Construction of Traf3 knockout liver cancer cell line using CRISPR/Cas9 system

Purpose The gene editing technology in CRISPR/Cas9 system was used to construct the Traf3 knockout HepG2 cell line to explore the role of Traf3 in the development of liver cancer. Methods Five sgRNA sites were designed for the exons of Traf3. The recombinant plasmid of Lentiviral vector2-Traf3-sgRNA was constructed and transformed into Stbl3 competent cells. The recombinants were screened and sequenced, and the effectiveness of the designed gRNA was verified by sequencing. The constructed vector was transfected into HepG2 cells by lentiviral, and the monoclonal antibody was selected to detect the knockout effect of Traf3 gene in HepG2 cells by Western blot. PCR amplification and gene sequencing were performed to obtain the cell line, which the Traf3 gene was knocked out. MTT and Transwell assays were used to detect the effect of Traf3-knockout on HepG2 cell proliferation and cell invasion, respectively. Results The Lentiviral vector2-sgRNA expression vector was successfully constructed. PCR amplification electrophoresis and gene sequencing showed that the Trep3-knockdown HepG2 cells were successfully constructed. Compared with the wild HepG2 cells group, the proliferation and invasion ability of HepG2 cells were enhanced in the Traf3 knockout group. Conclusion Knockout Traf3 gene by CRISPR/Cas9 system enhanced the proliferation, migration, and invasion of HepG2 cells, and provided an effective tool for studying the function and mechanism of Traf3.