MiR-182, miR-221 and miR-222 are potential urinary extracellular vesicle biomarkers for canine urothelial carcinoma
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ABSTRACT: Urothelial carcinoma (UC) is the most common tumour type in canine bladder cancer. Current diagnostic methods are technically challenging or can lack specificity, hence there is a need for novel biomarkers of UC. To this end, we analysed the microRNA (miRNA) cargo of extracellular vesicles (EVs) from urine samples of dogs with UC to identify miRNAs that could be utilised as biomarkers. Urine was fractionated using ultrafiltration combined with size-exclusion chromatography and small RNA sequencing analysis was performed on both the EV enriched and (EV free) protein fractions. A greater number of candidate miRNA biomarkers were detected in the EV fraction than the protein fraction, and further validation using droplet digital PCR (ddPCR) was performed on the EV enriched fraction of a second cohort of dogs with bladder cancer which validated three miRNAs as candidate biomarkers; miR-182, miR-221 and miR-222 as being significantly overrepresented in dogs with UC when compared with healthy dogs and dogs with urinary tract infections. Pathway analysis confirmed that these three miRNAs are involved in cancer. In addition, their potential downstream gene targets were predicted and PIK3R1, a well-known oncogene is likely to be a shared target between miRNA-182 and miRNA-221/222. In summary, this study highlights the potential of urinary EV-associated miRNAs as a source of biomarkers for the diagnosis of canine UC.
Project description:Hepatocellular carcinoma (HCC) is one of the most common causes of death worldwide and the fourth most prevalent type of cancer. Whereas curative treatments such as liver transplantation, ablation or surgery are optimal for early stages, only paliative treatments are given to intermediate and advanced stages of the disease. Despite the introduction of immune regulators as first-line treatments for advanced stages, Sorafenib is still the standard of care in the clinical practice. In cell lysates, anti-tumoral properties of Sorafenib were related to upregulation of miR-200c-3p (anti-tumoral miRNA) at 6 hours of treatment and downregulation of miR-222-5p and miR-512-3p (pro-tumoral miRNAs) at 24 hours. We have identified these miRNA biomarkers of Sorafenib treatment response in plasma of patients with advanced HCC treated with Sorafenib. In particular, miR-200c-3p has been related to increased survival benefit whereas miR-222-5p and miR-512-3p have been related to worse prognosis. Our study has sequenced HepG2 cells treated with Sorafenib and miR-200c-3p inhibitor, and transfected with miR-222-5p and miR-512-3p mimics to unravel the molecular pathways governing Sorafenib response
Project description:Extracellular vesicles (EVs) are known to be involved in inter-cellular communication during cancer progression, however, the biogenesis of EVs in cancer cells is not completely unveiled. It has been shown that microRNAs (miRNAs) regulated variety of physiological and pathological phenomena, thus, miRNAs could regulate the EV secretion. Here, we have performed high throughput miRNA-based screening to identify the regulators of EV secretion using ExoScreen assay. By this miRNA-based screening, we identified miR-26a, which was reported as tumor suppressive miRNA, was found to be an miRNA involved in EV secretion from prostate cancer (PCa) cells. To study the effect of miR-26a on gene expression in EV secretion, transcriptome analysis for miR-26a overexpressing PCa cell lines was performed.
Project description:Recent literature has documented the use of microRNAs (miRNAs) from circulating extracellular vesicles (EVs) as biomarkers for a plethora of diseases. The aim of this prospective study was to identify the diagnostic value of plasma EV-miRNAs in sepsis.Sepsis patients and healthy controls were matched for age and gender. EVs were separated from plasma of sepsis patients at admission as well as healthy controls. The expression of EV-miRNAs was evaluated by microarray and qRT-PCR.
