miR-28-5p showed a tumor suppressive activity in DU-145 prostate cancer cells and regulated E2F6

It is now well known that the dysregulations of miRNAs expression is one of the cause or contributory causes of cancer development. Several evidences have clearly demonstrated both that the downregulation of tumor suppressors (TS) miRNAs favors the expression of oncogenes and tumor growth and that the re-expression of TS-miRNAs in defective tumor cell lines reduces their proliferation. We have demonstrated that miR-28-5p induces apoptosis and senescence in mouse embryo broblasts (MEF) and that it is strongly downregulated during MEF immortalization. In this work we analyzed the role of miR-28-5p in human tumor cells and we investigated the miR-28-5p targets in prostate cancer (PCa) cell lines using the miRNA pull-out assay. miR-28-5p was transfected using the appropriate transfectant for each tumor cell lines. Apoptosis was measured by annexin assay and by the detection of PARP cleavage. miRNA pull-out assay was performed using biotinylated synthetic version of both miRNAs and the miRNA/targets complex isolated with streptvidine sepharose high performance (GE Health care). We evaluated the miR-28-5p expression level in several PCa as well as in other tumor cell lines and we found that it was strongly downregulated. Moreover, the miR-28-5p re-expression inhibited cell proliferation. In particular, miR- 28-5p was able to cause a G1 arrest and to affect the colony forming ability of DU- 145 PCa cells. To discover the miR-28-5p targets we performed the miRNA pull-out assay and we measured the enrichment of miR-28-5p targets already validated in other biological context using qRT-PCR. We found that not all validated targets were enriched by the pull-out procedure. The most enriched was E2F6. By overexpressing miR-28-5p we demonstrated that it was able to inhibit E2F6 also in DU-145 cells. Since it has been demonstrated that E2F6 plays an antiapoptotic role, we investigated and found that miR-28-5p overexpression induce apoptosis in DU-145 cells. Moreover, we showed that miR-28-5p re-expression enhanced the apoptosis induced by docetaxel even if at the same level of the miR-28-5p induced apoptosis. The overall results indicate that: i) the miR-28-5p plays a TS role in DU-145 cells; ii) its re-expression in DU-145 cells induces apoptosis through E2F6 even if it is not the only mediator. In addition, we reinforce the concept that miRNAs regulate different targets depending on the biological context.