Cognitive Function in Urban and Rural Postmenopausal Women.

BACKGROUND: Menopause is a well-known risk factor for decreasing cognitive function in women. Postmenopausal women are increasing in number but relevant studies are very scarce. This study compared the cognitive function between urban and rural postmenopausal women and assessed the influence of socio-demographic factors on cognitive function. OBJECTIVES: The aim of the study was to assess the association between the cognitive function of urban and rural postmenopausal women. METHODS: This comparative cross-sectional study was conducted among 87 urban and 87 rural postmenopausal women who were selected by purposive sampling method from the Nakhalpara and Dhamrai area of Dhaka district during the period from January to December 2020. Data were collected with a semi-structured questionnaire based on the Bengali version of the Mini-Mental State Examination (MMSE) scale through face-to-face interviews and record reviewing with a checklist. Statistical analyses of the results were obtained using Microsoft Excel (Microsoft Corporation, Redmond, WA) and SPSS version 24 (IBM Corp., Armonk, NY). RESULTS: The mean age of postmenopausal women was 58.09 ± 8.163 years in urban areas and 60.00 ± 7.562 years in rural areas. The majority (31, 35.6%) of urban women were primary school pass whereas 58 (66.7%) rural women were illiterate. The mean family income of the women was 43022.99 ± 10992.57 Bangladeshi taka (BDT) in the urban group and 14022.99 ± 5023.14 BDT in the rural group. The study revealed that 31 (35.6%) women in the urban group and 53 (60.9%) women in the rural group had abnormal cognitive function. CONCLUSION: The percentage of abnormal cognitive function was higher in rural postmenopausal women. Cognitive function has an association with monthly family income, housing condition, family type, age at the time of marriage, lifestyle, and co-morbidities. Policymakers can take the findings as a guide to formulate policies and programs for the improvement of cognitive function of postmenopausal women.

Upregulation and functional roles of miR-450b in canine oral melanoma.

Canine oral melanoma (COM) is a common and highly aggressive disease with the potential to model human melanomas. Dysregulated microRNAs represent an interesting line of research for COM because they are implicated in tumor progression. One example is miR-450b, which has been investigated for its molecular mechanisms and biological functions in multiple human cancers, but not human or canine melanoma. Here, we aimed to investigate miR-450b as a potential diagnostic biomarker of COM and its functional roles in metastatic and non-metastatic forms of the disease. We investigated the expression of miR-450b and its target mRNA genes in clinical (tumor tissue and plasma) samples and metastatic and primary-tumor cell lines. Knockdown and overexpression experiments were performed to determine the influence of miR-450b on cell proliferation, migration, colony formation, and apoptosis. miR-450b was significantly upregulated in COM and differentiated between metastatic and non-metastatic tumors, and its potential as a biomarker of metastatic and non-metastatic COM was further confirmed in ROC analysis. miR-450b knockdown promoted cell proliferation, migration, and clonogenicity and inhibited apoptosis, whereas its overexpression yielded the reverse pattern. miR-450b directly binds 3' UTR of PAX9 mRNA and modulates its function leading to BMP4 downregulation and MMP9 upregulation at the transcript level. Furthermore, we surmised that miR-450b activates the Wnt signaling pathway based on gene ontology and enrichment analyses. We concluded that miR-450b has the potential as a diagnostic biomarker and could be a target candidate for COM treatment.

Hypoxia-related Y RNA fragments as a novel potential biomarker for distinguishing metastatic oral melanoma from non-metastatic oral melanoma in dogs.

Hypoxia may promote tumor progression, and hypoxically altered noncoding RNA (ncRNA) expression may play a role in metastasis. Canine oral melanoma (COM) frequently metastasizes, and ncRNA expression under hypoxia may be clinically significant. We aimed to elucidate ncRNA fragments whose expression is altered by hypoxia in COM-derived primary KMeC and metastatic LMeC cell lines using next-generation sequencing to validate these results in qRT-PCR, and then compare expression between metastatic and non-metastatic COM. The NGS analysis and subsequent qRT-PCR validation were performed using hypoxic and normoxic KMeC and LMeC cells, and clinical samples [tumor tissue, plasma, and plasma-derived extracellular vesicles] obtained from dogs with metastatic or non-metastatic melanoma were analyzed with qRT-PCR. Y RNA was significantly decreased in metastatic LMeC cells versus primary KMeC cells in hypoxic and normoxic conditions. The expression of Y RNA was decreased in dogs with metastatic melanoma versus those with non-metastatic melanoma for all clinical sample types, reflecting the pattern found with hypoxia. Receiver operating characteristic analysis demonstrated that Y RNA level is a promising biomarker for discriminating metastatic from non-metastatic melanoma in plasma [area under the curve (AUC) = 0.993, p < 0.0001] and plasma-derived extracellular vesicles (AUC = 0.981, p = 0.0002). Overall, Y RNA may be more resistant to hypoxic stress in the metastatic than the non-metastatic state for COM. However, further investigation is required to elucidate the biological functions of Y RNA under hypoxic conditions.

YRNA and tRNA fragments can differentiate benign from malignant canine mammary gland tumors.

