Faculty Profiles - MIURA Naoki

Aritada Yoshimura, Takahiro Ohmori, Daiki Hirao, Miori Kishimoto, Tomoko Iwanaga, Naoki Miura, Kazuhiko Suzuki, Ryuji Fukushima . Protective Effect on Pancreatic Acinar Cell by Maintaining Cardiac Output in Canine Heart Failure Model With Decreased Pancreatic Blood Flow . Frontiers in Veterinary Science9 925847 2022.7Reviewed International journal

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Frontiers Media SA

Heart failure cause hypoperfusion-induced damage to abdominal organs due to decreased cardiac output (CO). Using a model dog with heart failure caused by rapid ventricular pacing (RVP), we have previously demonstrated that a decrease in CO reduces pancreatic blood flow (PBF). Furthermore, we have revealed that pancreatic acinar cell atrophy, which is a change in the pre-stage of pancreatitis was caused. However, the mechanism by which pancreatic acinar cell atrophy was caused in RVP dogs remains unknown. This study aimed to clarify the association between cardiac function, PBF, and histopathological changes in pancreatic acinar cells by administrating pimobendan, which increase CO, to RVP dogs. RVP dogs were divided into the control group (no medication, n = 5) and the pimobendan group (pimobendan at 0.25 mg/kg BID, n = 5). Non-invasive blood pressure measurement, echocardiography, and contrast-enhanced ultrasonography for PBF measurement were performed before initiating RVP and at 4 weeks after initiating RVP (4 weeks). At 4 weeks, the decreases in CO, mean blood pressure and PBF due to RVP were suppressed in pimobendan group. Furthermore, histopathological examination showed no changes in pancreatic acinar cells in the pimobendan group. Overall, it was clarified that the decrease in PBF due to cardiac dysfunction was a direct cause of pancreatic acinar cell atrophy. This suggests that maintaining PBF is clinically important for treating dogs with heart failure. In addition, these findings offer a reliable basis for developing new therapeutic strategies for heart failure in dogs, that is, pancreatic protection.

DOI： 10.3389/fvets.2022.925847

Yoneshige Ryuichi, Wada Mie, Honkawa Yusuke, Miura Naoki, Miyoshi Noriaki, Ando Takaaki . A clinical case of a subepiglottic cyst in a Japanese Black calf . 84 ( 7 ) 978 - 981 2022.7Reviewed International journal

HUI-WEN CHEN, YU-CHANG LAI, MD MAHFUZUR RAHMAN, AL ASMAUL HUSNA, MD NAZMUL HASAN, HITOSHI HATAI, NORIAKI MIYOSHI, OSAMU YAMATO, NAOKI MIURA . NGS-identified miRNAs in Canine Mammary Gland Tumors Show Unexpected Expression Alterations in qPCR Analysis . In Vivo36 ( 4 ) 1628 - 1636 2022.6Reviewed International journal

Authorship：Last author,　Corresponding author Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Anticancer Research USA Inc.

Background/Aim: Canine mammary gland tumors (MGTs), as a potential model of human breast cancer, have a well-defined histological classification system. MicroRNA (miRNA) expression is a key part of the molecular signatures of both MGTs and human breast cancer, although the signatures alone do not yet provide a sufficient basis for definitive diagnosis. In this study, we investigated the association between miRNA expression patterns and histological classification. Materials and Methods: Mammary gland tissue was collected from healthy dogs (n=7) and dog patients (n=80). Further samples (n=5) were obtained from established MGT cell lines. We targeted miRNAs differentially expressed in metastatic tumor tissue versus non-metastatic and normal tissue. A subset of samples was analyzed using small RNA next generation sequencing (NGS) with subsequent qPCR. Results: Six differentially expressed miRNAs were selected from the NGS analysis and submitted for large-scale qPCR. The large-scale qPCR analysis revealed greater alternations in miRNA expression. Large-scale analysis, based on 79 samples, revealed a hierarchical clustering based on selected miRNAs that did not strikingly match the histopathological subtype classification. Conclusion: We successfully investigated the large-scale miRNA expression pattern in canine MGT and provided the whole miRNA expression. The selected miRNA demonstrated that there is no straightforward mapping between molecular signatures and histological classification of canine MGTs at the miRNA level.

