Construction of forkhead box J2 gene knockout plasmids by CRISPR / Cas9 and the effects on expression of transforming growth factor￣β/ Smads and proliferation in hepatocellular carcinoma
10.16098/j.issn.0529-1356.2021.02.011
- Author:
Ming-Juan QU
1
;
Yu ZHENG
1
;
Yan-Min LI
1
;
Yang SONG
1
;
Lei WANG
1
;
Ju-Hua ZHOU
1
Author Information
1. School of Life Sciences, Ludong University
- Publication Type:Journal Article
- Keywords:
Clustered regularly interspaced short palindromic repeats/ associated protein 9;
Hepatocellular carcinoma cells;
Mouse;
MTT assay;
Smads protein;
Small guide RNA;
Tramsforming growth factor-β
- From:
Acta Anatomica Sinica
2021;52(2):231-235
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the clustered regularly interspaced short palindromic repeats / associated protein 9 (CRISPR/ Cas9) plasmid targeting forkhead box J2 (FOXJ2) gene and investigate the effects of FOXJ2 interference on the expression of transforming growth factor-β(TGF-β) / Smads and proliferation in hepatocellular carcinoma cells of mouse. Methods Small guide RNA(sgRNA) sequence of FOXJ2 was designed, linked with PX458 vector and transfected into competent E. coli for proliferation. The recombinant plasmids were sent for sequencing to confirm the accuracy of the sgRNA sequence. The PX458-FOXJ2-sgRNAs plasmids were transfected into Hepa1-6 cells by liposome transfection, respectively. The empty vectors of PX458 were transfected as control group. After 48 hours, the expression of FOXJ2, TGF-β and Smads were obtained by RT-PCR and agarose gel electrophoresis, respectively. The cell proliferation was detected by methylthio tetrazole (MTT) method . Results The CRISPR/ Cas9 plasmids of PX458-FOXJ2-sgRNAs were successfully constructed. The recombinant plasmid of PX458-FOXJ2-sgRNA2 could effectively inhibit FOXJ2 gene expression which induced increasing expression of TGF-β, Smad2 and Smad4 in Hepa1-6 cells comparing to the control group transfected with PX458 only. And the proliferation of Hepa1-6 was promoted in PX458-FOXJ2-sgRNA2 interference group. Conclusion In hepatocellular carcinoma cells of mouse, FOXJ2 gene inhibits the expression of TGF-β, Smad2, Smad4 and cell proliferation partially, which indicates the relationship between FOXJ2 and TGF-β signal pathway. The result provides the target molecule of FOXJ2 for the prevention and treatment of hepatocellular carcinoma.
