lncRNA HOTTIP通过miR-637/KLK4轴促进肺癌SPC-A-1细胞的恶性生物学行为  

Up-regulation of lncRNA HOTTIP promotes the malignant biological behaviors of lung cancer SPC-A-1 cells through miR-637/KLK4 axis

作　　者：张东伟[1] 蓝冰[1] 蔡双启[2] 钟家将 邹家威 张真强 ZHANG Dongwei;LAN Bing;CAI Shuangqi;ZHONG Jiajiang;ZOU Jiawei;ZHANG Zhenqiang(Department of Respiratory and Critical Care Medicine,Liuzhou People's Hospital,Liuzhou 545000,Guangxi,China;Department of Respiratory and Critical Care Medicine,the First Affiliated Hospital of Guangxi Medical University,Nanning 530021,Guangxi,China)

机构地区：[1]柳州市人民医院呼吸与危重症医学科,广西柳州545000 [2]广西医科大学第一附属医院呼吸与危重症医学科,广西南宁530021

出　　处：《中国肿瘤生物治疗杂志》2021年第10期961-968,共8页Chinese Journal of Cancer Biotherapy

基　　金：广西卫健委自筹经费科研项目(No.Z20190032);柳州市人民医院项目(No.lryjj201908)。

摘　　要：目的:探讨lncRNA HOTTIP对肺癌细胞增殖、凋亡及EMT的影响及其作用机制。方法:利用qPCR检测lncRNA HOTTIP、miR-637和KLK4在肺癌SPC-A-1、正常肺上皮BEAS-2B细胞中的表达量;siRNA干扰SPC-A-1细胞中lncRNA HOTTIP的表达后,分别通过CCK-8、Transwell、流式细胞术和WB法检测SPC-A-1细胞增殖、侵袭、凋亡和EMT能力的变化。miRanda软件和双荧光素酶报告基因实验分析lncRNA HOTTIP和miR-637之间的靶向关系,RNA pull-down实验检测lncRNA HOTTIP和miR-637的吸附作用,检测lncRNA HOTTIP通过miR-637对SPC-A-1细胞增殖、侵袭、凋亡和EMT的调控。利用TargetScan软件分析miR-637与KLK4的相关性,双荧光素酶报告基因实验检测miR-637与KLK4 mRNA之间的相互作用;检测miR-637通过KLK4 mRNA对SPC-A-1细胞增殖、侵袭、凋亡和EMT的调控。下调lncRNA HOTTIP和miR-637表达后,利用qPCR和WB检测KLK4 mRNA和蛋白表达水平的变化。结果:与BEAS-2B细胞比,在SPC-A-1细胞中lncRNA HOTTIP呈高表达(P<0.01),miR-637呈低表达(P<0.01),KLK4呈高表达(P<0.01)。下调lncRNA HOTTIP后,SPC-A-1细胞增殖、侵袭与EMT能力显著减弱,细胞凋亡率显著上升(P<0.01);lncRNA HOTTIP与miR-637具有靶向关系;下调miR-637表达后,SPC-A-1细胞增殖、侵袭与EMT能力显著上升,细胞凋亡率显著降低(P<0.01)。miR-637与KLK43’UTR特异性结合。miR-637通过KLK4显著促进了SPC-A-1细胞增殖、侵袭与EMT,细胞凋亡率显著上升(P<0.01)。下调lncRNA HOTTIP使KLK4表达显著降低,而下调miR-637可促进KLK4表达(P<0.05)。结论:上调lncRNA HOTTIP可通过miR-637/KLK4轴促进肺癌SPC-A-1细胞的增殖、侵袭与EMT而抑制癌细胞凋亡。Objective:To investigate the effect and mechanism of lncRNA HOTTIP on proliferation,apoptosis and EMT of lung cancer cells.Methods:The expressions of lncRNA HOTTIP,miR-637 and KLK4 in SPC-A-1,BEAS-2 B cells were detected by qPCR.After siRNA interference with the expression of lncRNA HOTTIP,the proliferation,invasion,apoptosis and EMT of SPC-A-1 cells were detected by CCK-8,Transwell,flow cytometry,and WB,respectively.The targeting relationship between lncRNA HOTTIP and miR-637 was analyzed by miRanda software and dual-luciferase reporter gene assay.RNA pull-down assay was used to detect the adsorption of lncRNA HOTTIP and miR-637,and to detect the effects of lncRNA HOTTIP regulating miR-637 on proliferation,invasion,apoptosis,and EMT of SPC-A-1 cells.The correlation between miR-637 and KLK4 was analyzed by TargetScan software,and the interaction between miR-637 and KLK4 was detected by dual-luciferase reporter gene assay.After siRNA interference with the expression of KLK4,the proliferation,invasion,apoptosis,and EMT of SPC-A-1 cells were detected.After down regulation of lncRNA HOTTIP and miR-637 expression,the levels of KLK4 mRNA and protein expression were detected by qPCR and WB.Results:Compared with BEAS-2 B cells,the expression of lncRNA HOTTIP in SPC-A-1 cells was significantly up-regulated(P<0.01),the expression of miR-637 was down-regulated(P<0.01),the KLK4 expression was up-regulated(P<0.01).Down-regulation of lncRNA HOTTIP could significantly reduce the proliferation,invasion,and EMT capacity of SPC-A-1 cells,and increase the apoptosis rate(P<0.01).lncRNA HOTTIP had a targeting relationship with miR-637.Down-regulation of miR-637 expression could significantly promote the proliferation,invasion and EMT capacity of SPC-A-1 cells,and inhibit the apoptosis rate(P<0.01).miR-637 specifically bound to KLK43’UTR.Down-regulation of KLK4 could significantly inhibit the proliferation,invasion,and EMT capacity of SPC-A-1 cells,and increase the apoptosis rate(P<0.01).Down-regulation of lncRNA HOTTIP could signif

关 键 词：lncRNA 同源基因A远端转录本 miR-637 激肽释放酶相关肽酶4 SPC-A-1细胞 增殖 上皮-间质转化 

分 类 号：R730.54[医药卫生—肿瘤] R734.2[医药卫生—临床医学]