MicroRNAs (miRNAs) post-transcriptionally regulate the expression of target genes, and may behave as oncogenes or tumor suppressors. Human malignant mesothelioma is an asbestos-related cancer, with poor prognosis and low median survival. Here we report, for the first time, a cross-evaluation of miRNA expression in mesothelioma (MPP-89, REN) and human mesothelial cells (HMC-telomerase reverse transcriptase). Microarray profiling, confirmed by real-time quantitative RT-PCR, revealed a differential expression of miRNAs between mesothelioma and mesothelial cells. In addition, a computational analysis combining miRNA and gene expression profiles allowed the accurate prediction of genes potentially targeted by dysregulated miRNAs. Several predicted genes belong to terms of Gene Ontology (GO) that are associated with the development and progression of mesothelioma. This suggests that miRNAs may be key players in mesothelioma oncogenesis. We further investigated miRNA expression on a panel of 24 mesothelioma specimens, representative of the three histotypes (epithelioid, biphasic, and sarcomatoid), by quantitative RT-PCR. The expression of miR-17-5p, miR-21, miR-29a, miR-30c, miR-30e-5p, miR-106a, and miR-143 was significantly associated with the histopathological subtypes. Notably, the reduced expression of two miRNAs (miR-17-5p and miR-30c) correlated with better survival of patients with sarcomatoid subtype. Our preliminary analysis points at miRNAs as potential diagnostic and prognostic markers of mesothelioma, and suggests novel tools for the therapy of this malignancy.

Exosomes are nano-sized, cell membrane surrounded structures that are released from many cell types. These exosomes are believed to transport a range of molecules, including mRNAs, miRNAs, and proteins; the contents depending on their cell of origin. The physiological and pathological relevance of exosomes has yet to be fully elucidated. Exosomes have been implicated in cell-to-cell communication. For example, in relation to the immune system, such exosomes may enable exchange of antigen or major histocompatibility complex-peptide complexes between antigen-bearing cells and antigen-presenting cells; in cancer, they may contain molecules that not only have relevance as biomarkers, but may also be taken up and cause adverse effects on secondary cells. Furthermore, exosomes have been proposed as autologous delivery systems that could be exploited for personalised delivery of therapeutics. In order to explore the contents and functional relevance of exosomes from medium conditioned by culture cells or from other biological fluids, prior to extensive molecular profiling, they must be isolated and purified. Here, we describe differential centrifugation methods suitable for isolating exosomes from conditioned medium and from other biological fluids, including serum, saliva, tumour ascites, and urine. We also detail Western blotting and transmission electron microscopy methods suitable for basic assessment of their presence, size, and purity, prior to progressing to global mRNA, miRNA, or protein profiling.Key words: Exosomes, Multivesicular bodies, Extracellular, Cell line, Conditioned medium, Serum, Plasma, Urine, Saliva Exosomes are membrane-bound nanoparticles (30-100 nm in diameter) that are released by many cell types. These small, rightside-out structures form intracellularly by inward budding of endosome membranes (1), resulting in vesicles-containing endosomes

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Breast cancer is the second leading cause of cancer deaths. This disease is estimated to be diagnosed in over one million people worldwide and to cause more than 400,000 deaths each year. This is a significant health problem in terms of both morbidity and mortality. Chemotherapy forms part of a successful treatment regime in many cases; however, as few as half of the patients treated may benefit from this, as a result of intrinsic or acquired multiple drug resistance (MDR). A range of mechanisms of MDR has been identified using in vitro cell culture models; many, if not all, of which may contribute to breast cancer resistance in the clinical setting. This phenomenon is complicated by the heterogenous nature of breast cancer and the likely multi-factorial nature of clinical resistance. It has been very well established that a "one treatment fits all" approach is not relevant and significant advances have been made through identifying and appropriately treating sub-groups of patients; particularly with newer rationally-targeted therapies, such as the HER2-targeted monoclonal antibody, Trastuzumab, and the dual HER2 and EGFR tyrosine kinase inhibitor, Lapatinab. Furthermore, large defined collaborative studies, using standardised global profiling approaches to study mRNA, microRNAs and proteins, followed by functional genomics studies, by ourselves and others, are underway in order to definitively establish the degree of complexity contributing to drug resistance. The overall vision is to identify the optimum therapeutic regime for individual patients -possibly involving novel targeted therapies, drug resistance modulators, and chemotherapy- to overcome breast cancer.

Background/Aims: A panel of prognostic and predictive biomarkers would contribute to personalised treatment of breast cancer patients. However, many such biomarkers have yet to be identified and evaluated. The aim of this study was to investigate the relevance of 3 such putative biomarkers. Methods: TMEM25, REPS2 and Meis 1 expression was investigated by qRT-PCR, in triplicate, in 103 breast tumour biopsies procured in 1993–1994. Normal breast tissue specimens were also analysed for comparative purposes. Univariate and multivariate analyses were used to identify associations between expression of these transcripts as well as patients’ clinicopathological and survival data. Results: TMEM25, REPS2 and Meis 1 transcripts were detected in approximately 52, 78 and 40% of tumour specimens, respectively. Expression of each of the 3 genes was indicative of extended survival times from diagnosis [association between relapse-free survival (RFS) and TMEM25, p = 0.0002; REPS2, p = 0.0287; association between overall survival (OS) and TMEM25, p = 0.001; REPS2, p = 0.0131; Meis 1, p = 0.0255]. Presence of TMEM25 and Meis 1 was associated with oestrogen receptor-positive (TMEM25, p < 0.0005; Meis 1, p = 0.011), lower-grade (TMEM25, p = 0.002; Meis 1, p = 0.001) tumours. Multivariate analysis indicated TMEM25 expression to be an independent prognostic factor for extended RFS (p = 0.011) and OS (p = 0.001). Furthermore, for patients who received adjuvant chemotherapy, significantly longer survival times were achieved if their tumours expressed TMEM25 (OS, p = 0.031; RFS, p = 0.0181) and REPS2 (OS, p = 0.011). While expression of these mRNAs was generally absent from triple-negative breast tumours, statistical significance was not achieved. Conclusion: Our results suggest that TMEM25, REPS2 and Meis 1 mRNAs may be useful members of a panel of favourable prognostic and predictive markers for breast cancer and an understanding of their function may provide useful information about this disease.