• Clinical and Experimental Reproductive Medicine
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• v.47 no.3
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• pp.194-206
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• 2020

Objective: The aim of this study was to investigate microRNAs (miRNAs) related to follicle-stimulating hormone (FSH) responsiveness using miRNA microarrays and to identify their target genes to determine the molecular regulatory pathways involved in FSH signaling in KGN cells. Methods: To change the cellular responsiveness to FSH, KGN cells were treated with FSH receptor (FSHR)-specific small interfering RNA (siRNA) followed by FSH. miRNA expression profiles were determined through miRNA microarray analysis. Potential target genes of selected miRNAs were predicted using bioinformatics tools, and their regulatory function was confirmed in KGN cells. Results: We found that six miRNAs (miR-1261, miR-130a-3p, miR-329-3p, miR-185-5p, miR-144-5p and miR-4463) were differentially expressed after FSHR siRNA treatment in KGN cells. Through a bioinformatics analysis, we showed that these miRNAs were predicted to regulate a large number of genes, which we narrowed down to cytochrome P450 family 19 subfamily A member 1 (CYP19A1) and estrogen receptor alpha (ESR1) as the main targets for miR-4463. Functional analysis revealed that miR-4463 is a regulatory factor for aromatase expression and function in KGN cells. Conclusion: In this study, we identified differentially expressed miRNAs related to FSH responsiveness. In particular, upregulation of miR-4463 expression by FSHR deficiency in human granulosa cells impaired 17β-estradiol synthesis by targeting CYP19A1 and ESR1. Therefore, our data might provide novel candidates for molecular biomarkers for use in research into poor responders.

• Molecules and Cells
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• v.39 no.2
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• pp.103-110
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• 2016

As a degenerative joint disease, osteoarthritis (OA) constitutes a major cause of disability that seriously affects the quality of life of a large population of people worldwide. However, effective treatment that can successfully reverse OA progression is lacking until now. The present study aimed to determine whether two small non-coding RNAs miR-29a and miR-140, which are significantly down-regulated in OA, can be applied together as potential therapeutic targets for OA treatment. MiRNA synergy score was used to screen the miRNA pairs that potentially synergistically regulate OA. An in vitro model of OA was established by treating murine chondrocytes with IL-$1{\beta}$. Transfection of miR-29a and miR-140 via plasmids was investigated on chondrocyte proliferation and expression of nine genes such as ADAMTS4, ADAMTS5, ACAN, COL2A1, COL10A1, MMP1, MMP3, MMP13 and TIMP metallopeptidase inhibitor 1 (TIMP1). Western blotting was used to determine the protein expression level of MMP13 and TIMP1, and ELISA was used to detect the content of type II collagen. Combined use of miR-29a and miR-140 successfully reversed the destructive effect of IL-$1{\beta}$ on chondrocyte proliferation, and notably affected the MMP13 and TIMP1 gene expression that regulates extracellular matrix. Although co-transfection of miR-29a and miR-140 did not show a synergistic effect on MMP13 protein expression and type II collagen release, but both of them can significantly suppress the protein abundance of MMP13 and restore the type II collagen release in IL-$1{\beta}$ treated chondrocytes. Compared with single miRNA transfection, cotransfection of both miRNAs exceedingly abrogated the suppressed the protein production of TIMP1 caused by IL-$1{\beta}$, thereby suggesting potent synergistic action. These results provided1novel insights into the important function of miRNAs' collaboration in OA pathological development. The reduced MMP13, and enhanced TIMP1 protein production and type II collagen release also implies that miR-29a and miR-140 combination treatment may be a possible treatment for OA.

