摘要
目的探讨miR-520通过激活内质网应激导致滋养细胞凋亡,从而参与复发性流产(RSA)的作用机制。方法利用miR-520慢病毒(实验组)和空载慢病毒(对照组)分别感染人滋养细胞系HTR8细胞,并用嘌呤霉素筛选出稳转株。以未感染细胞为空白对照,利用流式细胞术及荧光显微镜观察确定病毒转染效率、实时荧光定量PCR(qRT-PCR)检测各组细胞miR-520的表达水平;通过CCK-8法测定细胞增殖能力、流式细胞术分析细胞凋亡率、乳酸脱氢酶(LDH)试剂盒检测细胞损伤程度、Mito-Tracker Red CMXRos试剂盒检测线粒体膜电位变化;通过Western blot检测凋亡通路中cleaved Caspase-3、cleaved Caspase-9以及cleaved Caspase-12水平的变化,同时检测内质网应激相关蛋白CHOP、葡萄糖调节蛋白GRP78的水平变化。结果与对照组比较,miR-520在人滋养细胞系HTR8细胞中过表达后,HTR8细胞增殖能力显著下降(P<0.05),LDH活性升高(P<0.05),HTR8细胞线粒体膜电位降低(P<0.05),细胞凋亡数显著增加(P<0.05);凋亡相关蛋白cleaved Caspase-3、cleaved Caspase-9以及cleaved Caspase-12水平均有不同程度的增高(P<0.05),同时内质网应激相关蛋白CHOP、GRP78表达水平上调(P<0.05)。结论miR-520可能通过激活内质网应激诱导HTR8细胞发生凋亡,从而参与RSA的发生。
Objective:To explore the mechanism of miR-520 participating in recurrent spontaneous abortion(RSA)by activating endoplasmic reticulum stress to induce trophoblast apoptosis.Methods:HTR8 cells were infected with miR-520 lentivirus(experimental group)and no-load lentivirus(control group)respectively,and stable transformants were screened with puromycin.With uninfected cells as blank control,the virus transfection efficiency was determined by flow cytometry and fluorescence microscope.Real-time fluorescence quantification(qRT-PCR)was used to detect the expression level of miR-520 in each group.The ability of cell proliferation was measured by CCK-8 method.The apoptosis rate was analyzed by flow cytometry.The degree of cell injury was detected by lactate dehydrogenase(LDH)kit.The change of mitochondrial membrane potential was detected by Mito-Tracker Red CMXRos kit.The levels of cleaved-Caspase-3,cleaved Caspase-9 and cleaved Caspase-12 in apoptosis pathway were measured by Western blot,and the levels of CHOP and glucose-regulated protein GRP78 in endoplasmic reticulum stress were detected at the same time.Results:After miR-520 in human trophoblast line HTR8 cells was over-expressed,the proliferation ability of HTR8 cells decreased significantly(P<0.05),LDH activity increased(P<0.05),the mitochondrial membrane potential of HTR8 cells decreased and the number of apoptosis significantly increased(P<0.05).The levels of apoptosis-related proteins including cleaved-Caspase-3,cleaved Caspase-9 and cleaved Caspase-12 were increased in different degrees(P<0.05),and the expression levels of endoplasmic reticulum stress-related proteins CHOP and GRP78 were up-regulated(P<0.05).Conclusions:MiR-520 can induce apoptosis of HTR8 cells by activating endoplasmic reticulum stress to participate in the occurrence of recurrent abortion.
作者
陈欣
郭端英
付振琳
杨菁
CHEN Xin;GUO Duan-ying;FU Zhen-lin;YANG Jing(Reproductive Medical Center,Hubei Clinical Research Center for Assisted Reproductive Technology&Embryonic Development,Renmin Hospital of Wuhan University,Wuhan 430060;Longgang District People’s Hospital of Shenzhen,Shenzhen 518100)
出处
《生殖医学杂志》
CAS
2020年第8期1067-1073,共7页
Journal of Reproductive Medicine
基金
国家自然科学基金(81771618,81971356)。
关键词
miR-520
内质网应激
滋养细胞凋亡
复发性流产
miR-520
Mndoplasmic reticulum stress
Trophoblast apoptosis
Recurrent spontaneous abortion
