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OBJECTIVE: The objective of this study was to compare the DNA methylation profile in the longissimus dorsi muscle between Small Tailed Han and Dorper×Small Tailed Han crossbred sheep which were known to exhibit significant difference in meat-production. METHODS: Six samples (three in each group) were subjected to the methylated DNA immunoprecipitation sequencing (MeDIP-seq) and subsequent bioinformatics analyses to detect differentially methylated regions (DMRs) between the two groups. RESULTS: 23.08 Gb clean data from six samples were generated and 808 DMRs were identified in gene body or their neighboring up/downstream regions. Compared with Small Tailed Han sheep, we observed a tendency toward a global loss of DNA methylation in these DMRs in the crossbred group. Gene ontology enrichment analysis found several gene sets which were hypo-methylated in gene-body region, including nucleoside binding, motor activity, phospholipid binding and cell junction. Numerous genes were found to be differentially methylated between the two groups with several genes significantly differentially methylated, including transforming growth factor beta 3 (TGFB3), acyl-CoA synthetase long chain family member 1 (ACSL1), ryanodine receptor 1 (RYR1), acyl-CoA oxidase 2 (ACOX2), peroxisome proliferator activated receptor-gamma2 (PPARG2), netrin 1 (NTN1), ras and rab interactor 2 (RIN2), microtubule associated protein RP/EB family member 1 (MAPRE1), ADAM metallopeptidase with thrombospondin type 1 motif 2 (ADAMTS2), myomesin 1 (MYOM1), zinc finger, DHHC type containing 13 (ZDHHC13), and SH3 and PX domains 2B (SH3PXD2B). The real-time quantitative polymerase chain reaction validation showed that the 12 genes are differentially expressed between the two groups. CONCLUSION: In the current study, a tendency to a global loss of DNA methylation in these DMRs in the crossbred group was found. Twelve genes, TGFB3, ACSL1, RYR1, ACOX2, PPARG2, NTN1, RIN2, MAPRE1, ADAMTS2, MYOM1, ZDHHC13, and SH3PXD2B, were found to be differentially methylated between the two groups by gene ontology enrichment analysis. There are differences in the expression of 12 genes, of which ACSL1, RIN2, and ADAMTS2 have a negative correlation with methylation levels and the data suggest that DNA methylation levels in DMRs of the 3 genes may have an influence on the expression. These results will serve as a valuable resource for DNA methylation investigations on screening candidate genes which might be related to meat production in sheep.
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Colorectal cancer (CRC) continues to be one of the most malignant cancers with a high mortality rate to date. Promoting the radio-responsiveness of CRC is of great importance for local control and prognosis. In this study, we examined the roles of exosomal microRNA-19b (miR-19b) in CRC radioresistance. The regulatory role of miR-19b in CRC stem cells and radiotherapy-resistant cells was determined using miRNA microarray analysis, and its prognostic value was probed using the TCGA database. It was found that miR-19b was overexpressed in CRC tissues, which indicated a poor prognosis. CRC-derived exosomes (EXOs) enhanced the radio-resistance and stemness properties of CRC cells via delivery of miR-19b in vitro and in vivo. FBXW7 was identified as a putative target of miR-19b. On the contrary, reintroduction of FBXW7 reversed the effects of miR-19b on radioresistance and stemness properties. Furthermore, the Wnt/ß-catenin pathway activity was elevated in CRC cells upon EXOs treatment, decreased after miR-19b downregulation, and increased again after FBXW7 downregulation. These results suggest that miR-19b inhibition could enhance the efficacy of radiotherapy while reducing the stemness properties, thus presenting a promising strategy for sensitizing CRC cells to radiotherapy.
Assuntos
Neoplasias Colorretais/patologia , Neoplasias Colorretais/radioterapia , Exossomos , Proteína 7 com Repetições F-Box-WD/fisiologia , MicroRNAs/fisiologia , Células-Tronco Neoplásicas , Tolerância a Radiação , Humanos , Células Tumorais CultivadasRESUMO
Important candidate genes that regulate lipid metabolism have the potential to increase the content of intramuscular fat (IMF) and improve meat quality. Secreted protein acidic and rich in cysteine like 1(SPARCL1) is a secreted glycoprotein with important physiological functions and is involved in the proliferation and differentiation of various cells. However, the role of the SPARCL1 gene in sheep preadipocytes and its regulatory mechanism is still unclear. In this study, we explored the effect of SPARCL1 on the proliferation and differentiation of sheep preadipocytes. The results showed that the expression level of the SPARCL1 gene is higher in fat tissue than in other tissues, and the gene was significantly increased on the 6th day of preadipocyte differentiation. In the preadipocyte proliferation stage, interference of SPARCL1 gene reduced cell viability and increased cell apoptosis. In preadipocyte differentiation stage, SPARCL1 overexpression significantly inhibited lipid droplets accumulation and triglyceride content by increasing Wnt10b, Fzd8, IL6, and ß-catenin and inhibiting PPARγ, C/EBPα, LPL, and IGF1 genes expression, whereas SPARCL1 deficiency significantly promoted cell differentiation by inhibiting ß-catenin and increasing GSK3ß, PPARγ, C/EBPα, and LPL. The results of this study suggest that SPARCL1 plays a negative role during preadipocyte differentiation and may become a novel target for regulating preadipocyte differentiation and improving IMF.Abbreviations:IMF: Intramuscular fat SPARCL1: Secreted protein acidic and rich in cysteine like 1 PPARγ: Peroxisome proliferator-activated receptor γ C/EBPα: CCAAT/enhancer-binding protein-α LPL: Lipoprotein lipase IGF1: Insulin-like growth factor 1 Wnt10b: Wnt family member 10B Fzd8: Frizzled class receptor 8 IL6: Interleukin 6 ß-catenin: Catenin beta interacting protein 1 GSK3ß: Glycogen synthase kinase 3 beta LRP5/6: Low-density lipoprotein receptor-related protein 5/6.
