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目的研究食管鳞癌组织中Raf激酶抑制蛋白(RKIP)、miRNA224表达情况,miRNA224对RKIP的靶向作用,以及miRNA224基因上游启动子区甲基化水平。方法在食管鳞癌组织及癌旁对照组织临床标本中,用免疫组化法检测RKIP蛋白的表达;用荧光定量PCR法检测miRNA224的表达,荧光素酶报告基因检测miRNA224对RKIP的靶向作用;亚硫酸氰盐测序PCR(BSP)法检测食管鳞癌及癌旁对照组织miRNA224基因上游启动子区甲基化水平。结果在食管鳞癌组织中,RKIP低表达,miRNA224高表达;在癌旁对照组织中,RKIP高表达,miRNA224低表达。荧光素酶报告基因验证miRNA224可靶向抑制RKIP3′UTR表达。BSP法显示食管鳞癌组织miRNA224上游启动子区呈低甲基化状态,癌旁对照组织呈高甲基化状态。结论食管鳞癌组织RKIP表达下降、miRNA224基因启动子甲基化状态降低,miRNA224表达上升,RKIP是miRNA224下游作用的靶点,可能同食管鳞癌发生密切相关。
Objective To investigate the expression of Raf kinase inhibitor (RKIP) and miRNA224 in esophageal squamous cell carcinoma, the targeting effect of miRNA224 on RKIP, and the promoter methylation of miRNA224 upstream. Methods The expression of RKIP protein was detected by immunohistochemistry in esophageal squamous cell carcinoma tissues and adjacent non-cancerous adjacent tissues. The expression of miRNA224 was detected by fluorescence quantitative PCR, and the luciferase reporter gene was used to detect the targeting effect of miRNA224 on RKIP. Cytosulfite Sequencing PCR (BSP) was used to detect the promoter methylation of miRNA224 gene in esophageal squamous cell carcinoma and adjacent non-cancerous tissues. Results In esophageal squamous cell carcinoma, RKIP was lowly expressed and miRNA224 was highly expressed. In the adjacent non-cancerous tissues, RKIP was highly expressed and miRNA224 was lowly expressed. Luciferase Reporter Validation of miRNA224 Targets Inhibition of RKIP3’UTR Expression. BSP method showed that the upstream promoter region of miRNA224 in esophageal squamous cell carcinoma was hypomethylated and the paracancerous tissues were hypermethylated. Conclusion The expression of RKIP in esophageal squamous cell carcinoma is decreased, the methylation status of miRNA224 promoter is decreased and the expression of miRNA224 is up-regulated. RKIP is the downstream target of miRNA224 and may be closely related to the occurrence of esophageal squamous cell carcinoma.