CRISPR/CAS9靶向肝癌细胞Oct3/4增强化疗药物作用效果的研究

目的探讨CRISPR/CAS9靶向敲除Oct3/4基因后化疗药物索拉菲尼(Sorafenib)对肝癌细胞的作用影响。方法通过实时荧光定量PCR和免疫印迹检测人肝癌细胞Li-7、HepG2、Huh7、BEL-7405和人肝正常细胞LX-2中Oct3/4的表达水平。通过CRISPR设计工具(http://crispr.mit.edu/)设计靶向Oct3/4的gRNA,并通过CRISPR/CAS9技术敲除肝癌细胞HepG2的Oct3/4,评估Oct3/4缺失情况下,化疗药物Sorafenib对肝癌细胞HepG2的凋亡水平和DNA损伤水平的影响。结果Oct3/4在人肝癌细胞Li-7、HepG2、Huh7、BEL-7405中的mRNA表达水平显著高于人肝正常细胞LX-2(P<0.05);同时,Oct3/4在人肝癌细胞Li-7、HepG2、Huh7、BEL-7405中的蛋白表达水平也显著高于人肝正常细胞LX-2。使用CRISPR/CAS9对HepG2细胞进行基因编辑后,挑取嘌呤霉素筛选出的2个单克隆细胞株进行Oct3/4表达水平的检测,1#号单克隆细胞株Oct3/4表达水平低于野生型WT,2#号单克隆细胞株没有表达Oct3/4。测序后发现1#号基因组上的Oct3/4第1个外显子只有1条染色体缺失了2个碱基,另一条染色体并未缺失;2#号基因组上的Oct3/4第1个外显子2条染色体均存在不同程度的缺失,一条缺失2个碱基,另一条缺失4个碱基。使用Sorafenib处理野生型HepG2细胞和2#Oct3/4 KO HepG2细胞后,Oct3/4 KO细胞株的凋亡水平显著上升(P<0.05),DNA损伤水平显著上升(P<0.05)。结论Oct3/4基因的敲除能够显著提高化疗药物Sorafenib促肝癌细胞凋亡和DNA损伤的作用。

CRISPR/CAS9 Targeting Oct3/4 to Enhance the Effect of Chemotherapy Drugs

Objective To investigate the effect of Sorafenib(Sorafenib)on liver cancer cells after CRISPR/CAS9 targeted knockout of Oct3/4 gene.Methods The expression levels of Oct3/4 in human liver cancer cells Li-7,HepG2,Huh7,BEL-7405 and human liver normal cells LX-2 were detected by real-time fluorescent quantitative PCR and western blotting.The CRISPR design tool(http://crispr.mit.edu/)was used to design the gRNA targeting Oct3/4,and the Oct3/4 of the liver cancer cell HepG2 was knocked out by CRISPR/CAS9 technology.The chemotherapy drug Sorafenib was used to evaluate the effect of Oct3/4 deletion on the apoptosis level and DNA damage level of liver cancer cells HepG2.Results The mRNA expression level of Oct3/4 in human liver cancer cells Li-7,HepG2,Huh7,and BEL-7405 was significantly higher than that in normal human liver cells LX-2(P<0.05).At the same time,the protein expression level of Oct3/4 in human liver cancer cells Li-7,HepG2,Huh7,and BEL-7405 was significantly higher than that in normal human liver cells LX-2.After using CRISPR/CAS9 to perform gene editing on HepG2 cells,2 monoclonal cell lines selected by puromycin were selected to detect the expression level of Oct3/4.The expression level of Oct3/4 in the 1#monoclonal cell line was lower than that of the wild Type WT,2#monoclonal cell line does not express Oct3/4.After sequencing,it was found that only one chromosome of Oct3/4 on the 1#genome was missing 2 bases,and the other chromosome was not missing;the Oct3/4 on the 2#genome was the first exon Both chromosomes have different degrees of deletion,1 was missing 2 bases and the other was missing 4 bases.After Sorafenib was used to treat wild-type HepG2 cells and 2#Oct3/4 KO HepG2 cells,the apoptosis level of Oct3/4 KO cell line increased significantly(P<0.05),and the level of DNA damage increased significantly(P<0.05).Conclusion The knockout of Oct3/4 gene can significantly improve the effect of Sorafenib,a chemotherapy drug,on the apoptosis and DNA damage of liver cancer cells.