| The genomic loci of miR-26 a and miR-26 b are localized in the introns of genes encoding the protein family carboxy-terminal domain RNA polymerase ? polypeptide A small phosphatase(CTDSP).Transcription of miR-26 a and miR-26 b occurs via three genomic loci,miR-26a-1,miR-26a-2 and miR-26 b,which reside in the introns of genes coding for CTDSPL,CTDSP2 and CTDSPL,respectively.Mi R-26a-1 and miR-26a-2 share the same mature sequence.Mi R-26 inhibits the expression of the ATP-binding cassette transporter A1(ABCA1)and ADP-ribosylation factor-like 7(ARL7),two LXR target genes which play critical roles in cholesterol efflux.miR-26 b is regulated by tumor necrosis factor ?(TNF-?),leptin and resistin in human adipocytes,its inhibition can effectively suppress adipocyte differentiation,decrease lipid droplets,triglyceride accumulation and m RNA levels of adipocyte specific molecular markers: PPARG and C/EBP?.As a special white adipose tissue,the fatty acid metabolism is very active during lactation.Thus,investigating the function and regulatory effect of miR-26 on fatty acid metabolism is of great importance for revealing the mechanism of milk fat synthesis in ruminants,and laying the foundation for further research on miRNA regulatory mechanism in mammalian fatty acid metabolism network.Based on the analysis of the relationship between miR-26 family and their host genes,and the milk composition,and milk fatty acid composition,respectively,during the mid-lactation.We investigate the synergistic function of the miR-26 family and their host genes on fatty acid synthesis by using overexpression and down-regulation methods.Cloning and mutation analysis of the miR-26a/b promoter and investigating the effect of key transcription factors related lipid metabolism on the methylation level of miR-26 family provide the evidences for revealing the transcriptional regulation mechanism.The main results of this study are as follows: 1.Bioinformatics analysis of goat miR-26 family and its correlation with milk and milk1.Bioinformatics analysis of goat miR-26 family and its correlation with milk and milk fatty acid compositionThe miR-26 family homology analysis showed that the miR-26 family was highly conserved in goat,cattle,human,mice and sheep,and had the same seed sequence among these species.The potential targets of miR-26 family were predicted by the online software:Target Scan and Pic Tar,and the function of the targets was analyzed by GO and KEGG modules of the Kobas online software,the results showed that miR-26 family targets were involved in signaling pathway which are related to milk metabolism,such as Wnt Signaling pathway,PI3K-Akt signaling pathway,Hippo signaling pathway,m TOR signaling pathway,Fatty acid biosynthesis.By using RT-PCR,we analysis the expression level of miR-26 and their host genes at various time points during lactation cycle revealed that they are dynamically regulated,a significant upregulation of both miRNA were observed in early,peak and mid-lactation relative to the dry period,and CTDSP1/2/L displayed a similar trend of expression as miR-26a/b over the lactation cycle.To better evaluate the relationship between miR-26a/b and CTDSP1/2/L,data on m RNA expression in mammary gland tissue from goats at mid lactation was used for correlation analysis.There was a strong correlation between miR-26 a and miR-26 b expression,and a moderate correlation between miR-26a/b and their respective host genes in mid-lactation mammary gland.The results of correlation-ship analyses between the expression level of miR-26a/b and goat milk composition or milk fatty acid composition revealed that miR-26a/b as well as CTDSPL were significantly correlated with milk fat content,but had no correlation with milk lactose,milk protein and non-fat-solid.In addition,there was a moderate correlation between miR-26 a and C6: 0,C14: 1,C16: 0,C16: 1,C18: 0,C18: 1 and C18:3,between miR-26 b and C6:0,C14:1,C16:1,C18:0 and C18:3,between CTDSP1 and C10:0,C11:0,C12:0,C14:0,C15:0 and C18:3,and between CTDSP2 and C14:1,as well as between CTDSPL and C6:0,C14:1,C16:1,C18:0 and C18:1.2.Mi R-26 a function synergistically with miR-26 b to regulate fatty acid metabolismThe expression of miR-26 a and miR-26 b was significantly up-regulated in miR-26 a and miR-26 b mimic transfected goat mammary gland epithelial cells(GMEC),respectively.cells transfected with miR-26 a or miR-26 b mimic displayed marked up-regulation of genes responsible for de novo fatty acid(FA)synthesis(FASN,ACACA),triglyceride(TAG)synthesis(GPAM,DGAT1 and LPIN1),FA desaturation(SCD1 and FADS2),lipid droplet formation(PLIN2)and the key transcription regulators(PPARG,SREBP1 and LXRA).Cellular TAG content,lipid-droplet formation,and unsaturated fatty acid(UFA,C16:1 and C18:2)content were significantly increased by mimic treatment.While,the saturated fatty acid(SFA,C16: 0)and the DNA methylation level of PPARG,SREBP1,FASN and SCD1 were decreased.Compared with the miR-26 a treatment group,the expression level of FASN,GPAM,DGAT1,LPIN1,FADS2,SREBF1 and PIN2 were significantly up-regulated after miR-26 a and miR-26 b co-transfection,and the lipid droplet formation were also increased.Compared with the miR-26 b treatment group,the expression level of FASN,ACACA,LPIN1,SCD1,FADS2,PPARG and SREBF1 and the content of unsaturated fatty acid C18:1 were significantly increased in miR-26 a and miR-26 b co-treatment group,but saturated fatty aicd C16:0 content was significantly reduced.RT-q PCR and western blot analysis showed that overexpression of miR-26 family decreased the m RNA and protein level of INSIG1,luciferase reporter assays suggested that miR-26a/b cognate site is essential for negative regulation of luciferase expression driven by the INSIG1 3?