scholarly journals MiR-378 Plays an Important Role in the Differentiation of Bovine Preadipocytes

2015 ◽  
Vol 36 (4) ◽  
pp. 1552-1562 ◽  
Author(s):  
Si-Yuan Liu ◽  
Yang-Yang Zhang ◽  
Yan Gao ◽  
Lian-Jiang Zhang ◽  
Hong-Yan Chen ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Target Genes ◽  
Adipocyte Differentiation ◽  
Binding Protein ◽  
Farm Animals ◽  
Luciferase Reporter ◽  
Preadipocyte Differentiation ◽  
Expression Levels ◽  
Triglyceride Content ◽  
Mrna Expression Levels

Background: Adipocyte, the main cellular component of white adipose tissue, plays a vital role in energy balance in higher eukaryotes. In recent years, adipocytes have also been identified as a major endocrine organ involved in immunological responses, vascular diseases, and appetite regulation. In farm animals, fat content and categories are closely correlated with meat quality. MicroRNAs (miRNAs), a class of endogenous single-stranded non-coding RNA molecules, participate in the regulation of adipocyte differentiation and adipogenesis through regulating the transcription or translation of target mRNAs. MiR-378 plays an important role in a number of biological processes, including cell growth, cell differentiation, tumor cell survival and angiogenesis. Methods: In the present study, bioinformatics analysis and dual-luciferase reporter assay were used to identify and validate the target genes of miR-378. In vitro cell transfection, quantitative reverse transcription polymerase chain reaction (RT-qPCR), western blot analysis, Oil Red O staining, and triglyceride content measurement were conducted to analyze the effects of miR-378 on bovine preadipocyte differentiation. Results: MiR-378 was induced during adipocyte differentiation. In the differentiated adipocytes overexpressing miR-378, the volume of lipid droplets was enlarged, and the triglyceride content was increased. Moreover, the mRNA expression levels of the adipocyte differentiation marker genes, peroxisome proliferator-activated receptor gamma (PPARγ) and sterol regulatory element-binding protein (SREBP), were significantly elevated in the differentiated, mature adipocytes. In contrast, the mRNA expression level of preadipocyte factor 1 (Pref-1) was markedly reduced. E2F transcription factor 2 (E2F2) and Ras-related nuclear (RAN)-binding protein 10 (RANBP10) were the two target genes of miR-378. The mRNA expression levels of E2F2 and RANBP10 did not significantly change in bovine preadipocytes overexpressing miR-378. However, the protein expression levels of E2F2 and RANBP10 were markedly reduced. Conclusion: MiR-378 promoted the differentiation of bovine preadipocytes. E2F2 and RANBP10 were the two target genes of miR-378, and might involve in the effects of miR-378 on the bovine preadipocyte differentiation.

Fragile X-related protein 1 (FXR1) regulates cyclooxygenase-2 (COX-2) expression at the maternal–fetal interface

2018 ◽  
Vol 30 (11) ◽  
pp. 1566 ◽  
Author(s):  
Xiao-Cui Li ◽  
Meng-fan Song ◽  
Feng Sun ◽  
Fu-Ju Tian ◽  
Yu-mei Wang ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Recurrent Miscarriage ◽  
Fragile X ◽  
Firefly Luciferase ◽  
Cyclooxygenase 2 ◽  
Luciferase Reporter ◽  
Related Protein ◽  
Expression Levels ◽  
Cox 2 ◽  
Mrna Expression Levels

Cyclooxygenase-2 (COX-2) is regulated post-transcriptionally by the AU-rich element (ARE) in the 3′-untranslated region (UTR) of its mRNA. However, the mechanism of COX-2 induction in infertility has not been thoroughly elucidated to date. The aim of this study was to examine the association between COX-2 and fragile X-related protein 1 (FXR1) in trophoblasts. Using quantitative reverse transcription polymerase chain reaction, our results showed that FXR1 mRNA expression levels were significantly decreased in trophoblasts from recurrent miscarriage patients compared with healthy controls; conversely, COX-2 mRNA expression levels were increased in patient samples. We also observed that FXR1 was highly expressed in human placental villi during early pregnancy. Furthermore, we used western blotting and immunofluorescence to analyse the expression levels of FXR1 and COX-2 in HTR-8 cells that were treated with tumour necrosis factor α; we observed that the expression of COX-2 was clearly increased in HTR-8 cells treated with FXR1 small interfering RNA, whereas the expression of COX-2 was effectively decreased in HTR-8 cells with FXR1 overexpressed via a plasmid. Importantly, bioinformatics analysis identified FXR1 binding sites in the 3′-UTR region of COX-2 and firefly luciferase reporter assay analysis verified that FXR1 binds directly to the 3′-UTR region of COX-2. ELISA assays showed that overexpression of FXR1 enhanced vascular endothelial growth factor-A and interleukin-8 expression in HTR-8 cells, whereas conversely, knockdown of FXR1 effectively repressed these effects. In conclusion, the results of this study indicate that FXR1 is a novel COX-2 regulatory factor.

