RESUMEN
AIMS: To study the effect of thymine DNA glycosylase (TDG) gene knockdown on the differentiation of pig preadipocytes. METHODS: Preadipocytes were obtained from subcutaneous adipose tissue from the neck of 1- to 7-day-old pigs. The TDG gene was knocked down using siRNA, and cell differentiation was induced. The mRNA expression level was measured using fluorescence quantitative PCR, and the protein expression level was determined using Western blot analysis. The DNA methylation levels in promoter regions of differentiation-related genes were also evaluated. RESULTS: TDG gene knockdown decreased the mRNA expression levels of the peroxisome proliferator-activated receptorγ (PPARγ) and Fatty acid binding proteins 4(FABP4 Also known as aP2) genes (P<0.01), while the mRNA expression level of the CCAAT/enhancer binding protein alpha(C/EBPα) gene did not change significantly (P>0.05). In addition, after induced differentiation, the lipid droplet production significantly decreased, and the percentages of methylation in the promoter regions of C/EBPα, PPARγ, and aP2 genes were 0.9%, 80%, and 76%, respectively. In contrast, the percentages of methylation in the negative control groups were 0.5%, 67.5%, and 58%, respectively. CONCLUSION: TDG gene knockdown could inhibit the differentiation of pig preadipocytes and affect the DNA methylation levels of some transcription factors.
Asunto(s)
Adipocitos Blancos/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Epigénesis Genética , Proteínas de Unión a Ácidos Grasos/genética , PPAR gamma/genética , Timina ADN Glicosilasa/genética , Adipocitos Blancos/citología , Animales , Animales Recién Nacidos , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Diferenciación Celular , Metilación de ADN , Proteínas de Unión a Ácidos Grasos/metabolismo , Expresión Génica , Técnicas de Silenciamiento del Gen , Gotas Lipídicas/metabolismo , Masculino , PPAR gamma/metabolismo , Cultivo Primario de Células , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Grasa Subcutánea/citología , Grasa Subcutánea/metabolismo , Porcinos , Timina ADN Glicosilasa/deficienciaRESUMEN
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.
Asunto(s)
Adipocitos/citología , Adipogénesis , Bovinos/genética , Regulación de la Expresión Génica , MicroARNs/genética , Adipocitos/metabolismo , Animales , Secuencia de Bases , Bovinos/fisiología , Células Cultivadas , Factor de Transcripción E2F2/genética , Factores de Intercambio de Guanina Nucleótido/genética , RatonesRESUMEN
BACKGROUND: MBD4 (methyl-CpG binding domain protein 4) is an important G: T glycosylase that can identify T-G mismatches. It plays a role in active demethylation through base excision repair. Overexpression of MBD4 gene can cause the demethylation of numerous genes, and the remethylation of MBD4-associated genes can occur when the MBD4 gene is knocked out. To date, the functions and regulatory mechanisms of the MBD4 gene in the differentiation of porcine preadipocytes have not been clearly established. METHODS: Subcutaneous fat cells from 1- to 7-day-old Junmu-1 piglets were cultured in vitro, induced to differentiate, and then identified. A real-time fluorescence-based quantitative polymerase chain reaction (PCR) analysis was conducted to detect MBD4 messenger RNA (mRNA) expression. Cells were treated with MBD4-siRNA (small interfering RNA) and induced to differentiate. Changes in the lipid droplets were observed by oil red O staining. Changes in the mRNA and protein expression levels of MBD4 and the adipose differentiation-associated genes C/EBPα (CCAAT-enhancer-binding protein alpha), PPARγ (peroxisome proliferator-activated receptor gamma), and aP2 (adipocyte protein 2) were detected. In addition, the bisulfite sequencing method was used to detect changes in methylation in the promoters of certain genes associated with adipose differentiation. RESULTS: Levels of MBD4 mRNA and protein expression varied with time over the course of the porcine adipocyte differentiation, with the highest levels of this expression observed on day two of the differentiation process. After silencing MBD4 and inducing differentiation, the production of lipid droplets decreased, the mRNA expression levels of C/EBPα, PPARγ, and aP2 were significantly reduced, and DNA methylation modification levels were significantly elevated in the examined promoter regions. CONCLUSION: The silencing of the MBD4 gene can influence the DNA methylation levels of preadipocyte differentiation-related genes and subsequently inhibit the differentiation of porcine preadipocytes.
Asunto(s)
Adipocitos/fisiología , Diferenciación Celular/genética , beta-Defensinas/genética , Animales , Proteína alfa Potenciadora de Unión a CCAAT/genética , Diferenciación Celular/fisiología , Metilación de ADN/genética , PPAR gamma/genética , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Grasa Subcutánea/fisiología , PorcinosRESUMEN
PURPOSE: This study investigated proteins differentially expressed in the ovaries of menopausal women in comparison to childbearing women. METHODS: Differential protein expression was screened by difference gel electrophoresis and 2-D SDS-PAGE. Four differentially expressed proteins were excised manually, identified by mass spectrometry and confirmed by immunoblot and immunohistochemistry. RESULTS: The four proteins were identified as serum amyloid P, heat shock protein 27, Glyoxalase I and Ubiquitin carboxy-terminal hydrolase. Serum amyloid P expression was significantly up-regulated in the ovaries of menopausal women by immunoblot analysis (p < 0.05), Glyoxalase I and Ubiquitin carboxy-terminal hydrolase displayed an altered expression pattern, with higher expression in the atretic follicles of menopausal women. Weak Glyoxalase I and Ubiquitin carboxy-terminal hydrolase were observed in the granulosa and theca cells of the follicles of childbearing women. Heat shock protein 27 and serum amyloid P were clearly observed in the atretic follicles of menopausal women, while their expression was restricted to the theca cells and cytoplasm of primordial follicles in the ovaries of childbearing women. All four proteins were predominantly expressed in the atretic follicles of menopausal women. CONCLUSIONS: These data suggest that the identified proteins may play a role in the regulation of follicle atresia in menopausal women, although their functions and mechanism warrant further investigation.
