Construction of GSTZ1 knockout model based on CRISPR-Cas9 system and its effect on the proliferation and migration of hepatoma cells

Abstract:

Objective：To construct a human glutathione S-transferase zeta 1（GSTZ1） gene knockout model in the HepG2 cell line based on the CRISPR/Cas9 system and to investigate the effect of GSTZ1 knockout on the proliferation and migration of HepG2 cells. Methods：A 20-bp sgRNA oligonucleotide sequence targeting GSTZ1 was designed and synthesized. After annealing，it was cloned into the lentiCRISPR-V2 vector. And HEK293T cells were co-transfected with lentivirus packaging plasmids. Then the lentivirus was packaged and HepG2 cells were infected. The positive cell lines were screened for by an appropriate concentration of puromycin，and monoclonal cells were obtained by limited dilution method. All the experiments were divided into three groups，namely parental group，KO1 group，and KO2 group. MTS assay，colony-forming assay，and wound healing assay were used to determine cell proliferation and migration capacity. Results：The success－ful construction of GSTZ1 knockout monoclonal cells was veri－fied by Western blot and DNA sequencing，while MTS assay was used to determine changes in the proliferation capacity of HepG2 cells in each group at different time points. The results showed that the KO1 group had significantly increased proliferation capacity compared with the parental group at 48 h[（24.758±1.508）% vs. （20.185±0.720）%，P=0.002]. The proliferation capacity in the KO1 group was significantly higher than that in the parental group at 72 h[（23.903±0.840）% vs. （28.634±1.848）%，P=0.014]. The proliferation capacity in the KO1 group was also significantly elevated compared with the parental group at both 96 h and 120 h （P<0.05）. The same effects were observed in KO2 cells，indicating that GSTZ1 knockout could promote the proliferation of HepG2 cells. Colony-forming assay showed that the colony-forming numbers in GSTZ1 knockout cell lines（KO1 and KO2） were significantly higher than that in their parental cell line（73.330±1.528 and 82.330±4.163 vs. 42.330±2.517，P=0.000，P=0.000），suggest－ing that GSTZ1 knockout remarkably increased the proliferation capacity of hepatoma cells. In addition，wound healing assay showed that both GSTZ1 knockout cell lines had a significant increase in relative migration ratio compared with the parental cell line at 48 h（0.327±0.041 and 0.371±0.013 vs. 0.184±0.045，P=0.003，P=0.001），suggesting that GSTZ1 knockout enhanced the migration capacity of hepatoma cells. Conclusion：GSTZ1 knockout significantly promotes the proliferation and migration of human hepatocel－lular carcinoma cells，suggesting that GSTZ1 may regulate the development and progression of hepatocellular carcinoma as a tumor suppressor gene.