scholarly journals MiR-9-5p promotes rabbit preadipocyte differentiation by suppressing leptin gene expression

2020 ◽  
Author(s):  
Gang Luo ◽  
Shenqiang Hu ◽  
Tianfu Lai ◽  
Jie Wang ◽  
Li Wang ◽  
...  
Keyword(s):  
Binding Protein ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Marker Genes ◽  
Reporter Assay ◽  
Preadipocyte Differentiation ◽  
Fatty Acid Binding ◽  
Triglyceride Content ◽  
Oil Red O Staining ◽  
Oil Red O

Abstract Background: MicroRNAs (miRNAs) are a class of small non-coding RNAs, which participate in the regulation of cell differentiation. Previous studies have demonstrated that miR-9-5p plays a key role in cancer cell development, but the mechanisms by which miR-9-5p regulates adipogenesis remain poorly understood. The present study intended to investigate its significance in producing rabbits with high-quality meat by observing the regulatory effect of miR-9-5p in preadipocytes and finding the related targets. Methods: In this study, a dual-luciferase reporter assay was employed to validate the targeting relationship between miR-9-5p and leptin gene. We also utilized quantitative reverse transcription PCR (qRT-PCR), western blot, oil red-O staining assay, and determination of triglyceride content to analyze the regulation of miR-9-5p and leptin gene during adipocyte differentiation. Results: The analysis demonstrated that during preadipocyte differentiation, miR-9-5p was up-regulated and the fat formation related biomarkers, i.e., fatty acid-binding protein 4 (FABP4), CCAAT-enhancer binding protein α (C/EBPα), and peroxisome proliferator activated receptor γ (PPARγ) were also up-regulated. Meanwhile, the oil red-O staining assay revealed that the accumulation of lipid droplets increased. We also explored the expression pattern and role of miR-9-5p in adipogenesis using white pre-adipocytes. The results showed that miR-9-5p was up-regulated during preadipocyte differentiation, and overexpression of miR-9-5p enhanced lipid accumulation. Furthermore, we found that the overexpression of miR-9-5p significantly up- regulated the expression of marker genes, PPARγ, C/EBPα and FABP4, and increased the protein levels of PPARγ and triglyceride content. The results suggest that miR-9-5p might be involved in the regulation of rabbit preadipocyte differentiation. We predicted that leptin is the target gene of miR-9-5p, by using bioinformatics tools and the conclusion was validated by a luciferase reporter assay. Finally, we verified that the knock-down of leptin by si-leptin promoted preadipocyte differentiation in rabbits. Conclusion: The results of the present study indicate that miR-9-5p regulates white preadipocyte differentiation in rabbits by targeting the leptin gene.

scholarly journals miR-214-5p Regulating Differentiation of Intramuscular Preadipocytes in Goats via Targeting KLF12

2021 ◽  
Vol 12 ◽  
Author(s):  
Yu Du ◽  
Yong Wang ◽  
Yanyan Li ◽  
Quzhe Emu ◽  
Jiangjiang Zhu ◽  
...  
Keyword(s):  
Target Gene ◽  
Target Genes ◽  
Adipocyte Differentiation ◽  
Specific Binding ◽  
Intramuscular Fat ◽  
Hormone Sensitive Lipase ◽  
Expression Level ◽  
Luciferase Reporter ◽  
Peroxisome Proliferator ◽  
Preadipocyte Differentiation

Intramuscular fat (i.m.) is an adipose tissue that is deposited between muscle bundles. An important type of post-transcriptional regulatory factor, miRNAs, has been observed as an important regulator that can regulate gene expression and cell differentiation through specific binding with target genes, which is the pivotal way determining intramuscular fat deposition. Thus, this study intends to use RT-PCR, cell culture, liposome transfection, real-time fluorescent quantitative PCR (qPCR), dual luciferase reporter systems, and other biological methods clarifying the possible mechanisms on goat intramuscular preadipocyte differentiation that is regulated by miR-214-5p. Ultimately, our results showed that the expression level of miR-214-5p peaked at 48 h after the goat intramuscular preadipocytes were induced for adipogenesis. Furthermore, after inhibition of the expression of miR-214-5p, the accumulation of lipid droplets and adipocyte differentiation in goat intramuscular adipocytes were promoted by the way of up-regulation of the expression level of lipoprotein lipase (LPL) (p < 0.05) and peroxisome proliferator-activated receptor gamma (PPARγ) (p < 0.01) but inhibited the expression of hormone-sensitive lipase (HSL) (p < 0.01). Subsequently, our study confirmed that Krüppel-like factor 12 (KLF12) was the target gene of miR-214-5p. Inhibition of the expression of KLF12 promoted adipocyte differentiation and lipid accumulation by upregulation of the expression of LPL and CCAAT/enhancer binding protein (C/EBPα) (p < 0.01). Overall, these results indicated that miR-214-5p and its target gene KLF12 were negative regulators in progression of goat preadipocyte differentiation. Our research results provided an experimental basis for finally revealing the mechanism of miR-214-5p in adipocytes.

