| The aim of this thesis was to elucidate the molecular mechanism of lipid metabolism in sheep receiving diet supplemented with Allium mongolicum Regel extract from perspective of non-coding RNA.Extensive regulation of micro RNAs(miRNAs)that belong to a type of endogenous small noncoding RNAs on gene expression in sheep has been recognized.And thus this molecule regulates a variety of life activities by modulating gene expression.In present study,we firstly conducted the sheep feeding trial,in which water extract of Allium mongolicum Regel(WEA)was added to sheep’ s basal diet.Briefly,30 male small-tailed Han sheep(4.5 months old)were randomly assigned to two groups.A basal diet with 3.4 g·sheep-1·d-1 WEA supplementation(WEA group)and with no WEA supplementation(CON group)was fed to sheep,respectively.Subcutaneous and perirenal adipose tissue samples were collected during slaughter after 75-day feeding experiment(include a 15-day pre-feeding period and a 60-day trial period).We first determined the variations of fatty acid composition,a phenotype-related indicator,and then applied miRNA sequencing and mRNA sequencing methods to screen for differentially expressed miRNAs and mRNAs in adipose tissues of sheep after utilization of WEA in diet.Finally,we used bioinformatics analysis to explore functional enrichment of differentially expressed genes and screened for differentially expressed miRNA-target gene action pairs after WEA supplementation.To verify the specific regulatory mechanism,the function of miR-218 a,which was differentially expressed after WEA supplementation in subcutaneous adipose tissue as screened by sequencing,in 3T3-L1 adipocytes,and miR-200 b,which was differentially expressed in perirenal adipose tissue under the influence of WEA,were verified by transfected cell technology using overexpression/inhibitor of miRNA in adipocyte.This thesis contains five experiments,the details of which and the main results of the experiments are as follows:1.Effect of WEA on fatty acid composition in subcutaneous and perirenal adipose tissue of sheep.Whether in subcutaneous or perirenal adipose tissue,a significant decrease in total saturated fatty acids(SFAs)and a significant increase in monounsaturated fatty acids(MUFAs)were observed(P<0.05)in WEA group compared with CON group in both tissues.In addition,the content of myristic acid(C14:0)and palmitic acid(C16:0)in subcutaneous adipose tissue was significantly lower,while the content of α-linolenic acid(C18:3n3),eicosadienoic acid(C20:2n6),docosahexaenoic acid(C22:2n6),and polyunsaturated fatty acids(PUFAs)was significantly increased by dietary WEA,as compared with that in CON group(P<0.05).Fatty acid analysis in perirenal adipose tissue showed that the addition of WEA to sheep diet significantly decreased the content of pentadecanoic acid(C15:0)and significantly increased the content of palmitoleic acid(C16:1)and oleic acid(C18:1n9c)(P<0.05).2.Effect of WEA on miRNA and mRNA expression in subcutaneous adipose tissue of sheep.In subcutaneous adipose tissue of sheep,30 differentially expressed miRNAs(DE-miRNAs)were identified by sequencing between WEA group and CON group.A total of 746 differentially expressed mRNAs(DE-mRNAs)with 90 lipid-related DE-mRNAs in subcutaneous adipose tissue were identified after WEA supplementation.The intersection genes from predicted target genes of DE-miRNAs by databases and actual DE-mRNAs by mRNA sequencing were enriched in several lipid-related pathways(glycerophospholipid metabolism,PPAR signalling pathway)in subcutaneous adipose tissue.Likewise,we obtained differentially expressed miRNA-target gene action pairs with regard to lipid metabolism,including miR-218a-ELOVL5,in subcutaneous fat after WEA supplementation.3.Effect of WEA on miRNA and mRNA expression in perirenal adipose tissue of sheep.In perirenal adipose tissue of sheep,15 differentially expressed miRNAs(DEmiRNAs)were identified by sequencing between WEA group and CON group.A total of569 differentially expressed mRNAs(DE-mRNAs)with 73 lipid-related DE-mRNAs in perirenal adipose tissue were identified after WEA