The miR-28-5p targetome discovery identified SREBF2 as one of the mediators of the miR-28-5p tumor suppressor activity in prostate cancer cells

miR-28-5p is downregulated in some tumor tissues in which it has been demonstrated to have a 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 the 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 increasing importance in PCa. We validated miR-28-5p/SREBF2 interaction demonstrating that SREBF2 inhibition affects almost all the tumor processes affected by the 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. Overall design: The miRNA pull out assay was performed as described in Rizzo et al. [ref: doi:10.7150/jca.18396]. DU-145 were transfected using Lipofectamine 2000 (Thermo Fisher) with 60 nM of either miR-28-5p duplex (ds-miR-28CT) or a mix of 3' biotin-tagged miR-28-5p 8tU (nucleotide 8 was a thiouridine) and miR-28-5p 18tU duplexes (ds-miR-28BIO). All oligos were synthetized by Bio-Synthesis Inc. 24 hours after transfection cells were irradiated with UV (365nm, 2J/cm2) using the Bio-Link crosslinking (BLX) (Ambrose Lourmat) and total RNA extracted with TRIzol (Thermo Fisher) directly on adherent cells following the manufacturer's protocol. 15 µg of RNA was incubated 4 hours at 4°C with 100 µl of streptavidin-conjugated beads (Streptavidin Sepharose high performance, GE Healthcare) and the RNA complexed with the beads was recovered using TRIzol. We performed two biological replicates obtaining three miR-28CT (control) and two miR-28BIO (miR-28-5p) pull out samples consisting of background RNA and ds-miR-28BIO/interacting mRNA complexes respectively. The RNA isolated after the miRNA pullout procedure was used for the construction of the cDNA libraries using the TruSeq Stranded Total RNA Sample Preparation kit (Illumina) according to the manufacturer's suggestions. cDNA libraries were sequenced by HiSeq2000 (Illumina) in single-reads mode (50bp) by IGA Technology Service, Udine, Italy, obtaining about 20 million of reads for each samples.