Clinical and functional aspects of microRNA regulation in human cancers

miRNAs are short single-stranded non-coding RNAs which regulate gene expression at the posttranscriptional level in many biological processes, including proliferation, apoptosis and differentiation. A number of studies have shown the importance of miRNAs in carcinogenesis and accumulating evidence supports their role as diagnostic and prognostic biomarkers in human cancers. Moreover, dysregulation of miRNA processing factors, which are needed for miRNA maturation, have been recently shown to play an important role in tumor initiation and progression and to have a prognostic potential in different types of cancer. Despite all the achievements, our knowledge of the biological role of miRNAs and miRNA processing pathway in tumor development and progression is still in its infancy. The general purpose of this thesis was to improve our understanding of the clinical and functional implications of miRNA deregulation in human cancer. In paper I, we reported frequent deregulation of miRNA expression in lymph node metastases of malignant melanoma and melanoma cell lines as compared to normal melanocytes and showed its association to BRAF and NRAS mutational status. Moreover, we identified a two-miRNA signature that could predict survival in metastatic melanoma patients. In paper II, we performed genome-wide miRNA expression profiling of adrenocortical tumors and identified distinct miRNA expression patterns in adrenocortical carcinomas (ACC) compared to adenomas and normal adrenal cortex. Over-expression of miR-483-3p/-5p and down-regulation of miR-195 and miR-497 were the most common features of ACC. We also elucidated the functional consequences of deregulation of these 4 miRNAs on cell proliferation and apoptosis in ACC cells and, in addition, we demonstrated the potential involvement of the pro-apoptotic factor PUMA (a target of miR-483-3p) in adrenocortical tumors. Moreover, we found novel miRNAs associated with short survival in ACC. In paper III, we evaluated the expression and the potential role of the main components of the miRNA machinery in adrenocortical tumors. We observed frequent over-expression of TRBP2 in ACC and found that TRBP2 mRNA expression level is a reliable predictor of carcinoma among adrenocortical tumors. These data suggest that TRBP2 may be a novel and sensitive biomarker for adrenocortical tumors. Functionally, we unraveled the potential oncogenic role of TRBP2 in ACC and identified some of the molecular mechanisms involved in the regulation of TRBP2 expression in this tumor type. In paper IV, we analyzed the expression of miRNAs and miRNA machinery factors in diffuse large B-cell lymphoma (DLBCL). We identified miRNA signatures that could discriminate DLBCL tumors from non-neoplastic tissue and found subsets of miRNAs able to classify DLBCL sub-types. Moreover, we showed dysregulation of miRNA machinery factors in DLBCL and demonstrated that it could influence miRNA processing. We also showed, similarly to our observations in ACC, that deregulation of TRBP2 expression could affect cell proliferation and cell death in lymphoma cell lines, suggesting its potential oncogenic role in DLBCL development

Gene expression profiling of advanced ovarian cancer: characterization of a molecular signature involving fibroblast growth factor 2

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Targeting CDK2 overcomes melanoma resistance against BRAF and Hsp90 inhibitors

Novel therapies are undergoing clinical trials, for example, the Hsp90 inhibitor, XL888, in combination with BRAF inhibitors for the treatment of therapy-resistant melanomas. Unfortunately, our data show that this combination elicits a heterogeneous response in a panel of melanoma cell lines including PDX-derived models. We sought to understand the mechanisms underlying the differential responses and suggest a patient stratification strategy. Thermal proteome profiling (TPP) identified the protein targets of XL888 in a pair of sensitive and unresponsive cell lines. Unbiased proteomics and phosphoproteomics analyses identified CDK2 as a driver of resistance to both BRAF and Hsp90 inhibitors and its expression is regulated by the transcription factor MITF upon XL888 treatment. The CDK2 inhibitor, dinaciclib, attenuated resistance to both classes of inhibitors and combinations thereof. Notably, we found that MITF expression correlates with CDK2 upregulation in patients; thus, dinaciclib would warrant consideration for treatment of patients unresponsive to BRAF-MEK and/or Hsp90 inhibitors and/or harboring MITF amplification/overexpression

Демографический потенциал Республики Беларусь

Материалы III Междунар. науч. конф. студентов, аспирантов и молодых ученых, Гомель, 20 мая 2010 г

Proteomics identifies neddylation as a potential therapy target in small intestinal neuroendocrine tumors.