DOI： 10.21873/invivo.12873

Ijiri M, Odo K, Sato M, Kawaguchi M, Fujimoto Y, Miura N, Matsuo T, Hou DX, Yamato O, Tanabe T, Kawaguchi H . Potential biomarkers for chronic seasonal heat stress in Kagoshima Berkshire pigs reared in the subtropical region . Journal of Veterinary Research66 ( 2 ) in press - 214 2022.5Reviewed International journal

Language：English Publishing type：Research paper (scientific journal)

Potential biomarkers for chronic seasonal heat stress in Kagoshima Berkshire pigs reared in the subtropical region were investigated by comparing the biomarker changes in the summer (a period of chronic heat stress) and winter (a thermoneutral period) seasons.

Pigs were allocated to summer- and winter-finishing cohorts, 12 each. The evaluations included assessment of carcass traits and internal organs’ normality carried out at the time of slaughter, and measurement of biomarkers in whole blood: derivatives of reactive oxygen metabolites (d-ROMs) and biological antioxidant potential as markers of oxidative stress, and serum amyloid A and albumin/globulin (A/G) ratio as markers of acute and chronic inflammation, respectively.

The summer-finished pigs reared under subtropical field conditions showed lower carcass quality than the winter-finished pigs, indicating a potential adverse effect of summer temperatures on the swine industry. Marginal changes were observed in d-ROMs and the A/G ratio between the summer- and winter-finishing cohorts.

The results demonstrate that d-ROMs and the A/G ratio could be used as sensitive markers for heat stress under field conditions.

Keywords
albumin/globulin ratioderivatives of reactive oxygen metabolites (d-ROMs)biological antioxidant potential (BAP)heat stressKagoshima Berkshire pigoxidative stress

DOI： 10.2478/jvetres-2022-0024

YONESHIGE Ryuichi, WADA Mie, HONKAWA Yusuke, MIURA Naoki, MIYOSHI Noriaki, ANDO Takaaki . A clinical case of a subepiglottic cyst in a Japanese Black calf . Journal of Veterinary Medical Science84 ( 7 ) 978 - 981 2022.5Reviewed International journal

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPANESE SOCIETY OF VETERINARY SCIENCE

<p>A 36-day-old Japanese Black calf exhibited wheezing associated with dyspnea from birth. Arterial blood gas analysis revealed a low oxygen partial pressure of 51 mmHg, low oxygen saturation of 83%, and high carbon dioxide partial pressure of 58.8 mmHg. Computed tomography, endoscopy, and ultrasonography showed cyst formation under the epiglottis. When the cyst was aspirated under ultrasonic guidance to secure the airway, 30 ml of viscous white turbid content was aspirated. The cyst shrank immediately after aspiration, but the wheezing and respiratory symptoms resumed 7 days after aspiration. Therefore, the cyst was surgically removed from the ventral side of the neck. No cyst remodeling was observed 30 days after surgical removal.</p>

DOI： 10.1292/jvms.22-0183

Hui Wen Chen, Yu Chang Lai, Md Mahfuzur Rahman, Al Asmaul Husna, MD Nazmul Hasan, Naoki Miura . Micro RNA differential expression profile in canine mammary gland tumor by next generation sequencing . Gene818 146237 2022.4Reviewed International coauthorship International journal

Authorship：Last author,　Corresponding author Language：Japanese Publishing type：Research paper (scientific journal)

