In this study we have determined the expression status and regulatory mechanism of miR-31 in melanoma and performed functional characterization of its role in various oncogenic phenotypes and validated novel targets. Although miR-31 has been shown to act as either an oncomiR or tumor suppressor in various tumor types [13
], in this study we clearly establish miR-31 as a tumor suppressor, demonstrate that it targets multiple oncogenes such as SRC, MET, NIK and RAB27a and regulates the expression of EZH2 in melanoma.
The gene encoding miR-31 is located within the intron of a long noncoding RNA located on chromosome 9p21. Deletions of chromosome 9p21 are common in melanoma and other tumors and have been shown to include the miR-31 locus in cancers including mesothelioma and urothelial carcinoma [18
]. By array CGH we observed both heterozygous and homozygous deletions involving the miR-31 locus in 21% of melanomas. The expression of miR-31 was profoundly suppressed in most of the melanoma tissues and cell lines; the loss of expression is attributed to both genomic deletion and epigenetic silencing – a common mechanism of downregulation of tumor suppressors in cancer. Epigenetic modification of chromatin via
methylation of cytosine residues in CpG islands or by post-translational modification of histones marks transcriptional availability of genes. Polycomb repressor complex 2, of which EZH2 is the catalytic component, mediates trimethylation of histone 3 at lysine K27 (H3K27) and subsequent repression of target genes [24
] including miR-31 expression in adult T cell leukemia and prostate cancer [33
], and ENCODE ChipSeq data indicates an enrichment of trimethylated H3K27 at the miR-31 promoter region. In addition, miR-31 is silenced in triple-negative breast carcinoma by CpG island methylation [32
]. We observed a significant induction of miR-31 expression upon depletion of EZH2 either by RNA interference or by DZNep treatment as well as by treatment with the DNA methylation inhibitor, 5'aza-dC.
The inhibitory effect of miR-31 on cell proliferation has been reported for ATL and mesothelioma [18
], however cell growth of only a subset of melanoma cell lines were inhibited upon ectopic overexpression of miR-31 in our studies. Conversely, knockdown of miR-31 led to increased proliferation only in serum-free conditions. miR-31-mediated attenuation of local invasion and metastasis without affecting primary tumor growth was elegantly demonstrated in breast cancer [13
], and we observed a similar effect on migration and invasion upon miR-31 overexpression in all melanoma cell lines tested, supporting its role as a metastasis suppressor in melanomas.
In order to identify targets that are regulated by miR-31 to mediate tumor suppressive effects in melanoma, we employed an integrative approach utilizing gene expression microarrays, miR target prediction algorithms, and literature searches. We nominated SRC, MET, NIK and RAB27a as possible miR-31 targets in melanoma; these genes are known to play pro-tumorigenic roles in melanoma [37
]. The validation of these four melanoma oncogenes as a direct miR-31 target represents a significant finding and elucidates the molecular pathways affected by chromosome 9p21 deletion in melanoma. Moreover, NIK (NF-kB inducing kinase) which promotes cell proliferation and survival in melanoma has recently been validated as a miR-31 target in ATL [33
Downregulation of EZH2 may represent an additional tumor suppressive role for miR-31 since EZH2 plays a pro-tumorigenic role in melanoma [43
]. It is unlikely that EZH2 expression is directly targeted by miR-31 as EZH2 3'UTR lacks a miR-31 binding site. However, EZH2 expression and/or function can be regulated through an alternate mechanism such as MEK-ERK-Elk-1 activation pathway [26
]. We observed the presence of multiple cis-regulatory elements for transcriptional factors Elk-1, Sp1, AP1 (c-Jun) and NF-kB in the proximal promoter of EZH2 (Supplementary Figure 7
) that are known to be activated by miR-31 targets SRC, MET, and NIK proteins. Therefore, miR-31 may indirectly inhibit EZH2 via
repression of these upstream activators. Taken together, our findings indicate that EZH2 and miR-31 engage in a mutually antagonistic regulatory circuit that can accelerate tumorigenic progression (Figure ).
Model for mechanisms of interaction between miR-31 and EZH2 in melanoma cells
In conclusion, we found recurrent deletion and epigenetic silencing of miR-31 in melanoma. And miR-31 acts as a tumor-suppressive microRNA by exerting inhibitory effects on cell motility and invasion. Importantly, we identified SRC, MET, NIK and RAB27a to be direct targets of miR-31. Moreover, we found that miR-31 also engages in an antagonistic regulatory circuit with EZH2, and loss of miR-31 expression may contribute to EZH2 overexpression in melanoma and provides a potential therapeutic axis which can be targeted by small molecule inhibitor.