miRNA－25对人食管鳞状细胞癌细胞株TE1增殖的影响

摘要

目的：探讨过表达／沉默微小RNA－25（ microRNA－25，miRNA－25）对人食管鳞状细胞癌细胞株TE1增殖能力的影响及其作用机制。方法：RT－PCR检测各临床样品组织中miRNA－25的表达水平。建立miRNA－25过表达／沉默的TE1细胞株，采用CCK－8法、BrdU实验和流式细胞术检测TE1细胞增殖能力的变化及细胞周期状态；Western blot法和RT－PCR法检测细胞周期调控因子细胞周期蛋白E1（ cyclin E1）和细胞周期蛋白依赖性激酶2（CDK2）的蛋白与mRNA表达水平。结果：miRNA－25在食管黏膜组织中具有特异性表达并在TE1细胞中呈高表达。 CCK－8法和BrdU实验结果显示过表达miRNA－25的TE1细胞的增殖能力显著增加（ P＜0．05），而沉默miR－NA－25抑制TE1细胞的增殖；流式细胞术检测结果显示过表达miRNA－25可明显促进TE1细胞从G0／G1期向S期转换，沉默miRNA－25则抑制其转换。同时Western blot和RT－PCR实验结果显示过表达miRNA－25后，cyclin E1和CDK2的蛋白和mRNA表达水平均显著增加（P＜0．05），沉默miRNA－25后则显著减少（P＜0．05）。结论： miRNA－25能够促进人食管鳞状细胞癌细胞株TE1的增殖，其作用机制可能与促进细胞周期转换及上调cyclin E1和CDK2的表达水平有关，提示miRNA－25可作为治疗食管鳞状细胞癌的一个潜在靶点。%AIM:To investigate the effects and mechanisms of microRNA-25 (miRNA-25) on the proliferation of human esophageal squamous-cell carcinoma cell line TE1.METHODS: The abundance of miRNA-25 in different tis-sues was measured by RT-PCR.After silencing or over-expression of miRNA-25 with mimics or inhibitor in TE1 cells, the cell proliferation, cell cycle distribution and the expression of cyclin E1 and cyclin-dependent kinase 2 (CDK2) at mRNA and protein levels were measured by CCK-8 assay, BrdU detection, flow cytometry, RT-PCR and Western blot, respective-ly.RESULTS:miRNA-25 was prominent in esophageal mucosal tissue and highly expressed in TE1 cells (P<0.05).O-ver-expression of miRNA-25 increased TE1 cell proliferation, promoted the cell cycle progression and enhanced the en-trance of the cells into S phase (P<0.05).Inverse results were obtained after down-regulation of miRNA-25 (P<0.05). Furthermore, the expression of cyclin E1 and CDK2 at mRNA and protein levels was significantly increased after over-ex-pression of miRNA-25, but decreased after down-regulation of miRNA-25 (P<0.05).CONCLUSION: miRNA-25 en-hances cell cycle transition by increasing the expression of cyclin E1 and CDK2, thus accelerating TE1 cell proliferation. This study provides a novel mechanism by which miRNA-25 increases the proliferation of human esophageal squamous-cell carcinoma cell line TE1, suggesting that down-regulation of miRNA-25 may be a potential new therapeutic strategy for trea-ting esophageal squamous-cell carcinoma.