Canine mammary gland tumors are very common and represent a potential model of human breast cancer, and microRNA (miRNAs) are promising biomarkers and therapeutic targets for these tumors. Accordingly, we aimed to identify miRNAs differentially expressed in canine mammary gland tumors using next generation sequencing (NGS), with subsequent confirmatory qPCR and target gene analyses. Mammary gland tissue was collected from healthy dogs (n=7) and dogs with suspected tumors (n=80). A subset of samples was analyzed with NGS to identify differentially expressed miRNAs with CLC Genome Workbench. Normal (n=10), tumor-adjacent (n=6), and tumor-bearing (n=76) mammary gland tissue samples were analyzed for the identified miRNAs using qPCR. An in silico analysis (TargetScan) was performed to predict the miRNAs’ target genes using gene ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (DAVID). We identified four miRNAs (cfa-miR-1-3p, cfa-miR-133a-3p, cfa-miR-133b-3p, and cfa-miR-133c-3p) as down regulated in canine mammary gland tumor tissues relative to normal and tumor adjacent tissues. KEGG analysis revealed the potential target genes of cfa-miR-1-3p are related to the Rap1 signaling pathway, adherens junction, and Ras signaling pathway, and those of the miR-133 family are related to the TGF-beta signaling pathway, synaptic vesicle cycle, and sphingolipid signaling pathway. In combination, these target genes are related to the regulation of transcription and DNA binding transcription (GO analysis), and the Hippo signaling pathway, adherens junction, and endocytosis (KEGG analysis). Accordingly, we suggest these four miRNAs are promising potential biomarker candidates for canine mammary gland tumors warranting further investigation.

DOI： 10.1016/j.gene.2022.146237

Al Asmaul Husna, Md Mahfuzur Rahman, Hui Wen Chen, Md Nazmul Hasan, Takayuki Nakagawa, Naoki Miura . Long non-coding RNA and transfer RNA-derived small fragments in exosomes are potential biomarkers for canine oral melanoma . Veterinary and Comparative Oncology20 ( 3 ) 653 - 663 2022.4Reviewed International coauthorship International journal

Authorship：Last author,　Corresponding author Language：Japanese Publishing type：Research paper (scientific journal)

Novel small non-coding RNAs (sRNAs) represent an emerging line of research in both human and canine oncology, due to their diverse regulatory and functional roles. Novel sRNAs are regarded as distinct from microRNAs, although both are part of the exosomal cargo. Recently, we reported on exosomal miRNAs as biomarkers for canine melanoma; however, it is unknown if novel sRNAs hold similar potential. Accordingly, we aimed to identify and validate novel sRNAs as potential biomarkers of canine oral melanoma, as part of our larger project on sequencing small exosomal RNA for this disease. Next generation sequencing revealed several differentially expressed novel sRNAs in exosomes from two melanoma cell lines (KMeC and LMeC) when compared with reference exosomes (from tumour-free dogs). Among these novel sRNAs, long noncoding RNA fragments, tRNA-derived fragments, snoRNAs and snRNAs were abundantly expressed. We selected four novel sRNAs upregulated in each cell line, and validated their aberrant expression with qPCR. In analysis using plasma-derived exosomes from melanoma patients, six out of the eight selected novel sRNAs showed significantly elevated expression. Receiver operating curve (ROC) analysis showed that one long non-coding RNA-derived small fragment (ENSCAFT00000069599.1) and one transfer RNA-derived small fragment (tRNA-Ala-TGC-5-1) have more than 85% sensitivity and specificity for differentiating melanoma patients from tumour-free dogs. Therefore, we consider that novel sRNAs may serve as candidate biomarkers to facilitate more accurate diagnosis of canine oral melanoma in clinical settings.

DOI： 10.1111/vco.12818

T. Ozawa, N. Miura, H. Hasegawa, T. Uemura, Y. Nakamoto, M. Tsujio, T. Takeuchi, M. Shiraishi . Characteristics and outcome of suspected cerebrovascular disease in dogs: 66 cases (2009-2016) . Journal of Small Animal Practice63 ( 1 ) 45 - 51 2021.9Reviewed International journal

