MicroRNAs mediate metabolic stresses and angiogenesis

MicroRNAs are short endogenous RNA molecules that are able to regulate (mainly inhibiting) gene expression at the post-transcriptional level. The MicroRNA expression profile is cell-specific, but it is sensitive to perturbations produced by stresses and diseases. Endothelial cells subjected to metabolic stresses, such as calorie restriction, nutrients excess (glucose, cholesterol, lipids) and hypoxia may alter their functionality. This is predictive for the development of pathologies like atherosclerosis, diabetes, and hypertension. Moreover, cancer cells can activate a resting endothelium by secreting pro-angiogenic factors, in order to promote neoangiogenesis, which is essential for tumor growth. Endothelial altered phenotype is mirrored by altered mRNA, microRNA, and protein expression, with a microRNA being able to control pathways by regulating the expression of multiple mRNAs. In this review we will consider the involvement of microRNAs in modulating the response of endothelial cells to metabolic stresses and their role in promoting or halting angiogenesis

The analysis of the expression pattern of the intracellular and extracellular miRNAs in prostate tumor cell lines exposed to cytotoxic drugs.

It has been recently discovered that microRNAs are stably expressed in body fluids of several organisms and that the expression patterns in the plasma/serum of cancer patients could represent a diagnostic/predictive tool of the disease. Extracellular miRNAs have been also found in the growth medium of cells in culture and this prompted us to verify whether prostate tumor cell lines release miRNAs specific of prostate tumor patients and whether anticancer drugs affect the expression patterns of the extracellular miRNAs. First of all we determined the expression of either prostate specific (PS-miRNA) or non prostate specific (NPS-miRNA) miRNAs in the growth medium of PC3 and DU-145 cells (prostate metastatic tumor cell lines) in comparison to PNT1A (immortalized prostate cell line). We found that the levels of both the intracellular and extracellular PS-miRNAs were higher in PC3 and DU-145 tumor cell lines in comparison to the immortalized cell line PNT1A and that a positive correlation exists between the intracellular and the extracellular levels suggesting that the release of miRNA in the growth medium may be a manner to maintain the intracellular miRNAs at physiological level. Thereafter, we investigated whether the release of miRNAs is affected in cells upon their exposure to a cytotoxic drug. To address the point, PC-3 and DU-145 cells were exposed to a cytotoxic concentration of either fludarabine (10 μg/mL) or taxotere (30 nM) for 48 hours. At the end of the treatment both the intracellular and extracellular PS-miRNAs and NPS-miRNAs were quantified. Data showed that i) those miRNAs that were up regulated in cells surviving to either fludarabine or taxotere were not released and that ii) the up regulated miRNAs found in fludarabine-or in taxotere-surviving cells were not the same. Overall data indicate for the first time a possible involvement of miRNAs in the survival/resistance of tumor cells to cytotoxic drugs and suggest that the expression pattern of the intracellular and extracellular miRNAs could be an useful tool to identify in tumor cell lines miRNAs responsible of survival/resistance to cytotoxic drugs and in plasma/serum of cancer patients the efficacy of an anticancer treatment

The RNA Activator ds-p21 Potentiates the Cytotoxicity Induced by Fludarabine in Dohh2 Cells

Recently, it has been reported that, in several tumor cell lines, short double-stranded RNAs tailored for promoter regions of specific genes are able to activate their transcription. Such molecules (named RNA activators) act opposite to other double-stranded RNA molecules (named RNA inhibitors) in that the overexpression instead of underexpression of a given gene is triggered. In Dohh2 non-Hodgkin lymphoma cells, the transcriptional repressor BCL6, which negatively controls both p53 and p21, is overexpressed, so that the cells can escape the check point governed by p53 and proliferate. The aim of this work was to investigate whether the RNA activator p21 can represent a tool to circumvent the transcriptional control of BCL6 and induce the blockage of cell proliferation in Dohh2 non-Hodgkin lymphoma cells. For that, Dohh2 cells were transfected with either a control RNA activator (ds-NC) or an RNA activator specific for human p21 promoter (ds-p21). At various time points after transfection, the cells were collected and p21 was measured. Dohh2 cells transfected with ds-p21 showed a slight but significant overexpression of p21 at both mRNA and protein levels. Nonetheless, cell proliferation, cell cycle, and apoptosis were not significantly modified. In contrast, the exposure of Dohh2 cells transfected with ds-p21 to fludarabine potentiates the cytotoxicity of the drug, suggesting the RNA activator p21 complements the fludarabine-dependent cell death pathways

