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MiR-182, miR-221 and miR-222 are potential urinary extracellular vesicle biomarkers for canine urothelial carcinoma

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.

ORGANISM(S): Canis lupus familiaris

PROVIDER: GSE256461 | GEO | 2024/08/08

REPOSITORIES: GEO

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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:We investigated the expression profiles of microRNAs (miRNA) in 26 renal cell carcinoma (RCC) FFPE tissues (3 chRCC, 5 papRCC and 18 ccRCC), 4 urothelial cell carcinomas of the upper urinary tract (UT-UCs) and 20 normal kidneys by using the miRCURY LNA™ microRNA Array, 6th gen (Exiqon, Woburn MA), containing capture probes that target all miRNAs for all species registered in the miRBASE version 16.0. Real-time PCR (qRT-PCR) using appropriate endogenous controls was performed in order to validate the microarray results of the 27 most differentially expressed (DE) miRNAs. We identified 434 miRNAs that were significantly deregulated in all tumours compared to the normal kidney tissues. Overall, 126 miRNAs (29%) showed increased expression and 303 miRNAs (69.8%) had decreased expression in RCCs and UT-UCs vs. the normal kidneys. Specifically, 374, 420, 421 and 409 miRNAs were 90% consistently down-regulated in ccRCC, papRCC, chRCC and UT-UC, respectively. Unsupervised two-way hierarchical clustering (HCL) with Euclidian distance accurately discriminated between RCC and UT-UC. Apart from one chRCC sample that was clustered with ccRCCs, HCL also managed to successfully classify the 3 RCC subtypes among them. Furthermore, it showed that ccRCC is more closely related to papRCC and that both are distinct from chRCC or UT-UC. Ninety-four miRNAs were co-upregulated among ccRCC, papRCC and chRCC; and 11, 44 and 24 miRNAs were specifically up-regulated in each one of the three RCC subtypes (ccRCC, chRCC and papRCC), respectively. On the other hand, 222 miRNAs were co-down-regulated in the three RCC subtypes, whereas 16, 18 and 5 miRNAs were specifically down-regulated in ccRCC, chRCC and papRCC, respectively. When the DE miRNAs in each RCC subtype were combined with those in UT-UC, we identified 89 and 206 miRNAs, respectively, that were up- and down-regulated in all tumor types. miRNA profiling can distinguish between RCC and UT-UC as well as among distinct RCC subtypes. Our data validicate that miRNA expression tends to be down-regulated in RCC versus the normal kidney tissue.

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MiR-182, miR-221 and miR-222 are potential urinary extracellular vesicle biomarkers for canine urothelial carcinoma

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