m^(6)A去甲基化酶ALKBH5抑制胃癌细胞运动侵袭和Wnt信号通路活性的机制  

mA demethylase ALKBH5 restrains migration and invasion and Wnt signaling pathway activity of gastric cancer cells

作　　者：沈李婷 李金洋 李先峰 车林茸 刘科伟 刘琴[1] 王斌[1] 覃中毅 陈东风[1] SHEN Liting;LI Jinyang;LI Xianfeng;CHE Linrong;LIU Kewei;LIU Qin;WANG Bin;QIN Zhongyi;CHEN Dongfeng(Department of Gastroenterology,Daping Hospital,Army Medical University(Third Military Medical University),Chongqing,400042,China)

机构地区：[1]陆军军医大学(第三军医大学)大坪医院消化内科,重庆400042

出　　处：《第三军医大学学报》2021年第6期459-466,共8页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81672463);肿瘤免疫病理学教育部重点实验室开发课题(2018jsz107)。

摘　　要：目的研究RNA去甲基化酶ALKBH5对胃癌细胞迁移和侵袭能力的影响及机制。方法利用人类癌症基因库(the cancer genome Atlas, TCGA)对ALKBH5差异表达的胃癌患者进行总生存期分析;应用小鼠胃原位种植和转移模型分析ALKBH5的蛋白表达与胃癌细胞转移的相关性;通过构建敲低/过表达细胞株,结合Transwell实验研究ALKBH5对胃癌细胞迁移和侵袭能力的影响;利用m^(6)A修饰位点预测网站m^(6)AVAR(http://m^(6)Avar.renlab.org/index.html),结合蛋白免疫印迹、放线菌素D处理结合实时荧光定量PCR,检测Wnt信号通路下游基因表达和mRNA稳定性。结果 TCGA数据库提示ALKBH5的低表达提示胃癌患者更短的生存期;体内实验验证ALKBH5的低表达与胃癌细胞远处转移相关;ALKBH5的敲低可以促进胃癌细胞的侵袭和转移;Wnt下游分子MYC,CD44,c-Met的mRNA上存在m^(6)A修饰位点;ALKBH5缺失时,Wnt下游分子的蛋白表达增加及mRNA稳定性增强。结论 ALKBH5表达缺失促进胃癌细胞迁移和侵袭能力,维持Wnt信号通路的激活。Objective To investigate the effect of RNA demethylase ALKBH5 on migration and invasion of gastric cancer(GC) cells and its potential mechanism. Methods Cancer Genome Atlas(TCGA) was employed to determine the correlation of ALKBH5 expression variations with overall survival(OS) in GC patients(n=371). The mouse models of orthotopic transplantation and metastasis were established to evaluate the correlation of between ALKBH5 expression and GC cells metastasis. After ALKBH5 knockdown and overexpression cells were constructed, the effect of ALKBH5 on migration and invasion of GC cells was explored with Transwell chamber assay. Furthermore, m^(6)AVAR online database(http://m^(6)Avar.renlab.org/index.html) was used to predict the m^(6)A modification sites of Wnt activating targets;Western blotting and quantitative real-time PCR(qRT-PCR) combined with actinomycin D were applied to detect the protein expression and mRNA stability of downstream targets in Wnt signaling pathway. Results TCGA analysis showed that GC patients with lower ALKBH5 expression had shorter OS time. In vivo experiments confirmed that the low expression of ALKBH5 was involved in the distant metastasis of GC cells, and knockdown of ALKBH5 promoted the migration and invasion of GC cells. m^(6)A modification sites were found in the mRNAs of Wnt downstream targets MYC, CD44 and c-Met, and the absence of ALKBH5 indicated increased protein expression and mRNA stability of these molecules. Conclusion The loss of ALKBH5 expression promotes the migration and invasiveness of GC cells and sustains the activation of Wnt signaling pathway.

关 键 词：ALKBH5 RNA甲基化 WNT信号通路 胃癌侵袭 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R573.9[医药卫生—基础医学]