Effect of ARID2 knockout on proliferation gene expression of hepatocarcinoma cells Hep3B / 解放军医学杂志

ABSTRACT

Objective To construct ARID2 knockout human liver cancer cell line Hep3B by CRISPR/Cas9 system, explore the effect of ARID2 knockout on the proliferation of Hep3B, and the differences in gene expression between wild type and ARID2 knockout Hep3B cell lines. Methods The plasmid lentiCRISPRv2 was constructed with ARID2 knockout plasmid, and then transfected Hep3B cells; The positive cells were selected by puromycin, sorted by flow cytometry and cultured to obtain the monoclonal cell lines; ARID2 knockout Hep3B cell lines were identified by Western blotting and Sanger sequencing; The effect of ARID2 knockout on the proliferation of Hep3B cell line was detected by CCK-8 method; RNA-seq was used to analyze the differentially expressed genes between wild type Hep3B cells and ARID2 knockout Hep3B cells, and the results of RNA-seq were validated by Real-time quantitative PCR (RT-qPCR). The possible biology functions of ARID2 were explored through Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG Pathway), GO Biological Processes and Gene set enrichment analysis (GSEA). Results Two ARID2 knockout Hep3B cell lines were successfully constructed. Compared with the wild type cell line, the proliferation of ARID2 knockout cell lines was significantly accelerated (P<0.05). A total of 85 differentially expressed genes were identified by RNA-seq analysis between ARID2 knockout cell lines and wild type cell line, of which 17 genes were up-regulated and 68 down-regulated. The mRNA expression of 10 differentially expressed genes were validated by RT-qPCR, the verification results were consistent with that by RNA-seq. Results of KEGG Pathway, GO Biological Processes and GSEA indicated that ARID2 genes might be involved in such biological processes as protein processing and transport, chemokine signaling pathway, Wnt signaling pathway, complement and coagulation cascade reaction, epithelial-mesenchymal transition (EMT), glycolysis, TGF-β signaling pathway, and TNF-α/NF-KB signaling pathway, et al. Conclusion ARID2 knockout can promote proliferation of Hep3B cell line; and ARID2 may play an important role by variety of biological processes in tumor proliferation, invasion, metastasis and tumor microenvironment.