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  

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

作　　者：曲明娟[1] 李延敏[1] 谢静 秦苗苗 王静 周菊华[1] QU Ming-juan;LI Yan-min;XIE Jing;QIN Miao-miao;WANG Jing;ZHOU Ju-hua(School of Life Sciences,Ludong University,Shandong Yantai 264025,China)

机构地区：[1]鲁东大学生命科学学院,山东烟台264025

出　　处：《解剖学报》2021年第1期55-59,共5页Acta Anatomica Sinica

基　　金：国家自然科学基金(31100665);鲁东大学科技项目(72290401)。

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。Objective To construct the recombinant plasmids of knocking down Rho guanine dissociation inhibitorα(GDIα)gene by using clustered regularly interspaced short palindromic repeats/associated protein 9(CRISPR/Cas9)technique,and investigate the effect of Rho GDIαinterference 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 GDIαwas 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 GDIαpromoted the migration ability of Hepa1-6 cells.Conclusion The result is explicit that in vivo,Rho GDIαmay inhibit the migration of Hepa1-6 partially.Overexpression of Rho GDIαmight be used as an important method to prevent the metastasize of carcinoma.

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 

分 类 号：R73-37[医药卫生—肿瘤]