沉默ACLY基因可抑制结肠癌HCT116细胞的迁移和侵袭  

Silencing ACLY gene inhibits migration and invasion of colon cancer HCT116 cells

作　　者：文君[1] 闵雪洁 赵丽[1] 唐德伟 刘建军[1] 黄钢[3] 赵小平[1] WEN Jun;MIN Xuejie;ZHAO Li;TANG Dewei;LIU Jianjun;HUANG Gang;ZHAO Xiaoping(Department of Nuclear Medicine,Renji Hospital,School of Medicine,Shanghai Jiao Tong University,Shanghai 200127,China;Department of Oncology,The First Affiliated Hospital of Nanyang Medical College,Nanyang 473000,Henan Province,China;Shanghai University of Medicine and Health Sciences,Shanghai 201318,China)

机构地区：[1]上海交通大学医学院附属仁济医院核医学科,上海200127 [2]河南省南阳医学高等专科学校第一附属医院肿瘤科,河南南阳473000 [3]上海健康医学院,上海201318

出　　处：《肿瘤》2019年第6期460-468,共9页Tumor

摘　　要：目的:探讨沉默ATP柠檬酸裂解酶(ATP citrate lyase,ACLY)基因表达对结肠癌HCT116细胞增殖和迁移的抑制作用。方法:采用成簇规律间隔的短回文重复序列(clustered regulatory interspaced short palindromic repeats,CRISPR)/CRISPR相关蛋白9(CRISPR-associated protein 9,Cas9)技术特异性靶向ACLY基因,构建2株稳定沉默ACLY表达的结肠癌HCT116细胞(即ACLY-knockout-1和ACLY-knockout-2,简称KO-1和KO-2)。应用蛋白质印迹法检测KO-1和KO-2组及未敲除ACLY的对照组(Ctrl)HCT116细胞中ACLY蛋白的表达,应用CCK-8法和软琼脂克隆形成实验分别检测Ctrl、KO-1和KO-2组HCT116细胞的增殖能力,划痕愈合实验和Transwell小室法分别检测Ctrl、KO-1和KO-2组HCT116细胞的迁移和侵袭能力,应用实时荧光定量PCR法检测Ctrl、KO-1和KO-2组HCT116细胞中上皮-间质转化标志分子E-cadhrein、N-cadherin和波形蛋白(vimentin,VIM)以及相关转录因子Snail和锌指E盒增强子结合蛋白1(zinc-nger E-box binding homeobox 1,ZEB1)mRNA的表达水平。结果:KO-1和KO-2组HCT116细胞中ACLY蛋白不能正常表达,而Ctrl组HCT116细胞中可见ACLY蛋白表达。细胞培养72和96 h,KO-1和KO-2组HCT116细胞的增殖能力明显低于Ctrl组(P值均<0.01);KO-1组HCT116细胞形成的克隆数显著少于Ctrl组(P<0.01)。KO-1和KO-2组单克隆HCT116细胞的迁移(P值均<0.05)和侵袭(P值均<0.01)能力均低于Ctrl组。KO-1和KO-2组单克隆HCT116细胞中上皮-间质转化标志分子N-cadherin和VIM以及相关转录因子Snail和ZEB1 mRNA的表达水平均明显低于Ctrl组(P值均<0.01),E-cadherin mRNA的表达水平均明显高于Ctrl组(P值均<0.01)。结论:沉默ACLY基因可能逆转结肠癌HCT116细胞的上皮-间质转化,显著抑制结肠癌HCT116细胞的增殖和迁移。Objective:To investigate the effects of silencing ATP citrate lyase(ACLY)gene expression on the proliferation and migration abilities of colon cancer HCT116 cells.Methods:Two specific colon cancer HCT116 cell lines(ACLY-knockout-1 and ACLYknockout-2,herein after referred to as KO-1 and KO-2)with stable silencing of ACLY gene were constructed by using clustered regulatory interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)technique.The expression of ACLY in HCT116 cells of KO-1 and KO-2 groups as well as the control(Ctrl)group which the ACLY gene was not knocked out was detected by Western blotting.The proliferative capacity of HCT116 cells in KO-1,KO-2 and Ctrl groups was detected by CCK-8 and soft agar colony formation assay.The migration and invasion abilities of HCT116 cells in KO-1,KO-2 and Ctrl groups were detected by scratch healing assay and Transwell chamber assay,respectively.The expression levels of epithelial-mesenchymal transition markers including E-cadhrein,N-cadherin and vimentin(VIM)as well as the related transcription factors including snail and zinc-finger E-box binding homeobox 1(ZEB1)mRNAs in HCT116 cells of KO-1,KO-2 and Ctrl groups were detected by real-time fluorescent quantitative PCR.Results:The ACLY protein could not be expressed normally in HCT116 cells of KO-1 and KO-2 groups,while the ACLY protein could be expressed in Ctrl group.The proliferation activity of HCT116 cells in KO-1 and KO-2 groups was lower than that in Ctrl group at 72h and 96 h(all P<0.01).The number of clones formed by HCT116 cells in KO-1 and KO-2 groups was less than that in Ctrl group(both P<0.01).The migration and invasion abilities of HCT116 cells in KO-1 and KO-2 groups were lower than those in Ctrl group(both P<0.05,both P<0.01).The expression levels of epithelial-mesenchymal transition markers(N-cadherin and VIM)and the related transcription factors(Snail and ZEB1)in HCT116 cells of KO-1 and KO-2 groups were significantly lower than those in Ctrl group(all P<0.01),whereas the expre

关 键 词：结直肠肿瘤 细胞迁移分析 肿瘤侵润 上皮-间质转化 ACLY基因 成簇规律间隔的短回文重复序列/CRISPR相关蛋白9 

分 类 号：R735.34[医药卫生—肿瘤]