Mammary gland tumors (MGT) are the most common tumors in sexually intact female dogs. The functional regulation of miRNAs, a type of noncoding RNAs (ncRNAs), in canine MGT has been extensively investigated. However, the expression of other ncRNAs, such as YRNAs and transfer RNA-derived fragments (tRFs) in canine MGT is unknown. We investigated ncRNAs other than miRNAs from our small RNA project (PRJNA716131) in different canine MGT histologic subtypes. This study included benign tumors (benign mixed tumor, complex adenoma) and malignant tumors (carcinoma in benign tumor and carcinoma with metastasis) samples. Aberrantly expressed ncRNAs were examined by comparisons among MGT subtypes. The relative expression trends were validated in canine MGT tissues, plasma, extracellular vesicles, and MGT cell lines using quantitative reverse transcription PCR. Three aberrantly expressed ncRNAs were identified by comparisons among MGT subtypes. YRNA and tRNA-Gly-GCC distinguished benign mixed tumor from other MGT histologic subtypes, while tRNA-Val differentiated complex adenoma, carcinoma in benign tumors, and carcinoma with metastasis. The ROC curve of the three ncRNAs showed they might be potential biomarkers to discriminate malignant from benign MGT. YRNA and tRFs expression levels were decreased in metastatic compared with primary canine MGT cell lines. To the best of our knowledge, this is the first investigation of YRNA and tRFs in canine MGT. The three identified ncRNAs may be biomarkers for differentiating MGT histologic subtypes. Suggested Reviewers: Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporatio.

Elevated expression of miR-301a and its functional roles in canine oral melanoma.

miR-301a is one of numerous dysregulated microRNAs (miRNAs) in canine oral melanoma (COM), one of which is miR-301a (upregulated). Its biological role has been described in various human cancer types, including malignant melanoma, but not in COM. Accordingly, in this study, we investigated miR-301a expression in COM in greater detail to ascertain whether it could serve as a diagnostic biomarker, elucidate its functional roles in this cancer, and predict the possible pathways by which it exerts its effects. Relative expression of miR-301a was investigated in clinical oral tissue and plasma samples and COM cell (KMeC and LMeC) lines using qRT-PCR. Knockdown of miR-301a was also validated for KMeC and LMeC cells using qRT-PCR. We performed CCK-8 assays to assess cell proliferation, monolayer wound-healing, and transwell migration assays to assess cell migration, a colony-formation assay to assess clonogenicity, a TUNEL assay and flow cytometry to assess apoptosis-related effects, and gene enrichment analyses to predict possible related pathways. miR-301a was markedly upregulated in COM oral tissue and plasma clinically, suggesting its potential as a diagnostic biomarker for COM diagnosis. In vitro assays demonstrated that miR-301 significantly inhibited apoptosis in COM cells while promoting cell migration, proliferation, and clonogenicity. We also predicted that miR-301 exerts cancer-promoting effects through the Wnt signalling pathway for COM. Our findings suggest that miR-301a is a COM oncomiR that regulates several oncogenic phenotypes with the potential to be a diagnostic biomarker.

NGS-identified miRNAs in Canine Mammary Gland Tumors Show Unexpected Expression Alterations in qPCR Analysis.

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.

Long non-coding RNA and transfer RNA-derived small fragments in exosomes are potential biomarkers for canine oral melanoma.

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.

Micro RNA differential expression profile in canine mammary gland tumor by next generation sequencing.

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.

Identification of melanoma-specific exosomal miRNAs as the potential biomarker for canine oral melanoma.

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.

Hypoxic miRNAs expression are different between primary and metastatic melanoma cells.

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.

Bovine serum miR-21 expression affected by mastitis.

The expression levels of circulating microRNAs (miRNAs) can be affected by disease. The miRNA released from cells within exosomes can act as a remote communication tool and can participate in inflammatory response regulation. Therefore, circulating miRNA has the potential to be an indicator of local disease. The objective of this study was to investigate the serum level of bovine mastitis-related miRNAs. We found that miR-16 expression in serum was affected by hemolysis. The expression levels of miR-21 in serum were increased significantly in cows with mastitis compared with unaffected controls; however, the expression levels of miR-146a, miR-155, miR-222 and miR-383 in cows with mastitis were unchanged. We further verified the upregulation of miR-21 in the serum of cows with mastitis using a digital PCR system. Although the sensitivity and specificity of miR-21 in the serum to detect bovine mastitis was inferior to miRNA biomarkers in the milk, the significant increase of miR-21 in serum may reflect the impact of local inflammation on the systemic reaction.

Aberrantly expressed snoRNA, snRNA, piRNA and tRFs in canine melanoma.

