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目的:探讨食管鳞状细胞癌组织中微RNA-1(microRNA-1,miRNA-1)的表达及其可能的作用机制。方法:分别应用实时荧光定量PCR法和免疫组织化学法检测55例食管鳞状细胞癌组织及相应的癌旁组织中miRNA-1、LASP1(UM and SH3 domain protein 1)mRNA和LASPl蛋白的表达水平。应用生物信息学软件预测miRNA-1的潜在靶基因。将含有LASPl基因3’端非翻译区(3’-untranslated region,3’-UTR)的重组双荧光素酶报告载体psiCHECK-2-LASPl与miRNA-1模拟物(miRNA-1-mimic)共转染至食管癌Eca109细胞,通过双荧光素酶报告系统验证miRNA-1的靶基因。将miRNA-1 mimic或miRNA-1抑制物(miRNA-1-inhibitor)分别转染至Eca109细胞后,应用实时荧光定量PCR法检测细胞中miRNA-1的表达水平,应用蛋白质印迹法检测细胞中LASP1蛋白的表达水平。结果:食管鳞状细胞癌组织中miRNA-1的表达水平低于相应的癌旁组织(P<0.01),而LASP1 mRNA和蛋白的表达水平高于相应的癌旁组织(P值均<0.05)。并且,miRNA-1和LASP1的表达与食管癌患者的淋巴结转移、TNM分期及组织学分级等有关(P值均<0.05),食管鳞状细胞癌组织中miRNA-1与LASP1蛋白表达水平呈负相关(r=-0.45,P<0.05)。生物信息学软件预测LASP1是miRNA-1的潜在靶基因,双荧光素酶报告系统验证了miRNA-1可与LASP1基因的3’-UTR特异性结合。转染miRNA-1 mimic后,Eca109细胞中miRNA-1的表达水平上调(P<0.01),LASPl蛋白的表达水平下调(P<0.05);而转染miRNA-1 inhibitor后,Eca109细胞中miRNA-1的表达水平下调(P<0.05),LASP1蛋白的表达水平则上调(P<0.05)。结论:食管鳞状细胞癌组织中miRNA-1低表达,其机制可能与调控靶基因LASP1的表达有关。
Objective: To investigate the expression of microRNA-1 (miRNA-1) in esophageal squamous cell carcinoma and its possible mechanism. Methods: The expressions of miRNA-1, LASP1 mRNA and LASP1 protein in 55 cases of esophageal squamous cell carcinoma and its corresponding paracancerous tissues were detected by real-time fluorescence quantitative PCR and immunohistochemistry respectively . Bioinformatics software was used to predict the potential target genes of miRNA-1. The recombinant dual luciferase reporter vector psiCHECK-2-LASP1 containing the 3’-untranslated region (3’-UTR) of LASP1 gene was co-transfected with the miRNA-1 mimic Dyeing was performed on esophageal cancer Eca109 cells and the target gene of miRNA-1 was verified by dual luciferase reporter system. After transfection of miRNA-1 mimic or miRNA-1 inhibitor into Eca109 cells, the expression of miRNA-1 in cells was detected by real-time fluorescence quantitative PCR. The expression of LASP1 Protein expression levels. Results: The expression of miRNA-1 in esophageal squamous cell carcinoma was lower than that in corresponding adjacent tissues (P <0.01), while the expression of LASP1 mRNA and protein was higher than that in paracancerous tissues (all P <0.05) . Moreover, the expression of miRNA-1 and LASP1 was correlated with lymph node metastasis, TNM stage and histological grade in esophageal cancer patients (all P <0.05), while the expression of miRNA-1 and LASP1 protein in esophageal squamous cell carcinoma was negative (R = -0.45, P <0.05). Bioinformatics software predicts that LASP1 is a potential target gene for miRNA-1. Dual luciferase reporter system verified the specific binding of miRNA-1 to 3’-UTR of LASP1 gene. After transfection with miRNA-1 mimic, the expression of miRNA-1 in Eca109 cells was up-regulated (P <0.01) and the expression of LASP1 protein was down-regulated (P <0.05) 1 (P <0.05), while the expression of LASP1 protein was up-regulated (P <0.05). Conclusion: The low expression of miRNA-1 in esophageal squamous cell carcinoma may be related to the regulation of LASP1 gene expression.