supplementation.The intersection genes from predicted target genes of DE-miRNAs by databases and actual DE-mRNAs by mRNA sequencing were enriched in several lipid-related pathways(PI3K-Akt signalling pathway,regulation of lipolysis in adipocytes)in perirenal adipose tissue.Likewise,we obtained differentially expressed miRNA-target gene action pairs with regard to lipid metabolism,including miR-200b-KLF10,in perirenal fat after WEA supplementation.4.Functional characterisation of miR-218,which was differentially expressed in subcutaneous adipose tissue after WEA supplementation,in 3T3-L1 adipocytes.3T3-L1 preadipocytes were transfected with synthetic miR-218 mimics(mimics)and inhibitors(inhibitor)to overexpress and silence miR-218 in the cells,respectively.24 h after overexpression/inhibition treatments,miR-218 expression was measured.The results showed that miR-218 mimics and inhibitor groups successfully induced overexpression and inhibition of miR-218 in cells compared to their negative control groups,respectively.The expression of ELOVL5 gene(target gene of miR-218)in miR-218 inhibitor group was significantly increased(P<0.05)compared to its negative control(inhibitor NC)24 h after the transfection.miR-218 mimics/inhibitor groups decreased and increased ELOVL5 protein expression,respectively.After miR-218 overexpression or inhibition treatment,we induced 3T3-L1 cell differentiation,and at day 8 of differentiation,the overexpressed group significantly decreased triglyceride(TG)content,and the inhibition group significantly increased TG content(P<0.05).Oil red O staining showed that miR-218 mimics group decreased lipid droplet deposition,and miR-218 inhibitor group increased lipid droplet deposition.At day 8 of differentiation,miR-218 mimics group significantly decreased the adipocyte-specific marker genes for lipidogenic differentiation,PPARγ,FASN,FABP4,and LPL gene expression(P<0.05).Mi R-218 inhibitor group significantly increased PPARγ,C/EBPα,FASN,FABP4,SCD,LPL,and ACC gene expression(P<0.05).5.Functional characterisation of miR-200 b,which was differentially expressed in perirenal adipose tissue after WEA supplementation,in 3T3-L1 adipocytes.3T3-L1 preadipocytes were transfected with synthetic miR-200 b mimics(mimics)and inhibitors(inhibitor)to overexpress and silence miR-200 b in the cells,respectively.24 h after overexpression/inhibition treatments,miR-200 b expression was measured.The results showed that miR-200 b mimics and inhibitor groups successfully induced overexpression and inhibition of miR-200 b in cells compared to their negative control groups,respectively.The expression of KLF10 gene(predicted target gene of miR-200b)in miR-200 b mimics and inhibitor group was significantly decreased and increased(P<0.05)compared to its negative control 24 h after the transfection.miR-200 b mimics/inhibitor groups decreased and increased KLF10 protein expression,respectively.After miR-200 b overexpression or inhibition treatment,we induced 3T3-L1 cell differentiation,and at day 8 of differentiation,the overexpressed group significantly decreased triglyceride(TG)content,and the inhibition group significantly increased TG content(P<0.05).Oil red O staining showed that miR-200 b mimics group decreased lipid droplet deposition,and miR-200 b inhibitor group increased lipid droplet deposition.At day 8 of differentiation,miR-200 b mimics group significantly decreased the adipocyte-specific marker genes for lipidogenic differentiation,C/EBPα,FASN,FABP4,ACC,LPL and SCD gene expression(P<0.05).Mi R-200 b inhibitor group significantly increased PPARγ,C/EBPα,FASN,FABP4,SCD,LPL,and ACC gene expression(P<0.05).In summary,WEA improved fatty acid composition in mutton by affecting the expression of miRNAs and genes in subcutaneous and perirenal adipose tissue.In subcutaneous and perirenal adipose tissue of sheep,WEA may improve the composition and content of fatty acids in both types of adipose tissue,and ultimately improve nutritional value and flavour of mutton by regulating the expression of miR-218 a and miR-200 b,respectively. |
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