肺腺癌A549细胞中TNIK通过Rho/ROCK/LIMK1调控微丝骨架形态  

TNIK regulates actin cytoskeleton morphology via Rho/ROCK/LIMK1 pathway in lung adenocarcinoma A549 cells

作　　者：孙雪花 张莲 张春冬[1,3] 陈全[2] SUN Xuehua;ZHANG Lian;ZHANG Chundong;CHEN Quan(Molecular Medicine and Cancer Research Center,College of Basic Medical Sciences,Chongqing Medical University,Chongqing,400016,China;Department of Immunology,College of Basic Medical Sciences,Chongqing Medical University,Chongqing,400016,China;Department of Biochemistry and Molecular Biology,College of Basic Medical Sciences,Chongqing Medical University,Chongqing,400016,China)

机构地区：[1]重庆医科大学分子医学与肿瘤研究中心,重庆400016 [2]重庆医科大学免疫学教研室,重庆400016 [3]重庆医科大学生物化学与分子生物学教研室,重庆400016

出　　处：《陆军军医大学学报》2023年第7期659-666,共8页Journal of Army Medical University

基　　金：重庆市自然科学基金(cstc2021jcyj-msxmX0190)。

摘　　要：目的探讨肺腺癌A549细胞中TNIK调节微丝骨架形态的分子机制及其联合靶向有丝分裂化疗药物处理对癌细胞增殖与凋亡的影响。方法利用RNA-seq筛选稳定敲减TNIK表达后A549细胞差异表达基因,并分析微丝骨架组装相关基因的表达情况;通过RT-PCR、免疫印迹和双重免疫荧光染色分析TNIK调控微丝-微管骨架系统组装的分子机制,并联用靶向有丝分裂化疗药物紫杉醇处理癌细胞后,检测癌细胞的生长及凋亡情况。结果RNA-seq结果显示,敲减TNIK表达后的差异表达基因与有丝分裂进程密切相关;进一步分析发现TNIK表达可在转录水平显著抑制Rho通路相关基因RhoA/B、ROCK1/2和LIMK1表达(P<0.01),但对Rac与CDC42通路相关基因表达无显著影响。免疫荧光检测发现,敲减TNIK表达导致微丝骨架系统形态紊乱及有丝分裂异常。与对照细胞相比,联合紫杉醇处理的TNIK敲减组细胞增殖下调(P<0.01),而凋亡率显著增加(P<0.01)。结论肺腺癌A549细胞中TNIK通过Rho/ROCK/LIMK1调控细胞微丝-微管骨架系统形态,敲减TNIK可增强化疗药物对癌细胞增殖的抑制作用。ObjectiveTo investigate the molecular mechanism of TNIK regulating actin cytoskeleton morphology in lung adenocarcinoma A549 cells,and determine its effects on the proliferation and apoptosis of the cancer cells when combined with mitosis-targeted drug.MethodsRNA sequencing data were profiled to screen differentially expressed genes in the A549 cells with TNIK stable knockdown,and the expression of genes related to actin cytoskeleton assembly was subsequently detected.RT-PCR,Western blotting,and double immunofluorescence staining were preformed to analyze the molecular mechanism of TNIK regulating the assembly of actin-microtubule cytoskeleton system.Moreover,A549 cells with TNIK knockdown were treated with targeted antimitotic chemotherapeutic agent,paclitaxel,to observe the growth and apoptosis of the cancer cells.ResultsRNA-seq showed that the differentially expressed genes after TNIK knockdown were closely associated with mitotic progression.Further analysis revealed that TNIK knockdown significantly inhibited the expression of Rho pathway related genes including RhoA/B,ROCK1/2 and LIMK1 at the transcriptional level(P<0.01),but had no obvious effect on the expression of Rac and CDC42 pathway related genes.Immunofluorescence assay indicated that TNIK knockdown resulted in abnormal mitosis and morphology in actin cytoskeleton system.As compared with the control cells,paclitaxel treatment decreased the proliferation(P<0.01)while improved the apoptotic rate(P<0.01)in TNIK knockdown cells.ConclusionTNIK regulates the morphology of actin-microtubule cytoskeleton system in lung adenocarcinoma A549 cells through the Rho/ROCK/LIMK1 pathway,and TNIK knockdown can enhance the efficiency of mitosis-targeted chemotherapeutic drug.

关 键 词：肺腺癌细胞 微丝骨架 微管骨架 化疗 TNIK 

分 类 号：R394.3[医药卫生—医学遗传学] R730.23[医药卫生—基础医学] R734.2