Technical challenges of working with extracellular vesicles

Extracellular Vesicles (EVs) are gaining interest as central players in liquid biopsies, with potential applications in diagnosis, prognosis and therapeutic guidance in most pathological conditions. These nanosized particles transmit signals determined by their protein, lipid, nucleic acid and sugar content, and the unique molecular pattern of EVs dictates the type of signal to be transmitted to recipient cells. However, their small sizes and the limited quantities that can usually be obtained from patient-derived samples pose a number of challenges to their isolation, study and characterization. These challenges and some possible options to overcome them are discussed in this review

MicroRNA-100 Acts as a Tumor Suppressor in Human Bladder Carcinoma 5637 Cells

Bladder carcinoma is one of the most common tumors in the world and, despite the therapy currently available, most of the patients relapse. Better understanding of the factors involved in disease pathogenesis would provide insights for the development of more effective strategies in treatment. Recently, differential miRNA expression profiles in bladder urothelial carcinomas identified miR-100 down-regulation and miR-708 up-regulation among the most common alterations, although the possible influence of these miRNAs in the control of basic mechanisms in bladder tumors has not been addressed. In this context, the present study aimed to evaluate the in vitro effects of miR-100 forced expression and miR-708 inhibition in the bladder carcinoma cell line 5637. Our results showed that overexpression of miR-100 significantly inhibited growth when compared to controls at both times tested (72 and 96 hours, p<0.01) with a maximum effect at 72 hours reducing proliferation in 29.6 %. Conversely, no effects on cell growth were observed after inhibition of miR-708. MiR-100 also reduced colony formation capacity of 5637 cells by 24.4%. No alterations in cell cycle progression or apoptosis induction were observed. The effects of miR-100 on growth and clonogenicity capacity in 5637 cells evince a possible role of this miRNA in bladder carcinoma pathogenesis. Further studies are necessary to corroborate our findings and examine the potential use of this microRNA in future therapeutic interventions

Cytostatic in vitro Effects of DTCM-Glutarimide on Bladder Carcinoma Cells

Bladder cancer is a common malignancy worldwide. Despite the increased use of cisplatin-based combination therapy, the outcomes for patients with advanced disease remain poor. Recently, altered activation of the PI3K/Akt/mTOR pathway has been associated with reduced patient survival and advanced stage of bladder cancer, making its upstream or downstream components attractive targets for therapeutic intervention. In the present study, we showed that treatment with DTCM-glutaramide, a piperidine that targets PDK1, results in reduced proliferation, diminished cell migration and G1 arrest in 5637 and T24 bladder carcinoma cells. Conversely, no apoptosis, necrosis or autophagy were detected after treatment, suggesting that reduced cell numbers in vitro are a result of diminished proliferation rather than cell death. Furthermore previous exposure to 10 mu g/ml DTCM-glutarimide sensitized both cell lines to ionizing radiation. Although more studies are needed to corroborate our findings, our results indicate that PDK1 may be useful as a therapeutic target to prevent progression and abnormal tissue dissemination of urothelial carcinomas

<i>E2F1</i> and miRNA expression in a set of patient samples.

<p>A) <i>E2F1</i> expression in tumor relative to normal tissue was evaluated by qPCR; <i>PUM1</i> and <i>HMBS</i> expression were used for normalization. B) MIR205-5p and C) MIR136-5p tumor expression relative to normal expression was evaluated by qPCR; <i>RNU6b</i> expression was used for normalization. Red: patient #2 carries a somatic mutation at <i>E2F1</i>:MIR136-5p target site. Blue: patient #11 carries a germline alteration at <i>E2F1</i>:MIR205-5p target site. Dashed line represents the same expression value between normal and tumor tissues. Error bars indicate the SEM of experiments in triplicate.</p

List of genes screened by next-generation sequencing for genetic alterations at miRNA predicted target sites.

<p>List of genes screened by next-generation sequencing for genetic alterations at miRNA predicted target sites.</p

<i>E2F1</i> somatic mutation within miRNA target site impairs gene regulation in colorectal cancer

<div><p>Background</p><p>Genetic studies have largely concentrated on the impact of somatic mutations found in coding regions, and have neglected mutations outside of these. However, 3’ untranslated regions (3' UTR) mutations can also disrupt or create miRNA target sites, and trigger oncogene activation or tumor suppressor inactivation.</p><p>Methods</p><p>We used next-generation sequencing to widely screen for genetic alterations within predicted miRNA target sites of oncogenes associated with colorectal cancer, and evaluated the functional impact of a new somatic mutation. Target sequencing of 47 genes was performed for 29 primary colorectal tumor samples. For 71 independent samples, Sanger methodology was used to screen for <i>E2F1</i> mutations in miRNA predicted target sites, and the functional impact of these mutations was evaluated by luciferase reporter assays.</p><p>Results</p><p>We identified germline and somatic alterations in <i>E2F1</i>. Of the 100 samples evaluated, 3 had germline alterations at the MIR205-5p target site, while one had a somatic mutation at MIR136-5p target site. <i>E2F1</i> gene expression was similar between normal and tumor tissues bearing the germline alteration; however, expression was increased 4-fold in tumor tissue that harbored a somatic mutation compared to that in normal tissue. Luciferase reporter assays revealed both germline and somatic alterations increased <i>E2F1</i> activity relative to wild-type <i>E2F1</i>.</p><p>Conclusions</p><p>We demonstrated that somatic mutation within <i>E2F1</i>:MIR136-5p target site impairs miRNA-mediated regulation and leads to increased gene activity. We conclude that somatic mutations that disrupt miRNA target sites have the potential to impact gene regulation, highlighting an important mechanism of oncogene activation.</p></div

Position and sequencing characteristics of mutations found in miRNA predicted target sites.

<p>Position and sequencing characteristics of mutations found in miRNA predicted target sites.</p

Study overview.

<p>BWA: Burrows-Wheeler Aligner.</p