慢病毒转染SPOP基因对滋养层细胞HTR8-SVneo增殖和侵袭的影响及其机制  

The Effect of SPOP Gene Transfected by Lentivirus on the Proliferation and Invasion of HTR8-SVneo Cells and Its Mechanism

作　　者：袁冬 陈奕余 余秋波[1] Yuan Dong;Chen Yiyu;Yu Qiubo(Molecular Biology Laboratory,Institute of Life Sciences,Chongqing Medical University,Chongqing,400016)

机构地区：[1]重庆医科大学生命科学研究院分子检测中心,重庆400016

出　　处：《基因组学与应用生物学》2020年第5期2353-2360,共8页Genomics and Applied Biology

基　　金：国家自然科学基金项目(81100443)资助。

摘　　要：为了探讨慢病毒转染SPOP基因后对滋养层细胞HTR8-SVneo增殖和侵袭能力的影响,并阐明其可能的机制,分别构建过表达和敲低SPOP基因的慢病毒载体,转入到HTR8-SVneo细胞中。分别在荧光显微镜下观察转染的荧光效率。用qRT-PCR法检测感染病毒后的细胞中SPOP mRNA的表达量,Western blotting法检测各组细胞中SPOP、PTEN、AKT、p-AKT、GSK3β、p-GSK3β和GAPDH的表达量。采用CCK-8法检测各组细胞的增殖活力,Transwell小室法检测各组细胞的侵袭能力。结果显示,在HTR8-SVneo、JAR和JEG3这3种滋养层细胞中,HTR8-SVneo细胞中SPOP蛋白表达相对最高。HTR8-SVneo细胞经慢病毒转染后,与对照组比较,过表达组中SPOP mRNA和蛋白表达量明显升高(p<0.01),PTEN蛋白表达量升高,而p-AKT和p-GSK3β表达量降低,细胞的侵袭能力明显下降(p<0.05),细胞增殖活力无明显变化。相反,敲低组中SPOP mRNA和蛋白表达量明显下降(p<0.01),PTEN蛋白表达量降低,p-AKT和p-GSK3β表达量升高,细胞的侵袭能力和增殖活力均明显增强(p<0.05)。研究结果表明,慢病毒转染SPOP基因可以影响滋养层细胞HTR-8/SVneo的增殖和迁移能力,其机制可能与PTEN/AKT/GSK3β信号通路调节有关。To investigate the effects of SPOP gene transfected by lentivirus on the proliferation and invasion abilities of the trophoblast cells(HTR8-SVneo),and to explore the possible mechanisms.The lentiviral vectors of SPOP over-expression or knockdown were constructed and transferred into HTR8-SVneo cells.We observed the multiplicity of infection under fluorescence microscope.The expression level of SPOP mRNA in infected cells was detected by q RT-PCR.The protein expression levels of SPOP,PTEN,AKT,p-AKT,GSK3β,p-GSK3βand GAPDH were measured by Western blotting.CCK8 method was used to detect the proliferation abilities of HTR8-SVneo cells in the groups.Transwell chambers were used to detect the invasion abilities of cells.Among HTR8-SVneo,JAR and JEG3,the expression level of SPOP protein was highest in HTR8-SVneo cells.Compared with control group,the expression levels of SPOP mRNA and protein in over-expression group were significantly increased(p<0.01);the expression of PTEN was increased and the expressions of p-AKT and p-GSK3βwere decreased in over-expression group;Meanwhile,the invasion ability of HTR8-SVneo cells was markedly inhibited(p<0.05),but the proliferation activity of cells between control group and over-expression group did not change significantly.On the contrary,the expression levels of SPOP m RNA and protein in down-regulation group were significantly decreased(p<0.01);The expression of PTEN was decreased,the expressions of p-AKT and p-GSK3βwere increased(p<0.01),and proliferation ability and invasion ability of cells in down-regulation group were significantly increased(p<0.05).Our data indicated that SPOP gene transfected by lentivirus could affect the proliferation and migration of HTR-8/SVneo cells,and its underlying regulation mechanism may be related to PTEN/AKT/GSK3βsignaling pathway.

关 键 词：SPOP HTR8-SVneo 增殖 侵袭 PTEN/AKT/GSK3β 

分 类 号：R346[医药卫生—基础医学]