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

Abstract

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.

Transcriptional coactivator PGC-1α contributes to decidualization by forming a histone-modifying complex with C/EBPβ and p300

“…HepG2 cells, which highly express PGC-1α ( Fig. 5 C ) and are widely used for the experiment on genome editing ( 21 , 56 ) were used to examine the PGC-1α enhancer function by a genome editing approach. Single guide RNAs (sgRNAs) were designed to surround the C/EBPβ-binding sites in the two PGC-1α enhancer regions, respectively ( Fig.…”

A review of the literature on the use of CRISPR/Cas9 gene therapy to treat hepatocellular carcinoma

“…Hu et al found that CRISPR/Cas9-mediated gene deletion of Traf3 increased proliferation, migration, and invasion of HepG2 cells, providing valuable insights into Traf3’s function and mechanism [ 68 ].…”

“…In most cases, researchers conduct cell viability assays (such as MTT or MTS assays) after RP11-156P1.3 is knocked out in HCC cells [ 129 , 130 ]. As a result of RP11-156P1.3 knockout in HCC cells, they may report a significant drop in cell viability, quantified by a percentage.…”

CRISPR/Cas9: A powerful genome editing technique for the treatment of cancer cells with present challenges and future directions

“…The brown box represents the activation of different tumor suppressor genes. The orange box characterizes the inhibition/suppression of drug-resistance genes by CRISPR/Cas9 in solid tumors [ 31 , 35 , 45 , 70 , 74 , [115] , [116] , [117] , [118] , [119] , [120] , [121] , [122] , [123] , [124] , [125] , [126] , [127] , [128] ]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)…”