Next generation sequencing (NGS) provides a powerful tool in the field of medical genetics, allowing one to perform multi-gene analysis and to sequence entire exomes (WES), transcriptomes or genomes (WGS). The generated high-throughput data are particularly suitable for enhancing the understanding of the genetic bases of complex, multi-gene diseases, such as cancer. Among the various types of tumors, those with a familial predisposition are of great interest for the isolation of novel genes or gene variants, detectable at the germline level and involved in cancer pathogenesis. The identification of novel genetic factors would have great translational value, helping clinicians in defining risk and prevention strategies. In this regard, it is known that the majority of breast/ovarian cases with familial predisposition, lacking variants in the highly penetrant BRCA1 and BRCA2 genes (non-BRCA), remains unexplained, although several less penetrant genes (e.g., ATM, PALB2) have been identified. In this scenario, NGS technologies offer a powerful tool for the discovery of novel factors involved in familial breast/ovarian cancer. In this review, we summarize and discuss the state of the art applications of NGS gene panels, WES and WGS in the context of familial breast/ovarian cancer.
Breast cancer (BC) is a common and heterogeneous disease, of which six molecular subtypes, characterized by different biological features and clinical outcomes, were described. The identification of additional biomarkers able to further connote and distinguish the different BC subtypes is essential to improve the diagnostic, prognostic and therapeutic strategies in BC patients. MicroRNAs (miRNAs) are short non-coding RNA involved in several physiological and pathological processes, including cancer development and progression. In particular, circulating miRNAs, which can be found in an adequately stable structure in serum/plasma of cancer patients, are emerging as very promising non-invasive biomarkers. Several studies have analyzed the potential role of circulating miRNAs as prognostic and therapeutic markers in BC. In the present review we describe circulating miRNAs, identified as putative biomarker in BC, with special reference to different BC molecular subtypes.
The advent of Next Generation Sequencing technologies brought with it the discovery of several microRNA (miRNA) variants of heterogeneous lengths and/or sequences. Initially ascribed to sequencing errors/artifacts, these isoforms, named isomiRs, are now considered non-canonical variants that originate from physiological processes affecting the canonical miRNA biogenesis. To date, accurate IsomiRs abundance, biological activity, and functions are not completely understood; however, the study of isomiR biology is an area of great interest due to their high frequency in the human miRNome, their putative functions in cooperating with the canonical miRNAs, and potential for exhibiting novel functional roles. The discovery of isomiRs highlighted the complexity of the small RNA transcriptional landscape in several diseases, including cancer. In this field, the study of isomiRs could provide further insights into the miRNA biology and its implication in oncogenesis, possibly providing putative new cancer diagnostic, prognostic, and predictive biomarkers as well. In this review, a comprehensive overview of the state of research on isomiRs in different cancer types, including the most common tumors such as breast cancer, colorectal cancer, melanoma, and prostate cancer, as well as in the less frequent tumors, as for example brain tumors and hematological malignancies, will be summarized and discussed.
Extracellular vesicles (EVs), defined as intercellular messengers that carry their cargos between cells, are involved in several physiological and pathological processes. These small membranous vesicles are released by most cells and contain biological molecules, including nucleic acids, proteins and lipids, which can modulate signaling pathways of nearby or distant recipient cells. Exosomes, one the most characterized classes of EVs, include, among others, microRNAs (miRNAs), small non-coding RNAs able to regulate the expression of several genes at post-transcriptional level. In cancer, exosomal miRNAs have been shown to influence tumor behavior and reshape tumor microenvironment. Furthermore, their possible involvement in drug resistance mechanisms has become evident in recent years. Hepatocellular carcinoma (HCC) is the major type of liver cancer, accounting for 75-85% of all liver tumors. Although the improvement in HCC treatment approaches, low therapeutic efficacy in patients with intermediate-advanced HCC is mainly related to the development of tumor metastases, high risk of recurrence and drug resistance. Exosomes have been shown to be involved in pathogenesis and progression of HCC, as well as in drug resistance, by regulating processes such as cell proliferation, epithelial-mesenchymal transition and immune response. Herein, we summarize the current knowledge about the involvement of exosomal miRNAs in HCC therapy, highlighting their role as modulators of therapeutic response, particularly chemotherapy and immunotherapy, as well as possible therapeutic tools.
Non-alcoholic fatty liver disease (NAFLD) is considered a relevant liver chronic disease. Variable percentages of NAFLD cases progress from steatosis to steatohepatitis (NASH), cirrhosis and, eventually, hepatocellular carcinoma (HCC). In this study, we aimed to deepen our understanding of expression levels and functional relationships between miR-182-5p and Cyld-Foxo1 in hepatic tissues from C57BL/6J mouse models of diet-induced NAFL/NASH/HCC progression. A miR-182-5p increase was detected early in livers as NAFLD damage progressed, and in tumors compared to peritumor normal tissues. An in vitro assay on HepG2 cells confirmed Cyld and Foxo1, both tumor-suppressor, as miR-182-5p target genes. According to miR-182-5p expression, decreased protein levels were observed in tumors compared to peritumor tissues. Analysis of miR-182-5p, Cyld and Foxo1 expression levels, based on datasets from human HCC samples, showed results consistent with those from our mouse models, and also highlighted the ability of miR-182-5p to distinguish between normal and tumor tissues (AUC 0.83). Overall, this study shows, for the first time, miR-182-5p overexpression and Cyld-Foxo1 downregulation in hepatic tissues and tumors from a diet-induced NAFLD/HCC mouse model. These data were confirmed by the analysis of datasets from human HCC samples, highlighting miR-182-5p diagnostic accuracy and demonstrating the need for further studies to assess its potential role as a biomarker or therapeutic target.
Purpose. Nerve growth factor efficacy was demonstrated for corneal lesions treatment, and recombinant human NGF (rhNGF) was approved for neurotrophic keratitis therapy. However, NGF-induced molecular responses in cornea are still largely unknown. We analyzed microRNAs expression in human epithelial corneal cells after time-dependent rhNGF treatment. Methods. Nearly 700 microRNAs were analyzed by qRT-PCR. MicroRNAs showing significant expression differences were examined by DIANA-miRpath v.3.0 to identify target genes and pathways. Immunoblots were performed to preliminarily assess the strength of the in silico results. Results. Twenty-one microRNAs (miR-26a-1-3p, miR-30d-3p, miR-27b-5p, miR-146a-5p, miR-362-5p, mir-550a-5p, mir-34a-3p, mir-1227-3p, mir-27a-5p, mir-222-5p, mir-151a-5p, miR-449a, let7c-5p, miR-337-5p, mir-29b-3p, miR-200b-3p, miR-141-3p, miR-671-3p, miR-324-5p, mir-411-3p, and mir-425-3p) were significantly regulated in response to rhNGF. In silico analysis evidenced interesting target genes and pathways, including that of neurotrophin, when analyzed in depth. Almost 80 unique target genes (e.g., PI3K, AKT, MAPK, KRAS, BRAF, RhoA, Cdc42, Rac1, Bax, Bcl2, FasL) were identified as being among those most involved in neurotrophin signaling and in controlling cell proliferation, growth, and apoptosis. AKT and RhoA immunoblots demonstrated congruence with microRNA expression, providing preliminary validation of in silico data. Conclusions. MicroRNA levels in response to rhNGF were for the first time analyzed in corneal cells. Novel insights about microRNAs, target genes, pathways modulation, and possible biological responses were provided. Importantly, given the putative role of microRNAs as biomarkers or therapeutic targets, our results make available data which might be potentially exploitable for clinical applications.
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