652 can inhibit the proliferation of VSMCs,while inhibition of miRNA-652 can promote the proliferation of VSMCs.miRNA-652 has a negative regulatory effect on NPTN.Therefore,miRNA-652 may regulate the proliferation of VSMCs through the target gene NPTN.