MiR-130b-3p/5p Regulates Goat Intramuscular Adipocyte Differentiation By Targeting KLF3

The content of intramuscular fat(IMF)is mainly determined by the fat formation of intramuscular adipocytes and the lipid accumulation of mature adipocytes.Adipose deposition mainly depends on the degree of adipocyte proliferation and differentiation.miRNA(micro RNA)is a kind of endogenous noncoding RNA with regulatory functions found in eukaryotes,which binds to specific target genes to regulate cell differentiation at the post-transcriptional level.In recent years,goat meat has gradually been welcomed by consumers due to its high protein,low fat,and low cholesterol characteristics.However,there are few studies on the regulation mechanism of miRNAs on goat intramuscular adipocytes differentiation.In this study,two differentially expressed miRNAs,miR-130b-3p and miR-130b-5p,were screened from goat intramuscular adipocytes before and after differentiation obtained by RNA-seq in the previous experiment.By means of molecular and cellular biological technology,such as RT-PCR,real-time fluorescent quantitative PCR(qPCR),vector construction,cell culture,overexpression,RNA interference and dual-luciferase reporting detection system,the regulatory role and possible mechanism of miR-130b-3p and miR-130b-5p in regulating the differentiation of intramuscular adipocytes in goat were clarified.(1)The effect of miR-130b-3p and miR-130b-5p on the differentiation of goat intramuscular adipocytesThe relative expression levels of miR-130b-3p and miR-130b-5p in goat intramuscular adipocytes were stable at 0～36 h,and gradually increased at 36～60 h,and finally decreased at 60～96 h.After mimicking the expression of miR-130b-3p,the accumulation of lipid droplets in goat intramuscular adipocytes was weakened,and the amount of triglyceride synthesis decreased,the expression of CEBPα,PPARγ,SREBP1,ACC,FASN,LPL,ATGL,HSL,DGAT1,DGAT2,AGPAT6,GPAM and ADRP were extremely significantly down-regulated(P<0.01),the relative expression level of TIP47 was significantly reduced(P<0.05);after mimicking miR-130b-5p expression,the accumulation of lipid droplets in goat intramuscular adipocytes decreased,and the amount of triglyceride synthesis decreased,CEBPα,PPARγ,SREBP1,ACC,FASN,LPL,ATGL,HSL,DGAT1,DGAT2,AGPAT6,TIP47,GPAM and ADRP were very significantly down-regulated(P<0.01).After inhibiting the expression of miR-130b-3p,the accumulation of lipid droplets in goat intramuscular adipocytes increased,and the synthesis of triglycerides increased,the expression levels of a P2,CEBPα,CEBPβ,PPARγ,SREBP1,FASN,LPL,ATGL,HSL,DGAT1,DGAT2 AGPAT6,TIP47,GPAM and ADRP were extremely significantly upregulated(P<0.01),and the relative expression level of ACC was significantly upregulated(P<0.05);after miR-130b-5p expression being inhibited,the accumulation of lipid was stimulated,the synthesis of triglycerides increased,and the expression levels of a P2,CEBPα,CEBPβ,PPARγ,SREBP1,ACC,FASN,LPL,ATGL,HSL,DGAT1,DGAT2,AGPAT6,TIP47,GPAM and ADRP were extremely significantly up-regulated(P <0.01).(2)Identification of the targeting relationship between miR-130b-3p and miR-130b-5p and KLF3The expression pattern of goat KLF3 is opposite to miR-130b-3p and miR-130b-5p.The expression of miR-130b-3p/5p was mimicked/repressed in goat intramuscular adipocytes,and the expression level of KLF3 was highly significantly reduced/increased(P<0.01).The KLF3 3’UTR was cloned and a typical plus-tail signal was found,the binding sites of miR-130b-3p/5p were present.Based on the construction of KLF3 3’UTR wild-type(pmirGLO-KLF3 WT)and mutant-type(pmirGLO-KLF3 MT)reporter vectors,the dual luciferase reporter assay system showed highly significant inhibition of KLF3 3’UTR activity after mimicking the expression of miR-130b-3p and miR-130b-5p(P<0.01),respectively,however,its mutant 3’UTR activity was not inhibited(P>0.05).(3)The effect of target gene KLF3 on the differentiation of goat intramuscular adipocytesThe goat KLF3 CDS region sequence of 1038 bp was cloned and encoded 345 amino acids;KLF3 was widely expressed in goat tissues,and the highest expression in fat.After interference of KLF3 in goat intramuscular adipocytes,the accumulation of intracellular lipid droplets decreased,and the expression levels of a P2,CEBPα,CEBPβ,PPARγ,LPL,ATGL,TIP47,GPAM,ADRP,KLF2,KLF4,KLF5,KLF6,KLF8,and KLF13 were extremely significantly reduced(P<0.01),and the expression levels of HSL,KLF1,KLF7 and KLF16 were extremely significantly increased(P<0.01),and KLF12 were significantly increased(P<0.05).After overexpression of KLF3,the lipid accumulations in intramuscular adipocytes were increased,the expression levels of CEBPβ,PPARγ,SREBP1,ACC,FASN,LPL,ATGL,DGAT1,DGAT2,AGPAT6,TIP47,GPAM,ADRP,KLF2,KLF4,KLF5,KLF6,KLF7,KLF8,KLF9,KLF10,KLF11,KLF12,KLF13,KLF14,KLF16 and KLF17 were extremely significantly increased(P<0.01),and the relative expression level of KLF1 was extremely significantly down-regulated(P<0.01).The above results indicate that both miR-130b-3p and miR-130b-5p inhibit the differentiation of goat intramuscular adipocytes by targeting KLF3,while their target gene KLF3 may coordinate with KLF2,KLF4-6,KLF8 and KLF13 to regulate the expression of CEBPβ,PPARγ,LPL,ATGL,TIP47,GPAM,ADRP to promote intramuscular adipocyte differentiation in goats.The results of this study provide a theoretical basis for revealing the regulatory mechanism of miRNAs on goat fat differentiation,and provide a new reference for improving animal meat quality.