Patients with small intestinal neuroendocrine tumors (SI-NETs) frequently develop spread disease; however, the underlying molecular mechanisms of disease progression are not known and effective preventive treatment strategies are lacking. Here, protein expression profiling was performed by HiRIEF-LC-MS in 14 primary SI-NETs from patients with and without liver metastases detected at the time of surgery and initial treatment. Among differentially expressed proteins, overexpression of the ubiquitin-like protein NEDD8 was identified in samples from patients with liver metastasis. Further, NEDD8 correlation analysis indicated co-expression with RBX1, a key component in cullin-RING ubiquitin ligases (CRLs). In vitro inhibition of neddylation with the therapeutic agent pevonedistat (MLN4924) resulted in a dramatic decrease of proliferation in SI-NET cell lines. Subsequent mass spectrometry-based proteomics analysis of pevonedistat effects and effects of the proteasome inhibitor bortezomib revealed stabilization of multiple targets of CRLs including p27, an established tumor suppressor in SI-NET. Silencing of NEDD8 and RBX1 using siRNA resulted in a stabilization of p27, suggesting that the cellular levels of NEDD8 and RBX1 affect CRL activity. Inhibition of CRL activity, by either NEDD8/RBX1 silencing or pevonedistat treatment of cells resulted in induction of apoptosis that could be partially rescued by siRNA-based silencing of p27. Differential expression of both p27 and NEDD8 was confirmed in a second cohort of SI-NET using immunohistochemistry. Collectively, these findings suggest a role for CRLs and the ubiquitin proteasome system in suppression of p27 in SI-NET, and inhibition of neddylation as a putative therapeutic strategy in SI-NET

Потребительский экстремизм как правовое явление

Материалы XIII Междунар. науч. конф. студентов, магистрантов, аспирантов и молодых ученых, Гомель, 21–22 мая 2020 г

<em>miR-205</em> Expression Promotes Cell Proliferation and Migration of Human Cervical Cancer Cells

<div><p>MicroRNAs (miRNAs) are short non-coding RNA regulators that control gene expression mainly through post-transcriptional silencing. We previously identified <em>miR-205</em> in a signature for human cervical cancer using a deep sequencing approach. In this study, we confirmed that <em>miR-205</em> expression was frequently higher in human cervical cancer than their matched normal tissue samples. Functionally, we demonstrate that <em>miR-205</em> promotes cell proliferation and migration in human cervical cancer cells. To further understand the biological roles of <em>miR-205</em>, we performed <em>in vivo</em> crosslinking and Argonaute 2 immunoprecipitation of miRNA ribonucleoprotein complexes followed by microarray analysis (CLIP-Chip) to identify its potential mRNA targets. Applying CLIP-Chip on gain- and loss-of-function experiments, we identified a set of transcripts as potential targets of <em>miR-205</em>. Several targets are functionally involved in cellular proliferation and migration. Two of them, CYR61 and CTGF, were further validated by Western blot analysis and quantification of mRNA enrichment in the Ago2 immunoprecipitates using qRT-PCR. Furthermore, both <em>CYR61</em> and <em>CTGF</em> were downregulated in cervical cancer tissues. In summary, our findings reveal novel functional roles and targets of <em>miR-205</em> in human cervical cancer, which may provide new insights about its role in cervical carcinogenesis and its potential value for clinical diagnosis.</p> </div

Evaluation of <i>CYR61</i> and <i>CTGF</i> as targets of <i>miR-205</i>.

<p>(A) Representative Western blot showing the protein expression levels of CYR61 and CTGF in cells transfected with a <i>miR-205</i> mimic, <i>miR-205</i> inhibitor, or corresponding scramble and mock transfection controls. (B) CYR61 protein expression was significantly repressed in <i>miR-205</i>-overexpressing (treated with Pre-miR-205) cells and significantly increased in <i>miR-205</i>-depleting (treated with Anti-miR-205) cells as compared to their respective negative controls. CTGF protein expression was slightly repressed in HeLa cells treated with Pre-miR-205, and slightly increased in CaSki cells treated with Anti-miR-205, but the effect was not statistically significant. Data presented represent mean of at least four independent experiments. qRT-PCR analysis of <i>CYR61</i> (C) and <i>CTGF</i> (D) mRNA in the Ago2-immunoprecipitated RNAs of <i>miR-205</i>-overexpressing or -depleted cells as compared to mock-transfection control. Relative expression level of individual mRNAs was normalized to <i>miR-21</i> expression (as endogenous control for Ago2 IP RNA). Fold change was calculated by dividing the normalized expression values of Ago2-immunoprecipiated samples by the normalized expression values of its respective input samples. Data presented represent mean of at least three independent experiments. Error bars represent standard deviations from the mean. All comparisons were evaluated using <i>t</i>-test. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001; <i>n.s.</i> = not significant.</p

Functional analyses of <i>miR-205</i> regulation in cervical cancer cell lines.

<p>(A) Cell proliferation was assessed in human cervical cancer cell lines transfected with a <i>miR-205</i> mimic (Pre-miR-205), inhibitor (Anti-miR-205) or corresponding negative control (Anti-miR Neg control or Pre-miR Neg control) using WST-1 assay. Relative cell growth was normalized to its respective control-treated cells. (B) Graphs showing relative cell migration in both <i>miR-205</i> inhibition and overexpression experiments as evaluated by Transwell migration assay. (C) Representative images of cell migration evaluated by wound healing assay. Scratch wounds were made on confluent monolayer cultures after 48 h of transfection. Images of wound repair were taken at 0, 18 and 24 h after wound (left panel). The percentage of wound closure was normalized by wound area at 0 h (right panel). Data presented represent mean of three independent experiments. Error bars represent standard deviations from the mean. All comparisons were evaluated using t-test. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001; <i>n.s.</i> = not significant.</p