Among small non-coding RNAs (sncRNAs/sRNAs), the functional regulation of microRNAs (miRNAs) has been studied in canine oral melanoma (COM). However, the expression level of other sncRNAs, like small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), transfer RNA-derived fragments (tRFs) and PIWI-interacting RNAs (piRNAs), in COM is unknown. The aim of this study was to investigate sncRNAs other than miRNAs in COM from our small RNA sequencing project (PRJNA516252). We found that several snRNAs and piRNAs were upregulated, whereas tRFs and snoRNAs were downregulated in COM. Upregulation of U1 snRNA and piR-972, and downregulation of tRNA-ser (1) and snoRA24 was confirmed in dog melanoma tissue and cell lines by quantitative reverse transcription PCR. Consistently, the expression of tRNA-ser (1) and snoRA24 in plasma of COM cases was also decreased. Finally, we found a similar expression trend of U1 and snoRA24 in the human cutaneous melanoma cell line, MEWO, compared with human epidermal melanocyte cells (HEMa-Lp). In our study, snRNA, snoRNA, tRFs and piRNA were dysregulated during melanoma progression. Moreover, the melanoma-associated expression of U1 and snoRA24 was similar in human and dog melanoma.

Transcriptome analysis of dog oral melanoma and its oncogenic analogy with human melanoma.

Dogs have been considered as an excellent immunocompetent model for human melanoma due to the same tumor location and the common clinical and pathological features with human melanoma. However, the differences in the melanoma transcriptome between the two species have not been yet fully determined. Considering the role of oncogenes in melanoma development, in this study, we first characterized the transcriptome in canine oral melanoma and then compared the transcriptome with that of human melanoma. The global transcriptome from 8 canine oral melanoma samples and 3 healthy oral tissues were compared by RNA­Seq followed by RT­qPCR validation. The results revealed 2,555 annotated differentially expressed genes, as well as 364 novel differentially expressed genes. Dog chromosomes 1 and 9 were enriched with downregulated and upregulated genes, respectively. Along with 10 significant transcription site binding motifs; the NF­κB and ATF1 binding motifs were the most significant and 4 significant unknown motifs were indentified among the upregulated differentially expressed genes. Moreover, it was found that canine oral melanoma shared >80% significant oncogenes (upregulated genes) with human melanoma, and JAK­STAT was the most common significant pathway between the species. The results identified a 429 gene signature in melanoma, which was up­regulated in both species; these genes may be good candidates for therapeutic development. Furthermore, this study demonstrates that as regards oncogene expression, human melanoma contains an oncogene group that bears similarities with dog oral melanoma, which supports the use of dogs as a model for the development of novel therapeutics and experimental trials before human application.

Bovine milk transcriptome analysis reveals microRNAs and RNU2 involved in mastitis.

Mastitis is a common inflammatory infectious disease in dairy cows. To understand the microRNA (miRNA) expression profile changes during bovine mastitis, we undertook a genome-wide miRNA study of normal milk and milk that tested positive on the California mastitis test for bovine mastitis (CMT+). Twenty-five miRNAs were differentially expressed (23 miRNAs upregulated and two downregulated) during bovine mastitis relative to their expression in normal milk. Upregulated mature miR-1246 probably derived from a U2 small nuclear RNA rather than an miR-1246 precursor. The significantly upregulated miRNA precursors and RNU2 were significantly enriched on bovine chromosome 19, which is homologous to human chromosome 17. A gene ontology analysis of the putative mRNA targets of the significantly upregulated miRNAs showed that these miRNAs were involved in binding target mRNA transcripts and regulating target gene expression, and a Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the upregulated miRNAs were predominantly related to cancer and immune system pathways. Three novel miRNAs were associated with bovine mastitis and were relatively highly expressed in milk. We confirmed that one of the novel mastitis-related miRNAs was significantly upregulated using a digital PCR system. The differentially expressed miRNAs were involved in human cancers, infections, and immune-related diseases. The genome-wide analysis of miRNA profiles in this study provides insight into bovine mastitis and inflammatory diseases. DATABASES: The miRNAseq generated for this study can be found in the Sequence Read Archive (SRA) under BioProject Number PRJNA421075 and SRA Study Number SRP126134 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA421075).

Micro RNA Transcriptome Profile in Canine Oral Melanoma.

MicroRNAs (miRNAs) dysregulation contribute the cancer pathogenesis. However, the miRNA profile of canine oral melanoma (COM), one of the frequent malignant melanoma in dogs is still unrevealed. The aim of this study is to reveal the miRNA profile in canine oral melanoma. MiRNAs profile of oral tissues from normal healthy dogs and COM patients were compared by next-generation sequencing. Along with tumour suppressor miRNAs, we report 30 oncogenic miRNAs in COM. The expressions of miRNAs were further confirmed by quantitative real-time PCR (qPCR). Pathway analysis showed that deregulated miRNAs impact on cancer and signalling pathways. Three oncogenic miRNAs targets (miR-450b, 301a, and 223) from human study also were down-regulated in COM and had a significant negative correlation with their respective miRNA. Furthermore, we found that miR-450b expression is higher in metastatic cells and regulated MMP9 expression through a PAX9-BMP4-MMP9 axis. In silico analysis indicated that miR-126, miR-20b, and miR-106a regulated the highest numbers of differentially expressed transcription factors with respect to human melanoma. Chromosomal enrichment analysis revealed the X chromosome was enriched with oncogenic miRNAs. We comprehensively analyzed the miRNA's profile in COM which will be a useful resource for developing therapeutic interventions in both species.