摘要
目的转移和复发是导致非小细胞肺癌(non-small cell lung cancer,NSCLC)治疗失败的主要因素'明确影响转移和复发的相关机制对提高治疗效果及改善患者预后具有重要意义。本研究旨在检测转录因子激活增强子结合蛋白4(transcription factor activating enhancer-binding protein 4 ,TFAP4)在 NSCLC 组织中表达,并探讨下调该基因对 A549 增殖和侵袭的影响。方法选取2015-03-06-2018-03-04海南省人民医院行手术治疗的NSCLC标本及对应的癌旁组织(距肿瘤边缘≥5cm)78例,采用免疫组化法检测NSCLC和癌旁组织中TFAP4蛋白表达。细胞实验包括培养NSCLC细胞株,随机分为siRNA-TFAP4组(转染TFAP4基因干扰序列)、对照序列组(转染阴性对照序列)和对照组(不作任何处理),通过实时荧光定量聚合酶链反应法(quantitative real-time PCR, qRT-PCR)及蛋白质印迹法检测沉默的效率。四甲基偶氮唑盐(methyl thiazoly tetrazolim, MTT)比色法和Transwell小室分别检测细胞增殖、迁移和侵袭能力,蛋白质印迹法检测细胞中Snail、E-cadherin和Vimentin蛋白表达。结果 NSCLC组织中TFAP4蛋白阳性表达率为79.49%,高于癌旁组织的 30.77%,χ^2=37.419,P<0.001;TNM 分期Ⅲ~Ⅳ期(χ^2=6.395,P<0.05)、低分化(χ^2=4.577,P<0.05)和发生淋巴结转移的NSCLC组织中TFAP4(χ^2=7.386 ,P<0.05)蛋白阳性表达率升高;siRNA-TFAP4组细胞中TFAP4 mRNA相对表达量为0.31±0.07,低于对照序列组(0.98±0.13)和对照组(1.00±0.03),F=120.840, P<0.001;siRNA-TFAP4组迁移细胞数为 104.04±3.70,低于对照序列组(133.94±8.20)和对照组(130.98±7.26),F=6.555,P<0.001;siRNA-TFAP4组侵袭细胞数为96.12±4.36,低于对照序列组(120.31±4.67)和对照组(122.63±5.23),F=56.880,P<0.001;与对照序列组和对照组比较,24、48、72和96h siRNA-TFAP4组细胞的增殖能力降低,F值分别为9.924、16.429、18.156和16.970,均P<0.05;siRNA-TFAP4 组细胞中 TFAP4、 Snail和Vimentin 蛋白相对表达量低于对照序列组和对照组,F值分别为169.815、34.473和55.089,均P<0.05 ,而E-cadherin蛋白相对表达量高于对照序列组和对照组,F=33.780, P<0.05。结论 TFAP4蛋白在NSCLC组织中呈高表达,下调A549细胞中TFAP4基因表达可抑制细胞增殖、迁移和侵袭能力,其机制可能与抑制上皮-间质转化过程有关。
OBJECTIVE Metastasis and recurrence are the main factors of treatment failure for the patients with non-small cell lung cancer( NSCLC).This study aimed to investigate the expression of transcription factor activating enhancer- binding protein 4(TFAP4) in non-small cell lung cancer tissues,and study the effects of TFAP4 on cell proliferation and invasion of non-small cell lung cancer A549 cells.METHODS A total of 78 cases of patients with NSCLC underwent surgery in Hainan Provincial People s Hospital were selected from March 2015 to March 2018.The expressions of TFAP4 proteins in non-small cell lung cancer and adjacent tissues were detected by immunohistochemistry.Cell experiments included culture of NSCLC cell lines, which were randomly divided into siRNA-TFAP4 group(transfected with TFAP4 gene interference sequence), control sequence group (transfection negative control sequence) and control group ( without any treatment).Real-time qRT-PCR and western blotting were performed to assess TFAP4 knockdown efficiency.Methyl thiazoly tetrazolim( MTT) and transwell assays were used to detect the effect of down-regulation of TFAP4 on the proliferation, migration and invasion of non-small cell lung cancer A549 cells.Western blot was used to detect the expressions of Snail?E-cadherin and Vimentin proteins.RESULTS The positive expression rate of TFAP4 protein was 79.49 % in NSCLC tissues, which was lower than that of the adjacent tissues (30.77%),χ^2=37.419,P<0.001.The positive expression rate of TFAP4 protein in NSCLC tissues with stage Ⅲ一Ⅳ stage,poor differentiation and lymph node metastasis was increased,χ^2 values were 6.395,4.577 and 7.386,P<0.05.The relative expression level of TFAP4 mRNA in siRNA- TFAP4 group was 0.31±0.07, which was lower than that of the control sequence group and the control group .which were 0.98±0.13,1.00±0.03,respectively,F= 120.840,P<0.001.The number of migrated cells was 104.04±3.70 in the siRNA-TFAP4 group, which was lower than that of the control sequence group and the control group, which were 133.94±8.20,130.98±7.26 ,respectively,F=36.555 ,P<0.001.The number of invasive cells was 96.12±4.36 in the siRNA-TFAP4 group, which was lower than in the control sequence group and the control group, which were 120.31± 4.67,122.63±5.23, respectively.F=56.880, P<0.001.Compared with the control sequence group and the control group,the proliferation ability of the siRNA-TFAP4 group at 24,48,72 and 96 h was significantly decreased, F values were 9.924,16.429,18.156 and 16.970,respectively,P<0.05.The relative expression levels of TFAP4,Snail and Vimentin proteins in the siRNA-TFAP4 group were lower than those in the control sequence group and the control group, F values were 169.815,34.473 and 55.089,P<0.05 , while the relative expression of E-cadherin protein was higher than that in the control sequence group and the control group,F=33.780,P<0.05.CONCLUSIONS TFAP4 protein is high expression in non-small cell lung cancer tissues.Down-regulation of TFAP4 gene expression in A549 cells can inhibit cell proliferation, migration and invasion.The mechanism may be related to the inhibition of the epithelial-mesenchymal transition process.
作者
黄琰菁
王琳
李赛
盛莉
王海霞
杨生辉
HUANG Yan-jing;WANG Lin;LI Sai;SHENG Li;WANG Hai-xia;YANG Sheng-hui(Department of Medical Oncology,Hainan General People's Hospital,Haikou 570311,P.R.China)
出处
《中华肿瘤防治杂志》
CAS
北大核心
2019年第11期778-783,共6页
Chinese Journal of Cancer Prevention and Treatment
基金
海南省自然科学基金(814315)
关键词
非小细胞肺癌
转录因子激活增强子结合蛋白4
细胞增殖
细胞侵袭
non-small cell lung cancer
transcription factor activating enhancer-binding protein 4
cell proliferation
cell invasion
