Identification of dysregulated non-coding small RNA in canine tumor

犬の腫瘍における発現異常ノンコーディングスモールRNAの同定

Identification of dysregulated non-coding small RNA in canine tumor

Degree Grantors Yamaguchi University

In recent years, not only mRNA (messenger RNA) but also other small non-coding RNA have focused on molecular diagnosis and therapy in oncology fields. Especially in human medicine, many studies elucidate the ability and function of many microRNAs, which are small non-coding RNAs. However, there are still not many studies in the veterinary field. In my PhD study, I focused on the non-coding small RNA in canine oncology fields. In the first chapter, I studied the dysregulated micro RNA in canine oral melanoma. At first, I performed the microarray-based miRNA profiling of canine malignant melanoma (CMM) tissue obtained from the oral cavity. Then, I also confirmed the differentially expressed microRNA by quantitative reverse transcription-PCR (qRT-PCR). An analysis of the microarray data revealed 17 dysregulated miRNAs; 5 were up-regulated, and 12 were downregulated. qRT-PCR analysis was performed for 2 up-regulated (miR-204 and miR-383), 3 down-regulated (miR-122, miR-143, and miR-205) and 6 additional oncogenic miRNAs (oncomiRs; miR-16, miR-21, miR-29b, miR-92a, miR-125b and miR-222). The expression levels of seven of the miRNAs, miR16, miR-21, miR-29b, miR-122, miR-125b, miR-204, and miR-383 were significantly up-regulated, while the expression of miR-205 was down- 2 regulated in CMM tissues compared with normal oral tissues. The microarray and qRT-PCR analyses validated the up-regulation of two potential oncomiRs, miR-204 and miR-383. I also constructed a protein interaction network and a miRNA–target regulatory interaction network using STRING and Cytoscape. In the proposed network, was a target for miR-383, and were targets for miR-204, and was a target for both. The miR-383 and miR-204 were potential oncomiRs that may be involved in regulating melanoma development by evading DNA repair and apoptosis. In my second chapter, I focused on non-coding RNA other than microRNA, and I compared canine hepatocellular carcinomas (HCC) and hepatocellular adenomas (HCA). I elucidated the differential expression of Y RNA-derived fragments because Y RNA-derived fragments have yet to be investigated in canine HCC and HCA. I used qRT-PCR to determine Y RNA expression in clinical tissues, plasma, and plasma extracellular vesicles, and two HCC cell lines (95-1044 and AZACH). Y RNA was significantly decreased in tissue, plasma, and plasma extracellular vesicles for canine HCC versus canine HCA and healthy controls. Y RNA was decreased in 95-1044 and AZACH cells versus normal liver tissue and 3 in AZACH versus 95-1044 cells. In plasma samples, Y RNA levels were decreased in HCC versus HCA and Healthy controls and increased in HCA versus Healthy controls. Receiver operating characteristic analysis showed that Y RNA could be a promising biomarker for distinguishing HCC from HCA and healthy controls. Overall, the dysregulated expression of Y RNA can distinguish canine HCC from HCA. However, further research is necessary to elucidate the underlying Y RNA-related molecular mechanisms in hepatocellular neoplastic diseases. To the best of my knowledge, this is the first report on the relative expression of Y RNA in canine HCC and HCA. In conclusion, I have demonstrated the up-regulation of potential oncomiRs, miR-16, miR-21, miR-29b, miR-122, miR-125b, miR-204 and miR383 in CMM tissues. In particular, the strong up-regulation of miR-383 in CMM tissues compared with normal oral tissues identified by microarray screening was confirmed by qRT-PCR. I conclude that miR-383 and miR-204 may promote melanoma development by regulating the DNA repair/checkpoint and apoptosis. Then, I also demonstrated the Y RNA dysregulation in the cHCC. Especially to my knowledge, this is the first report on Y RNA in canine tumors. Interestingly, this ncRNA has distinctive characteristics and differentiates malignant tumors (HCC) from benign 4 tumors (HCA). The expression pattern of Y RNA is consistent across clinical samples and cell lines. Thus, Y RNA has promising potential for differentiating HCC from HCA. Further research is required to fully elucidate the role of Y RNA in the development and progression of canine HCC and HCA.

Creators Ushio Norio