-UTR.3.The coregulation of miR-26 family and their host genes in fatty acid metabolismInhibitor and si RNA respective specifically target miR-26 and CTDSP family were designed and transfected into GMEC,the expression of miR-26 and CTDSP family were decreased significantly for more than 70%,respectively.Inhibition of miR-26 a or miR-26 b decreased expression of various lipogenic genes,for instance,FASN,ACACA,GPAM,LPIN1,DGAT1,SCD1,PLIN2,CD36,PPARG,SREBP1 and LXRA,but increased the expression of INSIG1 and C16:0 and C18:0 content.Cellular TAG content,lipid-droplets formation and C16:1 and C18:1 content were decreased by miR-26 a or miR-26 b interference.In addition,the indicators detected above were synergistically inhibited by co-transfected with miR-26 a and miR-26 b inhibitor.Interference of CTDSP family resulted in a remarkable decrease in the expression of SREBP1,but increase in the expression of INSIG1 and C16:0 content.The content of C16:1,C18:1 and cellular TAG,and lipid-droplet formation were decreased significantly upon inhibition of CTDSP family.The downregulation of the expression of CD36,FASN,LXRA and SREBP1 were more evident when miR-26a/b and CTDSP1/2/L were co-suppressed.4.The relationship investigation between the miR-26 a promoter and the important transcription factors in fatty aicd metabolismThe 1274 bp sequence of goat miR-26a-1 and 1183 bp sequence of goat miR-26a-2 5? flanking region was cloned and sequenced,respectively.Bioinformatics analysis revealed that there are three PPRE elements,three SRE elements and one Cp G island in miR-26a-1 promoter,and three PPRE elements,three SRE elements,one LXRE element and two Cp G islands in miR-26a-2 promoter.Overexpression of SREPP1,PPARG or LXRA increased the expression of miR-26 a and CTDSP2/L,and decreased the DNA methylation level of miR-26a-1 and miR-26a-2 promoter.Luciferase activity analysis indicated that SREBP1,PPARG and LXRA activate the promoter activity of miR-26a-1 and miR-26a-2 significantly.Deleted mutation of PPRE1,SRE1 or SRE2 decreased the basal transcriptional activity of miR-26a-1 significantly,mutation of PPRE1 abolished the activation effect of PPARG on miR-26a-1 promoter,mutation either SRE1 or SRE2 weaken the activation effect of SREBP1 on miR-26a-1 promoter,while mutation SRE1 and SRE2 nearly abolished the activation effect of SREBP1 on miR-26a-1 promoter,the Ch IP assay showed that SREBP1 protein can directly bind to the SRE1 and SRE2 elements in miR-26a-1 promoter,indicated the direct regulation of miR-26a-1 by SREBP1.In addition,mutation SRE1 and SRE2 nearly abolished the activation effect of LXRA on miR-26a-1 promoter indicated that LXRA regulate the transcription of miR-26a-1 through an indirectly pathway.With the same assays,the PPRE3 and SRE1 elements were identified to have an important effects on the maintenance of the miR-26a-2 promoter activity.Mutation LXRE weaken the activation effect of LXRA on miR-26a-2 promoter.Furthermore,mutation SRE1 and LXRE nearly abolished the activation effect of LXRA on miR-26a-2 promoter,indicated that LXRA can regulate miR-26a-2 expression through direct or indirect pathway.5.The relationship investigation between the miR-26 b promoter and the important transcription factors in fatty aicd metabolismWe cloned and sequenced an 1102 bp fragment of miR-26 b gene flanking region,containing 1018 bp upstream of transcriptional start sites.Bioinformatics analysis revealed that there are four PPRE,four SRE,two LXRE elements and three Cp G islands in the miR-26 b promoter.Activation of PPARG,SREBP1 and LXRA significantly increased the expression of miR-26 b and CTDSP1,as well as miR-26 b promoter activity,and decreased the DNA methylation level of miR-26 b promoter.PPRE1 and PPRE2 mutated alone or simultaneously decreased the promoter activity dramatically,PPRE1 or PPRE2 mutation led to a decrease in the activation effects of PPARG,while the activation was nearly abolished when these two sites were mutated simultaneously.Similarly,SRE1 and SRE3 were identified as the main elements to maintain the basal promoter activity of miR-26 b.Furthermore,the results of Ch IP assay showed that SREBP1 interacted with the miR-26 b promoter at the SRE1 and SRE3 sites,indicating that these two elements in the miR-26 b promoter can bind to SREBP1 transcription factor.LXRA increased the promoter activity significantly when LXRE elements were mutated alone or simultaneously.In contrast,mutation constructs of SRE1 or SRE3 significantly decreased the activity,and the simultaneous mutation of SRE1 and SRE3 completely abolished the activation effects of LXRA,which suggested that LXR participates in the regulation of the miR-26 b promoter via an SRE binding sites,without the two LXRE having an influence on miR-26 b promoter activity.In summary,the miR-26 family is involved in the process of cellular triglyceride synthesis,lipid accumulation and the change of unsaturated fatty acid content through various mechanisms,which is an important regulator of milk fat metabolism in ruminants. |
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