• Experimental and Molecular Medicine
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• v.50 no.12
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• pp.13.1-13.12
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• 2018

We aimed to explore whether changes in circulating levels of miRNAs according to type 2 diabetes mellitus (T2DM) or prediabetes status could be used as biomarkers to evaluate the risk of developing the disease. The study included 462 patients without T2DM at baseline from the CORDIOPREV trial. After a median follow-up of 60 months, 107 of the subjects developed T2DM, 30 developed prediabetes, 223 maintained prediabetes and 78 remained disease-free. Plasma levels of four miRNAs related to insulin signaling and beta-cell function were measured by RT-PCR. We analyzed the relationship between miRNAs levels and insulin signaling and release indexes at baseline and after the follow-up period. The risk of developing disease based on tertiles (T1-T2-T3) of baseline miRNAs levels was evaluated by COX analysis. Thus, we observed higher miR-150 and miR-30a-5p and lower miR-15a and miR-375 baseline levels in subjects with T2DM than in disease-free subjects. Patients with high miR-150 and miR-30a-5p baseline levels had lower disposition index (p = 0.047 and p = 0.007, respectively). The higher risk of disease was associated with high levels (T3) of miR-150 and miR-30a-5p ($HR_{T3-T1}=4.218$ and $HR_{T3-T1}=2.527$, respectively) and low levels (T1) of miR-15a and miR-375 ($HR_{T1-T3}=3.269$ and $HR_{T1-T3}=1.604$, respectively). In conclusion, our study showed that deregulated plasma levels of miR-150, miR-30a-5p, miR-15a, and miR-375 were observed years before the onset of T2DM and pre-DM and could be used to evaluate the risk of developing the disease, which may improve prediction and prevention among individuals at high risk for T2DM.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.9
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• pp.1458-1468
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• 2019

Objective: As one of the most important metabolic organs, the liver plays vital roles in modulating the lipid metabolism. This study was to compare miRNA expression profiles of the Large White liver between two different developmental periods and to identify candidate miRNAs for lipid metabolism. Methods: Eight liver samples were collected from White Large of 70-day fetus (P70) and of 70-day piglets (D70) (with 4 biological repeats at each development period) to construct sRNA libraries. Then the eight prepared sRNA libraries were sequenced using Illumina next-generation sequencing technology on HiSeq 2500 platform. Results: As a result, we obtained 346 known and 187 novel miRNAs. Compared with the D70, 55 down- and 61 up-regulated miRNAs were shown to be significantly differentially expressed (DE). Gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis indicated that these DE miRNAs were mainly involved in growth, development and diverse metabolic processes. They were predicted to regulate lipid metabolism through adipocytokine signaling pathway, mitogen-activated protein kinase, AMP-activated protein kinase, cyclic adenosine monophosphate, phosphatidylinositol 3 kinase/protein kinase B, and Notch signaling pathway. The four most abundantly expressed miRNAs were miR-122, miR-26a and miR-30a-5p (miR-122 only in P70), which play important roles in lipid metabolism. Integration analysis (details of mRNAs sequencing data were shown in another unpublished paper) revealed that many target genes of the DE miRNAs (miR-181b, miR-145-5p, miR-199a-5p, and miR-98) might be critical regulators in lipid metabolic process, including acyl-CoA synthetase long chain family member 4, ATP-binding casette A4, and stearyl-CoA desaturase. Thus, these miRNAs were the promising candidates for lipid metabolism. Conclusion: Our study provides the main differences in the Large White at miRNA level between two different developmental stages. It supplies a valuable database for the further function and mechanism elucidation of miRNAs in porcine liver development and lipid metabolism.

• Animal Bioscience
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• v.37 no.6
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• pp.965-981
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• 2024

Objective: Milk composition varies considerably and depends on paratypical, genetic, and epigenetic factors. MiRNAs belong to the class of small non-coding RNAs; they are one of the key tools of epigenetic control because of their ability to regulate gene expression at the post-transcriptional level. We compared the relative expression levels of miR-106b, miR-191, and miR-30d in milk to demonstrate the relationship between the content of these miRNAs with protein and fat components of milk in Holstein and Ayrshire cattle. Methods: Milk fat, protein, and casein contents were determined in the obtained samples, as well as the content of the main fatty acids (g/100 g milk), including: saturated acids, such as myristic (C14:0), palmitic (C16:0), and stearic (C18:0) acids; monounsaturated acids, including oleic (C18:1) acid; as well as long-, medium- and short-chain, polyunsaturated, and trans fatty acids. Real-time stem-loop one-tube reverse transcription polymerase chain reaction with TaqMan probes was used to measure the miRNA expression levels. Results: The miRNA expression levels in milk samples were found to be decreased in the first two months in Holstein breed, and in the first four months in Ayrshire breed. Correlation analysis did not reveal any dependence between changes in the expression level of miRNA and milk fat content, but showed a multidirectional relationship with individual milk fatty acids. Positive associations between the expression levels of miR-106b and miR-30d and protein and casein content were found in the Ayrshire breed. Receiver operating characteristic curve analysis showed that miR-106b and miR-30d expression levels can cause changes in fatty acid and protein composition of milk in Ayrshire cows, whereas miR-106b expression level determines the fatty acid composition in Holsteins. Conclusion: The data obtained in this study showed that miR-106b, miR-191, and miR-30d expression levels in milk samples have peculiarities associated with breed affiliation and the lactation period.

