DDX6通过调控CKMT1A mRNA稳定性促进鼻咽癌细胞增殖和迁移的机制研究  

Study on the mechanism of DDX6 promoting proliferation and migration of nasopharyngeal carcinoma cells by regulating stability of CKMT1A mRNA

作　　者：熊茄妍 雷伟 游波[1,4] 张振新[1,4] 谢海静 单颖[1,4] 夏天 周勇 XIONG Jiayan;LEI Wei;YOU Bo;ZHANG Zhenxin;XIE Haijing;SHAN Ying;XIA Tian;ZHOU Yong(Research Institute of Otolaryngology Head and Neck Surgery,Affiliated Hospital of Nantong University,Nantong 226001,Jiangsu Province,China;Medical School of Nantong University,Nantong 226001,Jiangsu Province,China;Department of Otolaryngology Head and Neck Surgery,Second Affiliated Hospital of Soochow University,Suzhou 215000,Jiangsu Province,China;Department of Otolaryngology Head and Neck Surgery,Affiliated Hospital of Nantong University,Nantong 226001,Jiangsu Province,China)

机构地区：[1]南通大学附属医院耳鼻咽喉头颈外科研究所,江苏南通226001 [2]南通大学医学院,江苏南通226001 [3]苏州大学附属第二医院耳鼻咽喉头颈外科,江苏苏州215000 [4]南通大学附属医院耳鼻咽喉头颈外科,江苏南通226001

出　　处：《中国癌症杂志》2024年第5期451-459,共9页China Oncology

基　　金：国家自然科学基金(82103435,82203799)。

摘　　要：背景与目的:DDX是一类腺苷三磷酸(adenosine triphosphate,ATP)依赖的RNA解旋酶,与mRNA调控、肿瘤增殖及侵袭等密切相关。本研究旨在探讨DDX家族成员DDX6对CKMT1A mRNA稳定性的影响以及DDX6-CKMT1A轴对人鼻咽癌细胞CNE2增殖和迁移能力的影响及其分子机制。方法:检索癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库中DDX6和CKMT1A在人头颈部鳞状细胞癌中的表达情况并进行相关性分析,利用蛋白质印迹法(Western blot)检测南通大学附属医院保存的人体鼻咽癌组织和正常鼻咽部组织中CKMT1A和DDX6的表达,本研究通过了南通大学附属医院伦理委员会的审查(编号:2022-L114)。利用transwell实验检测细胞迁移能力,采用EdU试剂盒检测细胞增殖能力,采用细胞集落形成实验检测克隆形成能力。转染慢病毒、质粒,构建南通大学附属医院保存的人源鼻咽癌细胞CNE2的shDDX6、sh-CKMT1A、sh-CKMT1A+sh-DDX6和oe-CKMT1A细胞模型,明确DDX6和CKMT1A表达水平对鼻咽癌细胞恶性生物学表型的影响。构建BALC/c裸小鼠皮下移植瘤模型,检测DDX6和CKMT1A在小鼠体内对鼻咽癌细胞成瘤性的影响。采用RNA稳定性实验检测敲除DDX6对CKMT1A mRNA的影响,进一步明确DDX6的分子机制。结果:人鼻咽癌组织中DDX6高表达,CKMT1A低表达,DDX6与CKMT1A表达呈负相关。DDX6通过破环CKMT1A mRNA稳定性,抑制CKMT1A蛋白质翻译。在CNE2细胞中,CKMT1A低表达可增强细胞迁移和增殖能力,高表达则抑制细胞迁移和增殖能力,而DDX6的敲除可逆转CKMT1A下调导致的恶性行为进展。在裸鼠皮下移植瘤模型中,低表达CKMT1A促进肿瘤细胞生长,低表达DDX6抑制肿瘤细胞生长,而同时敲除DDX6与CKMT1A能恢复单独敲低DDX6导致的抑制效果。结论:鼻咽癌细胞中DDX6通过破坏CKMT1A mRNA稳定性,负调控CKMT1A蛋白质翻译,增强鼻咽癌细胞增殖和迁移能力,从而促进鼻咽癌恶性进展。Background and purpose:DDX is an adenosine triphosphate(ATP)-dependent RNA helicase closely related to mRNA regulation,tumor proliferation and invasion.This article aimed to explore the effect of DDX6,a member of the DDX family,on the stability of CKMT1A mRNA,as well as the effect of the DDX6 CKMT1A axis on the proliferation and migration ability of human nasopharyngeal carcinoma cell CNE2 and its molecular mechanism.Methods:We retrieved the data of expressions of DDX6 and CKMT1A in human head and neck squamous cell carcinoma from The Cancer Genome Atlas(TCGA) database and performed a correlation analysis.Western blot was performed to detect the expressions of CKMT1A and DDX6 in human nasopharyngeal carcinoma tissues and normal nasopharyngeal tissues preserved by Affiliated Hospital of Nantong University.This study was approved by the Ethics Committee of Affiliated Hospital of Nantong University(Number:2022-L114).We used transwell assay to detect cell migration ability,EdU assay to detect cell proliferation ability,and colony formation assay to detect clone formation ability.We transfect with lentivirus and plasmids to construct sh-DDX6,sh-CKMT1A,sh-CKMT1A+sh-DDX6 and oe-CKMT1A cell models derived from the human nasopharyngeal carcinoma cell line CNE2,preserved by Affiliated Hospital of Nantong University,to clarify the impact of DDX6 and CKMT1A expression levels on the malignant biological phenotypes of nasopharyngeal carcinoma cells.BALB/c nude mice subcutaneous xenograft tumor model was constructed to detect the effects of DDX6 and CKMT1A on nasopharyngeal carcinoma cells in mice.RNA stability assay was used to detect the effect of DDX6 knockout on CKMT1A mRNA and further clarify the molecular mechanism of DDX6.Results:DDX6 was highly expressed,CKMT1A level was low in human nasopharyngeal carcinoma tissue,and DDX6 was negatively correlated with CKMT1A expression.DDX6 inhibited protein translation of CKMT1A by disrupting its mRNA stability.Low expression of CKMT1A in CNE2 cells enhanced cell migration and proli

关 键 词：鼻咽癌 CKMT1A DDX6 细胞增殖 细胞迁移 

分 类 号：R739.6[医药卫生—肿瘤]