微小RNA-381在前列腺癌中的表达及作用机制

Expression and role of microRNA - 381 in prostate cancer

目的探讨微小RNA（miRNA，miR）-381在前列腺癌（PCa）中的表达及其作用机制。方法应用实时定量聚合酶链反应（Real—timePCR）检测miR．381在44例PCa组织中的表达，并分析其与临床病理特征之间的关系。利用脂质体Lipofectamine2000将miR-381模拟物（mimic）分别转染人人PCa细胞株Pc-3和DU145，应用细胞计数试剂盒（CCK-8）法检测细胞增殖能力的变化，流式细胞仪检测细胞周期的变化，双荧光素酶报告基因系统验证miR481的靶基因双孔钾离子通道TREK-1，Westernblot检测TREK-1、周期蛋白依赖性激酶4（CDK4）和周期蛋白依赖性蛋白激酶抑制剂p21Cipl表达的变化。结果miR-381在PCa组织中呈低表达，在Gleason评分≥8分的患者中显著低于≤7分的患者（-13．01±3．33比-8．99±3．77，P〈0．01），在病理分期pT3期的患者中显著低于pT2期（-13．87±3．51比一10．56±4．11，P〈0．05）。Real-timePCR结果显示，miR-381-mimic转染组细胞中miR-381水平明显高于对照组；miR-381过表达显著抑制了PCa细胞增殖，以72h和96h较为明显（P〈0．05），且诱导了G，／S期细胞周期阻滞；双荧光素酶报告基因系统检测证实TREK．1是miR-381的靶基因；miR-381过表达抑制了TREK-1、CDK4的表达，上调了p21Cipl的表达。结论MiR-381参与了PCa的发生发展，其机制之一可能是通过抑制其靶基因TREK-1的表达来实现。

Objective To investigate the expression and role of microRNA （miRNA, miR） -381 in prostate cancer （PCa）. Methods The expression levels of miR -381 were detected in 44 PCa tissues using real- time quantitative polymerase chain reaction （Real -time PCR） , and their clinieopathological significance was analyzed. MiR - 381 mimic was transfected into PC - 3 cells and DU145 cells by lipo- fectamine 2000 reagent. Cell counting kit - 8 （ CCK - 8 ） assay and flow cytometry were performed to eval- uate cell proliferation and cell cycle. Lueiferase assay was carried out to validate the target gene of miR -381. The protein expression levels of TREK - 1, eyclin dependent kinase （CDK） 4 and p21 clpl were detected using Western blotting. Results Low levels of miR -381 were detected in PCa tissues. Com- pared with those with Gleason score ∽〈7, miR -381 levels were lower in patients with Gleason score 〉18 （ - 13.01 _＋ 3.33 vs. - 8.99 ＋_ 3.77,P 〈 0.01 ）. In addition, miR - 381 levels were significantly lower in patient with pT3 disease than those with pT2 disease （ - 13.87 _＋ 3.51 vs. - 10. 56 _＋ 4. 11, P 〈 0. 05 ）. The expression of miR - 381 was remarkably elevated after transfeetion of miR - 381 - mimic compared with the control. Overexpression of miR- 381 significantly inhibited cell proliferation and hindered the Gl to S phase transition. TREK - 1 was demonstrated to be a target gene of miR - 381 by lueiferase assay. Reduced protein levels of TREK- 1 and CDK4, as well as elevated p21cipl level, were observed after miR -381 overexpression. Conclusion Our results suggest that miR -381 participate in PCa carcinogen- esis via targeting TREK - 1.