靶向叉头框蛋白J2的CRISPR/Cas9基因敲除质粒的构建及其对肝癌细胞转化生长因子β/Smads表达和增殖的影响

Construction of forkhead box J2 gene knockout plasmids by CRISPR/Cas9 and the effects on expression of transforming growth factor-β/Smads and proliferation in hepatocellular carcinoma

目的构建靶向叉头框蛋白J2(FOXJ2)的规律间隔成簇短回文重复序列/相关蛋白9(CRISPR/Cas9)基因敲除质粒,探讨干扰FOXJ2基因后对小鼠肝癌细胞Hepa1-6的信号通路转化生长因子β/Smads家族蛋白(TGF-β/Smads)表达和增殖的影响。方法设计小鼠FOXJ2的小向导RNA(sgRNA)序列,与PX458载体连接并转化感受态大肠埃希菌,挑取单克隆扩增提质粒。利用脂质体将重组质粒PX458-FOXJ2-sgRNAs转染入肝癌细胞Hepa1-6,对照组只转染空载体,48 h后RT-PCR检测对Hepa1-6细胞FOXJ2的抑制作用,同时检测FOXJ2被抑制后TGF-β和Smads的表达情况;MTT法检测干扰FOXJ2后对肝癌细胞增殖能力的影响。结果成功构建3个FOXJ2的CRISPR/Cas9基因敲除质粒PX458-FOXJ2-sgRNAs,其中质粒PX458-FOXJ2-sgRNA2可以有效抑制FOXJ2的表达,同时检测到干扰了FOXJ2的细胞的TGF-β、Smad2和Smad4的表达均高于对照组,差异显著;并且FOXJ2被抑制组肝癌细胞的增殖能力明显提高。结论小鼠肝癌细胞中的FOXJ2基因在一定程度上抑制TGF-β、Smad2和Smad4基因的表达,抑制细胞的增殖,推测在体情况下,肝癌细胞的增殖和TGF-β信号通路的激活与FOXJ2基因的负调控有一定的相关性,FOXJ2可以作为肝癌预防和治疗的靶点分子进行干预。

Objective To construct the clustered regularly interspaced short palindromic repeats/associated protein 9(CRISPR/Cas9)plasmid targeting forkhead box J2(FOXJ2)gene and investigate the effects of FOXJ2 interference on the expression of transforming growth factor-β(TGF-β)/Smads and proliferation in hepatocellular carcinoma cells of mouse.Methods Small guide RNA(sgRNA)sequence of FOXJ2 was designed,linked with PX458 vector and transfected into competent E.coli for proliferation.The recombinant plasmids were sent for sequencing to confirm the accuracy of the sgRNA sequence.The PX458-FOXJ2-sgRNAs plasmids were transfected into Hepa1-6 cells by liposome transfection,respectively.The empty vectors of PX458 were transfected as control group.After 48 hours,the expression of FOXJ2,TGF-βand Smads were obtained by RT-PCR and agarose gel electrophoresis,respectively.The cell proliferation was detected by methylthio tetrazole(MTT)method.Results The CRISPR/Cas9 plasmids of PX458-FOXJ2-sgRNAs were successfully constructed.The recombinant plasmid of PX458-FOXJ2-sgRNA2 could effectively inhibit FOXJ2 gene expression which induced increasing expression of TGF-β,Smad2 and Smad4 in Hepa1-6 cells comparing to the control group transfected with PX458 only.And the proliferation of Hepa1-6 was promoted in PX458-FOXJ2-sgRNA2 interference group.Conclusion In hepatocellular carcinoma cells of mouse,FOXJ2 gene inhibits the expression of TGF-β,Smad2,Smad4 and cell proliferation partially,which indicates the relationship between FOXJ2 and TGF-βsignal pathway.The result provides the target molecule of FOXJ2 for the prevention and treatment of hepatocellular carcinoma.