miR-199a通过Wnt3a/β-catenin信号通路调控非小细胞肺癌细胞的生物学活动  

miR-199a regulates the biological activity of non-small cell lung cancer cells via Wnt3a/β-catenin signaling pathway

作　　者：庄焕伟[1] 白树堂[1] 符洪犊[1] 曹贤君[1] 梁丽明 陈知群 羊家慧 欧阳华 程弦 张亮[2] ZHUANG Huanwei;BAI Shutang;FU Hongdu;CAO Xianjun;LIANG Liming;CHEN Zhiqun;YANG Jiahui;OUYANG Hua;CHENG Xian;ZHANG Liang(Department of Cardiothoracic Surgery,Haikou Hospital Affiliated to Central South University Xiangya School of Medicine,Haikou 570208,China)

机构地区：[1]中南大学湘雅医学院附属海口医院心胸外科,海口570208 [2]中山大学附属第七医院胸外科,广东深圳518107

出　　处：《实用医学杂志》2020年第18期2515-2521,共7页The Journal of Practical Medicine

基　　金：海南省国际科技合作专项(编号:KJHZ2014-06)。

摘　　要：目的探讨miR-199a调控非小细胞肺癌A549细胞的增殖、凋亡、转移和侵袭等生物学功能及作用机制。方法RT-PCR检测正常肺上皮细胞BEAS-2B和非小细胞肺癌细胞A549内miR-199a的表达;分别用miR-199a-mimics、miR-199a-NC和miR-199a-inhibitor转染至A549细胞内,转染48 h后CCK-8法检测细胞各组细胞增殖率,Annexin V-FITC/PI联合流式细胞仪检测细胞凋亡率,划痕实验和Transwell小室实验分别检测细胞迁移、侵袭能力,采用双荧光素酶报告实验验证Wnt3a与miR-199a的靶向关系,Western blot检测Wnt3a、β-catenin、C-myc、Survivin、E-cadherin、Vimentin蛋白表达。结果miR-199a在肺癌细胞中的表达低于正常肺上皮细胞(P<0.05);miR-199a-mimics组细胞增殖、迁移、侵袭活性显著低于其他两组(P<0.05),而凋亡率显著高于其他两组(P<0.05),miR-199a-inhibitor组细胞增殖、迁移、侵袭活性显著高于其他两组(P<0.05),而凋亡率显著低于其他两组(P<0.05);双荧光素酶报告实验证实miR-199a靶向抑制A549细胞中Wnt3a的表达;miR-199a-mimics组细胞Wnt3a、β-catenin、C-myc、Survivin、Vimentin蛋白表达显著低于其他两组(P<0.05),而E-cadherin表达在3组中最强(P<0.05),miR-199a-inhibitor组细胞Wnt3a、β-catenin、C-myc、Survivin、Vimentin蛋白表达显著高于其他两组(P<0.05),而E-cadherin表达在3组中最弱(P<0.05)。结论miR-199a能抑制非小细胞肺癌A549细胞的增殖、迁移和侵袭,同时刺激其凋亡,这种作用是通过靶向抑制Wnt3a/β-catenin信号通路的活性而实现的。Objective To investigate whether miR-199a can regulate the biological functions of non-small cell lung cancer A549,including proliferation,apoptosis,metastasis and invasion,and its possible mechanism.Methods RT-PCR was used to detect the expression of miR-199a in normal lung epithelial cells BEAS-2B and non-small cell lung cancer cells A549;miR-199a-mimics,miR-199a-NC,and miR-199a-inhibitor were transfected into A549 cells,respectively.After 48 hours of transfection,the cell proliferation rate of each cell group was detected by CCK-8 method.Apoptosis rate was detected by Annexin V-FITC/PI combined with flow cytometry.Scratch test and Transwell chamber test were used to detect cell migration and invasion,respectively.Double luciferase reporter assay was used to verify the targeting relationship between Wnt3a and miR-199a.Western Blot was used to detect the expression of Wnt3a,β-catenin,C-myc,Survivin,E-cadherin and Vimentin.Results The expression of miR-199a in lung cancer cells was lower than that in normal lung epithelial cells(P<0.05).The proliferation,migra-tion and invasion activities of miR-199a-mimics group were significantly lower than those of the other two groups(P<0.05),while the apoptotic rate was significantly higher than that of the other two groups(P<0.05).The cell proliferation,migration and invasion activities of miR-199a-inhibitor group were significantly higher than the other two groups(P<0.05),and the apoptotic rate was significantly lower than the other two groups(P<0.05);The double luciferase gene reporting experiment confirmed that Wnt3a was the downstream target gene of miR-199a.The expressions of Wnt3a,β-catenin,C-myc,Survivin and Vimentin in miR-199a-mimics group were significantly lower than those in other groups(P<0.05),while E-cadherin expression was the strongest among the three groups(P<0.05).The expression of Wnt3a,β-catenin,C-myc,Survivin and Vimentin in the miR-199a-inhibitor group was significantly higher than that in the other two groups(P<0.05),while the expression of E-cadh

关 键 词：miR-199a 非小细胞肺癌A549 增殖 迁移 侵袭 凋亡 Wnt3a/β-catenin信号通路 

分 类 号：R363.14[医药卫生—病理学]