Identification of canine circulating miRNAs as tumor biospecific markers using Next-Generation Sequencing and Q-RT-PCR

Payal Agarwal; Melissa P Crepps; Natalie A Stahr; Will P Kretzschmar; Hannah C Harris; Nripesh Prasad; Shawn E Levy; Bruce F Smith

doi:10.1016/j.bbrep.2021.101106

Identification of canine circulating miRNAs as tumor biospecific markers using Next-Generation Sequencing and Q-RT-PCR

Biochem Biophys Rep. 2021 Aug 19:28:101106. doi: 10.1016/j.bbrep.2021.101106. eCollection 2021 Dec.

Authors

Payal Agarwal^{1

2}, Melissa P Crepps¹, Natalie A Stahr¹, Will P Kretzschmar¹, Hannah C Harris¹, Nripesh Prasad³, Shawn E Levy³, Bruce F Smith^{1

2}

Affiliations

¹ Scott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, USA.
² Department of Pathobiology, College of Veterinary Medicine, Auburn University, USA.
³ HudsonAlpha Discovery Life Science, Huntsville, AL 35806, USA.

Abstract

Delay in cancer diagnosis often results in metastasis and an inability to successfully treat the tumor. The use of broadly cancer-specific biomarkers at an early stage may improve cancer treatment and staging. This study has explored circulatory exosomal miRNAs as potential diagnostic biomarkers to identify cancer patients. Secretory exosomal miRNAs were isolated from 13 canine cancer cell lines (lymphoma, mast cell tumor, histiocytic cell line, osteosarcoma, melanoma, and breast tumor) and were sequenced by Next-Generation sequencing (NGS). We have identified 6 miRNAs (cfa-miR-9, -1841, -1306, -345, -132, and -26b) by NGS that were elevated in all cancer cell types. The miRNAs identified by NGS were then examined by Q-RT-PCR. The PCR data demonstrated similar expression patterns to those seen with NGS but provided fold differences that were much lower than those seen for NGS. Cfa-miR-9 was found to be the most consistently elevated miRNA in NGS and PCR, making it the most likely miRNA to prove diagnostic. In this study, we have demonstrated that it is possible to identify exosomal miRNAs with elevated secretion across multiple tumor types that could be used as circulatory diagnostic biomarkers for liquid biopsy in the future.

Keywords: Cancer; Circulatory miRNAs; Diagnostic biomarker; Dog; Next-generation sequencing.