miR-204-5p in vivo inhibition cause diminished CD45RO cells rate in lungs of melanoma B16-bearing mice.

The treatment of melanoma remains a challenge, despite novel approaches recently becoming available for disseminated tumors. RNA targeting is being intensively studied in various types of disease. The aim of the present study was to explore whether the in vivo use of a microRNA (miR)-204-5p inhibitor affected melanoma progression, and whether its metastasis affects target organ remodeling. CD45RO+, CD3+, CD8+, forkhead box P3+, smooth muscle α-actin+ cells in the lungs of B16 melanoma-bearing mice were evaluated using immunohistochemistry following miR-204-5p inhibitor transfection. Next, CD45RO expression in peripheral blood mononuclear cells (PBMCs), as well as the apoptosis of these cells, were measured by flow cytometry. The results revealed that the number of CD45RO+ cells was decreased in the lungs of B16 melanoma-bearing mice and CD45RO+ PBMCs following the use of an miR-204-5p inhibitor, which was associated with increased levels of PBMC apoptosis. In conclusion, the findings of the present study suggested that targeting miR-204-5p in melanoma metastasis target organs could be used to develop novel approaches for the treatment of disseminated forms of the disease.

Semaphorin-5A downregulation is associated with enhanced migration and invasion of BRAF-positive melanoma cells under vemurafenib treatment in melanomas with heterogeneous BRAF status.

Tumor heterogeneity affects the efficacy of anticancer treatment as tumor subclones with distinct molecular patterns may be present within one tumor, leading to differing sensitivities to chemotherapeutic agents. In the present study, six melanoma tissue fragments were obtained from different parts of tumor of four patients and then the effect of vemurafenib treatment on biological characteristics and molecular processes of cell cultures was estimated by using MTT-test, apoptosis, migration and invasion assays, PCR real time. There was different BRAF status determined between cells derived from the central and peripheral regions of primary melanoma tumors. BRAF-positive melanoma cells showed an increased apoptotic rate under vemurafenib treatment, as well as increased migration and invasion rates, whereas BRAF-negative melanoma cells did not exhibit such tendency. Furthermore, semaphorin-5A levels were diminished in BRAF-positive cells, but not in BRAF-negative ones, which could be related to increased migration and invasion. Melanoma cells derived from different regions of the same tumor may differ by mutations status, molecular processes and biological response to target therapy. The downregulation of semaphorin-5A may be involved in divergent effects of anticancer agents on tumor cell biology.

Differences in microRNA expression between melanoma and healthy adjacent skin.

BACKGROUND: The tumor microenvironment is composed of cancer-associated fibroblasts, tumor-associated macrophages, endothelial cells, immune cells, signaling molecules and extracellular matrix structures, which closelycommunicate with the tumor via multiple mechanisms. MicroRNAs are paracrine regulators that provide a direct interaction between the microenvironment and cancer cells. In the presentstudy, we aimed to identify the microRNA expression profile in melanoma compared with thatin healthy adjacent skin, with a further assessment of altered microRNA signaling pathways and target genes. METHODS: Formalin-fixed paraffin-embedded (FFPE) melanoma tissue samples were separated by dissection into tumor and surrounding health tissue fragments. MicroRNA expression profiles were obtained by microarray using Gene Atlas Microarray System (Affymetrix, California, USA). To confirm microarray results real-time PCR was carried out. Bioinformatic analysis was performed using the DIANA-miRPath v.3.0 database. Target genes for miR-146a-5p were determined using three algorithms: TargetScan 7.0, miRWalk 2.0 and miRTarBase v.4.5. RESULTS: A microarray profiling revealed 143 microRNAs asdifferent in tumor versus adjacent tissues. Expression level of hsa-miR-146a-5p showedto be higher in melanoma cells as compared to thehealthy adjacent skin. The bioinformatic study has determined several signaling cascades associated with miR-146a-5p:Toll-like receptor pathway, NF-κB pathway, ErB pathway, and measles signaling pathway. The 38 target genes have been shown for miR-146a-5p of which NRAS gene is known asone of the most frequent mutated in melanoma. CONCLUSIONS: Elucidation of the role of miR-146-a-5p in complex interactions between the tumor and the cells of healthy adjacent skin is necessary for our understanding of the mechanisms oftumor progression. Significant differences found between cancer cells and adjacent tissues in microRNA expression profile corresponding to divergent mRNA/protein levels in these structures should be taken into account when tumor samples characterization estimatedby high-throughput methods.

miR-155 overexpression is followed by downregulation of its target gene, NFE2L2, and altered pattern of VEGFA expression in the liver of melanoma B16-bearing mice at the premetastatic stage.

