基于CRISPR-Cas9系统构建GSTZ1基因敲除模型并探索其对肝癌细胞增殖和迁移能力的影响

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

摘要:

目的：通过CRISPR/Cas9系统在HepG2肝癌细胞系中构建人谷胱甘肽S-转移酶ζ1（glutathione S-transferase zeta，GSTZ1）基因敲除模型并探索GSTZ1敲除对肝癌细胞增殖和迁移能力的影响。方法：设计并合成长度20 bp的靶向GSTZ1的sgRNA寡核苷酸序列，退火后克隆到LentiCRISPR-V2载体上，与慢病毒包装质粒共转染HEK293T细胞，包装慢病毒并感染HepG2细胞，用合适浓度的嘌呤霉素筛选阳性细胞株，有限稀释法获得单克隆细胞。所有实验均分成3组：parental组、KO1组和KO2组，采用MTS实验、克隆形成实验和划痕实验分别检测细胞增殖和迁移能力。结果：Western blot和DNA测序鉴定GSTZ1敲除的单克隆细胞构建成功；MTS检测不同时间点下各组HepG2细胞的增殖能力变化情况。结果显示，48 h时，KO1组[（24.758±1.508）%]相比亲本组[（20.185±0.720）%]，增殖能力明显增强，差异有统计学意义（P=0.002）。72 h时，相比亲本组[（23.903±0.840）%]，KO1组[（28.634±1.848）%]增殖能力明显增强（P=0.014）。96 h和120 h时，KO1组相比亲本对照组，其增殖能力也明显增强（P<0.05）。在KO2细胞中观察到同样现象，说明敲除GSTZ1后能够促进HepG2细胞的增殖能力。克隆形成实验表明，GSTZ1敲除细胞株（KO1和KO2）克隆形成数（73.330±1.528、82.330±4.163）相比亲本细胞克隆形成数（42.330±2.517）增多，差异有统计学意义（P=0.000，P=0.000），提示GSTZ1基因敲除可以明显促进肝癌细胞的增殖能力。划痕实验结果显示，GSTZ1敲除细胞株KO1和KO2在48 h的迁移相对比分别为0.327±0.041、0.371±0.013，与亲本细胞（0.184±0.045）相比明显增加（P=0.003，P=0.001），提示GSTZ1基因敲除可明显促进肝癌细胞的迁移能力。结论：GSTZ1缺失明显促进肝癌细胞的增殖和迁移能力，提示GSTZ1可能作为抑癌基因调控肝癌的发生和进程。

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.