微小RNA-223-3p靶向调控TGFBR3影响肺癌细胞上皮间质转化及Wnt/β-catenin通路  

Targeting regulation of TGFBR3 by microRNA-223-3p affects epithelial mesenchymal transformation of lung cancer cells and Wnt/β-catenin pathway

作　　者：姚菲 石玮[2] 李春姗[2] 蒋锐沅[2] 林丽珠 YAO Fei;SHI Wei;LI Chunshan;JIANG Ruiyuan;LIN Lizhu(First Clinical Medical College,Guangzhou University of Chinese Medical,Guangdong Guangzhou 510006,China;Department of Oncology,the First Affiliated Hospital of Guangxi University of Chinese Medical,Guangxi Nanning 530200,China.)

机构地区：[1]广州中医药大学第一临床医学院,广东广州510006 [2]广西中医药大学第一附属医院,广西南宁530200

出　　处：《现代肿瘤医学》2022年第15期2673-2679,共7页Journal of Modern Oncology

基　　金：国家自然科学基金(编号:82160926);广西自然科学青年基金项目(编号:2019JJB140156);广西自然科学面上基金项目(编号:2020JJA140364);广西中医药大学自然科学研究课题项目(编号:2018MS017);北京希思科临床肿瘤学研究基金项目(编号:Y-L2020-0019)。

摘　　要：目的:探讨微小RNA-223-3p(miR-223-3p)对转化生长因子Ⅲ型受体(TGFBR3)的靶向调控及对肺癌上皮间质转化(EMT)及Wnt/β-catenin通路相关指标的影响。方法:采用实时荧光定量PCR(qPCR)检测肺癌细胞(95D、LTEP-α-2、A549及NCI-H460)以及人肺上皮细胞BEAS-2B的miR-223-3p和TGFBR3水平,经双荧光素酶报告基因实验验证miR-223-3p和TGFBR3的靶向关系。将A549细胞分成3组:对照组、miR-223-3p NC组(转染miR-223-3p NC)、miR-223-3p mimic组(转染miR-223-3p mimic),MTT法、划痕实验及Transwell小室实验分别检测3组细胞的增殖及迁移能力。接着收集3组转染后的细胞,分别制备裸鼠移植瘤,处死裸鼠并剥离肿瘤组织,测量肿瘤体积及重量,免疫组化法对比各组镜下TGFBR3蛋白的表达情况,Western blotting实验检测EMT相关指标上皮钙黏蛋白(E-Cadherin)、神经型钙黏蛋白(N-Cadherin)、波形蛋白(Vimentin)及Wnt/β-catenin通路相关因子(Wnt1和β-catenin)的表达。结果:与BEAS-2B细胞对比,肺癌细胞miR-223-3p及TGFBR3的表达水平均降低,差异有统计学意义(P<0.05);miR-223-3p能靶向调控TGFBR3的表达。miR-223-3p mimic组A549细胞的增殖活力、划痕愈合率及穿膜细胞数均较miR-223-3p NC组明显降低(P<0.05);此外,miR-223-3p mimic组裸鼠瘤体体积及重量均明显低于miR-223-3p NC组。免疫组化结果显示,miR-223-3p mimic组TGFBR3阳性表达率上升,与miR-223-3p NC组相比,差异具有统计学意义(P<0.05);Western blotting结果提示,与miR-223-3p NC组相比,miR-223-3p mimic组肿瘤组织中TGFBR3、E-Cadherin表达水平升高,而N-Cadherin、Vimentin、Wnt1和β-catenin表达水平均下降(P<0.05)。对照组与miR-223-3p NC组的增殖活力、划痕愈合能力以及EMT和Wnt/β-catenin通路相关因子水平的差异无统计学意义(P>0.05)。结论:miR-223-3p在肺癌中异常低表达,miR-223-3p通过靶向调控TGFBR3来抑制肺癌A549细胞的增殖、迁移侵袭、EMT过程并阻断Wnt/β-catenin通路,在肺Objective:To investigate the targeted regulation of microRNA-223-3p(miR-223-3p)on transforming growth factor typeⅢreceptor(TGFBR3)and its effects on epithelial mesenchymal transformation(EMT)of lung cancer cells and Wnt/β-catenin pathway related indicators.Methods:Real-time quantitative PCR(qPCR)was used to detect the levels of miR-223-3p and TGFBR3 in lung cancer cells(95D,LTEP-α-2,A549 and NCI-H460)and human lung epithelial cells BEAS-2B.The dual-luciferase reporter gene experiment verified the targeting relationship between miR-223-3p and TGFBR3.A549 cells were divided into three groups:Control group,miR-223-3p NC group(transfected with miR-223-3p NC)and miR-223-3p mimic group(transfected with miR-223-3p mimic).MTT assay,scratch test and Transwell chamber test were used to detect the proliferation and migration of the three groups of cells.Then,the transfected cells in each group were collected to prepare transplanted tumors in nude mice.The nude mice were killed and the tumor tissue was stripped,and the tumor volume and weight were measured.The expression of TGFBR3 protein in each group was compared by immunohistochemistry.The EMT related indexes including E-Cadherin,N-Cadherin,Vimentin and Wnt/β-catenin pathway related factors(Wnt1 andβ-catenin)were detected by Western blotting.Results:Compared with BEAS-2B cells,the expression level of miR-223-3p and TGFBR3 in lung cancer cells decreased(P<0.05).miR-223-3p can target and regulate the expression of TGFBR3.The proliferation activity,scratch healing rate and the number of membrane penetrating cells of A549 cells in miR-223-3p mimic group were significantly lower than those in miR-223-3p NC group(P<0.05).In addition,the tumor volume and weight of nude mice in the miR-223-3p mimic group were significantly lower than those in the miR-223-3p NC group.The result of immunohistochemistry showed that the positive expression rate of TGFBR3 in the miR-223-3p mimic group increased,and the difference was statistically significant compared with the miR-223-3p NC grou

关 键 词：miR-223-3p TGFBR3 肺癌 侵袭迁移 

分 类 号：R734.2[医药卫生—肿瘤]