整合素连接激酶基因敲除骨肉瘤细胞系的建立及其对上皮-间质转化的影响  

Establishment of human ILK knockout osteosarcoma cell line and the effect of ILK loss on epithelial-mesenchymal transition

作　　者：赖永洁 由万宁 金炼驰 金明哲 Lai Yongjie;You Wanning;Jin Lianchi;Jin Mingzhe(Department of Immunology and Pathogenic Biology,Zhuhai Campus of Zunyi Medical University,Zhuhai 519041,Guangdong,China;Zhuhai Campus of Zunyi Medical University/Education and Innovation Base for Immunology Graduate Students in Guizhou Province,Zhuhai 519041,Guangdong China)

机构地区：[1]遵义医科大学珠海校区免疫学与病原生物学教研室,广东珠海519041 [2]遵义医科大学珠海校区/贵州省免疫学研究生创新研究基地,广东珠海519041

出　　处：《贵州医药》2023年第3期339-342,349,共5页Guizhou Medical Journal

基　　金：贵州省教育厅自然科学基金(黔教合KY字[2017]184);遵义医科大学校级项目(2017-F823)。

摘　　要：目的利用CRISPR/Cas9基因编辑技术构建人整合素连接激酶ILK基因敲除的人骨肉瘤U2OS细胞系,探讨ILK敲除对骨肉瘤细胞上皮-间充质转化(EMT)的影响。方法首先在人ILK基因的第2,4,6外显子区域选取序列,分别设计3组靶向ILK的sgRNA,退火后与酶切lentiCRISPRv2慢病毒质粒连接,构建含有sgRNA的重组质粒,重组载体与病毒包装质粒共转染293T细胞后获得慢病毒颗粒;将病毒颗粒感染U2OS细胞,经嘌呤霉素筛选及有限稀释法获得单克隆细胞株,通过测序、qRT-PCR及Western blot鉴定ILK基因稳定敲除细胞系,并进一步通过Western blot检测E-钙黏蛋白、N-钙黏蛋白、Snail1、Snail2、Twist等EMT相关标记分子以及Akt的磷酸化水平。结果构建了3组lentiCRISPRv2-ILK-sgRNA敲除质粒,经慢病毒感染U2OS细胞后及单细胞克隆株的筛选鉴定后得到了一株含有ILK等位基因突变的细胞,Western blot证实ILK^(-/-)细胞中ILK蛋白表达缺失;相比野生型,ILK^(-/-)细胞中N-钙黏蛋白、Snail1、Snail2及Twist蛋白表达水平均显著降低(P<0.05),而E-钙黏蛋白表达则显著升高(P<0.05);与野生型细胞相比,ILK^(-/-)细胞Akt通路的磷酸化水平显著降低(P<0.05),而经TGF-β1处理后,相比野生型细胞,ILK^(-/-)细胞中Akt激活水平亦显著下降(P<0.01)。结论成功构建ILK^(-/-)U2OS细胞系,敲除ILK可能通过下调Akt信号通路抑制U2OS细胞的EMT进程。Objective To knock out integrin-linked kinase(ILK)gene in osteosarcoma U2OS cell line using CRISPR/Cas9 system and investigate the effect of ILK knockout on the epithelial-mesenchymal transition(EMT).Methods Three sgRNAs targeted the second,the fourth and the sixth exons of ILK gene were designed using online software.The synthesized sgRNAs were annealed and ligated with linear lentiCRISPRv2 vector to construct three lentiCRISPRv2-ILK-sgRNA plasmids.Lentivirus particles were harvested via the cotransfection of the recombinant plasmids and package plasmids into 293T cells.Next,U2OS cells were infected with lentivirus,single cell clones were obtained by puromycin selection and limiting dilution.The ILK homozygous knockout cell clones were determined by sequencing and the loss of ILK expression was confirmed by qRT-PCR and Western blot.The levels of EMT related makers including E-cadherin,N-cadherin,Snail1,Snail2 and Twist in the ILK^(-/-)cells as well as the phosphorylation status of Akt were detected via Western Blot.Results Three different ILK knockout plasmids were constructed,and the ILK homozygous knockout cell line was generated.Compared with wild-type U2OS cells,the levels of N-cadherin,Snail1,Snail2,Twist were significantly decreased,while E-cadherin level was increased(P<0.05);compared with wild-type cells,the phosphorylated Akt in the ILK^(-/-)cells were markedly downregulated(P<0.05),while in the context of TGF-β1 treatment,the phosphorylation level of Akt induced by TGF-β1 was also reduced compared with wild-type cells(P<0.01).Conclusion ILK knockout U2OS cell line was generated by using CRISPR-/Cas9 strategy,and the loss of ILK expression inhibited the EMT progress via downregulating Akt signaling pathway,which may provide a basis for further translational research of ILK on osteosarcoma.

关 键 词：骨肉瘤 ILK 上皮-间充质转化 AKT信号通路 

分 类 号：R738.1[医药卫生—肿瘤]