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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 construct the NPAS2-knockout HepG2 hepatoma cell line using CRISPR / Cas9 gene editing technology and to investigate the effect of NPAS2 knock-out on hepatocellular carcinoma cell apoptosis. METHODS: Two sgRNAs were designed for NPAS2 using sgRNA online design tool. PX459-sgRNA1 and PX459-sgRNA2 knockout vectors containing two sgRNAs were constructed by PX459 plasmid. The two sgRNAs were detected by T7 endonuclease I. HepG2 cells were transiently transfected into HepG2 cells with high activity and NPAS2 knockdown after drug screening, cloning and gene sequencing. Western blot was used to detect the expression of NPAS2 and activation of Caspase3, Flow cytometry was used to detect the level of apoptosis in knockdown cell lines. Results: NPAS2 targeting vector was successfully constructed and targeted active vector was screened. NPAS2 protein was not detected in NPAS2 knockdown hepatoma cell line after drug screening and cloning culture. NPAS2 knockout was further found Of liver cancer cells Caspase3 significantly activated, the level of apoptosis was significantly increased. Conclusion: NPAS2 gene knockout HepG2 hepatoma cell line was successfully constructed by using CRISPR / Cas9 gene editing technology. NPAS2 knockdown was found to promote apoptosis of hepatocellular carcinoma cells. In order to further study the occurrence and development of NPAS2 and other related genes The mechanism of action provides a powerful tool.