摘要
目的 探讨miR-30c-5p对人绒毛滋养层细胞线粒体自噬、间充质转化及PEG10水平影响.方法 人绒毛滋养层细胞HTR-8/SVneo分为空白组(HTR-8/SVneo细胞正常培养)、低氧组(HTR-8/SVneo细胞低氧培养)、miR-30c-5p mimics 组(HTR-8/SVneo 细胞低氧培养转染 miR-30c-5p mimics)、NC-mimics 组(HTR-8/SVneo 细胞低氧培养转染 NC-mimics)、miR-30c-5p inhibitor 组(HTR-8/SVneo 细胞低氧培养转染 miR-30c-5p inhibitor)、NC-inhibitor 组(HTR-8/SVneo 细胞低氧培养转染 NC-inhibitor).qRT-PCR 检测各组细胞miR-30c-5p表达;Transwell小室检测细胞侵袭数目;划痕试剂盒检测细胞划痕愈合率;相关试剂盒检测ROS水平;免疫印记检测PEG10、Parkin、PINK1蛋白水平.结果 空白组、低氧组、miR-30c-5p mimics组、NC-mimics组、miR-30c-5p inhibitor组及 NC-inhibitor组的 miR-30c-5p表达分别为 1.00±0.00、1.00±0.00、1.61±0.15、1.03±0.13、0.75±0.08 及 0.96±0.10,差异有统计学意义(F=45.750,P＜0.05);与空白组相比,低氧组 HTR-8/SV-neo细胞侵袭数目、划痕愈合率、PEG10、Parkin、PINK1降低,ROS升高,差异均有统计学意义(P＜0.05);低氧组与NC-mimics组、NC-inhibitor组比较上述指标,差异均无统计学意义(P＞0.05);与低氧组相比,miR-30c-5p mimics组细胞侵袭数目、划痕愈合率、PEG10、Parkin、PINK1降低,ROS升高,差异均有统计学意义(P＜0.05),miR-30c-5p inhibitor组细胞侵袭数目、划痕愈合率、PEG 10、Parkin、PINK 1升高,ROS降低,差异均有统计学意义(P＜0.05).结论 抑制miR-30c-5p可加快低氧状态下的人绒毛滋养层细胞侵袭及迁移,并通过促进线粒体自噬降低ROS表达,机制与抑制PEG10水平相关.
Abstract
Objective To investigate the effects of miR-30c-5p on mitophagy,mesenchymal transition,and PEG10 levels in human trophoblast cells.Methods Human trophoblast cell line HTR-8/SVneo was divided in-to the following groups:blank group(HTR-8/SVneo cells cultured normally),hypoxia group(HTR-8/SVneo cells cultured under hypoxic conditions),miR-30c-5p mimics group(HTR-8/SVneo cells cultured under hypoxic condi-tions transfected with miR-30c-5p mimics),NC-mimics group(HTR-8/SVneo cells cultured under hypoxic condi-tions transfected with NC-mimics),miR-30c-5p inhibitor group(HTR-8/SVneo cells cultured under hypoxic condi-tions transfected with miR-30c-5p inhibitor),and NC-inhibitor group(HTR-8/SVneo cells cultured under hypoxic conditions transfected with NC-inhibitor).qRT-PCR was used to detect the expression of miR-30c-5p in each group of cells.Transwell chamber was used to measure cell invasion.Scratch test kit was used to measure the cell scratch healing rate.ROS level was measured using relevant assay kits.Immunostaining was used to measure the protein lev-els of PEG10,Parkin,and PINK1.Results The expression of miR-30c-5p in the blank group,hypoxia group,miR-30c-5p mimics group,NC-mimics group,miR-30c-5p inhibitor group,and NC-inhibitor group were 1.00±0.00,1.00±0.00,1.61±0.15,1.03±0.13,0.75±0.08,and 0.96±0.10,respectively,showing significant differences(F=45.750,P＜0.05).Compared with the blank group,the number of invasive cells,scratch healing rate,PEG10,Par-kin,and PINK1 levels were decreased(P＜0.05),while ROS level was increased(P＜0.05)in the hypoxia group.There was no significant difference in these parameters between the hypoxia group and the NC-mimics group or the NC-inhibitor group(P＞0.05).Compared with the hypoxia group,the number of invasive cells,scratch healing rate,PEG10,Parkin,and PINK1 levels were decreased(P＜0.05),while ROS level was increased(P＜0.05)in the miR-30c-5p mimics group.Conversely,the number of invasive cells,scratch healing rate,PEG10,Parkin,and PINK1 levels were increased(P＜0.05),while ROS level was decreased(P＜0.05)in the miR-30c-5p inhibitor group.Con-clusion Inhibition of miR-30c-5p can promote invasion and migration of hypoxic human trophoblasts and reduce ROS expression by promoting mitochondrial autophagy,and this mechanism is associated with the inhibition of PEG10 level.
基金项目
贵州省科学技术基金(黔科合J字[2013]2056号)
NETL
NSTL
万方数据