MiR-214-5p Regulating Differentiation Of Intramuscular Preadipocytes In Goat Via Targeting KLF12

Intramuscular fat(IMF)is deposited in muscle fibers and between muscle bundles.Intramuscular adipocyte differentiation is the key to determine intramuscular fat deposition.Furthermore,abundant intramuscular fat deposits are the main factor that has a positive impact on meat quality,and can also give producers higher profits.As an important type of post-transcriptional regulatory factor,miRNA can regulate gene expression and regulate cell differentiation by specifically binding to target genes.A large number of studies have shown that miRNA is closely related to lipid metabolism and fat deposition.Therefore,this study used the differentially expressed miR-214-5p obtained by RNA-Seq screening in the early stage of the laboratory as the research objectives.Using RT-PCR,cell culture,vector construction,liposome transfection,qPCR,overexpression,RNA interference,dual luciferase reporter system,oil red O and Bodipy staining and other biological methods to clarify the possible mechanism in intramuscular adipocyte differentiation.(1)The effect of miR-214-5p on the differentiation of goat intramuscular adipocytes.During the induction and differentiation of goat intramuscular adipocytes for 0-120 h,the expression level of miR-214-5p was peaked at the 48 h,which was significantly higher than the expression level at the 0 h.Inhibiting the expression of miR-214-5p promotes the accumulation of lipid droplets in goat intramuscular adipocytes,and promotes adipocyte differentiation by up-regulating the expression of LPL(P<0.05)and PPARγ(P<0.01)while inhibiting the expression of HSL(P<0.01).Overexpression miR-214-5p inhibited the differentiation of goat intramuscular adipocytes by down-regulating the expression levels of LPL(P<0.05),AP2,FASN and ACC(P<0.01)while up-regulating the expression of HSL(P<0.01).(2)Identification the relationship between miR-214-5p and its target gene KLF12According to the four online database Target Scan,microRNAseq,miRDB and miRTCDs,the target gene of miR-214-5p was predicted,and KLF12 was selected as the potential target gene of miR-214-5p.Using Target Scan database to reversely predict miRNAs that may target to KLF12,the results showed that the KLF12 3′UTR sequence has miR-214-5p binding sites.The KLF12 3’UTR sequence was amplified by RT-PCR.Then the dual luciferase reporter vector KLF12 3’UTR wild type(PmirGLO-KLF12WT)and mutant type(PmirGLO-KLF12 MT)were constructed.The results showed that overexpression of miR-214-5p can significantly inhibit the luciferase activity of KLF12(P<0.01),while the mutant activity was not inhibited(P>0.05).We confirmed that KLF12 is the target gene of miR-214-5p.(3)Goat KLF12 gene cloning and bioinformatics analysisRT-PCR amplification obtained KLF12 gene sequence 1315 bp,including 5’UTR sequence 52 bp,3’UTR sequence 54 bp and CDS sequence 1209 bp.The gene encodes402 amino acids.Bioinformatics analysis indicated that KLF12 may interact with proteins such as FSBP,SRBD1,MAGEB10,TYWS,TANC2,PARD3 B and TFAP2 A.Moreover,KLF12 is highly conservative among different species and KLF12 is expressed at high levels in tissues such as heart,kidney,muscle,subcutaneous and abdominal fat.(4)The effect of KLF12 on the differentiation of goat intramuscular adipocytesConstruct KLF12-PcDNA 3.1 overexpression vector and chemically synthesize goat KLF12 siRNA sequence.Interference/overexpression of KLF12 in goat intramuscular preadipocytes promotes/inhibits lipid droplet accumulation,and this effect may be achieved by up-regulating or down-regulating the expression of LPL and CEBPα(P<0.01).In addition,interference or overexpression of KLF12 significantly inhibited or up-regulated the expression levels of KLF8 and KLF11,respectively.Based the above research,we speculated that KLF12 may have an antagonistic or synergy effect with KLF8 and KLF11,respectively.In conclusion,miR-214-5p can inhibit the differentiation of goat intramuscular adipocytes by targeting the expression of KLF12.Its target gene KLF12 may inhibit the differentiation of goat intramuscular adipocytes by regulating the expression of LPL.The results of this study provide an experimental basis for finally revealing the mechanism of miR-214-5p in adipocytes.