BackgroundMetastatic melanoma is a devastating disease with limited therapeutic options. MicroRNAs (miRNAs) are small non coding RNA molecules with important roles in post-transcriptional gene expression regulation, whose aberrant expression has been implicated in cancer.ResultsWe show that the expression of miRNAs from a large cluster on human chromosome 14q32 is significantly down-regulated in melanoma cell lines, benign nevi and melanoma samples relative to normal melanocytes. This miRNA cluster resides within a parentally imprinted chromosomal region known to be important in development and differentiation. In some melanoma cell lines, a chromosomal deletion or loss-of-heterozygosity was observed in the cis-acting regulatory region of this cluster. In several cell lines we were able to re-express two maternally-induced genes and several miRNAs from the cluster with a combination of de-methylating agents and histone de-acetylase inhibitors, suggesting that epigenetic modifications take part in their silencing. Stable over-expression of mir-376a and mir-376c, two miRNAs from this cluster that could be re-expressed following epigenetic manipulation, led to modest growth retardation and to a significant decrease in migration in-vitro. Bioinformatic analysis predicted that both miRNAs could potentially target the 3'UTR of IGF1R. Indeed, stable expression of mir-376a and mir-376c in melanoma cells led to a decrease in IGF1R mRNA and protein, and a luciferase reporter assay indicated that the 3'UTR of IGF1R is a target of both mir-376a and mir-376c.ConclusionsOur work is the first to show that the large miRNA cluster on chromosome 14q32 is silenced in melanoma. Our results suggest that down-regulation of mir-376a and mir-376c may contribute to IGF1R over-expression and to aberrant negative regulation of this signaling pathway in melanoma, thus promoting tumorigenesis and metastasis.
BackgroundThe incidence of cutaneous malignant melanoma continues to rise, and once the disease metastasizes it is almost inevitably fatal. We recently reported that a large miRNAs cluster on human chromosome 14q32, implicated in many types of cancers, is significantly down-regulated in melanoma. miR-377, one of the miRNAs located within this cluster, was studied here.MethodsqRT-pCR was used to quantify miR-377 levels in melanoma cell lines and samples. Melanoma cell lines ectopically expressing miR-377 were generated by stable transfection, mRNA expression was assessed using mRNA arrays and protein expression was assessed by Western blot analysis. Potential targets of miR-377 were identified through luciferase reporter assays. Cellular proliferation, migration and soft-agar colony formation were monitored in control and miR-377-expressing cells using cell biology techniques.ResultsmiR-377 is expressed in normal melanocytes but not in melanoma cell lines or samples. Its ectopic stable expression in melanoma cell lines decreased their proliferative and migratory capacity and their colony-forming capability. mRNA arrays of melanoma cells over-expressing miR-377 pointed to several down-regulated mRNAs that have putative binding sites for miR-377 in their 3′UTR, of which both E2F3 and MAP3K7 were found to be direct targets of miR-377.E2F3, a potent transcriptional inducer of cell-cycle progression, was found to be elevated in melanoma cell lines, but decreased following ectopic expression of miR-377. Ectopic miR-377 also led to a decrease in the activity of a reporter plasmid containing three E2F DNA-binding sites linked to a luciferase cDNA sequence, demonstrating that miR-377 down-regulates E2F3-induced transcription.MAP3K7 (known as TAK1), a serine/threonine kinase along the MAPK signaling pathway, was over-expressed in melanoma but decreased following ectopic expression of miR-377. MAP3K7 is involved in the activation of NF-κB. MiR-377 over-expression led to decreased activity of a reporter plasmid containing two NF-κB DNA-binding sites and to decreased output along the NF-kB signaling pathway.ConclusionOur results suggest that miR-377 is an important negative regulator of E2F and MAP3K7/NF-kB signaling pathway in melanoma cells; it is tempting to speculate that its silencing in melanoma promotes the tumorigenic and metastatic potential of the cells through activation of these pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0338-9) contains supplementary material, which is available to authorized users.
