摘要
目的:研究慢病毒介导的TPX2基因沉默对人宫颈癌He La细胞系增殖、迁移和细胞周期的影响及其机制。方法:构建4种靶向TPX2基因的慢病毒表达载体(LV-TPX2-sh RNA-1/2/3/4),同时构建阴性对照质粒,将5种质粒分别转染到293T细胞中制备慢病毒。慢病毒感染宫颈癌He La细胞后,实时荧光定量PCR和Western blot分别检测TPX2 m RNA和蛋白的沉默效果。选择沉默效果最佳的重组慢病毒进行后续功能实验。分别采用CCK-8法、Transwell迁移实验及流式细胞术检测各组细胞的增殖、迁移及细胞周期分布情况。Western blot检测TPX2-sh RNA转染前后Ki-67、cyclin B2、Aurora-A及P53的表达。结果:与对照组比较,构建的靶向TPX2基因的RNA干扰慢病毒载体均可持续稳定的抑制He La细胞TPX2基因的表达,尤其以LV-TPX2-sh RNA-1最为明显(P<0.01),故选择LV-TPX2-sh RNA-1进行后续实验。与对照组比较,沉默TPX2基因的表达能降低He La细胞增殖及迁移能力(P<0.05),使G2及S期细胞比例明显增加(P<0.05),且上调P53蛋白的表达水平,下调Ki-67、cyclin B2及Aurora-A蛋白的表达水平(P均<0.05)。结论:沉默TPX2基因蛋白表达能抑制宫颈癌细胞的增殖及迁移能力,可能与其改变细胞周期分布及下调Ki-67、cyclin B2、AuroraA蛋白表达水平,上调P53蛋白表达水平有关。
OBJECTIVE:To investigate the effect and mechanism of silencing of TPX2gene expression bylentivirus on cell proliferation,migration and cell cycle in human cervical cancer HeLa cells.METHODS:Fourlentivirus expression vectors carrying TPX2-shRNA1/2/3/4which target TPX2gene(named as LV-TPX2-shRNA-1/2/3/4)were constructed,while vector TPX2-NC-shRNA was used as the negative control.The five plasmids were separatelytransfected into293T cells to prepare the recombinant lentivirus.The recombinant lentivirus carrying TPX2-shRNA1/2/3/4and TPX2-NC-shRNA were infected into HeLa cells,respectively.Then,expression levels of TPX2mRNA and proteinin HeLa cells were determined by real-time fluorescent quantitative PCR and Western blotting,respectively.The effect ofTPX2gene silencing on the proliferation of HeLa cells was detected using the CCK-8method.Cell migration and cellcycledistribution were detected by transwell migration experiment and flow cytometry,respectively,in each group ofHeLa cells.Changes of Ki-67,cyclin B2,P53and Aurora-A expressions were detected by Western blotting.RESULTS:Compared with the negative control group,expressions of TPX2mRNA and protein in HeLa cells weresignificantly inhibited by LV-TPX2-shRNA,especially LV-TPX2-shRNA-1(P<0.01).Subsequently,LV-TPX2-shRNA-1was selected for follow-up experiments.Silencing TPX2gene expression reduced the proliferation and migration(P<0.05),increased the percentage of G2and S phase cell in the cell cycle(P<0.05),up-regulated theexpression of P53,and down-regulated the expression of Ki-67,cyclin B2and Aurora-A in HeLa cells(all P<0.05).CONCLUSION:Our results show that silencing TPX2gene expression reduced the proliferation and migration in HeLacells,which may be related to changes in cell cycle distribution,up-regulating the expression of P53,and downregulatethe expression of Ki-67,cyclin B2and Aurora-A in HeLa cells.
作者
宋淑芳
常海平
杨彩容
赵荣伟
张龙
任杰
郑健
SONG Shufang;CHANG Haiping;YANG Cairong;ZHAO Rongwei;ZHANG Long;REN Jie;ZHENG Jian(Department of Obstetrics and Gynecology,Affiliated Hospital of Inner Mongolia Medical College, Hohhot 010050, Mongolia, China)
出处
《癌变．畸变．突变》
CAS
CSCD
2017年第5期329-334,339,共7页
Carcinogenesis,Teratogenesis & Mutagenesis
基金
国家自然科学基金资助项目(81360385)
作者简介
宋淑芳，E-mail：ssf43@sohu.com;通信作者，常海平，E-mail：haipingchang@163.com