Project description:Currently, micro RNAs (miRNAs) constitute a promising models for cell-to-cell communication since they are transferred between cells to execute essential roles in many processes. Their structure and size, in addition to various transport mechanisms, allow them to remain stable within various biological fluids, surviving in extremely adverse conditions, including low pH, boiling, and freezing. The presence of miRNAs in reproductive fluids, such as follicular, uterine and seminal fluid, indicate potential roles in the reproductive system. These extracellular miRNAs can be transported by lipoproteins (both HDL and LDL) or other proteins, including Argonaute2 (AGO2) and nucleophosmin1 (NPM1). Another transport system is mediated by extracellular vesicles (EVs), such as apoptotic bodies, microvesicles (MVs) and/or exosome-like vesicles. EVs protect miRNAs from degradation and contribute to their stability within biological fluids. Furthermore, EVs can transport a wide range of components packaged in a selective way. For instance, Squadrito et al. (2014), used macrophages and endothelial cells to demonstrate that the sorting of miRNAs into EVs for heterotrophic cell communication is altered by both, the presence of target transcripts and the self-presence of the respectively miRNA. In addition, the signature of miRNAs found in the exosomes significantly differed for those detected in the parent cells. To date, mechanisms controlling the specific loading of miRNAs into exosomes remain unclear. Indeed, several mechanisms may govern exosome sorting of specific subsets of miRNAs. MiRNA sorting appears to be influenced by different pathways and molecules in different cell types and tissues, and miRNAs contain well defined motifs (i.e., EXOmotifs), that direct the miRNA allocation into exosomes before delivery into recipient cells. A recent study showed that this RNA sequence can be recognized by the sumoylated form of the heterogeneous ribonucleoprotein A2B1 (hnRNPA2B1). Moreover, a terminal 30 nucleotide addition in miRNAs affects their selective sorting in B cells. Another hypothesis suggests that RNAs are selectively sorted depending on the differential affinity of RNA motifs towards the raft-like region of the cytoplasmic surface of microvesicular body (MVB) limiting membranes. Current data indicate that cells can communicate with each other through the transfer of miRNA-loaded exosomes. For example, monocyte-derived exosomes deliver miR-150 to endothelial cells and enhance endothelial cell migration by reducing c-myb expression. The miRNA content of exosomes plays a critical role in such cell-to-cell communication and determines the fate of recipient cells. Thus, exosomes derived from the bone marrow mesenchymal stromal cells of myeloma patients promote tumor growth depending on the content of miR-15a in exosomes. Our group has described a novel cell-to-cell communication mechanism involving the delivery of endometrial miRNAs from the maternal endometrium to the trophectoderm cells of preimplantation embryos. Specifically, in B6C3 derived mouse embryos, we found EV-associated and free miR-30d to cause overexpression of genes involved in embryonic adhesion processes, including Itb3, Itga7 and Cdh5. Furthermore, supplementing murine embryos with miR-30d significantly improved embryo adhesion, suggesting that external miRNAs may have a functional role as transcriptomic modifiers of preimplantation embryos. Based on profiling of miRNAs in endometrial fluid, maternally-derived miRNAs are present within EVs in the uterine microenvironment. The internalization of maternally-derived exosomes has been visualized, but the mechanism by which miR-30d becomes incorporated into exosomes remains unknown. The present study aimed to elucidate the underlying mechanism of hsa-miR-30d transfer from human endometrial epithelial cells (hEECs) to the interior of exosomes and eventually to early-stage blastocysts, using a mir-30d knockout murine mode.
Project description:Purpose: The goals of this study are to compare the serum extracellular vesicle (EV) delivered miRNA levels of patients with bone-metastatic prostate cancer (PCa), non-bone -metastatic PCa and benign prostatic hyperplasia (BPH), and to identify EV-delivered microRNAs in patient’s serum as indicators for bone-metastatic PCa. Methods:Serum extracellular vesicle delivered miRNA profiles of patients with bone-metastatic PCa or non-bone -metastatic PCa or BPH were generated by miRNA chip array, using Agilent-070156 Human_miRNA_V21.0_Microarray plateform. Results: Differential analysis showed the expressions of 27 EV delivered miRNAs were significantly different between serum of patients with bone-metastatic PCa and non-bone-metastatic PCa with a p value <0.05. the expressions of 5 EV delivered miRNAs were confirmed with qRT–PCR. Conclusions: Serum EV-delivered miR-181a-5p is a promising diagnostic biomarker for bone-metastatic PCa.