• Korean Journal of Clinical Laboratory Science
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• v.47 no.4
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• pp.292-297
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• 2015

Research of thyroid cancer, liver cancer, and lung cancer has been reported in Korea. However microRNA research of multiple myeloma has never been reported. Hence we intended to confirm whether microRNA can be utilized as a diagnostic marker to patients of multiple myeloma. We also intended to evaluate whether microRNA can be detected in paraffin-embedded tissue (FFPE). This research was conducted targeting 8 samples from patients of multiple myeloma who do not have any other diseases, and 2 control samples. From January 2010 to July 2012, we selected miR-15a, miR-16, miR-21, miR-181a and miR-221 as microRNA target genes. It was decided that for a sample to be significant, the results should show values more than 1.5 or less than -1.5. Our findings of fold change were highly significant in miR-15a with a value of 37.5% (3/8). From these studies, we learned that miR-15a is useful with westerners. miR-221, on the other hand, shows conflicts with westerners, so more research will be needed in this area. In addition, it was confirmed that microRNA can be detected in paraffin embedded tissue (FFPE).

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.10
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• pp.5915-5920
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• 2013

Aim: Although aberrant miRNA expression has been documented, altered miR-101 expression in cervical cancer and its carcinogenic effects and mechanisms remain unexplored. The aim of our study was to investigate the role of miR-101 alteration in cervical carcinogenesis. Methods: Expression of miR-101 was examined by quantitative real-time reverse transcriptase PCR (qRT-PCR) in Hela cells. After modulating miR-101 expression using miR-101 mimics, cell growth, apoptosis and proliferation, and migration were tested separately by MTT or flow cytometry and cell wound healing assay and protein expression was detected by qRT-PCR. The expression of COX-2 in Hela cell was also examined by immunohistochemical staining and the correlation with miR-101 expression was analysed. Results: The miR-101 demonstrated significantly low expression in Hela cell. When we transfected miR-101 mimics into Hela cells, the modulation of miR-101 expression remarkably influenced cell proliferation, cycling and apoptosis: 1) The expression of microRNA-101 tended to increase after transfection; 2) Overexpression of miR-101 was able to promote cell apoptosis, the apoptosis rate being markedly higher (97.6%) than that seen pre-transfection (12.2%) (P<0.05); 3) The miR-101 negatively regulates cell migration and invasion, scratch results being lower ($42.7um{\pm}2um$) than that observed pre-transfection ($181.4um{\pm}2um$); 4) miRNA-101 inhibits the proliferation of Hela cells as well as the level of COX-2 protein, which was negatively correlated with miR-101 expression. Conclusions: Overexpression of miR-101 has obvious inhibitory effects on cell proliferation, migration and invasion. Thus reduced miR-101 expression could participate in the development of cervical cancer at least partly through loss of inhibition of target gene COX-2, which probably occurs in a relative late phase of carcinogenesis. Our data suggest an important role of miR-101 in the molecular etiology of cancer and indicate potential application of miR-101 in cancer therapy.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.12
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• pp.1873-1884
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• 2020