The main goal of continuing medical education (CME) is to help healthcare providers (HCP) improve their knowledge and levels of competency with an ultimate enhancement of their performance in practice. Despite the long and well-intentional history of CME, the proof of success (based on improved clinical outcomes) is difficult to obtain objectively. In the past several years, the traditional CME world has been disrupted by replacing multiple-choice questions with virtual simulation. We utilised an innovative, next-generation virtual patient simulation (VPS) platform to develop objective measures to assess the success of educational activities that can be applied to the CME. This VPS platform was used at five distinct educational events designed to assess learners’ knowledge and competency in the guideline-driven management of Type 2 diabetes, hyperlipidaemia, and hypertension. A total of 432 learners (medical doctors, nurse practitioners, and clinical pharmacists) participated in these educational events of whom 149 went through two consecutive cases with a similar clinical picture and educational goals. Their ability to achieve glycaemic, lipid, and blood pressure control improved significantly as they moved from the first to the second case. The participants improved their test performance in all categories – between 5 and 38%, achieving statistically significant increases in the many goals examined. In conclusion, this study employed the pioneering application of technology to produce, collect and analyse the VPS data to evaluate objectively educational activities. This VPS platform allows not only an objective assessment of the effectiveness of the CME activity but also provides timely and helpful feedback to both learners and providers of a given educational event.
Metastatic melanoma is a devastating disease with limited treatment options. miRNAs are small RNA species within cells whose aberrant expression has been implicated in cancerous transformation, in some cases by a mechanism of cellular de-differentiation. The role of miRNA in melanoma is still not known. We recently observed that the expression of miRNAs from a large intergenic miRNA cluster is significantly down-regulated or absent in melanoma cell lines in comparison to normal melanocytes. This cluster resides within a parentally imprinted chromosomal region implicated in development and differentiation, and the miRNAs in this region are normally expressed selectively only in brain and skin. This chromosomal region was shown to be involved in the pathogenesis of ovarian carcinoma, and several of the miRNAs within it were shown to have tumor suppressor functions in other cellular models. Our results demonstrate that in some melanoma cell lines, there is a chromosomal deletion or loss of heterozygosity in the cis-acting differentially-methylated regulatory region of this cluster. Conversely, in other cell lines we were able to re-express some of these miRNAs using epigenetic modifiers (de-methylating agents and histone de-acetylase inhibitors), suggesting that epigenetic aberrations take part in their silencing. We are now assessing whether miRNA re-expression or ectopic expression decreases the tumorigenic and metastatic potential of melanoma cells. Our results suggest that aberrant regulation of this chromosomal region has a role in the pathogenesis of melanoma, and may even lead, in the future, to the development of new therapeutic strategies and bio-markers in this yet incurable disease. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A17
Background: The incidence of cutaneous malignant melanoma continues to rise, and once the disease metastasizes it is almost inevitably fatal. We were the first to report that a large micro-RNA (miRNA) cluster on human chromosome 14q32, implicated in many types of cancers, is significantly down-regulated in melanoma, and have been studying this ‘tumor-suppressor miRNA cluster’ for several years now. MiR-377, one of the miRNAs located within this cluster, was studied in this work. Methods: qRT-pCR was used to quantify miR-377 levels in melanoma cell lines and samples. Melanoma cell lines ectopically expressing miR-377 were generated by stable transfection and mRNA expression was assessed using mRNA arrays. Potential targets of miR-377 were identified through luciferase reporter assays. Cellular proliferation, migration and soft-agar colony formation were monitored in control and miR-377-expressing cells using cell biology techniques and protein expression was assessed by western blot. Results: miR-377 is expressed in normal melanocytes but not in melanoma cell lines or samples. Its ectopic stable expression in melanoma cell lines decreased their proliferative and migratory capacity and their colony-forming capability. mRNA arrays of melanoma cells over-expressing miR-377 pointed to several down-regulated mRNAs that have putative binding sites for miR-377 in their 3'UTR, of which both E2F3 and MAP3K7 were found to be direct targets of miR-377. E2F3, a potent transcriptional inducer of cell-cycle progression, was found to be elevated in melanoma cell lines, but decreased following ectopic expression of miR-377. Ectopic miR-377 also led to a decrease in the activity of a reporter plasmid containing three E2F DNA-binding sites linked to a luciferase cDNA sequence, demonstrating that miR-377 down-regulates E2F3-induced transcription. MAP3K7, a serine/threonine kinase along the MAPK signaling pathway, was over-expressed in melanoma but decreased following ectopic expression of miR-377. MAP3K7 is known to be involved in the activation of NF-κB. MiR-377 over-expression led to decreased activity of a reporter plasmid containing two NF-κB DNA-binding sites and to decreased output along the NF-κB signaling pathway. Conclusion Our results suggest that miR-377 is an important negative regulator of E2F and of the MAP3K7/NF-κB signaling pathway in melanoma cells. The NF-κB signaling has been implicated in the acquisition of resistance to B-Raf inhibition. It is tempting to speculate that silencing of miR-377 in melanoma promotes the tumorigenic and metastatic potential of the cells through activation of these pathways. Indeed we found in preliminary results that overexpression of miR-377 in melanoma cells resistance to PLX-4032 (Zelboraf), re-sensitized the cells to the drug. Citation Format: Liron Zehavi, Adi Layani, Jasmine Jacob-Hirsch, Yechezkel Sidi, Raya Leibowitz-Amit, Dror Avni. MiR-377 targets E2F3 and alters the NF-κB signaling pathway through MAP3K7 in malignant melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A15.
