The MicroRNA Expression Signature of Bladder Cancer by Deep Sequencing: The Functional Significance of the <i>miR-195/497</i> Cluster

Current genome-wide microRNA (miRNA) expression signature analysis using deep sequencing technologies can drive the discovery of novel cancer pathways regulated by oncogenic and/or tumor suppressive miRNAs. We determined the genome-wide miRNA expression signature in bladder cancer (BC) by deep sequencing technology. A total of ten small RNA libraries were sequenced (five BCs and five samples of histologically normal bladder epithelia (NBE)), and 13,190,619 to 18,559,060 clean small RNA reads were obtained. A total of 933 known miRNAs and 17 new miRNA candidates were detected in this analysis. Among the known miRNAs, a total of 60 miRNAs were significantly downregulated in BC compared with NBE. We also found that several miRNAs, such as miR-1/133a, miR-206/133b, let-7c/miR-99a, miR-143/145 and miR-195/497, were located close together at five distinct loci and constituted clustered miRNAs. Among these clustered miRNAs, we focused on the miR-195/497 cluster because this clustered miRNA had not been analyzed in BC. Transfection of mature miR-195 or miR-497 in two BC cell lines (BOY and T24) significantly inhibited cancer cell proliferation, migration and invasion, suggesting that the miR-195/497 cluster functioned as tumor suppressors in BC. Regarding the genes targeted by the miR-195/497 cluster, the TargetScan algorithm showed that 6,730 genes were putative miR-195/497 targets, and 113 significantly enriched signaling pathways were identified in this analysis. The “Pathways in cancer” category was the most enriched, involving 104 candidate target genes. Gene expression data revealed that 27 of 104 candidate target genes were actually upregulated in BC clinical specimens. Luciferase reporter assays and Western blotting demonstrated that BIRC5 and WNT7A were directly targeted by miR-195/497. In conclusion, aberrant expression of clustered miRNAs was identified by deep sequencing, and downregulation of miR-195/497 contributed to BC progression and metastasis. Tumor suppressive miRNA-mediated cancer pathways provide new insights into the potential mechanisms of BC oncogenesis.当前，基于深度测序技术的全基因组微小RNA（microRNA，miRNA）表达谱分析，可助力发现受致癌性和/或抑癌性miRNA调控的新型癌症通路。本研究通过深度测序技术解析了膀胱癌（bladder cancer，BC）的全基因组miRNA表达谱。本研究共构建10个小RNA文库并进行测序，其中包含5例膀胱癌组织样本与5例组织学正常膀胱上皮（histologically normal bladder epithelia，NBE）样本，最终获得13,190,619至18,559,060条清洁小RNA读段。本次分析共检测到933种已知miRNA以及17个新的miRNA候选体。与正常膀胱上皮相比，膀胱癌组织中共60种已知miRNA呈现显著下调表达。研究还发现，miR-1/133a、miR-206/133b、let-7c/miR-99a、miR-143/145及miR-195/497等多种miRNA在5个不同基因座上紧密聚集，构成成簇miRNA。在这些成簇miRNA中，我们重点关注miR-195/497簇，因此前尚未有针对该成簇miRNA在膀胱癌中的相关研究。将成熟miR-195或miR-497转染至两株膀胱癌细胞系（BOY与T24）后，可显著抑制癌细胞的增殖、迁移与侵袭能力，这表明miR-195/497簇在膀胱癌中发挥抑癌功能。针对miR-195/497簇的靶基因，通过TargetScan算法预测得到6730个潜在靶基因，本分析中共鉴定出113条显著富集的信号通路。其中富集程度最高的为"癌症通路"类别，共涉及104个候选靶基因。基因表达数据显示，104个候选靶基因中有27个在膀胱癌临床样本中呈现显著上调表达。荧光素酶报告基因实验与蛋白质印迹法（Western blotting）证实，BIRC5与WNT7A为miR-195/497的直接靶基因。综上，本研究通过深度测序鉴定出成簇miRNA的异常表达模式，且miR-195/497簇的下调表达与膀胱癌的进展及转移密切相关。抑癌miRNA介导的癌症通路为膀胱癌发生的潜在机制提供了新的研究视角。