scholarly journals Effect of mir-129 on the Sensitivity Enhancement of Methotrexate and Migration Inhibition in Osteosarcoma Cancer Cells

2021 ◽  
Keyword(s):  
Mrna Expression ◽  
Cancer Cells ◽  
Target Genes ◽  
Osteosarcoma Cells ◽  
Cytotoxic Effects ◽  
Growth And Survival ◽  
Expression Levels ◽  
Cell Death Assay ◽  
Underlying Mechanisms ◽  
Mrna Expression Levels

Background: MicroRNAs have been recently declared to be contributed to the various aspects of osteosarcoma cells, including growth and survival, apoptosis, invasion, and chemoresistance. Objectives: The present study aimed to investigate the potentiating effects of miR-129 on the chemosensitivity of Saose-2 osteosarcoma cells to methotrexate (MTX) and underlying mechanisms. Methods: Saose-2 cells were transfected with miR-129 mimics using Lipofectamine. The cytotoxic effects of miR-129 and MTX on Saose-2 cells were measured using MTT assay. Scratch wound healing assay was used to evaluate cell migration. The apoptosis rate of cancer cells was also measured using ELISA Cell Death Assay and flow cytometry. The mRNA expression levels of target genes were measured using quantitative RT-PCR. Results: miR-129 mimic transfection significantly increased the expression levels of this miRNA in Saose-2 cells (P<0.05). The combination of MTX with miR-129 transfection led to enhanced cytotoxic effects of MTX in lower concentrations. In addition, miR-129 significantly increased MTX-induced apoptosis levels and decreased invasion behavior in Saose-2 cells. The mRNA expression levels of c-Myc, K-Ras, CXCR4, MMP9, and ADAMTS, as main genes involved in chemoresistance and invasion, were downregulated in miR-129 transfected cells. Conclusion: The obtained results revealed the importance of miR-129 in the sensitivity of osteosarcoma cells to MTX and its underlying mechanisms. Therefore, miR-129 might be an appropriate candidate for reversing MTX resistance in osteosarcoma cells.

scholarly journals Tissue-specific Nutrient Metabolism in the Liver and Muscle of Juvenile Blunt Snout Bream (Megalobrama Amblycephala) in Response to Dietary Methionine Levels

2021 ◽  
Author(s):  
Ke Ji ◽  
Hualiang Liang ◽  
Mingchun Ren ◽  
Xianping Ge ◽  
Lu Zhang ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Mrna Expression ◽  
Binding Protein ◽  
Whole Body ◽  
Megalobrama Amblycephala ◽  
Mrna Levels ◽  
Nutrient Metabolism ◽  
Tissue Specific ◽  
Expression Levels ◽  
Mrna Expression Levels

Abstract BackgroundMethionine is an essential amino acid, that affects the metabolism of protein, lipid and glucose. However, the metabolic polytrophic response in the liver and muscle of juvenile Megalobrama amblycephala to dietary methionine levels is unclear.ResultsThe 0.84% methionine diet significantly improved the growth performance compared with the 0.40% diet. Dietary methionine levels had no marked effects on plasma parameters or whole body composition of juveniles. The protein levels of phospho-phosphatidylinositol 3-kinase, protein kinase B, phospho-eukaryotic initiation factor 4E binding protein-1 (p-4E-BP1), 4E-BP1 and ribosomal protein S6 kinase 1, in the liver of fish fed the 0.84% diet were higher than those in fish fed the 0.40% diet. While in muscle, these proteins showed the opposite trend. The mRNA levels of the muscular lipid synthesis associated genes: sterol regulatory element-binding protein 1 (SREBP1), fatty acid synthetase (FAS) and acetyl-CoA carboxylase (ACC), were significantly upregulated by the 1.28% methionine diet; while hepatic SREBP1, FAS and ACC mRNA expression levels were increased by 0.40% methionine. In addition, 1.28% dietary methionine significantly induced fatty acid β-oxidation and lipolysis of the liver and muscle via increased carnitine palmitoyl transferase 1, peroxisome proliferator activated receptor alpha, lipoprotein lipase and lipase expression levels. Compared with 0.40% dietary methionine, 1.28% methionine enhanced the mRNA levels of the hepatic gluconeogenesis related genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase, and the muscular glycolysis related genes phosphofructokinase (PFK) and pyruvate kinase (PK). The mRNA expression levels of hepatic PFK, PK and glucokinase were markedly upregulated by the 0.84% methionine diet compared with the 1.28% diet. In addition, muscular PEPCK and glycogen synthase, and hepatic glucose transporters 2 mRNA levels were induced by 1.28% methionine. ConclusionThe study showed that optimal methionine levels could enhance the growth of juvenile Megalobrama amblycephala, and the nutrient metabolism response to dietary methionine in the liver and muscle was tissue-specific.