MicroRNAs are involved in the control of tumour progression and in metastatic cascade dynamics. However, the role of microRNAs in distant organ reorganization at the premetastatic stage is less clear, although the process of premetastatic niche formation is a crucial event according to modern concepts of tumour dissemination. The role of the present study was to investigate the expression levels of miR-155, miR-21, miR-205 and miR-let7b, as well as that of their target genes, in target organs of melanoma metastasis at the premetastatic stage. The expression levels of both the pro-oncogenic miR-155 and the tumour suppressive miR-205 were found to be altered in the premetastatic liver of melanoma B16-bearing mice. Bioinformatics analysis identified the target genes of miR-155 to be nuclear factor, erythroid 2 like 2 (NFE2L2), secretogranin II, miR-205, semaphorin 5A and vascular endothelial growth factor A (VEGFA). Among those, the redox status regulatory factor NFE2L2 was downregulated, which corresponded to increased levels of miR-155. Due to the ability of pro-oxidative events to initiate angiogenesis, VEGFA levels were evaluated in the premetastatic liver by immunohistochemistry, which revealed increased VEGFA expression in the central parts of the organ and diminished expression in the periphery. Taken together, these findings may support the concept of functional organ reorganization due to melanoma progression.

Antiproliferative and Pro-Apoptotic Effects of MiR-4286 Inhibition in Melanoma Cells.

INTRODUCTION: MicroRNAs are essential regulators of gene expression at the post-transcriptional level. Their expression is altered in cancer tissues, and evaluation of these alterations is considered a promising tool used to diagnose and identify prognostic markers. MATERIALS AND METHODS: The microRNA expression profiles of formalin-fixed, paraffin-embedded melanoma and melanocytic nevi samples were estimated with a microarray and subsequently validated by real-time PCR. Melanoma cells were transfected with miR-4286 inhibitor to evaluate the influence of this microRNA on the viability, proliferation, apoptosis, migration, and invasion of melanoma cells. RESULTS: The microarray revealed that the expression of 1,171 microRNAs was altered in melanoma samples compared to melanocytic nevi. Real-time PCR validation experiments found the microRNA expression levels to correspond to the melanoma/melanocytic nevi microarray results. The pathway analysis identified 52 modulated pathways in melanoma. Moreover, the application of miR-4286 inhibitor to BRO melanoma cells resulted in a 2.6-fold increase in the apoptosis rate and a 1.7-fold decrease in the cell proliferation/viability but did not affect the invasiveness and migration of these cells. Furthermore, the use of miR-4286 inhibitor altered the mRNA expression of several miR-4286 gene targets: folylpolyglutamate synthase, RNA polymerase I-specific transcription initiation factor, apelin, G-protein-coupled receptor 55, and high-mobility group A1 protein, which have been implicated in cell proliferation/apoptosis regulation. Lastly, the transiently transfected SK-MEL-1 cells with miR-4286 inhibitor decreased proliferation rate and modulated folylpolyglutamate synthase rates of these cells. CONCLUSION: Our results demonstrate that miR-4286 mediates proliferation and apoptosis in melanoma cells, these findings may represent a novel mechanism underlying these processes.

Melanoma incidence mortality rates and clinico-pathological types in the Siberian area of the Russian Federation.

Russian rates for melanoma incidence and mortality are relatively low as compared to some other white populations but the tumor is of increasing importance. In this paper, data are based on a retrospective descriptive analysis of melanoma epidemiology and clinicopathological characteristics in Krasnoyarsk Territory belonging to the Siberian Federal District of the Russian Federation. The age-adjusted incidence and mortality rates for the period 1996-2009 were determined with subsequent retrospective analysis of clinicopathological data of 103 primary melanoma cases. Our results showed that incidence and mortality rates in the region under consideration match the Russian national trends and correspond to epidemiological data of the countries of Eastern Europe. Stratification of melanoma cases by age, sex, clinicopathological state and localization revealed a prevalence of lesions on the trunk and lower extremities. Most melanomas diagnosed were of superficial spreading type and the third Clark's level of tumor invasion and stage II according to AJCC. In spite of comparatively low rates of incidence and mortality the trend to increase of melanoma cases in the region under consideration obviously calls for more attention and further investigation.