抑制miR-151表达对低氧条件下人绒毛膜滋养层细胞生物学行为的影响

Effect of inhibition of miR-151 expression on biological behavior of human chorionic trophoblast cells under hypoxia condition

目的:探讨微小RNA-151(miR-151)对低氧条件下人绒毛膜滋养层细胞HTR-8/SVneo生物学行为的影响,并阐明其可能的作用机制。方法:选择47例子痫前期产妇(子痫前期组)和36例正常产妇(正常组)作为研究对象,采用实时荧光定量PCR(RT-qPCR)法检测2组产妇胎盘组织中miR-151表达水平。将miR-151 inhibitor及其阴性对照inhibitor NC转染至HTR-8/SVneo细胞中,进行低氧(1%O_(2))干预48 h,设立对照组、低氧组、低氧+inhibitor NC组和低氧+inhibitor组。采用RT-qPCR法检测各组细胞中miR-151表达水平,MTT法检测各组细胞存活率,Transwell小室实验检测各组迁移细胞数和侵袭细胞数,Western blotting法检测各组细胞中基质金属蛋白酶(MMP)-2和MMP-9及上皮间质转化(EMT)相关蛋白表达水平。采用生物信息学分析预测miR-151下游靶基因,并结合STRING数据库对交集靶基因进行蛋白-蛋白互作(PPI)网络分析。结果:与正常组比较,子痫前期组产妇胚胎组织中miR-151表达水平明显升高(P<0.05)。与对照组比较,低氧组HTR-8/SVneo细胞存活率、迁移细胞数和侵袭细胞数及细胞中MMP-2、MMP-9、N-钙黏蛋白和波形蛋白表达水平明显降低(P<0.05),miR-151和E-钙黏蛋白表达水平明显升高(P<0.05)。与低氧组比较,低氧+inhibitor组HTR-8/SVneo细胞中MMP-2、MMP-9、N-钙黏蛋白和波形蛋白表达水平明显升高(P<0.05),miR-151和E-钙黏蛋白水平明显降低(P<0.05),而低氧+inhibitor NC组上述指标差异无统计学意义(P>0.05)。生物信息学分析,miR-151下游有34个潜在靶基因,其中环指CCCH-型锌指蛋白域蛋白1(RC3H1)、AGO_(2)、AGO3、脆性X相关蛋白1(FXR1)和转化因子2β(TRA2B)可能是关键潜在靶基因。结论:miR-151在子痫前期患者胎盘组织中高表达,下调miR-151表达可促进低氧条件下滋养层细胞的增殖、迁移和侵袭。

Objective:To discuss the effect of microRNA-151(miR-151)on the biological behavior of the human trophoblast cells HTR-8/SVneo under hypoxic conditions,and to clarify the potential mechanism.Methods:A total of 47 parturients with preeclampsia(preeclampsia group)and 36 parturients(normal group)were selected as the research subjects.The expression level of miR-151 in placenta tissue of the subjects in two groups was detected by real-time fluorescence quantitative PCR(RT-qPCR)method.The miR-151 inhibitor and its negative control inhibitor NC were transfected into the HTR-8/SVneo cells,followed by exposure to hypoxia(1%O_(2))for 48 h to establish control,hypoxia,hypoxia+inhibitor NC,and hypoxia+inhibitor groups.RT-qPCR method was used to detect the expression levels of miR-151 in the cells in various groups;MTT assay was used to detect the survival rates of the cells in various groups;Transwell chamber assay was used to detect the migration and invasion numbers of the cells in various groups;Western blotting method was used to detect the expression levels of matrix metalloproteinases(MMP)-2 and MMP-9,and epithelial-mesenchymal transition(EMT)related proteins in the cells in various groups;Bioinformatics analysis was used to predict the downstream target genes of miR-151,and the intersection target genes were further analyzed for protein-protein interaction(PPI)network by STRING Database.Results:Compared with normal group,the expression level of miR-151 in placenta tissue of the patients in preeclampsia group was significantly increased(P<0.05).Compared with control group,the proliferation activity,number of invasion cells,and number of migration cells of HTR-8/SVneo cells in hypoxia group were significantly decreased(P<0.05),the expression levels of MMP-2,MMP-9,N-cadherin,and vimentin in the cells were significantly decreased(P<0.05),and the expression levels of miR-151 and E-cadherin were significantly increased(P<0.05).Compared with hypoxia group,the expression levels of MMP-2,MMP-9,N-cadherin,and vimentin in the cells in hypoxia+inhibitor group were significantly increased(P<0.05);the levels of miR-151 and E-cadherin were significantly decreased(P<0.05);while there were no significant differences in the above indexes in hypoxia+inhibitor NC group(P>0.05).The bioinformatics analysis results showed that 34 potential target genes of miR-151,among which RCCCH-type zinc finger protein 1(RC3H1),AGO_(2),AGO3,Fragile X related protein 1(FXR1),and transformer 2β(TRA2B)may be the key potential target genes.Conclusion:miR-151 is highly expressed in placenta tissue of the patients with preeclampsia.The downregulation of miR-151 expression can promote the proliferation,invasion,and migration of the trophoblast cells under hypoxic conditions.