scholarly journals The role of CXCR3 and its ligands expression in Brucellar spondylitis

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Xin Hu ◽  
Xiaoqian Shang ◽  
Liang Wang ◽  
Jiahui Fan ◽  
Yue Wang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Mononuclear Cells ◽  
Healthy Controls ◽  
Inflammatory Infiltration ◽  
Peripheral Blood Mononuclear ◽  
Expression Levels ◽  
Inflammatory Damage ◽  
Ifn Γ ◽  
Mrna Expression Levels

Abstract Aim Brucellar spondylitis (BS) is one of the most serious complications of brucellosis. CXCR3 is closely related to the severity of disease infection. This research aimed to study the degree of BS inflammatory damage through analyzing the expression levels of CXCR3 and its ligands (CXCL9 and CXCL10) in patients with BS. Methods A total of 29 BS patients and 15 healthy controls were enrolled. Real-Time PCR was used to detect the mRNA expression levels of IFN-γ, CXCR3, CXCL9 and CXCL10 in peripheral blood mononuclear cells (PBMCs) of BS patients and healthy controls. Hematoxylin-Eosin staining was used to show the pathological changes in BS lesion tissues. Immunohistochemistry staining was used to show the protein expression levels of Brucella-Ab, IFN-γ, CXCR3, CXCL9 and CXCL10 in BS lesion tissues. At the same time, ELISA was used to detect the serum levels of IFN-γ, CXCL9 CXCL10 and autoantibodies against CXCR3 in patients with BS. Results In lesion tissue of BS patients, it showed necrosis of cartilage, acute or chronic inflammatory infiltration. Brucella-Ab protein was abundantly expressed in close lesion tissue. And the protein expression levels of IFN-γ, CXCR3 and CXCL10 were highly expressed in close lesion tissue and serum of BS patients. At the same time, the mRNA expression levels of IFN-γ, CXCR3 and CXCL10 in PBMCs of BS patients were significantly higher than those in controls. Conclusion In our research, the expression levels of IFN-γ, CXCR3 and its ligands were significantly higher than those in controls. It suggested that high expression levels of IFN-γ, CXCR3 and its ligands indicated a serious inflammatory damage in patients with BS.

Unilateral labyrinthectomy induced changes on GDNF receptor complex proteins in the rat medial vestibular nuclei

2007 ◽  
Vol 16 (4-5) ◽  
pp. 171-177
Author(s):  
Adrian Lozada ◽  
Kaj Karlstedt ◽  
Pertti Panula ◽  
Antti A. Aarnisalo
Keyword(s):  
Mrna Expression ◽  
Vestibular Nucleus ◽  
Medial Vestibular Nucleus ◽  
Receptor Complex ◽  
Trophic Effect ◽  
Expression Levels ◽  
Protein Levels ◽  
Unilateral Labyrinthectomy ◽  
Ganglion Neurons ◽  
Mrna Expression Levels

In the auditory periphery, GDNF has been shown to have a trophic effect to spiral ganglion neurons, both during development and in adult animals. We have studied the effect of unilateral labyrinthectomy (UL) on protein levels and expression of GDNF multicomponent receptor complex: the ret tyrosine kinase and coreceptor GFRα-1 in the medial vestibular nucleus of the adult rat. GFRα-1 protein levels display an increasing trend in ipsilateral medial vestibular nucleus culminating at 48 h post UL. On the other hand, GFRα-1 mRNA expression levels in ipsi- and contralateral medial vestibular nucleus show a steadily decreasing trend that is significant at 1 week post-lesion. Protein levels for c-Ret isoforms also show an initial bilateral decreasing trend that ceases at 48 h in ipsilateral medial vestibular nucleus but persists on the contralateral side. c-Ret mRNA expression levels show a significant decrease at 4 h post UL followed by another significant decrease 1 week post UL. Our data would suggest that neurotrophins belonging to the GDNF family are involved in this model of post-lesional CNS plasticity.

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