喉鳞状细胞癌SMAD3和hsa-miR-193b表达与靶向关系验证

Expression and the verification of targeted relationship of SMAD3 and hsa-miR-193b in laryngeal squamous cell carcinoma

目的 Hsa-miR-193b与LSCC的发生发展密切相关。本研究探讨LSCC中miR-193b与SMAD3的表达及二者的靶向调控关系。方法 qRT-PCR检测山西医科大学第一医院2013-01-03-2014-06-30来源的20例喉鳞状细胞癌(laryngal squamous cell carcinoma,LSCC)组织及癌旁正常黏膜组织中miR-193b的表达,qRT-PCR、蛋白质印迹法检测SMAD3的表达。构建野生型和突变型SMAD3 3′UTR载体,与miR-193bmimics或NC共转染HEK-293T细胞,双荧光素酶报告系统检测各组荧光素酶活性。结果 qRT-PCR检测结果,癌组织miR-193b总表达为11.50±6.289,癌旁正常组织为1.00±0.000,与癌旁正常组织相比,miR-193b在LSCC组织中的表达上调且差异有统计学意义,t=7.466,P<0.001;癌组织SMAD3表达为0.402±0.201,癌旁正常组织表达为1.00±0.000,SMAD3在LSCC组织中的表达降低且差异有统计学意义,t=13.31,P<0.001。蛋白质印迹法检测结果,癌组织SMAD3表达为1.57±0.127,癌旁正常组织表达为3.68±0.198,SMAD3在癌组织的表达降低且差异有统计学意义,t=48.40,P<0.001。经测序,成功构建野生型与突变型SMAD3 3′UTR载体,共转染后各组样品经检测显示,miR-193b+mutSMAD3组荧光素酶活性变化倍数为1.042±0.100 8,NC+mutSMAD3组为1.102±0.070 6,差异无统计学意义(t=0.854,P=0.442),miR-193b+SMAD3组荧光素酶活性变化倍数为0.449±0.047 2,NC+SMAD3组为1.124±0.014 1,miR-193b+SMAD3组比NC+SMAD3组荧光素酶活性明显降低(t=31.89,P=0.001),说明miR-193b可以与SMAD3 3′UTR结合。结论 LSCC组织中miR-193b表达上调,SMAD3表达明显降低。SMAD3是miR-193b的直接调控靶基因。

OBJECTIVE The SMAD3 and hsa-miR-193 bwere closely associated with tumorgenesis and development in patients with laryngeal squamous cell carcinoma.The aim of this study was to investigate the expression and the verification of targeted relationship of SMAD3 and hsa-miR-193 bin laryngeal squamous cell carcinoma.METHODS From January 3 rd 2013 to June 30 th 2014,a total of 20 pairs of laryngeal cancer and adjacent normal tissues were collected and the expression of miR-193 bwas detected by qRT-PCR.The expression level of SMAD3 was detected by qRT-PCR and Western blot.The 3′UTR of the wild type SMAD3 and mutant SMAD3 sequence were cloned into luciferase reporter vector,which was co-transfected into HEK293 Tcells with miR-193 bmimics or NC respectively.The luciferase activity was detected by dual luciferase reporter gene system.RESULTS In laryngeal carcinoma tissue qRT-PCR showed that the expression of miR-193 bwas 11.50±6.289,while the expression of the tissue adjacent to carcinoma was 1.00±0.000.Compared with normal tissues,qRT-PCR showed miR-193 bwas upregulated significantly（t=7.466,P〈0.001）in laryngeal carcinoma tissue.The expression of SMAD3 in laryngeal carcinoma tissue was 0.402±0.201,while the SMAD3 expression of the tissue adjacent to carcinoma was 1.00±0.000.Compared with normal tissues,qRT-PCR showed SMAD3 was reduced significantly（t=13.31,P〈0.001）in laryngeal carcinoma tissue.Western blot indicated that the expression of SMAD3 in laryngeal carcinoma tissue was 1.57±0.127,while the expression of the tissue adjacent to carcinoma was 3.68±0.198.Western blot also indicated SMAD3 was also reduced significantly（t=48.40,P〈0.001）.Dual-luciferase reporter assay system revealed luciferase activity of miR-193 b＋SMAD3 group（0.449±0.047 2）was lower than that of NC＋SMAD3 group（1.124±0.014 1）significantly（t=31.89,P=0.001）.CONCLUSIONS The expression of miR-193 bin LSCC is up-regulated,and the expression of SMAD3 is significantly reduced.SMAD3 is the direct target gene of miR-193 b.