ROCK1基因敲除对乳腺癌细胞系迁移及侵袭的影响

doi:10.3969/j.issn.1004-616x.2019.03.004

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

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. 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. 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/Abstract

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。

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

中图分类号:

王晓杰, 滕宇, 顾勐, 王子宇, 赵晓婷, 岳文涛. ROCK1基因敲除对乳腺癌细胞系迁移及侵袭的影响[J]. 癌变·畸变·突变, 2019, 31(3): 193-197,202.

WANG Xiaojie, TENG Yu, GU Meng, WANG Ziyu, ZHAO Xiaoting, YUE Wentao. Effects of ROCK1 knockout on breast cancer cell migration and invasion[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2019, 31(3): 193-197,202.

参考文献

[1] RIVERA-FRANCO M M,LEON-RODRIGUEZ E. Delays in breast cancer detection and treatment in developing countries[J]. Breast Cancer (Auckl),2018,12:117822341775267.
[2] LI T, MELLOTHOMS C, BRENNAN P. Descriptive epidemiology of breast cancer in China:incidence, mortality, survival and prevalence[J]. Breast Cancer Res Treat,2016,159(3):395-406.
[3] TOT T. Metastasis of breast cancer prior to invasion[J]. Breast Cancer Res Treat,2018,170(1):197.
[4] MIZUKAMI Y,FUJIKI K,DUERR E M,et al. Hypoxic regulation of vascular endothelial growth factor through the induction of phosphatidylinositol 3-kinase/Rho/ROCK and c-Myc[J]. J Biol Chem, 2006, 281(20):13957-13963.
[5] RIDLEY A J. Rho GTPase signalling in cell migration[J]. Curr Opin Cell Biol,2015,36:103-112.
[6] PARRI M, CHIARUGI P. Rac and Rho GTPases in cancer cell motility control[J]. Cell Commun Signal, 2010,8:23.
[7] 卜强,汤华明,谈健,等. 前列腺癌中RhoC和Rho激酶1的表达及其对丝裂原活化蛋白激酶和Akt蛋白的影响[J]. 中华肿瘤杂志,2011,33(3):202-206.
[8] HAHMANN C,SCHROETER T. Rho-kinase inhibitors as therapeutics:from pan inhibition to isoform selectivity[J]. Cell Mol Life Sci,2010,67(2):171-177.
[9] LIAO J K, SETO M, NOMA K. Rho kinase (ROCK) inhibitors[J]. J Cardiovasc Pharmacol,2007,50(1):17-24.
[10] ZHAO X Y,WANG X F,LI L,et al. Effects of high glucose on human umbilical vein endothelial cell permeability and myosin light chain phosphorylation[J]. Diabetol Metab Syndr,2015,7:98.
[11] RAN F A, HSU P D, WRIGHT J, et al. Genome engineering using the CRISPR-Cas9 system[J]. Nat Protoc,2013,8(11):2281-2308.
[12] LEE D J, KWON M J, NAM E S, et al. Histopathologic predictors of lymph node metastasis and prognosis in tonsillar squamous cell carcinoma[J]. Korean J Pathol,2013,47(3):203-210.
[13] FRITZ G, BRACHETTI C, BAHLMANN F, et al. Rho GTPases in human breast tumours:expression and mutation analyses and correlation with clinical parameters[J]. Br J Cancer,2002,87(6):635-644.
[14] BOTTINO J, GELALETI G B, MASCHIO L B, et al. Immunoexpression of ROCK-1 and MMP-9 as prognostic markers in breast cancer[J]. Acta Histochem, 2014,116(8):1367-1373.
[15] VIGIL D,KIM T Y,PLACHCO A,et al. ROCK1 and ROCK2 are required for non-small cell lung cancer anchorage-independent growth and invasion[J]. Cancer Res, 2012,72(20):5338-5347.
[16] SAHAI E, MARSHALL C J. Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis[J]. Nat Cell Biol, 2003,5(8):711-719.
[17] CHEN Y, WANG D, GUO Z, et al. Rho kinase phosphorylation promotes Ezrin-mediated metastasis in hepatocellular carcinoma[J]. Cancer Res, 2011, 71(5):1721-1729.

ROCK1基因敲除对乳腺癌细胞系迁移及侵袭的影响

Effects of ROCK1 knockout on breast cancer cell migration and invasion

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