NPAS2基因敲除的HepG2细胞系构建及其对肝癌细胞凋亡的影响

Construction of NPAS2 Knockout HepG2 Cell Line and Its Effect on the Apoptosis of Hepatocarcinoma Cells

目的:利用CRISPR/Cas9基因编辑技术构建生物节律基因NPAS2敲除的HepG2肝癌细胞系,并初步探讨NPAS2基因敲除对肝癌细胞凋亡的影响。方法:利用sgRNA在线设计工具,针对NPAS2设计两条sgRNA;利用PX459质粒构建分别含有两条sgRNA的敲除载体PX459-sgRNA1;PX459-sgRNA2;利用T7核酸内切酶I检测两条sgRNA活性;将活性较高的打靶载体瞬时转染HepG2细胞,经过药物筛选,克隆化培养及基因测序后得到NPAS2敲除的HepG2肝癌细胞系;利用Western blot检测NPAS2蛋白的表达和凋亡相关蛋白Caspase3的活化;利用流式细胞仪检测敲除细胞系的凋亡水平。结果:成功构建了针对NPAS2的打靶载体;并筛选得到了活性较高的打靶载体;经过药物筛选和克隆化培养得到的NPAS2敲除肝癌细胞系未检测到NPAS2蛋白的表达;进一步发现NPAS2敲除的肝癌细胞Caspase3明显活化,凋亡水平显著升高。结论:利用CRISPR/Cas9基因编辑技术成功构建了NPAS2基因敲除的HepG2肝癌细胞系,并发现NPAS2敲除可以促进肝癌细胞凋亡,为进一步研究生物节律基因NPAS2及其它相关基因在肝癌发生发展中的作用机制提供了有力的工具。

Objective： To explore the effect of NPAS2 knockout on apoptosis of hepatocarcinoma cells, the circadian gene NPAS2 knockout HepG2 cell line was constructed by CRISPR/Cas9 gene editing technology. Methods： Using sgR.NA online design tools to design two sgRNAs for NPA$2; Using PX459 plasmid to construct knockout vector respectively containing two sgRNAs, which are PX459-sgRNA1 and PX459-sgRNA2; The activity of two sgRNAs was detected by T7 endonuclease I; The high activity targeting vector was transfected into HepG2 cells; Then, through drug screening, cloning and sequencing, We finally got the NPAS2 knockout HepG2 cell line; Using Western blot to detect the expression of NPAS2 protein and the apoptosis associated protein Caspase3; Using flow cy- tometry to analyze apoptosis of hepatocarcinoma cells. Results： We successfully constructed the target vector of NPAS2, screened out the high activity vector. Moreover, we found that the expression of NPAS2 protein was not detected in NPAS2 knockout HepG2 cell lines by drug screening and cloning culture, the activation of Caspase3 and the level of apoptosis were significantly increased. Conclusion： NPAS2 gene knockout HepG2 cell line was successfully constructed using CRISPR/Cas9 gene editing technology, and NPAS2 knockout could promote apoptosis of hepatocarcinoma cells. These results will be a foundation for the further study ofNPAS2 gene related to the role of biological rhythm in the mechanism of the occurrence and development of hepatocellular carcinoma.