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

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。

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.