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Adipócitos , Proteínas de Ligação ao Cálcio/metabolismo , Proliferação de Células , Proteínas da Matriz Extracelular/metabolismo , Adipócitos/citologia , Adipogenia , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Diferenciação Celular , OvinosRESUMO
Many local sheep breeds in China have poor meat quality. Increasing intramuscular fat (IMF) content can significantly improve the quality of mutton. However, the molecular mechanisms of intramuscular adipocyte formation and differentiation remain unclear. This study compared differences between preadipocytes and mature adipocytes by whole-transcriptome sequencing and constructed systematically regulatory networks according to the relationship predicted among the differentially expressed RNAs (DERs). Sequencing results showed that in this process, there were 1,196, 754, 100, and 17 differentially expressed messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs), respectively. Gene Ontology analysis showed that most DERs enriched in Cell Part, Cellular Process, Biological Regulation, and Binding terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the DERs primarily focused on Focal adhesion, phosphoinositide 3-kinase (PI3K)-Akt, mitogen-activated protein kinase (MAPK), peroxisome proliferator-activated receptor (PPAR) signaling pathways. Forty (40) DERs were randomly selected from the core regulatory network to verify the accuracy of the sequence data. The results of qPCR showed that the DER expression trend was consistent with sequence data. Four novel promising candidate miRNAs (miR-336, miR-422, miR-578, and miR-722) played crucial roles in adipocyte differentiation, and they also participated in multiple and important regulatory networks. We verified the expression pattern of the miRNAs and related pathways' members at five time points in the adipocyte differentiation process (0, 2, 4, 6, 8, 10 days) by qPCR, including miR-336/ACSL4/LncRNA-MSTRG71379/circRNA0002331, miR-422/FOXO4/LncRNA-MSTRG54995/circRNA0000520, miR-578/IGF1/LncRNA-MSTRG102235/circRNA0002971, and miR-722/PDK4/LncRNA-MSTRG107440/circ RNA0002909. In this study, our data provided plenty of valuable candidate DERs and regulatory networks for researching the molecular mechanisms of sheep adipocyte differentiation and will assist studies in improving the IMF.
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Single nucleotide polymorphism in exon 1 of the estrogen receptor (ESR) gene was detected by PCR-SSCP in both high fecundity sheep breeds (Small Tail Han sheep, Hu sheep and German Mutton Merino sheep) and low fecundity sheep breeds (Dorset sheep,Suffolk sheep). Results indicated that there were three genotypes (AA, AB and BB) in all three high fecundity sheep breeds, but only two genotypes (AA, AB) in both low fecundity breeds. In Hu sheep,German Mutton Merino sheep, Small Tail Han sheep, Suffolk sheep and Dorset sheep,the frequency of allele A was 0.672, 0.786, 0.846, 0.857 and 0.867, respectively, and the frequency of allele B was 0.328, 0.214, 0.154, 0.143, and 0.133, respectively. Sequencing revealed a C-->G mutation at 363 bp of exon 1 of ESR gene in the BB genotype in comparison to the AA genotype. The genotype distribution was significantly different between Small Tail Han sheep and Hu sheep (P<0.01) and between Dorset sheep and Hu sheep (P <0.05). There was no difference in genotype distribution between other sheep breeds. The Small Tail Han sheep ewes with genotypes AB or BB had 0.51 (P < 0.05) and 0.7 (P < 0.05) more lambs than those with genotype AA, respectively. These results showed that the estrogen receptor locus is either a major gene that influences the prolificacy in Small Tail Han sheep or in close linkage with such a gene. In view of our results, marker-assisted selection using ESR is warranted to increase litter size in sheep and will be of considerable economic value to mutton producers.
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Polimorfismo de Nucleotídeo Único , Receptores de Estrogênio/genética , Carneiro Doméstico/genética , Alelos , Animais , Sequência de Bases , Cruzamento , China , Éxons , Feminino , Fertilidade/genética , Frequência do Gene , Genótipo , Tamanho da Ninhada de Vivíparos/genética , Masculino , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Análise de Sequência de DNA , Carneiro Doméstico/classificaçãoRESUMO
Genetic variation of seven microsatellite loci BM1818, BM1258, BM1443, BM1905, BM302, BM4505 and CYP21 which were closely linked to somatic cell score (SCS) was analyzed in 240 Beijing Holstein cows with nondenaturing polyacrylamide gel electrophoresis. Allele frequencies, heterozygosity, polymorphic information content, the effective number of alleles of seven microsatellite loci were calculated. Relationships between seven microsatellite loci and somatic cell score in Beijing Holstein cows were primarily analyzed by least squares linear model. Least squares means of SCS for BM1818 (284 bp/284 bp), BM1258 (106 bp/92 bp), BM1443 (166 bp/160 bp), BM1905 (187 bp/187 bp), BM302 (142 bp/140 bp), BM4505 (240 bp/236 bp) and CYP21 (215 bp/198 bp) were relatively lower,and their genotypes were the most favorable genotypes in respective locus for mastitis resistance. Least squares means of SCS for BM1818 (286 bp/286 bp), BM1258 (102 bp/102 bp), BM1443 (170 bp/160 bp), BM1905 (197 bp/195 bp), BM302 (154 bp/145 bp), BM4505 (240 bp/238 bp) and CYP21 (204 bp/192 bp) were relatively higher,and their genotypes were the most unfavorable genotypes in respective locus for mastitis resistance. The information found in the present study would be very important for improving mastitis resistance in dairy cattle by marker assisted selection.