RT-qPCR Expression Profiles of Selected Oncogenic and Oncosuppressor miRNAs in Formalin-Fixed, Paraffin-Embedded Canine Mammary Tumors

Abstract

MicroRNAs (miRNAs) can act as oncogenes or oncosuppressor genes, and their involvement in nearly all cancer-associated processes makes these small molecules promising diagnostic and prognostic biomarkers in cancer, as well as specific targets for cancer therapy. This study aimed to investigate the expression of 7 miRNAs (miR-18a, miR-18b, miR-22, miR-124, miR-145, miR-21, miR-146b) in formalin-fixed, paraffin-embedded canine mammary tumors (CMTs) by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Twenty-six mammary samples were selected, including 22 CMTs (7 benign; 15 malignant) and 4 control samples (3 normal mammary gland and 1 case of lobular hyperplasia). Oncogenic miR-18a, miR-18b and miR-21 were significantly upregulated in malignant tumors compared with control tissues (p < 0.05). Conversely, oncosuppressor miR-146b was significantly downregulated in benign and malignant mammary tumors compared with control samples (p < 0.05) while, no group-related differences in the expression levels of miR-22, miR-124 and miR-145 were found (p > 0.05). Upregulated miRNAs found here, may regulate genes involved in receptor-mediated carcinogenesis and proteoglycan remodeling in cancer; while miRNA with reduced expression can regulate genes involved in Toll-like receptor and MAPK signaling pathways. According to the results obtained in the current study, the oncogenic and oncosuppressor miRNAs analyzed here are dysregulated in CMTs and the dysregulation of miRNA targets may lead to specific altered cellular processes and key pathways involved in carcinogenesis. Of note, since oncogenic miRNAs predicted to regulate neoplastic cell proliferation and hormonal activities, they may play an active role in neoplastic transformation and/or progression, having mechanistic and prognostic relevance in CMTs.

Epigenetics in Canine Mammary Tumors: Upregulation of miR-18a and miR-18b Oncogenes Is Associated with Decreased ERS1 Target mRNA Expression and ERα Immunoexpression in Highly Proliferating Carcinomas

“…Notably, mir-18a and miR-18b were significantly overexpressed in malignant tumors (Group M1–3) compared with the normal/hyperplastic mammary gland (Group C). Furthermore, miR-18a expression was also upregulated in malignant CMTs compared with benign tumors (Group B) ( Figure 2 ) [ 30 ].…”

“…Based on the target gene analysis, ESR1 mRNA, known to be associated with a risk for CMTs [ 42 ], most likely appears to be the main target gene for both miR-18a and miR-18b (miRDB score 99) [ 30 , 31 ]. In addition, these miRNAs are also involved in other levels of epigenetic regulation and many of their gene targets are related to chromatin remodeling processes [ 30 , 31 ].…”

“…The reverse transcription (RT) reaction and RT-qPCR for miR-18a and miR-18b have been described in detail in our previous publication [ 30 ]. Briefly, RT was performed using the miRCURY ® LNA RT Kit (QIAGEN, Milan, Italy; cat.…”

“…Recently, we have demonstrated that oncogenes miR-18a and miR-18b are overexpressed in malignant CMTs compared with normal mammary tissues, and it is noteworthy that both miRNAs are predicted to target ESR1 mRNA with a very high target score (miRDB target score of 99) [ 30 ]. In agreement, both miR-18a and miR-18b have been found to be upregulated in canine-mammary-cancer-derived cell lines [ 31 ], and in the sera of dogs with metastatic mammary carcinomas [ 32 ], and also, they have been predicted to target the ESR1 gene.…”

“…However, to the best of the authors’ knowledge, no study has previously investigated the expression of miRNAs and ESR1 target mRNA in canine mammary tumors. Therefore, in this study we aimed to investigate the expression of ESR1 mRNA via real-time quantitative polymerase chain reaction (RT-qPCR) in formalin-fixed, paraffin-embedded (FFPE) CMTs and to compare the expression data with those of miR-18a and miR-18b, as previously assessed [ 30 ]. Furthermore, in order to evaluate the prognostic value of these miRNAs, this study aimed to correlate for the first time miRNA gene expression with ERα immunoexpression and the Ki67 proliferative index, as major immunohistochemical prognostic factors.…”