The microRNA expression signature of bladder cancer by deep sequencing: the functional significance of the miR-195/497 cluster

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.

Figure 1. Schematic representation of the human miR-195/497 cluster location.

miR-195 and miR-497 are in an intron of the MIR497HG gene on human chromosome 17q13.1, where they are separated by 209 bp.

Figure 2. Expression levels of miR-195 and miR-497 in BC clinical specimens, and noncancerous bladder tissues.

A, B) The expression of miR-195 and miR-497 was significantly lower in 20 clinical BC specimens than in 29 adjacent noncancerous specimens. RNU48 was used as the internal control. *, P<0.0001. C) Significant positive correlation between miRNA expression patterns of miR-195 and miR-497 (rank test Spearman's correlation coefficient r = 0.984, P<0.0001).

Figure 3. Effect of mature miR-195 and miR-497 transfection of BC cell lines (BOY, T24).

Gain-of-function studies were performed by using miR-195 and miR-497-transfected BOY and T24 in comparison with the miR-control transfectants. A) Cell proliferation determined by the XTT assay. B) Cell invasion activity demonstrated by the Matrigel invasion assay. C) Cell migration activity determined by the wound healing assay. *, P<0.0001; **, P = 0.009.

Figure 4. Identification of miR-195/497 cluster-mediated molecular pathways and putative molecular targets.

A total of 6730 genes were identified by the TargetScan algorithm. Then, 113 significantly enriched signaling pathways were identified by using the KEGG and GeneCodis3 programs. Among them, the top 21 enriched pathways are shown in Table S3. The expression levels of 104 genes in the top enriched pathway (Pathways in cancer) were finally evaluated. Among them, 27 putative target genes were upregulated in BC clinical specimens based on expression data of GEO database (accession numbers; GSE11783 and GSE31684) (Table S4).

Figure 5. Genes upregulated by the miR-195/497 cluster in BC specimens.

Heat map diagram shows the expression of 27 candidate genes involved in “Pathways in cancer” in 90 BC specimens and six adjacent noncancerous bladder specimens.

Figure 6. BIRC5 and WNT7A mRNA and protein expression levels were suppressed by miR-195 and miR-497 transfection in comparison with the miR-control transfection in BC cell lines (BOY and T24).

A) BIRC5 and WNT7A mRNA expression 72 h after transfection with miR-195, miR-497, and the miR-control. GUSB expression was used for normalization. B) BIRC5 and WNT7A protein expression 72 h after transfection with miR-195, miR-497, and the miR-control. GAPDH was used as a loading control. The ratio of BIRC5 or WNT7A to GAPDH expression was evaluated using ImageJ software (ver. 1.43; http://rsbweb.nih.gov/ij/index.htmL). *P<0.01.

Figure 7. BIRC5 and WNT7A as target genes of the miR-195/497 cluster in BC.

Luminescence intensity was measured in miR-195- and miR-497-transfectants in comparison with the miR-control transfectant. A) miR-195/497 cluster binding sites in the 3′-UTR of BIRC5 mRNA. Luciferase reporter assay vector used the encoding 3′-UTR region of BIRC5 including putative miR-195/497 target sites. Renilla luciferase values were normalized to firefly luciferase values. *P<0.01. B) miR-195/497 cluster binding sites in the 3′-UTR of WNT7A mRNA. Luciferase reporter assay vector is the encoding 3′-UTR region of WNT7A including putative miR-195/497 target site. *P<0.01.