Abstract
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.
Highlights
In developed countries, bladder cancer (BC) is the fifth most commonly diagnosed tumor and the second most common cause of death in patients with genitourinary tract malignancies
We previously reported that miR-1-1/133a-2 and miR-1-2/133a-1 formed clusters in different chromosomal loci in the human genome, 20q13.33 and 18q11.2, respectively, and these clusters function as tumor suppressors, targeting several oncogenes in human cancers, including BC [18,19,20,21,22,23,24]
We found that the expression levels of miR-195 and miR-497 in BCs were significantly lower than those in Normal bladder epithelium (NBE) (Figure 2A, B)
Summary
Bladder cancer (BC) is the fifth most commonly diagnosed tumor and the second most common cause of death in patients with genitourinary tract malignancies. In Japan, the number of new BC patients was estimated at 17,461 in 2007 and the number of deaths was estimated at 7,008 in 2011 [2]. BCs can be classified into two categories, non-muscle-invasive tumors and muscle-invasive tumors. 70%–80% of patients are diagnosed with non-muscle-invasive tumors, high recurrence rates (50%–70%) are observed in these patients. Among recurrent cases, 15% of BCs progress to muscle-invasive disease [3]. The five-year survival rate for patients with non-muscle-invasive BC is close to 90%, whereas that of patients with muscle-invasive BC is approximately 60% [4]. Since most clinical trials of chemotherapeutics for advanced BC have shown limited benefits, new prognostic markers and effective treatment strategies based on current cancer-genome analyses are necessary
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