通过Crispr-cas9介导抑制KIAA0101基因可诱导肝癌细胞凋亡

Inhibition of KIAA0101 gene by Crispr-cas9 induces apoptosis of hepatoma cells

目的构建肝癌细胞KIAA0101基因敲除的稳转株,研究抑制KIAA0101表达后肝癌细胞的细胞行为学改变。方法通过生物信息学筛选sgRNA,通过Crispr-cas9技术,构建抑制KIAA0101基因表达的sgRNA-cas9共转染慢病毒,并对KIAA0101基因敲除的实验组与空白对照组进行细胞增殖、凋亡、细胞周期及克隆形成实验。结果通过生物信息学筛选获得3条sgRNA,其中,sg1、sg3组具有明显的抑制KIAA0101基因表达的功能。通过crispr-cas9技术,获得了基于crispr-cas9技术介导的KIAA0101低表达的Huh-7肝癌细胞稳转株,结果显示,抑制KIAA0101基因表达后,相比对照组,实验组细胞增殖、迁移能力无显著变化,对细胞周期影响不显著(P>0.05),细胞凋亡率显著增加(P<0.05)。结论本研究通过Crispr-cas9技术,构建了Huh-7肝癌细胞KIAA0101基因敲除的稳转株,证实抑制KIAA0101基因表达后,Huh-7细胞凋亡显著增加。

Objective To construct a stable transgenic strain of KIAA0101 knockout gene in hepatocellular carcinoma cells,and to study the cell behavioral changes of hepatoma cells after inhibiting the expression of KIAA0101.Methods SgRNA was screened by bioinformatics,and sgRNA-cas9,which inhibits KIAA0101 gene expression,was co-transfected with lentivirus by Cristr-cas9 technology.The cell function experiments were performed between experimental gene knockout group and control group.Results Three sgRNAs were obtained by bioinformatics screening.Among them,sg1 and sg3 had obvious functions of inhibiting KIAA0101 gene expression.Using Crispr-cas9 technology,a stable transgenic strain of Huh-7 hepatoma cells with crispr-cas9-mediated low expression of KIAA0101 was obtained.After inhibiting the expression of KIAA0101 gene,no significant changes of cell proliferation and migration ability were found in gene knockout group compared with control group.And there was no significant difference in the effect on the cell cycle between two groups.The apoptosis rate was significantly increased in gene knockout group compared with control group(P<0.05).Conclusion The sgRNA sequence of human KIAA0101 gene is successfully designed by Crispr-cas9 technology,and a stable transgenic strain of KiaA0101 gene knockout of Huh-7 hepatoma cells is successfully constructed.It is confirmed that the apoptosis of Huh-7 cells is significantly increased after inhibiting the expression of KIAA0101 gene.