miR-28-5p is an intragenic miRNA which is underexpressed in several tumor types showing a tumor suppressor (TS) activity. Routinely, the known miR-28-5p targets are validated in specific tumor contexts but it is unclear whether these targets are also being regulated in other tumor types. To this end, we adopted the miRNA pull out assay to capture the miR-28-5p targets in DU-145 prostate cancer (PCa) cells. Firstly, we demonstrated that miR-28-5p acts as a TS-miRNA in PCa, affecting cell proliferation, survival, and apoptosis. Secondly, we evaluated the enrichment of the 10 validated miR-28-5p targets in the pull out sample. We showed that E2F6, TEX-261, MAPK1, MPL, N4BP1, and RAP1B but not BAG1, OTUB1, MAD2L1, and p21 were significantly enriched, suggesting that not all the miR-28-5p targets are regulated by this miRNA in PCa. We then verified whether the miR-28-5p-interacting targets were regulated by this miRNA. We selected E2F6, the most enriched target in the pull out sample, and demonstrated that miR-28-5p downregulated E2F6 at the protein level suggesting that our approach was effective. In general terms, these findings support the miRNA pull out assay as a useful method to identify context-specific miRNA targets.
It is well known that the deregulation of miRNA expression is one of the causes or contributory causes of cancer development. miRNAs may act as tumor suppressors (TS), oncogenes, or both depending on the tumor context [
The molecular targets through which miR-28-5p exerts its anti- or proproliferative role are only partially known. For example, miR-28-5p reduced cell growth and migration in hepatocellular carcinoma [
To date, there are no data either on the role of miR-28-5p in prostate cancer (PCa) or on the targets regulated by this miRNA in PCa cells. In this work, we evaluated whether the miR-28-5p targets validated in other types of tumors were regulated in PCa cells using the miRNA pull out assay, a technique that allows the isolation of all the targets of a given miRNA in specific biological contexts. We demonstrated that miR-28-5p exerted TS activity in PCa cells and that not all validated miR-28-5p targets are regulated by this miRNA in the PCa context.
DU-145 and A-549 cells were grown in RPMI Medium 1640 (EuroClone) whereas PC-3 cells were grown in HAM’s medium (EuroClone) and MCF-7 cells in DMEM low glucose (EuroClone). 10% FBS (fetal bovine serum, EuroClone), 1% penicillin/streptomycin (2 mM, EuroClone), and 1% L-glutamine (2 mM, Sigma-Aldrich) were added to the medium. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2.
Transient transfections of double-stranded miRNA mimics (miR-28a-5p) or negative control (miR-NC) (GenePharma) in DU-145 cells were carried out using Lipofectamine 2000 (Thermo Fisher): 1.5 × 105 cells were seeded in P30 dishes and after 48 hours, cells were transfected with miRNA mimic using 10
1 × 105 cells were seeded in a series of 30 mm diameter dishes and grown for 96 hours. At 24-hour intervals, cells were collected and counted.
Cell cycle analysis was performed as follows: 5 × 105 cells were fixed with 95% cold ethanol and labelled with 300
Survival was measured as follows: cells were collected and seeded at cell density of 200 cells/60 mm diameter culture dish to allow colony formation. After 10–12 days, dishes were stained with 0.1% CV and the ratio (number of colonies/number of seeded cells) was used to calculate the fraction of surviving cells.
Apoptosis was measured as follows: 1 × 106 cells were suspended in 300
The miRNA pull out assay was performed as described in Rizzo et al. [
Total RNA was extracted from 1 × 106 cells using the miRNeasy mini kit following the manufacturer’s recommendations. 1
Proteins were extracted from cell pellets using lysis buffer (1 M Tris HCl pH 8, Triton X-100 1%, and Na deoxycholate 0.25%) with the addition of PMSF 1 mM. The proteins were then quantified colorimetrically using the BioRad protein Assay Reagent (Bio-Rad). Absorbance was measured at 595 nm with ChroMate microplate reader (Awareness Technology). The proteins were separated on polyacrylamide gels SDS-PAGE (10%, gel precast Mini-PROTEAN® TGX Stain-Free™, Bio-Rad) and transferred to 0.2
Results are expressed as mean SD of at least three independent experiments, and data are analyzed using Student’s
To investigate the role of miR-28-5p in PCa, we first evaluated its expression in two PCa cell lines (DU-145 and PC-3) compared to normal cells (Figure
miR-28-5p expression and effect on tumor cells. (a) Analysis of the miR-28-5p expression level with qRT-PCR in prostate (PC-3 and DU-145), lung (A-549), and breast (MCF-7) cancer cell lines compared to the normal cell RNA. Cell proliferation of DU-145 (b), PC-3 (c), A-549 (d), and MCF-7 (e) cells at different time points or at 96 hours after the miR-28-5p reexpression.
