慢病毒介导的TPX2基因沉默对人宫颈癌HeLa细胞系增殖、迁移和细胞周期的影响及机制

doi:10.3969/j.issn.1004-616x.2017.05.002

癌变·畸变·突变 ›› 2017, Vol. 29 ›› Issue (5): 329-334,339.doi: 10.3969/j.issn.1004-616x.2017.05.002

慢病毒介导的TPX2基因沉默对人宫颈癌HeLa细胞系增殖、迁移和细胞周期的影响及机制

宋淑芳, 常海平, 杨彩容, 赵荣伟, 张龙, 任杰, 郑健

内蒙古医科大学附属医院妇产科, 内蒙古呼和浩特 010050

收稿日期:2017-05-31 修回日期:2017-08-16 出版日期:2017-09-30 发布日期:2017-09-30
通讯作者: 常海平,E-mail:haipingchang@163.com E-mail:haipingchang@163.com
作者简介:宋淑芳,E-mail:ssf43@sohu.com。
基金资助:
国家自然科学基金资助项目（81360385）

Effect and mechanism of lentivirus-mediated silencing of TPX2 gene expression on proliferation, migration and cell cycle of HeLa cells

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

Received:2017-05-31 Revised:2017-08-16 Online:2017-09-30 Published:2017-09-30

摘要/Abstract

摘要： 目的：研究慢病毒介导的TPX2基因沉默对人宫颈癌HeLa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-shRNA-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HeLa细胞后，实时荧光定量PCR和Western blot分别检测TPX2 mRNA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-shRNA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HeLa细胞TPX2基因的表达，尤其以LV-TPX2-shRNA-1最为明显（P < 0.01），故选择LV-TPX2-shRNA-1进行后续实验。与对照组比较，沉默TPX2基因的表达能降低HeLa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、Aurora-A蛋白表达水平，上调P53蛋白表达水平有关。

关键词: 宫颈癌, TPX2基因, 慢病毒, 细胞增殖, 细胞周期

Abstract: OBJECTIVE: To investigate the effect and mechanism of silencing of TPX2 gene expression by lentivirus on cell proliferation,migration and cell cycle in human cervical cancer HeLa cells. METHODS: Four lentivirus expression vectors carrying TPX2-shRNA1/2/3/4 which target TPX2 gene (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 separately transfected into 293T cells to prepare the recombinant lentivirus. The recombinant lentivirus carrying TPX2-shRNA1/2/3/4 and TPX2-NC-shRNA were infected into HeLa cells,respectively. Then,expression levels of TPX2 mRNA and protein in HeLa cells were determined by real-time fluorescent quantitative PCR and Western blotting,respectively. The effect of TPX2 gene silencing on the proliferation of HeLa cells was detected using the CCK-8 method. Cell migration and cell-cycle distribution were detected by transwell migration experiment and flow cytometry,respectively,in each group of HeLa cells. Changes of Ki-67,cyclin B2,P53 and Aurora-A expressions were detected by Western blotting. RESULTS: Compared with the negative control group,expressions of TPX2 mRNA and protein in HeLa cells were significantly inhibited by LV-TPX2-shRNA,especially LV-TPX2-shRNA-1 (P < 0.01). Subsequently,LV-TPX2-shRNA-1 was selected for follow-up experiments. Silencing TPX2 gene expression reduced the proliferation and migration(P < 0.05),increased the percentage of G2 and S phase cell in the cell cycle(P < 0.05),up-regulated the expression of P53,and down-regulated the expression of Ki-67,cyclin B2 and Aurora-A in HeLa cells (all P < 0.05). CONCLUSION: Our results show that silencing TPX2 gene expression reduced the proliferation and migration in HeLa cells,which may be related to changes in cell cycle distribution,up-regulating the expression of P53,and down-regulate the expression of Ki-67,cyclin B2 and Aurora-A in HeLa cells.

Key words: cervical cancer, TPX2 gene, lentivirus, cell proliferation, cell cycle

中图分类号:

R737.3

宋淑芳, 常海平, 杨彩容, 赵荣伟, 张龙, 任杰, 郑健. 慢病毒介导的TPX2基因沉默对人宫颈癌HeLa细胞系增殖、迁移和细胞周期的影响及机制[J]. 癌变·畸变·突变, 2017, 29(5): 329-334,339.

SONG Shufang, CHANG Haiping, YANG Cairong, ZHAO Rongwei, ZHANG Long, REN Jie, ZHENG Jian. Effect and mechanism of lentivirus-mediated silencing of TPX2 gene expression on proliferation, migration and cell cycle of HeLa cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2017, 29(5): 329-334,339.

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慢病毒介导的TPX2基因沉默对人宫颈癌HeLa细胞系增殖、迁移和细胞周期的影响及机制

Effect and mechanism of lentivirus-mediated silencing of TPX2 gene expression on proliferation, migration and cell cycle of HeLa cells

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