尿液多种microRNA检测方法的建立及其在膀胱癌诊断中的应用研究

Establishment of urinary micro-RNA detection method and its application in diagnosis of bladder cancer

目的:建立尿液中8种微小RNA(micro RNA,miRNA)的检测方法 ,明确其分布规律及其在膀胱癌中的变化。方法:选取15名健康人的晨尿及随机尿液,以及15例确诊膀胱癌患者的随机尿液,进行总RNA提取,然后反转录得到cDNA,用荧光实时PCR法扩增8种膀胱癌相关miRNA,分别为miR-126、miR-200a、miR-200b、miR-200c、miR-182、miR-183、miR-429和miR-141,探讨晨尿与随机尿、尿液浓缩前与浓缩后对尿液miRNA浓度的影响。比较正常对照组与膀胱癌患者尿液中上述8种miRNA的表达差异,并采用受试者工作特征曲线分析尿液miRNA检测在膀胱癌诊断中的价值。结果:浓缩尿液组的6种miRNA(miR-126、miR-200a、miR-200b、miR-200c、miR-429和miR-141)浓度显著高于未浓缩组(P<0.05),而浓缩晨尿组的8种miRNA浓度与浓缩随机尿组间比较无统计学差异(P>0.05)。与正常对照相比较,miR-126、miR-182、miR-183及miR-141的浓度在膀胱癌组中显著升高(P<0.05),而miR-200a浓度在膀胱癌组中显著降低(P<0.05)。受试者工作特征曲线分析显示,单独检测尿液miRNA对于膀胱癌的诊断缺乏有效性,而对5种尿液miRNA(miR-126、miR-200a、miR-182、miR-183和miR-141)进行联合检测则可显著提高膀胱癌诊断的灵敏度和特异度。结论:尿液中稳定存在多种miRNA,尿液中多项miRNA的联合检测可能有望成为诊断膀胱癌的有效指标。

Objective： To establish a method for detection of 8 urinary micro-RNA and to detect their distribution in urine and evaluating its significance in detection of bladder cancer. Methods： Early morning and random urine samples from 15 healthy controls and random urine samples from 15 patients with bladder cancer were collected. The total RNA were extracted and then were reversely transcripted to cDNA, and 8 micro-RNAs （miR-126, miR-200a, miR-200b, miR- 200c, miR-182, miR-183, miR-429 and miR-141） were amplified with real-time polymerase chain reaction （PCR）. Diffe- rences in miRNA levels were compared between early morning and random urine samples as well as between non-concen- trated and concentrated samples. Different extraction and purification methods for miRNAs were compared and the diffe- rences of miRNA concentrations between controls and bladder cancer patients were analyzed. The diagnostic efficacy of multiple urinary miRNA combinations for bladder cancer was analyzed using ROC curve. Results： The relative concentra- tions of 6 miRNAs （miR-126, miR-200a, miR-200b, miR-200c, miR-429 and miR-141） were significantly higher in the concentrated urine than those in non-concentrated urine. There was no significant difference in relative miRNA concentra- tion between early morning and randomized urine collection. Compared with normal controls, the relative concentrations of 4 miRNAs （miR-126, miR-182, miR-183 and miR-141） were significantly increased in bladder cancer group while miR- NA-200a was significantly decreased. ROC analysis showed that detection of single miRNA had limited diagnostic efficacy for bladder cancer, while combined analysis of 5 miRNA had better diagnostic value for bladder cancer. Conclusions： Multiple micro-RNAs are stably distributed in urine, and combined detection of multiple miRNAs might be used as a dia- gnostic marker for bladder cancer.