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Wiley

Objectives : To characterise the clinical signs of suspected cerebrovascular disease in dogs. Materials and Methods : Medical records of one hospital were searched from November 2009 to December 2016 for dogs that suffered of cerebrovascular disease. We diagnosed cerebrovascular disease based on acute onset, clinical signs and magnetic resonance imaging findings. The medical history, clinical signs, concurrent disease, area of infarction, cerebrospinal fluid results, month at onset and outcome were investigated in the cerebrovascular disease group and in a control group (dogs with brain disorders other than cerebrovascular disease). Results : A total of 122 CVD cases were extracted from the 5312 patients that visited during the study period. Of these 122 cases, 66 (1.2%) matched the subject selection criteria of our study and were included in the analysis. Forebrain infarction was observed in 51 of 66 cases, of which 24 (47.1%) suffered from seizures. The number of dogs diagnosed with cerebrovascular disease was disproportionately high in August (nine of 59 cases) and December (13 of 59 cases). In the outcome survey, deterioration was observed in 11 of 55 cases. Clinical Significance : Seizure is an important clinical sign of cerebrovascular disease in dogs. There was a significant seasonal variation in the number of dogs diagnosed with cerebrovascular disease in Japan. Clinical features observed in this report differ from those of previous reports and highlight the need for additional research in this area.

DOI： 10.1111/jsap.13422

Al Asmaul Husna, Md Mahfuzur Rahman, Yu-Chang Lai, Hui-Wen Chen, Md Nazmul Hasan, Takayuki Nakagawa, Naoki Miura . Identification of melanoma-specific exosomal miRNAs as the potential biomarker for canine oral melanoma. . Pigment cell & melanoma research34 ( 6 ) 1062 - 1073 2021.7Reviewed International journal

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

Considering the importance of the canine cancer model of human disease, as well as the need for strategies for canine cancer management, the properties of exosomes are an emerging topic in canine oncology. In our study, exosomal RNA was isolated and investigated by next-generation sequencing. We identified several differentially expressed microRNAs (miRNAs/miRs) in the exosomes of two melanoma cell lines compared with non-tumor reference exosomes. We explored these potential melanoma-specific exosomal miRNAs further and found that miR-143 and let-7b increased in primary, whereas miR-210, 708, 221, and 222 increased in metastatic site originated melanoma cells. Further analysis showed miR-143 and 221 significantly increased in plasma exosomes of metastatic melanoma patients. Moreover, the sensitivity and specificity are >85% for differentiating the non-metastatic and metastatic patients. Therefore, these miRNAs can be an incredible biomarker candidate to identify metastatic melanoma and facilitate a better prognosis.

DOI： 10.1111/pcmr.13000

Yasunori Hino, Md Mahfuzur Rahman, Yu-Chang Lai, Al Asmaul Husna, Hui-Wen Chen, Md Nazmul Hasan, Takayuki Nakagawa, Naoki Miura . Hypoxic miRNAs expression are different between primary and metastatic melanoma cells. . Gene782 145552 - 145552 2021.5Reviewed International coauthorship International journal

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

MicroRNAs (miRNAs) can rapidly respond to cellular stresses, such as hypoxia. This immediate miRNA response regulates numerous genes and influences multiple signaling pathways. Therefore, identifying hypoxia-regulated miRNAs (HRMs) is important in canine oral melanoma (COM) to investigate their clinical significance. The hypoxic and normoxic miRNA profiles of two COM cell lines were investigated by next generation sequencing. HRMs were identified by comparing miRNA expression profiles in these cell lines with that in COM tissue. The HRM profile was different between cell lines of primary and metastatic origin, except for miR-301a and miR-8884. The time course of miRNA expression determined by qRT-PCR, especially for miR-210 and miR-301a, showed that metastatic cells are more resistant to hypoxia than primary cells. Analysis of an experimentally validated human miRNA target database revealed that miR-21 and miR-301a control a complex gene regulatory network in response to hypoxia, which includes pathways of well-known oncogenes, such as VEGF, PTEN, and TGFBR2. In conclusions, we revealed the HRM of COM. Moreover, our study shows the difference in regulation and response of hypoxic miRNAs between primary and metastatic originated melanoma cells.

DOI： 10.1016/j.gene.2021.145552