癌变·畸变·突变 ›› 2019, Vol. 31 ›› Issue (3): 193-197,202.doi: 10.3969/j.issn.1004-616x.2019.03.004

• 论著 • 上一篇    下一篇

ROCK1基因敲除对乳腺癌细胞系迁移及侵袭的影响

王晓杰1, 滕宇1, 顾勐1, 王子宇1, 赵晓婷1, 岳文涛2   

  1. 1. 首都医科大学附属北京胸科医院/北京市结核病胸部肿瘤研究所细胞生物学研究室, 北京 101149;
    2. 首都医科大学附属北京妇产医院中心实验室, 北京 100026
  • 收稿日期:2019-02-01 修回日期:2019-04-22 出版日期:2019-05-31 发布日期:2019-05-31
  • 通讯作者: 岳文涛,E-mail:yuewt@ccmu.edu.cn E-mail:yuewt@ccmu.edu.cn
  • 作者简介:王晓杰,E-mail:xiaojiewang93@163.com。
  • 基金资助:
    北京市医院管理局临床技术创新项目(XMLX201705);国家自然科学基金(81672838)

Effects of ROCK1 knockout on breast cancer cell migration and invasion

WANG Xiaojie1, TENG Yu1, GU Meng1, WANG Ziyu1, ZHAO Xiaoting1, YUE Wentao2   

  1. 1. Department of Cellular and Molecular Biology, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149;
    2. Central Laboratory, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing 100026, China
  • Received:2019-02-01 Revised:2019-04-22 Online:2019-05-31 Published:2019-05-31

摘要: 目的:通过成簇规律间隔的短回文重复序列(CRISPR/Cas9)系统在MCF-7乳腺癌细胞系中构建Rho相关蛋白激酶1(ROCK1)基因敲除模型,研究ROCK1敲除对乳腺癌细胞迁移及侵袭能力的影响。方法:设计并合成靶向ROCK1的向导RNA (sgRNA)寡核苷酸序列,长度为20 bp,构建到CRISPR单载体慢病毒上,感染并筛选稳定细胞株,采用划痕实验和Transwell实验分别检测细胞迁移和侵袭能力。结果:目的sgRNA寡核苷酸双链成功插入酶切后的GV392质粒载体中且测序正确;经Western blot鉴定ROCK1敲除的细胞株构建成功;ROCK1基因敲除后,与对照组细胞相比,其蛋白表达缺失。划痕实验结果显示ROCK1敲除细胞株24 h划痕愈合度为(60.600±0.047)%,对照组细胞划痕愈合度为(80.404±0.018)%,差异有统计学意义(P=0.003)。Transwell实验结果显示ROCK1敲除细胞株在24 h的迁移细胞数为(271.3±5.0)个,对照组的迁移细胞数为(448.3±5.5)个;48 h的迁移细胞数在实验组和对照组分别为(1.7±2.9)个和(298.3±5.7)个,差异有统计学意义(P=0.000)。结论:ROCK1基因敲除可明显抑制乳腺癌细胞的迁移及侵袭能力,提示ROCK1基因在乳腺癌侵袭和转移中可能发挥重要作用。

关键词: Rho相关蛋白激酶1, CRISPR/Cas9, 迁移, 侵袭

Abstract: OBJECTIVE: To construct a ROCK1 gene-knockout model using the CRISPR/Cas9 system and to investigate effects of the ROCK1 knockout on MCF-7 breast cancer cell migration and invasion. METHODS: Three pairs of 20 bp sgRNA targeting ROCK1 were chemically synthesized and inserted into CRISPR expression vectors. The ROCK1 knockout cells were selected with lentivirus infection of MCF-7 cells, and were identified by gene sequencing and Western blot. Effects of the knockout on MCF-7 cell migration and invasion were evaluated by wound-healing and Transwell assays. RESULTS:Western blot data show that ROCK1 knockout MCF-7 cells were successfully and stably established by usage of the CRISPR/Cas9 system. Wound-healing assays show that the wound closure of ROCK1 knockout cells was significantly lower than that of their parental cells[(60.600±0.047)% and (80.404±0.018)%,respectively;P=0.003]. Transwell assays without matrigel show that the migration capability of ROCK1 knockout cells was significantly decreased compared with the control group(271.3 ±5.0 and 448.3 ±5.5, respectively; P=0.000) by 24 h. Transwell assays with matrigel show the invasion capability of ROCK1 knockout cells at 48 h was deeply suppressed compared to that of the controls (1.7±2.9 and 298.3±5.7,respectively;P=0.000). CONCLUSION:Knockout of ROCK1 gene caused significant inhibition of the migration and invasion ability of breast cancer cells,suggesting that ROCK1 may play an important role in the invasion and metastasis of breast cancer.

Key words: Rho-associated protein kinase 1, CRISPER/Cas9, migration, invasion

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