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

doi:10.16098/j.issn.0529-1356.2021.01.008

Abstract

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

CLC Number:

R73-37

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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