| The quality,flavor,juiciness and freshness of mutton were positively correlated with intramuscular fat content.Intramuscular fat content is related to intramuscular fat deposition rate,which is the result of adipocyte differentiation and lipid metabolism.Adipocyte differentiation is a complex process regulated by non-coding RNA,functional genes and other factors.The size,morphology,structure and function of adipocytes change significantly in this process.Micro RNA(micro RNA,miRNA)is a kind of non-coding RNA,which is about the length of 22 nt.As a special RNA molecule,it can regulate a series of life activities by combining with target genes.In recent years,a large number of studies have shown that miRNA can regulate many important processes,such as fat decomposition and deposition,adipocyte proliferation and differentiation.At present,some studies have shown that some miRNAs can regulate the differentiation of goat intramuscular adipocytes,but whether other miRNA can affect the differentiation of goat intramuscular adipocytes and the specific mechanism need to be further studied.Therefore,according to the previous high-throughput sequencing results of the laboratory,the differentially expressed miR-28-5p and miR-92a-3p were selected as the research objects of this experiment,and the mimics and inhibitor of miR-28-5p/miR-92a-3p were synthesized by chemical synthesis.The effects and mechanisms of miR-28-5p and miR-92a-3p and their target genes on goat adipocyte differentiation were elucidated by biotechnology methods such as RT-PCR,real-time fluorescence quantitative PCR(Real-time Quantitative PCR,q PCR),overexpression vector construction,RNA interference,cell isolation and culture,transient transfection,oil red O staining,Bodipy staining and double luciferase reporter gene detection system.(1)The effects of miR-28-5p or miR-92a-3p on the differentiation of intramuscular adipocytes in goatThe expression of miR-28-5p and miR-92a-3p decreased at first and then increased and then decreased during the differentiation of goat intramuscular adipocytes.After imitating miR-28-5p in goat intramuscular adipocytes,the intracellular lipid droplets and OD values decreased significantly,and the expression levels of PPARγ,LPL,C/EBPα,AP2,SREBP1 and C/EBPβ decreased significantly(P<0.05);after the inhibition of miR-28-5p,the intracellular lipid droplets,OD value and the relative expression of PPARγ,LPL,C/EBPα,AP2,SREBP1 and C/EBPβ increased significantly(P<0.05).After upregulating the expression of miR-92a-3p,the intracellular lipid droplets and OD value were significantly decreased,the expression levels of PPARγ,LPL,C/EBPβ,AP2 and SREBP1 were significantly decreased,while the expression of C/EBPα was significantly increased(P<0.05);after the inhibition of miR-92a-3p expression,the intracellular lipid droplets increased,the OD value increased significantly,and the expression levels of PPARγ,LPL,C/EBPα,C/EBPβ and SREBP1 increased significantly(P<0.05).(2)Identification the targeted relationship between miR-28-5p or miR-92a-3p and APOL6The goat APOL6 3’UTR sequence was cloned and analyzed,and it was found that there was a paired binding sequence of miR-28-5p and miR-92a-3p on the 3’UTR of APOL6 m RNA.The reporter vectors of APOL6 3’UTR wild type(pmri GLO-APOL6 WT)and mutant(pmri GLO-APOL6 28 MT,pmri GLO-APOL6 92 a MT)were successfully constructed.Dual luciferase reporter gene detection system showed that the expression of simulated miR-28-5p/miR-92a-3p significantly inhibited the activity of APOL6 3’UTR(P<0.01),while the expression of simulated miR-28-5p/miR-92a-3p did not affect the activity of mutant 3’UTR.The time sequence expression profile of APOL6 increased at first and then decreased.After transfection of miR-28-5p/miR-92a-3p mimics in goat intramuscular cells,the expression level of APOL6 was extremely significantly decreased,while the expression of APOL6 increased significantly when the expression of miR-28-5p/miR-92a-3p was inhibited in goat muscle cells(P<0.01).(3)Effect of target Gene APOL6 on differentiation of Goat intramuscular adipocytesAccording to the goat APOL6 CDS region provided by NCBI and pc DNA-3.1 vector sequence,the APOL6 overexpression vector(pc DNA-APOL6)was successfully constructed by double enzyme digestion.After overexpression of APOL6 in goat intramuscular adipocytes,compared with the control group,the intracellular lipid droplets increased significantly,the OD value increased significantly(P<0.05),the relative expression levels of PPARγ,C/EBPα,C/EBPβ,AP2 and SREBP1 increased significantly(P<0.05),while the expression of LPL decreased significantly(P<0.05);after interfering with APOL6 in goat intramuscular adipocytes,the intracellular lipid droplets and OD value decreased significantly(P<0.05),and the relative expression levels of differentiation-related marker genes PPARγ,LPL,C/EBPα,C/EBPβ,AP2 and SREBP1 decreased significantly.(4)The recovery experiment further confirmed that miR-28-5p or miR-92a-3p play a regulatory role by inhibiting APOL6APOL6 overexpression vector was transfected with miR-28-5p/miR-92a-3p mimics in intramuscular precursor adipocytes of goats,and the expression of APOL6 was increased compared with miR-28-5p/miR-92a-3p mimics alone(P<0.05),the intracellular lipid accumulation increased,and the expression levels of C/EBPα,C/EBPβ,AP2,SREBP1,PPARγ and LPL were up-regulated(P<0.05).After inhibiting miR-28-5p/miR-92a-3P and interfering with APOL6 in goat intramuscular preadipocytes,compared with miR-28-5p and miR-92a-3p alone,the expression of APOL6 decreased(P<0.05),intracellular lipid droplets increased,and the expression levels of C/EBPα,C/EBPβ,AP2,SREBP1,PPARγ and LPL were down-regulated(P<0.05).Combined with the previous experimental results,it was further verified that miR-28-5p and miR-92a-3p regulate the differentiation of goat intramuscular adipocytes by inhibiting APOL6.These results suggest that both miR-28-5p and miR-92a-3p inhibit the differentiation of goat intramuscular adipocytes by inhibiting the activity of APOL6 3’UTR,while its downstream target gene APOL6 may promote the differentiation of goat intramuscular adipocytes by regulating the expression of PPARγ,C/EBPα,C/EBPβ,AP2 and SREBP1.This study provides reference materials for elucidating the effects of miR-28-5p and miR-92a-3p on the differentiation of goat intramuscular preadipocytes and exploring the ways by which they may play a role,and provides important data support for further revealing the molecular mechanism of APOL6 in goat fat deposition and candidate genes for goat molecular breeding. |
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