The demand for food is increasing day by day because of the increasing global population. Therefore, meat, the easiest and largely available source of protein, needs to be produced in large amounts with good quality. The pork industry is a significant shareholder in fulfilling the global meat demands. Notably, myogenesis- development of muscles during embryogenesis- is a complex mechanism which culminates in meat production. But the molecular mechanisms which govern the myogenesis are less known. The involvement of miRNAs in myogenesis and meat quality, which depends on factors such as myofiber composition and intramuscular fat contents which determine the meat color, flavor, juiciness, and water holding capacity, are being extrapolated to increase both the quantity and quality of pork. Various kinds of microRNAs (miRNAs), miR-1, miR-21, miR22, miR-27, miR-34, miR-127, miR-133, miR-143, miR-155, miR-199, miR-206, miR-208, miR-378, and miR-432 play important roles in pig skeletal muscle development. Further, the quality of meat also depends upon myofiber which is developed through the expression of different kinds of miRNAs at different stages. This review will focus on the mechanism of myogenesis, the role of miRNAs in myogenesis, and meat quality with a focus on the pig.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.4
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• pp.1801-1810
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• 2016

Recent discovery showing the presence of microRNAs (miRNAs) in the circulation sparked interest in their use as potential biomarkers. Our previous studies showed the diagnostic potential of miR-451 as a serological marker for inflammatory breast cancer (IBC), miR-337-5p and miR-30b for non-inflammatory breast cancer (non-IBC). The aim of this study is to investigate the prognostic values of circulating miRNAs by comparing the amounts of 12 circulating miRNAs in the serum of IBC and non-IBC from Tunisian breast cancer patients, and by determinating whether correlated pairs of miRNAs could provide useful information in the diagnosis of IBC and non-IBC patients. TaqMan qPCR was performed to detect circulating expression of miRNAs in serum of 20 IBC, 20 non-IBC and 20 healthy controls. Nonparametric rank Spearman rho correlation coefficient was used to examine the prognostic value of miRNAs and to assess the correlation profile between miRNAs expression. Further, a large number of miRNAs were highly correlated (rho>0.5) in both patients groups and controls. Also, the correlations profiles were different between IBC, non-IBC and healthy controls indicating important changes in molecular pathways in cancer cells. Our results showed that miR-335 was significantly overexpressed in premenopausal non-IBC patients; miR-24 was significantly overexpressed in non-IBC postmenopausal patients. Patients with previous parity had higher serum of miR-342-5p levels than those without. Furthermore, patients with HER2+ IBC present lower serum levels of miR-15a than patients with HER2-disease. Together, these results underline the potential of miRNAs to function as diagnostic and prognostic markers for IBC and non-IBC, with links to the menopausal state, Her2 status and parity.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.8
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• pp.1103-1109
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• 2018

Objective: This study aimed to screen and identify the target genes of miR-375 in pig Sertoli (ST) cells and to elucidate the effect of miR-375 on the proliferation of ST cells. Methods: In this study, bioinformatics software was used to predict and verify miR-375 target genes. Quantitative polymerase chain reaction (PCR) was used to detect the relationship between miR-375 and its target genes in ST cells. Enzyme-linked immunosorbent assay (ELISA) of rearranged L-myc fusion (RLF) and hypoxia-induced gene domain protein 1A (HIGD1A) was performed on porcine ST cells, which were transfected with a miR-375 mimics and inhibitor to verify the results. Dual luciferase reporter gene assays were performed to assess the interactions among miR-375, RLF, and HIGD1A. The effect of miR-375 on the proliferation of ST cells was analyzed by CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS). Results: Five possible target genes of miR-375, including RLF, HIGD1A, colorectal cancer associated 2, POU class 3 homeobox 1, and WW domain binding protein 1 like, were found. The results of quantitative PCR suggested that mRNA expression of RLF and HIGD1A had a negative correlation with miR-375, indicating that RLF and HIGD1A are likely the target genes of miR-375. The ELISA results revealed that RLF and HIGD1A were negatively correlated with the miR-375 protein level. The luminescence results for the miR-375 group cotransfected with wild-type RLF and HIGD1A vector were significantly lower than those of the miR-375 group co-transfected with the blank vector or mutant RLF and HIGD1A vectors. The present findings suggest that RLF and HIGD1A are target genes of miR-375 and that miR-375 inhibits ST cell proliferation according to MTS analysis. Conclusion: It was speculated that miR-375 affects cell proliferation through its target genes, which play an important role in the development of testicular tissue.