CMTM6基因对肝内胆管癌侵袭转移的影响  

Effect of CMTM6 gene on invasion and metastasis of intrahepatic cholangiocarcinoma

作　　者：刘承祥 董严 梁后杰 LIU Chengxiang;DONG Yan;LIANG Houjie(Department of Oncology,The First Affiliated Hospital of Army Medical University,Chongqing 400038,China)

机构地区：[1]陆军军医大学第一附属医院肿瘤科,重庆400038

出　　处：《重庆医学》2023年第18期2726-2732,共7页Chongqing medicine

基　　金：重庆市英才计划项目(4139Z3395)。

摘　　要：目的 探讨CMTM6基因对肝内胆管癌细胞(ICC)侵袭转移的影响。方法 通过慢病毒转染构建敲低含家族成员6的趋化因子样奇迹跨膜结构域(CMTM6)基因的ICC RBE细胞(RBE-CMTM6-KD)及相应对照细胞(RBE-Con),实时荧光定量PCR(qPCR)及Western blot分别在RNA及蛋白层面验证两种细胞CMTM6的表达水平,平板克隆形成、Transwell、CCK-8及划痕实验分别检测两种细胞生长曲线及细胞存活状况,两种细胞构建裸鼠皮下成瘤并模拟肺转移观察CMTM6对肿瘤生长及转移能力的影响,免疫组织化学技术检测人ICC组织芯片中癌旁组织及不同分化程度ICC组织中CMTM6的表达强度,并分析CMTM6不同表达强度ICC患者的预后。结果 Western blot结果显示,RBE-CMTM6-KD中CMTM6、Snail蛋白表达水平低于RBE-Con, β-catenin表达水平高于RBE-Con(P<0.05)。qPCR结果显示,RBE-CMTM6-KD中CMTM6 mRNA表达水平低于RBE-Con(P<0.05)。与RBE-Con比较,24、48 h RBE-CMTM6-KD细胞存活率更低,增殖能力、侵袭迁移能力减弱(P<0.05)。RBE-KD-CMTM6构建的裸鼠皮下成瘤最终质量低于RBE-Con[(26.4±0.7)mg vs.(173.9±12.8)mg],差异有统计学意义(P<0.05)。第11、14、17、20天,RBE-CMTM6-KD构建的裸鼠肿瘤体积小于RBE-Con(P<0.05)。第7、14天,RBE-KD-CMTM6构建的裸鼠肺部转移瘤的转移能力低于RBE-Con(P<0.05)。CMTM6在癌旁正常胆管组织中几乎不表达,在ICC组织表达水平明显高于癌旁正常胆管组织,且在低分化ICC组织中表达水平高于高分化ICC组织。与CMTM6低表达患者比较,CMTM6高表达患者OS明显缩短(P<0.05)。结论 成功建立了CMTM6敲低的ICC细胞模型,CMTM6能促进ICC生长并增强侵袭转移能力,CMTM6高表达水平与ICC预后呈负相关。Objective To investigate the effect of CMTM6 gene on invasion and metastasis of intrahepatic cholangiocarcinoma(ICC).Methods ICC RBE cells(RBE-CMTM6-KD)and corresponding control cells(RBE-Con)were constructed by lentivirus transfection to knock down the chemokine-like miracle transmembrane domain(CMTM6)gene containing family member 6.Real-time fluorescence quantitative PCR(qPCR)and Western blot were used to detect the expression of CMTM6 at the RNA and protein levels in both groups.The growth curves and cell survival status of RBE-CMTM6-KD and RBE-Con were detected by plate cloning,Transwell,CCK-8 and scratch tests.The effect of CMTM6 on tumor growth and metastasis was verified by subcutaneous tumor formation and tail vein simulated lung metastasis experiments in nude mice with RBE-CMTM6-KD and RBE-Con.The expression intensity of CMTM6 in human ICC tissue microarray and ICC tissues with different degrees of differentiation was detected b y immunohistochemistry.The prognosis of patients with ICC with different expression intensities o f CMTM6 was analyzed.Results The Western blot showed that the expression of CMTM6 and Snail protein in RBE-CMTM6-KD cells was significantly lower than that in RBE-Con cells(P<0.05).The expression ofβ-catenin protein in RBE-CMTM6-KD cells was significantly higher than that in RBE-Con cells(P<0.05).qPCR results showed that the mRNA expression level of CMTM6 in RBE-CMTM6-KD was lower than that in RBE-Con(P<0.05).Compared with RBE-Con,the survival rate of RBE-CMTM6-KD cells at 24 and 48h was lower,and the proliferation ability,invasion and migration ability were weakened(P<0.05).The final mass of subcutaneous tumor formation in nude mice constructed with RBE-KD-CMTM6 was lower than that of RBE-Con[(26.4±0.7)mg vs.(173.9±12.8)mg],and the difference was statistically significant(P<0.05).On days 11,14,17 and 20,the tumor volume of nude mice constructed by RBE-CMTM6-KD was smaller than that of RBE-Con(P<0.05).On days 7 and 14,the metastasis ability of lung metastases in nude mice construc

关 键 词：肝内胆管细胞癌 含家族成员6的趋化因子样奇迹跨膜结构域 肿瘤侵袭 肿瘤转移 预后 

分 类 号：R735.8[医药卫生—肿瘤]