BCL6 and LRF crosstalk in follicular lymphoma

B cell Lymphoma 6 (BCL6) and Leukaemia/Lymphoma related factor (LRF) are Pokproteins over-expressed in some types of Non-Hodgkin\u27s lymphoma. BCL6 is located on chromosome 3 in the breakpoint affecting 3q27 band which is the most frequent translocation in Non-Hodgkin\u27s lymphomas. This gene is a transcriptional repressor whose principal effect is to thwart the response to DNA damage by directly inhibiting both p53 and the cell cycle inhibitor p21. In lymphoma cells BCL6 prevents apoptosis induced by DNA damage. LRF, encoded by the Zbtb7a gene, also known as Pokemon, is a transcriptional repressor involved in many cellular processes as viral infection, differentiation, inflammation and oncogenesis. LRF plays an important role as proto-oncogene in Non- Hodgkin\u27s lymphomas: LRF indirectly inhibits p53, by repressing p14/ARF with consequent activation of MDM2 followed by p53 degradation. LRF is often aberrantly over-expressed in association with BCL6 in diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL), the commonest types of Non- Hodgkin\u27s lymphomas. It has been demonstrated that microRNAs, a class of endogenous 22-25 nt single stranded RNA molecules, regulate target gene expression at the post-transcriptional level by binding with imperfect complementarity to specific regions of the 3\u27UTR of the target mRNA and act by repressing its translation. Recently miRNAs were shown to participate in the network of oncogenes and tumour suppressors, behaving either as tumour suppressors or as oncogenes, with the consequent implication of their potential use as novel anti tumourigenic drug. For these reason we investigated the role of miRNAs in the complex network connecting BCL6 and LRF in follicular lymphoma. Results In this work we focused our attention on the possible correlation between BCL6 and LRF in DOHH2 cells taking into consideration miRNAs which potentially link these two oncogenes. Using our data and data present in the literature, we reconstructed a hypothetical circuit connecting BCL6 and LRF. A negative feedback loop appears to link BCL6 to LRF, however in DLBCL and FL both genes are over expressed; these data strongly indicate that the negative feedback loop is bypassed in tumour cells to favour high proliferation rate. Different approaches were used to modulate the expression of BCL6 and LRF in Dohh2, a cell line derived from Follicular Lymphoma. A) Dohh2 cells were treated with etoposide, a chemotherapeutic agent that is able to reduce both BCL6 and LRF. Our results show that the reduction of BCL-6/LRF cells is followed by cell proliferation block and over-expression of miR-145, a tumour suppressor miRNA, under p53 control, which targets c-myc. Interestingly while c-myc was down regulated as expected, E2F1 resulted to be up regulated. B) To verify whether miR-145 could substitute for etoposide treatment miR-145 was transiently over expressed in Dohh2 cells. Although the network was influenced by miR-145 and both BCL6 and LRF were down regulated, no change in the proliferation rate of Dohh2 cells was found. Unexpectedly p53 was also markedly down regulated which could in part explain why cell proliferation was not inhibited notwithstanding BCL6/LRF decrease. C) In order to see the effect of a prolonged increase of miR-145, stably BCL6 silenced Dohh2 cells were used. In this case miR-145 was stably up regulated due to p53 increase and LRF was down regulated, yet again no effect on cell proliferation was observed. Our results clearly show that in the tumour Dohh2 cells BCL6 and LRF are always co-regulated (contrary to the hypothetical network); however their concomitant down regulation is not sufficient to determine cell proliferation block as cells appear to find a new equilibrium to counteract these anti-proliferative signals. We suggest that the down regulation of BCL6/LRF must be concomitant to the upregulation of the tumour suppressor p53 and the proto-oncogene E2F1 to influence the cell cycle and that miRNAs (such as miR-145 and miR-20) are part of these regulatory mechanisms

