In order to find tissue-based BC-specific miRNA signatures, a comparative study was performed using a qRT-PCR array platform to profile 19 BC tissue samples and 19 matched adjacent normal bladder tissue samples from patients with BC.
The study revealed that 70 aberrant miRNAs (Table 2), hsa-miR-96, hsa-miR-182, hsa-miR-183, hsa-miR-429, hsa-miR-425, hsa-miR-431 are overexpressed in BC comparing to the normal controls. Among these miRNAs, miR-96, miR-182 and miR-183 are clustered in one locus of chromosome 7 (15). miR-429 belongs to the miR-200 family, which is clustered in the chromosomes 12, we further found that other members (200a/b/c) of miR200 family are also overexpressed in BC compared with in normal controls (Supplement Table 1). Both two miRNA clusters are well-known oncogenic miRNA clusters that have been extensively reported to be involved in tumor genesis in ovarian cancer and in other cancers including in BC (16-19).
The ten best groups of miRNAs signatures have been selected to discriminate the BC from the normal controls with 100% sensitivity and 100% specificity, suggesting their potential value for the diagnosis and prognosis of BC (Table 3, Fig. 3). These selected signatures all contain hsa-miR-133a that means hsa-miR-133a may play a key role in discriminating BC from normal controls. The number one signature contains three miRNAs, hsa-miR-133a, hsa-miR-431 and hsa-miR-4251. Hsa-miR-133a was significantly down-regulated and hsa-miR-431 was significantly up-regulated in BC. However, the expression level of hsa-miR-4251 was slightly higher in BC than that in normal controls. Furthermore, these signatures can predict BC with significantly high accuracy over 95% in a blind test. More importantly, these miRNA signatures could effectively distinguish early-stage (25 cases in stage I) BC from normal controls, suggesting their potential value in the detection of BC at an early stage.
A previous study (20) has reported the expression levels of miR-133a, miR-133b, miR-1, and miR-99a are down-regulated, however, miR-182 is up-regulated in BC. These miRNAs might be involved in the tumorigenesis and deterioration of BC. Our results confirmed the previous findings, and further demonstrated that the powers to distinguish BC from normal control of the selected signatures containing miR-133a are significantly better than that of miR-133a alone (Supplemental Fig.2). The signatures containing miR-133a identified in our study can diagnose BC even at an early stage with high sensitivity and high specificity and could be used as biomarkers in the diagnosis and prognosis of BC.
It has been shown that miR-133a-3p acts as a tumor suppressor in BC.Numerous studies demonstrated that miR-133a inhibits cell proliferation, migration, and invasion in various tumors by targeting to different signaling pathways, such as caspase signaling pathway, insulin/IGF signaling pathway, and EGFR signaling pathway (21-28). The connecting between the importance of miR-133a in tumorigenesis and significant downregulation of miR-133a in BC has shed light on the molecular mechanism of miRNA-133a in the tumorigenesis of BC.
Cystoscopy is the gold standard for diagnosis and surveillance of BC, while the procedure is invasive, uncomfortable and costly. Many potential BC biomarkers, like protein, miRNA, mRNA, DNA methylation, miRNA and miRNA signature have been reported, however, their successful applications in the diagnosis and prognosis of BC have not been demonstrated yet. As reported from other investigators (29, 30), our results also showed that miR-1, miR-133a, miR-133b, miR-143, miR-145, and miR-10b, are downregulated in BC (Supplement Table 1), suggesting a major role of these tumor suppressor miRNAs in bladder carcinoma. Of all these downregulated miRNAs, the miR-133a is the only one miRNA shown up in all ten best BC diagnosis signatures (Table 3).
miR-133a was one member of the miR-133 family, which was first experimentally characterized in mice (31). miR-133 and miR-1 are clustered in the same chromosomal locus in the human genome (18q11.2) and share the same transcriptional unit, which has shown their essential functions in controlling skeletal muscle proliferation and differentiation (32). Genes encoding miR-133 (miR-133a-1, miR-133a-2 and miR-133b) are transcribed as bicistronic transcripts together with miR-1-2 and miR-1-1. We had analyzed the potential target gene and the function of miR-133a by using www.mirnet.ca. We recognized that there is a correlation between miR-133a and miR-1 by targeting TAGLN2 and LASP1 (30, 33). In agreement with our study, miR-133a has been reported to be down-regulated in BC and in other cancers (30, 34-36). miR-133a seems to function as a tumor suppressor by inhibiting cell proliferation, invasion, migration, and apoptosis (30, 33).