In order to test whether miR-28-5p behaves as a TS in PCa cells, we first measured cell proliferation of DU-145 and PC-3 cells after miR-28-5p reexpression. Data showed a significant inhibition of cell proliferation of both PCa cell lines (Figures
To further investigate the biological effects of the miR-28-5p in PCa, we checked the colony-forming ability (CFA) and the cell cycle after miRNA reexpression in DU-145 cells. Data showed that miR-28-5p reexpression resulted in both a significant reduction of CFA (Figure
Effects of miR-28-5p reexpression on DU-145 cells. Cell survival (a) and cell cycle (b) in DU-145 cells after miR-28-5p reexpression. Apoptosis analysis measured with both annexin assay (c) and western blot of PARP-1 and cleaved PARP-1 (d) in miR-28-5p-transfected DU-145 cells.
To investigate which targets were regulated by miR-28-5p in PCa, we transfected this miRNA in DU-145 cells and the miRNA pull out assay was performed [
miR-28-5p targets validated with gene reporter assay according to miRTarBase.
miR-28-5p target | Tumor type | Reference |
---|---|---|
p21 | Choriocarcinoma cells | [ |
MPL | Myeloproliferative neoplasms | [ |
N4BP1 | Myeloproliferative neoplasms/ovarian cancer | [ |
OTUB1 | Myeloproliferative neoplasms | [ |
TEX-261 | Myeloproliferative neoplasms | [ |
MAPK1 | Myeloproliferative neoplasms | [ |
E2F6 | Myeloproliferative neoplasms | [ |
MAD2L1 | B-cell lymphomas | [ |
BAG1 | B-cell lymphomas | [ |
RAP1B | B-cell lymphomas/renal cell carcinoma | [ |
Using qRT-PCR, we checked the enrichment of these targets in the pool of miR-28-5p-captured targets (miR-28 pull out sample) and found that RAP1B, N4BP1, MPL, MAPK1, TEX-261, and E2F6 were enriched by more than 2-fold in the miR-28-5p pull out sample (Figure
miR-28-5p target validation. (a) miR-28-5p-validated target enrichment quantified by qRT-PCR in miR-28-5p pull out sample compared to miR-28-5p control pull out sample (CT). E2F6 mRNA (b) and protein (c) quantification in DU-145 cells transfected with miR-28-5p or miR-NC.
To verify whether the enrichment of the selected targets in the miR-28-5p pull out sample was indicative of the miRNA regulatory function, we selected the most enriched one, that is, E2F6, and determined its expression after the miR-28-5p reexpression in DU-145 cells. We demonstrated that E2F6 was inhibited by miR-28-5p reexpression only at the protein level (Figures
miRNAs are key inhibitors of gene expression that play a pivotal role in tumor development and progression affecting genes and pathways involved in all the hallmarks of cancer [
In this work, performing the miR-28-5p pull out assay, we have explored whether the known miR-28-5p targets, deposited in miRTarBase and validated with at least the luciferase reporter assay, interacted and were regulated by miR-28-5p in PCa. Under this strategy was that, among all miR-28-5p-validated targets, the ones that interact with miR-28-5p in PCa have higher chance to be regulated by the miRNA in this tumor. We first evaluated the miR-28-5p role in PCa, and we demonstrated for the first time that this miRNA is underexpressed in these cells and that its reexpression inhibited cell proliferation and survival. These data led us to conclude that miR-28-5p acted as a TS-miRNA in PCa. As it has been demonstrated that miR-28-5p negatively regulated genes involved in tumor cell growth in lung [
Using the miRNA pull out assay, we found that not all the validated miR-28-5p targets were enriched in the miR-28 pull out sample. Among the enriched targets that are more strongly associated with cancer (i.e., RAP1B, N4BP1, MAPK1, and E2F6), almost all are protumoral. Indeed, both MAPK1 and RAP1B, a Ras-related small GTP-binding protein that acts as GTPase in several signaling cascades, are proproliferative proteins involved as oncogenes in the development and progression of several tumor types (e.g., [
In conclusion, in this work, we demonstrated that the capture of the targets that interact with a given miRNA in a specific tumor is a suitable approach to identify the subset of targets that have a higher probability of being regulated by that miRNA in the context under evaluation. In the future, the identification of all the miR-28-5p targets (miR-28-5p targetome) could help to decipher the genes and pathways affected by the regulation of this miRNA in PCa.
The authors declare that there are no conflicts of interest.
The authors would like to thank Dr. Marcella Simili for critical reading and Dr. Mike Minks for the revision of the manuscript. This work was supported by the Istituto Toscano Tumori (Grant 2010, Giuseppe Rainaldi; Grant 2013, Milena Rizzo).