Screening Of Differential CircRNAs In Canine Mammary Tumors

Canine mammary tumors are one of the common diseases of dogs and seriously endanger their health.A large number of studies have shown that circular RNA(circRNA)is involved in the occurrence and development of tumors,and may be used as a disease-related molecular marker.However,the expression profile and clinical significance of circRNA in canine mammary tumors remains unclear.Therefore,we explored the expression profile of circRNA in canine mammary tumors by high-throughput sequencing technology.We used bioinformatics analysis software and consulted a large number of literature to lay the foundation for further research on canine mammary tumor-related genes or proteins.In our study,we analyzed the expression profiles of 3 pairs of canine mammary tumors and their adjacent normal tissues.The total RNA was extracted and RNA library was constructed.GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)analysis displayed that these genes were mainly concentrated on 14 biological pathways.We selected 11 validated circRNAs,and further confirmed the existence of these circRNAs by RT-qPCR method.The circRNA-miRNA network diagram was constructed by using Cytoscape software.Thus,circRNA expression profiles in canine mammary tumors and the functions and involved pathways of related differential genes were explored.We found that a total of 14851 circRNAs and 106 differentially expressed circRNAs in canine mammary tumors and their adjacent normal tissues(fold change ? 2,P ? 0.05).There are 64 circRNAs up-regulation and 42 circRNAs down-regulated.The main GO functions are regulation of regulated secretory pathway,regulation of neurotransmitter secretion and positive regulation of phagocytosis.Most of these pathways are related to cGMP-PKG(cyclic guanosine monophosphate)signaling pathway,cAMP(Cyclic Adenosine monophosphat)signaling pathway and OXYTOCIN signaling pathway.CircRNAs have the function of adsorbing miRNA as a sponge,and we further constructed the interaction network of circRNAs and miRNAs.The screened source-genes closely related to canine mammary tumors include RYR2,PDE4 D,ROCK2,CREB3L2 and UBA3.The screened circRNAs related to canine mammary tumors include chr27:26618544-26687235-,chr26:8194880-8201833+,chr17:7960861-7967766-,and so on.Validation of the sequencing samples revealed that the trend of gene change detected by high-throughput sequencing and low-throughput RT-qPCR was consistent,which proved the high reliability of the sequencing results.Further expansion of the sample size revealed that the expression of chr13-62685728-62729175+ was significantly down-regulated in clinical samples,showed that the gene chr13-62685728-62729175+ has certain reference value as a potential biomarker for canine mammary tumors.In different tumor cells,the expression of chr13-62685728-62729175+ in cell culture fluid changes with the expression level in tumor cells,suggesting that chr13-62685728-62729175+ is released from the tumor cells into the cell culture fluid.The RNase R enzyme assay showed that the circRNA was not easily degraded by the RNase R enzyme.Tissue-specific detection results showed that chr13-62685728-62729175+ and chr25-42146647-42163429-were expressed in various tissues of dogs and were particularly enriched in mammary gland tissue.Our study uncovered circRNA expression profiles in canine mammary tumors.It can provide a basis for the veterinary clinic to further study the key circRNAs related to the pathogenesis of canine mammary tumors.