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

doi:10.16098/j.issn.0529-1356.2021.01.008

摘要/Abstract

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

中图分类号:

R73-37

曲明娟李延敏谢静秦苗苗王静周菊华. 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[J]. 解剖学报, 2021, 52(1): 55-59.

QU Ming-juan LI Yan-min XIE Jing QIN Miao-miao WANG Jing ZHOU Ju-hua. 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[J]. Acta Anatomica Sinica, 2021, 52(1): 55-59.

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