解剖学报 ›› 2021, Vol. 52 ›› Issue (1): 55-59.doi: 10.16098/j.issn.0529-1356.2021.01.008

• 肿瘤生物学 • 上一篇    下一篇

规律间隔成簇短回文重复序列/相关蛋白9靶向Rho GDIα基因敲除质粒的构建及其对小鼠肝癌细胞系Hepa 1-6迁移的影响

曲明娟* 李延敏 谢静 秦苗苗 王静 周菊华   

  1. 鲁东大学生命科学学院,山东 烟台  264025
  • 收稿日期:2019-08-06 修回日期:2019-11-11 出版日期:2021-02-06 发布日期:2021-02-06
  • 通讯作者: 曲明娟 E-mail:shqmjyg@163.com
  • 基金资助:
    国家自然科学基金项目;鲁东大学科技项目

Construction of Rho GDIα-sgRNAs plasmids by clustered regularly interspaced short palindromic repeats/associated protein 9 and the effect on migration of Hepa 1-6 cell line in mouse

QU Ming-juan*  LI Yan-min  XIE Jing  QIN Miao-miao  WANG Jing  ZHOU Ju-hua   

  1. School of Life Sciences, Ludong University, Shandong Yantai  264025, China
  • Received:2019-08-06 Revised:2019-11-11 Online:2021-02-06 Published:2021-02-06
  • Contact: QU Ming-juan E-mail:shqmjyg@163.com

摘要:

目的 利用规律间隔成簇短回文重复序列/相关蛋白9(CRISPR/Cas9)技术构建Rho鸟苷酸解离抑制因子α(GDIα)的基因敲除载体,并探讨干扰Rho GDIα后对小鼠肝癌细胞Hepa 1-6迁移的影响。  方法  根据CRISPR/Cas9靶点设计原则,设计Rho GDIα的向导RNA(sgRNA)序列,并与PX458载体相连,构建Rho GDIα基因敲除重组质粒PX458-Rho GDIα-sgRNA1和PX458-Rho GDIα-sgRNA2;脂质体转染法将重组质粒转染入Hepa 1-6细胞抑制Rho GDIα的基因表达;RT-PCR法检测Rho GDIα被抑制后的mRNA表达情况;划痕法检测Rho GDIα干扰后Hepa 1-6细胞迁移距离的差异;迁移小室法检测抑制Rho GDIα后Hepa 1-6细胞迁移数量的差异。  结果  成功构建了Rho GDIα的CRISPR/Cas9基因敲除质粒PX458-Rho GDIα-sgRNAs;转染有PX458-Rho GDIα-sgRNA1的Hepa 1-6 细胞与对照组只转染空载体PX458的细胞相比,Rho GDIα的表达被明显抑制(P<0.05),而且该组细胞从划痕起始处迁移到空白处距离较远,从迁移小室上端迁移到底端数目明显增多,差异极其显著(P<0.01),说明Rho GDIα被抑制后肝癌细胞的迁移能力提高。  结论  CRISPR/Cas9法构建的重组质粒PX458-Rho GDIα-sgRNA1可以有效抑制Rho GDIα的基因表达;Rho GDIα被抑制后明显促进小鼠肝癌细胞Hepa 1-6的迁移,提示在体情况下,Rho GDIα的表达可能起到抑制细胞迁移的重要作用,在肿瘤组织中,可以利用过表达Rho GDIα来抑制肿瘤细胞的转移。

关键词: 规律间隔成簇短回文重复序列/相关蛋白9, 鸟苷酸解离抑制因子α, 向导RNA, PX458质粒, 肝癌细胞系Hepa 1-6, 反转录聚合酶链反应, 小鼠

Abstract:

Objective  To construct the recombinant plasmids of knocking down Rho guanine dissociation inhibitor α (GDIα) gene by using clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) technique, and investigate the effect of Rho GDIα interference on the migration of Hepa 1-6 cells of mouse in order to provide the method  of prevention and treatment of liver cancer.   Methods  To construct and identify the PX458-Rho GDIα-single guide(sg)RNAs by using CRISPR/Cas9 technique. And the Hepa 1-6 cells were transfected by liposomes with PX458-Rho GDIα-sgRNAs for 48 hours respectively, and cells treated with PX458 plasmids were used as control. The migration ability of Hepa 1-6 was checked by wound healing assay and Transwell assay, respectively.  Results  The expression of Rho GDIα was depressed in group of PX458-Rho GDIα-sgRNA1 transfection which was detected by using RT-PCR. The migration distance of Hepa 1-6 in PX458-Rho GDIα-sgRNA1 transfection group was significantly promoted comparing with the control group which was transfected with PX458 only, and the cell number of PX458-Rho GDIα-sgRNA1 group was more than that in control group by using transwell assay, indicating concluded that knocking down of Rho GDIα promoted the migration ability of Hepa1-6 cells.
  Conclusion  The result  is explicit that in vivo, Rho GDIα may inhibit the migration of Hepa1-6 partially. Overexpression of Rho GDIα  might be used as an important method  to prevent the metastasize of carcinoma.

Key words: Clustered regularly interspaced , short palindromic repeats/associated protein 9, Rho guanine dissociation inhibitor α, Single guide RNA, PX458 plasmid, Hepatoma Hepa1-6 cell line, RT-PCR, Mouse

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