The ‘melanoma-enriched’ microRNA miR-4731-5p acts as a tumour suppressor

Abstract

We previously identified miR-4731-5p (miR-4731) as a melanoma-enriched microRNA following comparison of melanoma with other cell lines from solid malignancies. Additionally, miR-4731 has been found in serum from melanoma patients and expressed less abundantly in metastatic melanoma tissues from stage IV patients relative to stage III patients. As miR-4731 has no known function, we used biotin-labelled miRNA duplex pull-down to identify binding targets of miR-4731 in three melanoma cell lines (HT144, MM96L and MM253). Using the miRanda miRNA binding algorithm, all pulled-down transcripts common to the three cell lines (n=1092) had potential to be targets of miR-4731 and gene-set enrichment analysis of these (via STRING v9.1) highlighted significantly associated genes related to the ‘cell cycle’ pathway and the ‘melanosome’. Following miR-4731 overexpression, a selection (n=81) of pull-down transcripts underwent validation using a custom qRT-PCR array. These data revealed that miR-4731 regulates multiple genes associated with the cell cycle (e.g. CCNA2, ORC5L, and PCNA) and the melanosome (e.g. RAB7A, CTSD, and GNA13). Furthermore, members of the synovial sarcoma X breakpoint family (SSX) (melanoma growth promoters) were also down-regulated (e.g. SSX2, SSX4, and SSX4B) as a result of miR-4731 overexpression. Moreover, this down-regulation of mRNA expression resulted in ablation or reduction of SSX4 protein, which, in keeping with previous studies, resulted in loss of 2D colony formation. We therefore speculate that loss of miR-4731 expression in stage IV patient tumours supports melanoma growth by, in part; reducing its regulatory control of SSX expression levels.

Comparing two approaches of miR-34a target identification, biotinylated-miRNA pulldown vs miRNA overexpression

“…Collectively, based on our data and a recent study, 26 we can conclude that biotinylated miRNAs can be used to identify miRNA targets. The sensitivity of this method can be increased with the addition of a scramble control mimic.…”

“…18 Affinity purification, another method to identify miRNA targets, has been used in many studies, where synthetic miRNA duplexes typically biotinylated at the 3 0 end of the guide strand are used. [21][22][23][24][25][26][27] The duplexes are transfected into appropriate cell type, where biotinylated miRNA (Bio-miRNA) presumably gets incorporated into RISC to form miRNA-mRNA complex. Following cell lysis, tagged miRNAs are captured on streptavidin beads followed by mRNA isolation and identification.…”

Transplacental innate immune training via maternal microbial exposure: the XBP1-ERN1 axis in programming dendritic cell precursors

“…Identification of ERN1 is noteworthy given that during the ER stress response, this gene encodes the ER stress sensor inositol-requiring enzyme 1 (IRE1a), responsible for the unconventional cleavage of a 26 nucleotide fragment from Xbp1 mRNA, resulting in the generation of the active spliced variant of XBP1 (XBP1s) and enabling it to function as a potent transcription factor (17). Additional candidate drivers in fetal bone marrow following maternal OM-85 treatment included CD38, IL5 and an array of microRNAs (miR) ( Figure 3A right panel; Supplemental Table 5) recognised principally in the context of cancer-associated functions (18)(19)(20). It is important to note however, that miR-149-3p has been shown to negatively regulate Toll-like receptor (TLR) 4 expression in murine monocytic cells in vitro (21) and it is possible that other miRs may have similar (but as yet undefined) innate immune regulatory functions (22).…”

Circulating cell‐free microRNAs in cutaneous melanoma staging and recurrence or survival prognosis

“…Of the cf-miRNAs in this panel, miR-16-5p and miR-211-5p have been reported to act as tumour suppressors in different reports (Guo et al, 2016;Mazar et al, 2016;Poell et al, 2012;Yu & Yang, 2016), as well as miR-4731 in a single report (Stark et al, 2016). Lastly, both the miR-509 filaments, -3p and -5p, were found to control the expression of proteins involved in epithelial-mesenchymal transition (EMT) in melanoma cells (Wang et al, 2015).…”