Project description:The majority of metastatic clear cell renal cell carcinoma (ccRCC) patients are treated with tyrosine kinase inhibitors (TKIs) in first line, however, a fraction are refractory to these drugs. MicroRNAs (miRNA) are regulatory molecules that have proven to be accurate biomarkers in cancer. Here we identified miRNA predictive of progressive disease under TKI treatment. Whole miRNA expression was quantified by deep-sequencing in a discovery set of 74 metastatic ccRCC cases uniformly treated with TKIs. Twenty nine miRNAs were found to be differentially expressed in the tumors of patients who progressed under TKI therapy. Among six miRNAs selected for validation, an over-expression of miR-1307-3p, miR-155-5p, miR-221-3p, miR-425-5p and miR-222-3p was confirmed in patients with progressive disease as best response. A 2 miRNA-based classifier could discriminate individuals with progressive disease upon TKI treatment with a better predictive value than clinicopathological risk factors commonly used. miRNAs significantly associated with progression-free survival and overall survival were identified, and 7 miRNAs were found to overlap as predictive for early progressive disease, PFS and OS.
Project description:Background: Dilated cardiomyopathy (DCM) is the most common acquired heart disease in large- and giant-breed dogs with Doberman Pinschers representing one of the most frequently affected breeds. MicroRNAs (miRNAs) are short non-coding RNAs which play important roles in gene regulation. Different miRNA expression patterns have been described for human DCM and might represent potential diagnostic markers. There are no studies to date investigating miRNA expression profiles in canine DCM. Goals: The goals of this study were to screen the miRNAs expression profile of canine serum by using a miRNA microarray platform and to compare the miRNA expression patterns of a group of Doberman Pinschers with DCM and healthy controls. Results: Although total RNA concentrations were very low in canine serum samples, 421 different miRNAs were detectable with sufficient signal intensity on the miRNA microarrays. About 30 miRNAs were differentially expressed in the two groups, but did not reach statistical significance. No significant differences were found using specific miRNA PCR assays. Conclusions: More than 400 miRNAs can be detected in canine serum samples. Changes in expression levels of various miRNAs between healthy and DCM dogs could be detected, but the results did not reach statistical significance most probably due to the small group size. miRNAs are potential new circulating biomarkers in veterinary medicine and should be investigated in larger patient groups and additional canine diseases Blood was drawn from two groups of Doberman Pinschers: 4 healthy dogs and 4 dogs suffering from dilated cardiomyopathy. After clotting, samples were centrifuged and total mRNA was extracted from serum. These 8 serum samples were analyzed and the groups were compared
Project description:Patients with ulcerative colitis (UC) are at increased risk for colorectal cancer although mechanisms remain poorly understood. We sought to evaluate the role of miRNAs in neoplasia development in this high-risk population RNA was extracted from formalin fixed paraffin embedded tissue from controls (n=12), UC without neoplasia (UC, n=9), UC-associated neoplastic tissue (UCN, n=10; HGD-4, CRC-6) and adjacent tissue (n=9). miRNA array analysis was performed using the Nanostring nCounter System v1
Project description:Extracellular vesicles (EV) has been shown to deliver potential microRNA (miRNA) as cargo for specific target cells; effectively, the EV unique nature of the bilayer allows miRNA to be protected from degradation. We used microarray analysis to compare the miRNA profiles of EV isolated from acute antibody-mediated allograft rejection patients (AAMR) and chronic antibody-mediated allograft rejection (CAMR) compared to Tx Controls patients. By microarray miRNA profiling we detected 42 miRNAs downregulated and 34 miRNAs upregulated with FDR < 0.05 and Fold change >2. Principal Component analysis showed that these 76 miRNAs were able to distinguish AMR-derived EV from healthy TX patients. We also investigated whether differences in EV miRNA content could separate AAMR and CAMR patients. We found 9 miRNAs differentially expressed in the two AMR groups (eight downregulated and only one upregulated in AAMR compared to CAMR; effectively, these miRNAs could distinguish AAMR-derived EV from CAMR-derived EV. We then investigated the possible target genes of these miRNAs according to their expression, fold change and the p value; interestingly, several targets were associated to CDKN1A and CDKN2A genes regulation.