非小细胞肺癌组织WIF1过表达对A549细胞迁移和侵袭影响机制

目的 WNT抑制因子1(WNT inhibitory factor 1,WIF1)与肿瘤的发展与转移密切相关,在多种肿瘤中均发现WIF1表达降低或缺失,但其在非小细胞肺癌中的表达及功能尚不清楚。本研究探讨WIF1在非小细胞肺癌组织的表达及过表达WIF1对A549肺腺癌细胞迁移和侵袭的作用。方法选取安徽医科大学第一附属医院干部呼吸科2010-08-01-2014-08-01经病理确诊的50例肺癌及癌旁组织,Real-time PCR、蛋白质印迹检测2种组织中WIF1 mRNA和蛋白的表达水平,同时用癌症基因组图谱(The Cancer Atlas,TCGA)公共数据库的数据进行辅助验证。分析WIF1mRNA表达与临床病理、预后的关系。WIF1过表达载体(WIF1组)或空载转染A549(Vector组),划痕和Transwell实验检测WIF1过表达对细胞迁移和侵袭的影响;蛋白质印迹检测MMP-2、MMP-9、β-catenin和Myc蛋白表达水平。结果 WIF1 mRNA和蛋白的表达在癌组织中表达值分别为0.25±0.06、0.44±0.08,低于癌旁组织的0.56±0.12、0.96±0.12,差异有统计学意义,t值分别为15.509和1.615,均P<0.05。同时TCGA共数据库的数据显示,WIF1在肺腺癌和肺鳞癌中表达丰度分别为1.94(0.39~6.12)和0.69(0.12~2.99),均低于正常肺组织的137.94(88.98~189.25)和99.94(68.51~138.97),均P<0.05。WIF1表达在年龄、性别、组织病理类型及切取淋巴结数比较差异无统计学意义,均P>0.05;但在淋巴结转移(χ^2=19.100,P<0.001)、TNM分期为Ⅲ期(χ^2=8.333,P=0.004)及N分期为N1+N2组(χ^2=8.681,P=0.007),差异有统计学意义;且WIF1高表达组生存期为为38.00(19.00~51.50)个月,高于低表达组的24.00(17.00~35.50)个月,差异有统计学意义,χ^2=4.413,P=0.038。过表达WIF1抑制A549细胞的迁移和侵袭能力,t值分别为6.607和15.650,均P<0.05;与Vector组(0.90±0.05、0.90±0.03、0.60±0.05、0.31±0.04)比较,过表达WIF1能下调肿瘤转移相关标志物MMP-2(0.30±0.05)和MMP-9(0.28±0.02),差异有统计学意义,t值分别为15.459、27.391,均P<0.05;并能抑制Wnt/β-catenin信号通路中关键蛋白β-catenin(0.16±0.08)和Myc(0.12±0.03)的表达,差异有统计学意义,t值分别为7.962和6.124,P值分别为0.001和0.004。结论 WIF1在非小细胞肺癌组织中表达降低,过表达WIF1可通过抑制Wnt/β-catenin信号通路抑制A459肺腺癌细胞迁移和侵袭。

Over expression of WIF1 in non-small cell lung cancer and its influence on the migration and invasion of A549 lung adenocarcinoma cells

OBJECTIVE Wnt inhibitory factor 1(WIF1)is closely related to the development and metastasis of tumor.It was found that the expression of WIF1 was decreased or absent in many tumors.However,its expression and function in NSCLC are not clear.In this study,we investigated the role of overexpression of WIF1 in the regulation of migration and invasion of NSCLC cells.METHODS From August 1,2010 to August 1,2014,50 cases lung cancer tissues and paracancerous tissues were selected from Department of Cadres Respiratory,First Affiliated Hospital of Anhui Medical University.The WIF1 mRNA and protein expression in two groups of tissues were detected by Real-time PCR and Western blot respectively,and the data of The Cancer Atlas(TCGA)public database was used for auxiliary verification.The relationship between WIF1 mRNA expression and clinicopathology and prognosis was also analyzed.A549 cells were transfected with WIF1 overexpression vector(WIF1 group)or negative control vector(empty vector)respectively,and the effects of WIF1 on migration and invasion of lung cancer cells were performed with wound-healing assay and transwell assay.Protein expression of MMP-2,MMP-9,β-Catenin and myc was detected by Western blot.RESULTS The expression values of WIF1 mRNA and protein in cancer tissues(0.25±0.06,0.44±0.08)were lower than those in paracancer tissues(0.56±0.12,0.96±0.12),the difference was statistically significant(t values were 15.509 and 1.615,both P<0.05).At the same time,TCGA data showed that the expression abundance of WIF1 in lung adenocarcinoma and squamous cell carcinoma[1.94(0.39-6.12)],[0.69(0.12-2.99)]was lower than that in normal lung tissue [137.94(88.98-189.25),99.94(68.51-138.97)],P<0.05.There was no significant difference in the expression of WIF1 in age,sex,histopathological type and number of lymph nodes removed(P>0.05),but the difference was statistically significant in lymph node metastasis(χ^2=19.100,P<0.001),TNM stageⅢ (χ^2=8.333,P=0.004)and N stage N1+N2(χ^2=8.681,P=0.007),and the median survival time of high expression group 38.00(19.00-51.50)months was significantly higher than that of low expression group 24.00(17.00-35.50)months,the difference was statistically significant(χ^2=4.413,P=0.038).Overexpression of WIF1 inhibited the migration and invasion of A549 cells,t values were 6.607 and 15.650,P<0.05.Compared with vector group(0.90±0.05,0.90±0.03,0.60±0.05,0.31±0.04),overexpression of WIF1 can down regulate the expression of MMP-2(0.30±0.05)and MMP-9(0.28±0.02),the difference was statistically significant(t=15.459,P<0.05;t=27.391,P<0.05),and can inhibit the expression of key proteinsβ-Catenin(0.16±0.08)and Myc(0.12±0.03)in Wnt/β-catenin signaling pathway,the difference was statistically significant(t=7.962,P=0.001;t=6.124,P=0.004).CONCLUSIONS The expression of WIF1 decreased in NSCLC.Overexpression of WIF1 can inhibit migration and invasion of A459 lung adenocarcinoma cells by regulating Wnt/β-catenin signaling pathway.