Abstract
miR-28-5p is downregulated in some tumor tissues in which it has been demonstrated to have tumor suppressor (TS) activity. Here, we demonstrate that miR-28-5p acts as a TS in prostate cancer (PCa) cells affecting cell proliferation/survival, as well as migration and invasion. Using the miRNA pull out assay and next generation sequencing, we collected the complete repertoire of miR-28-5p targets, obtaining a data set (miR-28-5p targetome) of 191 mRNAs. Filtering the targetome with TargetScan 7, PITA and RNA22, we found that 61% of the transcripts had miR-28-5p binding sites. To assign a functional value to the captured transcripts, we grouped the miR-28-5p targets into gene families with annotated function and showed that six transcripts belong to the transcription factor category. Among them we selected SREBF2, a gene with an important role in PCa. We validated miR-28-5p/SREBF2 interaction, demonstrating that SREBF2 inhibition affects almost all the tumor processes altered by miR-28-5p re-expression, suggesting that SREBF2 is an important mediator of miR-28-5p TS activity. Our findings support the identification of the targetome of cancer-related miRNAs as a tool to discover genes and pathways fundamental for tumor development, and potential new targets for anti-tumor therapy.
Highlights
MiR-28-5p is a miRNA with a tumor suppressor (TS) activity downregulated in several tumor tissues [1,2,3,4,5,6]
We already demonstrated that miR-28-5p is involved in both tumor cell proliferation and survival by inhibiting cell proliferation and colony forming ability, and by inducing apoptosis and increasing the percentage of DU-145 cells in G1 phase [10]
Plays a role in the capability of the tumor cells to migrate and invade, finding that miR-28-5p re-expression determined a reduction of the prostate cancer (PCa) cells capability to grow unattached to a matrix and to migrate and invade
Summary
MiR-28-5p is a miRNA with a tumor suppressor (TS) activity downregulated in several tumor tissues [1,2,3,4,5,6]. Some reports about the molecular targets through which the miR-28-5p exerts its TS activity in different tumor types are available, most experimental studies validate only one or a few miRNA targets at once. We performed an miRNA pull out assay [15] that is based on the capture of the specific miRNA/target complexes through the transfection of a biotinylated version of the miRNA of interest We already used this technique associated with the microarray for target identifications [16], and more recently we combined the miRNA pull out assay with NGS technology to identify the miR-26a-5p targetome in PCa cells [17]. We focused on SREBF2, validating miR-28-5p/SREBF2 interaction and demonstrating that SREBF2 inhibition exerts TS activity in PCa cells
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