299 Background: Urothelial carcinoma of the bladder is among the 5 most common cancers in the US. Despite several clinical trials attempting to determine the best approach to muscle-invasive disease, the optimal treatment modalities and their sequence have not been established. Specifically, the decision to administer neo-adjuvant chemotherapy is currently based solely on clinical parameters, with no validated biomarkers. Micro-RNAs (miRNAs) are short RNA molecules that have roles in post-transcriptional gene expression regulation by binding to mRNAs. They were shown to have cardinal roles in many cancers, and their potential to serve as biomarkers is extensively studied. Our goal was to study whether miRNAs can serve as predictive biomarkers for response to neo-adjuvant chemotherapy in urothelial carcinoma. Methods: miRNAs were extracted from paraffin-embedded pre-operative muscle-invasive tumor biopsies of patients diagnosed with urothelial carcinoma, whose pathological surgical specimen was later found to either show complete or no-response to neo-adjuvant chemotherapy (termed 'responders' and 'non-responders', respectively). The expression pattern of approximately 900 miRNAs was compared using a commercial miRNA array, and the levels of candidate miRNAs was further assessed by quantitative real-time PCR (qRT-PCR). Results: The vast majority of miRNAs exhibited a similar expression pattern in the two patient groups, but two miRNAs were significantly lower in the responders (p <0.001 and q<0.1 using the false detection rate (FDR) method). Interestingly, both miRNAs can potentially target the mRNA of PTCH1 and SP5, two genes with known tumor-suppressor functions. qRT-PCR showed that high levels of one of the miRNAs correlated with lack of response to chemotherapy. Conclusions: This retrospective analysis identified two miRNAs that are differentially expressed between chemotherapy responders and non-responders. One of these miRNAs was confirmed to correlate with lack of response to neo-adjuvant chemotherapy. A prospective trial assessing the predictive values of these miRNAs is currently underway. Future research directions and potential implications will be discussed.
Introduction: A high level of medical knowledge of Advanced Practice Providers is extremely important to secure their role in the contemporary healthcare environment. Continuing Medical Education is the key element in improving their skills and level of competency. Hypothesis: We hypothesized that a mobile and desktop VPS application that provides a self-propelled simulation of disease progression and the effects of treatments, would allow precise assessment of providers’ medical knowledge. Methods: We used the Edocate® virtual patient simulation (VPS) platform to compare the level of competency in treating patients with type 2 diabetes and cardiovascular problems in two groups of nurse practitioners: diabetes-trained (DNP) and family medicine-trained (FNP). All participants completed the same VPS case before being asked to address a second case with similar problems, thus enabling us to assess improvement in their knowledge as well. For each case, the NPs received personalized and detailed feedback on their actions. Results: DNP were significantly more likely to achieve good glycemic control (78.6% vs. 46.8% in the FNP cohort), control of hyperlipidemia (57.1% vs. 40.0%), control of blood pressure (78.5% vs. 46.6%), and were more likely to use SGLT2i or GLP1 RA in patients with cardiovascular problems (78.4% vs. 66.7%). Both groups improved their performance markedly while working with their second patient, reaching levels of competency in key clinical areas between 83 and 100% in both groups. Conclusion: Working with the VPS, DNP demonstrated a higher level of medical knowledge in diabetes and cardiovascular disease management than FNP. However, training with the VPS platform significantly improved the levels of competency in both groups supporting the notion that virtual simulation provides excellent tools for enhancing providers’ knowledge, critical for their real practice skills. Disclosure I.Iancu: None. R.Shental: None. L.Zehavi: None. B.Draznin: None.
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