目的研究膀胱尿路上皮癌miRNAs的差异表达。方法取手术切除新鲜膀胱尿路上皮癌及正常膀胱黏膜组织各3例保存于液氮中;Trizol试剂提取总RNA;使用Nanodrop及变性琼脂糖凝胶电泳进行RNA质量检测;用标记酶Hy3TM荧光基团标记RNA的探针和miRCURYTM芯片杂交;使用GenePix4000B芯片扫描仪及GenePix pro V6.0完成图像采集和数据分析;荧光定量RT-PCR方法验证。结果膀胱尿路上皮癌与正常膀胱黏膜上皮组织共有192个差异表达基因,其中71个上调,124个下调;差异表达miRNAs经随机RT-PCR验证而证实。结论膀胱尿路上皮癌与正常膀胱黏膜组织中存在差异miRNAs基因,这种miRNAs差异可能导致膀胱尿路上皮癌的发生及发展。
Objective:The aim of the study was to investigate the differential expression of microRNAs (miRNAs) in bladder transitional cell carcinoma (BTC). Methods:Fresh tissues were obtained from patients with BTC (9 cases; 3 cases with grade I, 3 cases with grade II, 3 cases with grade III) and those with normal bladder mucosa (3 cases) and stored in liquid nitrogen. Total RNA was extracted using TRizol reagent and RNA was quantified and quality control was performed. miRNA probes were labeled with Hy3 fluorescence, then hybridized with a miRCURYTM array labeling kit. miRNA arrays were scanned and analyzed and the scanned result was validated using reverse transcription-polymerase chain reaction (RT-PCR). Results:In four groups of differentially expressed genes obtained from grade I, grade II, grade III, and grade I + grade II + grade III BTC tissues compared with normal bladder mucosa, hsa-miR-29b-1* was upregulated, and hsa-miR-923 and hsa-miR-300 were downregulated. The hsa-miR-29b-1*, hsa-miR-300, and hsa-miR-923 findings were confirmed by real-time RT-PCR. Conclusion:Genes that were differentially expressed between BTC and normal bladder mucosa may be involved in the pathogenesis and development of BTC, and may be useful for further studies of BTC-related genes.