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Proteínas de Choque Térmico HSP27/metabolismo , Lactoilglutatión Liasa/metabolismo , Menopausia/fisiología , Ovario/metabolismo , Componente Amiloide P Sérico/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Adulto , Femenino , Colorantes Fluorescentes , Atresia Folicular , Perfilación de la Expresión Génica/instrumentación , Perfilación de la Expresión Génica/métodos , Humanos , Immunoblotting , Inmunohistoquímica , Espectrometría de Masas , Folículo Ovárico/metabolismo , Células Tecales/metabolismo , Electroforesis Bidimensional Diferencial en GelRESUMEN
microRNAs (miRNAs/miRs) are a class of singlestranded noncoding RNA molecules of 1924 nucleotides (nt) in length. They are widely expressed in animals, plants, bacteria and viruses. Via specific mRNA complementary pairing of target genes, miRNAs are able to regulate the expression of mRNA levels or inhibit protein translation following transcription. miRNA expression has a time and space specificity, and it is involved in cell proliferation and differentiation, apoptosis, development, tumor metastasis occurrence and other biological processes. miR26b is an miRNA of 22 nt and is important in the regulation of cellular processes. With the advancement of molecular biology techniques in recent years, there have been extensive investigations into miR26b. Numerous studies have observed that miR26b is involved in early embryonic development, cell proliferation regulation, pituitary hormone secretion and other physiological activities. miRNAs are associated with the function of propagation. The present study used reverse transcription quantitative polymerase chain reaction to detect the relative expression levels of miR26b in the pituitary tissue of Yanbian cattle at different developmental stages. The 2∆∆Ct method was used to calculate the relative gene expression levels. The miRNA target gene database TargetScan and RNA22 were used for prediction of the miR26b target gene and selective recognition was also performed. The results demonstrated that miR26b is expressed in the pituitary tissues of Yanbian cattle at 6 and 24 months of age. The relative expression levels of miR26b in the pituitary tissues of 24monthold Yanbian cattle were 2.41 times that of those in the sixmonthold Yanbian cattle, demonstrating significant differences in the relative expression (P<0.01). The relative expression of the candidate target genes, EphA2 and miR26b, exhibited the opposite expression pattern. The relative expression levels in the pituitary tissues of sixmonthold Yanbian cattle were 3.34 times that of those in 24monthold Yanbian cattle (P<0.01). There are miR26b binding sites in the 3'untranslated region (3'UTR) of EphA2 in bovine, human, murine and other mammalian mRNAs, suggesting that the EphA2 gene may be a target gene of miR26b. The results of a Luciferase reporter system assay revealed that miR26b is able to suppress EphA2 expression at the transcription level. Following the sitedirected mutagenesis of plasmid EphA2 3'UTR pmirGLOMUT and miR26b mimictransfected HeLa cells, the dualluciferase reporter gene assay revealed that there were three consecutive nucleotide mutations in the 3'UTR, binding with the predicted seed region. This may have caused the miR26b inhibition of luciferase activity to decrease from 60% in the wildtype to 26%, suggesting that miR26b achieved its function via binding with the TACTTGAA sequence of the 3'UTR in EphA2. In conclusion, the present study successfully assessed the expression pattern of miR26b in the pituitary tissue of Yanbian cattle, and also confirmed that EphA2 was a target gene of miR26b in Yanbian cattle in vitro. The present study provided the theoretical basis to further investigate the role of miR26b in early embryonic development, pituitary hormone secretion and other reproductive functions.
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Regulación del Desarrollo de la Expresión Génica , MicroARNs/genética , Hipófisis/metabolismo , Receptor EphA2/genética , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Sitios de Unión , Bovinos , Genes Reporteros , Células HeLa , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Ratones , MicroARNs/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Hipófisis/crecimiento & desarrollo , Plásmidos/química , Plásmidos/metabolismo , Receptor EphA2/metabolismo , Transducción de SeñalRESUMEN
MicroRNAs (miRNAs) are a class of small, non-coding RNA molecules that act as a negative regulator of most mRNAs. miRNAs influence the gene expression as transcriptional regulators and play an important role in many fundamental biological processes. It is generally acknowledged that miRNAs have a very important affection on mammalian pituitary. However, the answers of which role miRNAs play in the development of sexual function or how much they contribute to the pituitary function are not exactly. In our study, we used three female 21-day-old rats and three female 12-month-old rats to analysis the function of miRNAs. By the analyses of microarray data, we finished the stem-loop real-time RT-PCR for the differentially expressed miRNAs. We detected a total of 93 differentially expressed miRNAs between 21-day-old rats' pituitary and 12-month-old rats'. Stem-loop real-time RT-PCR suggests that the obtained data is of high credibility. Among these miRNAs, 7 miRNAs' expression (rno-miR-880, rno-miR-503, rno-miR-125a-3p, rno-miR-3596b, rno-miR-30e, rno-miR-214 and rno-miR-22) are significant different (P≤0.05). In a word, this study identified a number of specific changes in the expression of miRNAs, in rats by detecting the expression profile of miRNAs in rat's pituitary, and all of that lay the foundation for elucidating the regulatory mechanisms of miRNAs in rat's reproduction process. These differentially expressed miRNAs may play a very important role in rat's reproduction process.