A new method for discovering disease-specific miRNA-target regulatory networks

Genes and their expression regulation are among the key factors in the comprehension of the genesis and development of complex diseases. In this context, microRNAs (miRNAs) are post-transcriptional regulators that play an important role in gene expression since they are frequently deregulated in pathologies like cardiovascular disease and cancer. In vitro validation of miRNA - targets regulation is often too expensive and time consuming to be carried out for every possible alternative. As a result, a tool able to provide some criteria to prioritize trials is becoming a pressing need. Moreover, before planning in vitro experiments, the scientist needs to evaluate the miRNA-target genes interaction network. In this paper we describe the miRable method whose purpose is to identify new potentially relevant genes and their interaction networks associate to a specific pathology. To achieve this goal miRable follows a system biology approach integrating together general-purpose medical knowledge (literature, Protein-Protein Interaction networks, prediction tools) and pathology specific data (gene expression data). A case study on Prostate Cancer has shown that miRable is able to: 1) find new potential miRNA-targets pairs, 2) highlight novel genes potentially involved in a disease but never or little studied before, 3) reconstruct all possible regulatory subnetworks starting from the literature to expand the knowledge on the regulation of miRNA regulatory mechanisms

The miRNA Pull Out Assay as a Method to Validate the miR-28-5p Targets Identified in Other Tumor Contexts in Prostate Cancer

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

Immortalization of MEF is characterized by the deregulation of specific miRNAs with potential tumor suppressor activity

The life span (Hayflick limit) of primary mouse embryo fibroblasts (MEF) in culture is variable but it is still unclear if the escape of the Hayflick limit is also variable. To address this point MEF were expanded every fifteen days (6T15) instead of every three days (6T3) until they became immortal. With this protocol MEF lifespan was extended and immortalization accordingly delayed. By testing a panel of genes (p19ARF, p16, p21) and miRNAs (miR-20a, miR-21, miR-28, miR-290) related to primary MEF senescence, a switch of p21 from up to down regulation, the down regulation of specific miRNAs as well as a massive shift from diploidy to hyperdiploidy were observed in coincidence with the resumption of cell proliferation. Collectively, these data indicate that the inactivation of genes and miRNAs, important in controlling cell proliferation, might be determinant for the escape from the Hayflick limit. In support of this hypothesis was the finding that some of the down regulated miRNAs transfected in immortalized MEF inhibited cell proliferation thus displaying a tumor suppressor-like activity

Immortalized mouse embryo fibroblasts are resistant to miR-290-induced senescence regardless of p53 status

Immortalized mouse embryo fibroblasts are resistant to miR-290-induced senescence regardless of p53 status. Physiol Genomics 43: 1153-1159, 2011. First published August 16, 2011; doi:10.1152/physiolgenomics.00064.2011.-The prosenescence role of miR-290 and nocodazole has been documented in primary mouse embryo fibroblasts (MEF), while it is not clear whether immortal murine fibroblasts are still responsive to these senescence inducing stimuli. To establish this point, immortal murine fibroblasts with functional (NIH3T3) or nonfunctional p53 (I-MEF) and low levels of miR-290 were tested for their capability to undergo senescence after exposure to either nocodazole or miR-290. Our results clearly indicate that nocodazole induces senescence only in NIH3T3 cells with a functional p53 but not in I-MEF lacking a functional p53. miR-290 overexpression is unable to address any of the tested immortalized clones toward senescence, regardless of the p53 status, suggesting that the prosenescence role of miR-290 is specific for primary but not for immortal murine fibroblasts. Moreover our findings suggest that the mere downregulation of a potential tumor suppressor miRNA in a given cell type does not necessarily imply that it behaves as a tumor suppressor

The zebrafish/tumor xenograft angiogenesis assay as a tool for screening anti-angiogenic miRNAs

The zebrafish/tumor xenograft angiogenesis assay is used to approach tumor angiogenesis, a pivotal step in cancer progression and target for anti-tumor therapies. Here, we evaluated whether the assay could allow the identification of microRNAs having an anti-angiogenic potential. For that, we transfected DU-145 prostate cancer cells with four microRNAs (miR-125a, miR-320, miR-487b, miR-492) responsive to both anti- and pro-angiogenic stimuli applied to human umbilical vein endothelial cells. After transfection, DU-145 cells were injected close to the developing subintestinal vessels of transgenic Tg(Kdrl:eGFP)s843 zebrafish embryos that express green fluorescent protein under the control of Kdrl promoter. At 72 h post-fertilization, we observed that green fluorescent protein–positive neo-vessels infiltrated the graft of DU-145 transfected with miR-125a, miR-320, and miR-487b. Vice versa, neo-vessel formation and tumor cell infiltration were inhibited when DU-145 cells transfected with miR-492 were used. These results indicated that the zebrafish/tumor xenograft assay was adequate to identify microRNAs able to suppress the release of angiogenic growth factors by angiogenic tumor cells