ALKBH5靶向调控Titin基因介导Wnt/β-catenin信号通路参与宫颈癌生物学行为的机制研究  

Mechanism of ALKBH5 on the biological behavior of cervical cancer via targeted-regulating of Titin gene and mediation of Wnt/β-catenin signaling pathway

作　　者：胡益飞 吴淋淋[2] 郑顺杰[3] HU Yifei;WU Linlin;ZHENG Shunjie(Department of Clinical Laboratory,JinHua Municipal Central Hospital,Jinhua 321000,China;不详)

机构地区：[1]金华市中心医院检验科,321000 [2]金华市人民医院妇科,321000 [3]金华市中心医院妇科

出　　处：《浙江医学》2025年第1期32-39,共8页Zhejiang Medical Journal

基　　金：浙江省医药卫生科技计划项目(2021KY1184);金华市科技计划项目(2023-3-149)。

摘　　要：目的探讨ALKB同源物5(ALKBH5)靶向调控肌联蛋白(Titin)基因表达,并介导无翅型小鼠乳房肿瘤病毒整合位点家族蛋白(Wnt)/β-连锁蛋白(β-catenin)信号通路参与宫颈癌生物学行为的相关机制。方法回顾性选取2022年10月至2023年10月经金华市中心医院病理诊断为宫颈良性肿瘤、上皮不典型增生以及浸润性癌患者各30例并收集其手术切除获取的新鲜宫颈组织,采用实时荧光定量聚合酶链反应(qRT-PCR)检测ALKBH5和Titin mRNA表达水平。体外选择宫颈癌细胞株Hela并分为5组,即正常对照(NC)组、ALKBH5-小干扰RNA(siRNA)组、ALKBH5-NC组、ALKBH5-siRNA+Titin-模拟物(mimic)组和ALKBH5-siRNA+Titin-NC组;连续培养72 h,分别采用四甲基偶氮唑蓝法、流式细胞术、划痕实验、Transwell法检测细胞增殖率、凋亡率、迁移率以及侵袭细胞数,蛋白质印迹法检测上皮间质转化相关蛋白波形蛋白(Vimentin)、N-钙黏蛋白(N-cadherin)和E-钙黏蛋白(E-cadherin)以及Wnt、β-catenin蛋白表达水平,qRT-PCR检测细胞ALKBH5和Titin m RNA表达水平。采用荧光素酶法检测ALKBH5与Titin基因的靶向作用。结果与宫颈良性肿瘤组织、上皮不典型增生组织相比,浸润性癌组织中ALKBH5和Titin mRNA表达水平均明显升高(均P<0.01)。与NC组、ALKBH5-NC组相比,ALKBH5-siRNA组细胞增殖率、迁移率均下降(均P<0.05),侵袭细胞数均减少(均P<0.05),凋亡率均升高(均P<0.05),E-cadherin蛋白表达水平均上调(均P<0.05),Vimentin、N-cadherin、Wnt、β-catenin蛋白表达水平以及ALKBH5、Titin mRNA表达水平均下调(均P<0.05);与ALKBH5-siRNA组、ALKBH5-siRNA+Titin-NC组相比,ALKBH5-siRNA+Titin-mimic组细胞增殖率、迁移率均升高(均P<0.05),侵袭细胞数均增加(均P<0.05),凋亡率均下降(均P<0.05),E-cadherin蛋白表达水平均下调(均P<0.05),Vimentin、N-cadherin、Wnt、β-catenin蛋白表达水平以及Titin mRNA表达水平均上调(均P<0.05)。荧光素酶报告显示了ALKBH5Objective To explore the mechanisms of ALKB homology 5(ALKBH5)on tumor biological behaviors of cervical cancer via targeted regulation of muscle associated protein Titin gene expression and mediation of Wnt/β-catenin signaling pathway.Methods Fresh cervical tissues from 30 patients with benign cervical tumors,30 patients with atypical cervical epithelial hyperplasia,and 30 patients with invasive cervical cancer diagnosed at JinHua Municipal Central Hospital from October 2022 to October 2023 were obtained.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression levels of ALKBH5 and Titin mRNA.Cervical cancer cell line Hela was randomly divided into 5 groups in in vitro experiment,namely normal control(NC)group,ALKBH5-small interfering RNA(siRNA)group,ALKBH5-NC group,ALKBH5 siRNA+Titin-mimic group,and ALKBH5-siRNA+Titin-NC group.After continuous cell cultivation for 72 hours,methyl thiazolyl tetrazolium,flow cytometry,scratch assay,Transwell chamber were used to detect cell proliferation,apoptosis,migration,and invasion.Western blot was used to detect expressions of epithelial mesenchymal transition associated proteins Vimentin,N-cadherin,E-cadherin,and Wnt/β-catenin proteins.qRT-PCR was used to detect expressions of ALKBH5 and Titin mRNA in cells.Fluorescence enzyme method was used to detect the targeting effect of ALKBH5 on Titin gene.Results Compared with benign cervical tumors and atypical proliferative tissues,the expression levels of ALKBH5 and Titin mRNA in invasive cancer tissues were significantly higher(all P<0.01).Compared with NC group and ALKBH5-NC group,the ALKBH5-siRNA group showed decreases in cell proliferation and migration rates and number of invasive cells,increase in apoptosis rate,up-regulation of E-cadherin protein expression,and down-regulation of Vimentin,N-cadherin,and Wnt/β-catenin protein,ALKBH5 and Titin mRNA(all P<0.05).Compared with ALKBH5-siRNA and ALKBH5-siRNA+Titin-NC groups,ALKBH5-siRNA+Titin-mimic group showed increases in cell proliferation a

关 键 词：宫颈癌 ALKB同源物5 肌联蛋白 无翅型小鼠乳房肿瘤病毒整合位点家族蛋白/β-连锁蛋白信号通路 增殖 凋亡 迁移 侵袭 上皮间质转化 

分 类 号：R73[医药卫生—肿瘤]