基于CRISPR/Cas9系统构建ARID2基因敲除模型并探索其对肝癌细胞增殖和迁移能力的影响

高庆祝; 汪凯; 梁利; 张韵致; 郑亚秋; 唐霓

doi:10.13406/j.cnki.cyxb.001310

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基于CRISPR/Cas9系统构建ARID2基因敲除模型并探索其对肝癌细胞增殖和迁移能力的影响

肝癌分子机制研究 | 更新时间：2024-01-17

- 基于CRISPR/Cas9系统构建ARID2基因敲除模型并探索其对肝癌细胞增殖和迁移能力的影响
- Effects of ARID2 knockout on hepatoma cells proliferation and migration based on CRISPR/Cas9
- 重庆医科大学学报 2017年42卷第7期页码：850-855
- 作者机构：
  
  重庆医科大学感染性疾病分子生物学教育部重点实验室
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.001310
  中图分类号： R735.7
- 纸质出版日期：2017，
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高庆祝, 汪凯, 梁利, 等. 基于CRISPR/Cas9系统构建ARID2基因敲除模型并探索其对肝癌细胞增殖和迁移能力的影响[J]. 重庆医科大学学报, 2017,42(7):850-855.

Gao Qingzhu, Wang Kai, Liang Li, et al. Effects of ARID2 knockout on hepatoma cells proliferation and migration based on CRISPR/Cas9[J]. Journal of Chongqing Medical University, 2017,42(7):850-855.
高庆祝, 汪凯, 梁利, 等. 基于CRISPR/Cas9系统构建ARID2基因敲除模型并探索其对肝癌细胞增殖和迁移能力的影响[J]. 重庆医科大学学报, 2017,42(7):850-855. DOI： 10.13406/j.cnki.cyxb.001310.

Gao Qingzhu, Wang Kai, Liang Li, et al. Effects of ARID2 knockout on hepatoma cells proliferation and migration based on CRISPR/Cas9[J]. Journal of Chongqing Medical University, 2017,42(7):850-855. DOI： 10.13406/j.cnki.cyxb.001310.

摘要

目的:通过成簇的规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats/Cas9

CRISPR/Cas9)系统在SK-Hep1肝癌细胞系中构建AT富集作用域2(AT-rich interaction domain 2

ARID2)基因敲除模型

并初步观察ARID2敲除对肝癌细胞增殖和迁移能力的影响。方法:设计并合成靶向ARID2的向导RNA(single guide RNA

sg RNA)寡核苷酸序列

长度为20 bp

构建到CRISPR表达载体上

转染HEK293T细胞包装慢病毒并筛选稳定细胞株

采用克隆形成、Transwell和划痕实验分别检测细胞增殖和迁移能力。结果:目的 sg RNA寡核苷酸双链成功插入酶切后的Lenti CRISPER-V2质粒载体中且测序正确;经Western blot鉴定ARID2敲除的细胞株筛选成功;ARID2基因敲除后

与亲本细胞系相比

其蛋白表达缺失;克隆形成实验结果示ARID2敲除细胞株(1#6)培养至10 d时克隆形成数为(125.600±5.131)个

亲本细胞株克隆形成数为(69.000±5.567)个(t=12.962

P=0.000);ARID2敲除细胞株(2#1)培养至8 d时克隆形成数为(94.000±8.737)个

亲本细胞株克隆形成数为(67.000±5.292)个(t=4.635

P=0.010)

差异有统计学意义。Transwell结果示ARID2敲除细胞株(1#6)在24 h的迁移细胞数为(180.000±5.542)个

亲本细胞组的迁移细胞数为(90.400±5.031)个(t=20.731

P=0.000);ARID2敲除细胞株(2#1)在24 h的迁移细胞数为(138.600±5.749)个

亲本细胞组的迁移细胞数为(78.400±5.703)个(t=12.891

P=0.000)

差异有统计学意义。划痕实验结果示ARID2敲除细胞株(1#6)16 h划痕愈合度为(88.000±0.011)%

亲本细胞组划痕愈合度为(52.500±0.047)%(t=12.490

P=0.000);ARID2敲除细胞株(2#1)16 h划痕愈合度为(71.900±0.020)%

亲本细胞组划痕愈合度为(55.200±0.024)%(t=9.190

P=0.001)

差异有统计学意义。结论:ARID2敲除可明显促进肝癌细胞的增殖和迁移能力

证实ARID2作为抑癌基因参与肝癌的发生和进程。ARID2敲除细胞模型为肝癌的深入研究提供新的手段。

Abstract

Objective:To construct ARID2 gene knockout model using CRISPR-Cas9 system and to investigate the effects of ARID2 knockout on SK-Hep1 cell proliferation and migration. Methods:Three pairs of 20 bp sg RNAs targeting ARID2 were chemically synthesized and inserted into CRISPR expression vectors. The lentiviral was then packaged in HEK293 T cell lines. The ARID2 knockout cell lines were selected in SK-Hep1 cells and identified by gene sequencing and Western blot. The effects of ARID2 knockout on proliferation and migration of SK-Hep1 cells were evaluated by colony formation

Transwell and Wound-Healing assays. Results:Western blot showed that SK-Hep1 cell lines with ARID2 knockout were successfully established by the usage of CRISPR-Cas9 system. Colony formation assay showed that the colony number of ARID2 knockout cell lines（1#6） were significantly increased on10 d（125.600 ±5.131）

compared with parental cell lines（69.000±5.567）（t=12.962

P=0.000）

meanwhile

the colony number of ARID2 knockout cell lines（2#1）were increased on 8 d（94.000±8.737）

compared with parental cell lines（67.000 ±5.292）（t =4.635

P =0.010）. Transwell assay showed that the migration number of ARID2 knockout cell lines（1#6）（180.000±5. 542）was significantly increased than that of parental cell lines（90.400±5.031）（t=20.731

P=0.000）;the migration number of ARID2 knockout cell lines（2#1）（138.600±5.749）was increased than that of parental cell lines（78.400 ±5.703）（t =12.891

P=0.000）. Wound-healing assay showed that the wound closure of ARID2 knockout cell lines（1#6）（88.000±0.011）% was higher than that of parental cell lines（52.500±0.047）%（t=12.490

P=0.000）

meanwhile

the wound closure of ARID2 knockout cell lines（2#1）（71.900±0.020）% was higher than that of parental cell lines（55.200±0.024）%（t=9.190

P=0.001）. Conclusion:ARID2 knockout results in an enhanced proliferation and migration of SK-Hep1 cells

suggesting that ARID2 may be involved with the initiation and progress of hepatocarcinoma. Our study provides a new tool for further investigation of HCC.

关键词

AT富集作用域2CRISPER/Cas9SK-Hep1增殖迁移

Keywords

AT-rich interaction domain 2clustered regularly interspaced short palindromic repeats/Cas9SK-Hep1proliferationmigration

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