Ssc-miR-26a Targets ACSL3 To Regulate Adipogenic Differentiation Of Porcine Intramuscular Fat Cells

The content of intramuscular fat(IMF)is one of the main indicators for evaluating the quality of meat.It is very important in the process of pork production.Studying the fat deposition mechanism of intramuscular fat cells can provide data support for livestock production.In recent years,many miRNAs have played a role in the proliferation and differentiation of adipocytes.In the laboratory,high-throughput sequencing was used to screen out many miRNAs related to fat deposition,combined with the relative expression of ACSL family members in muscle.Bidirectional prediction by target gene online prediction software: differentially expressed miR-26a-5p and miR-223-3p may have a targeting relationship with acetyl-CoA synthetase 3(ACSL3).In this experiment,the target relationship was verified by the dual fluorescein reporter enzyme system,and the intramuscular fat cells derived from the longissimus dorsi muscle were isolated and cultured by differential adherence method,and the miRNA was analyzed by overexpression,RT-qPCR and other techniques.Regulation of the differentiation process of intramuscular precursor adipose cells.The main results of this study:(1)Analysis of mRNA tissue expression in Yimeng black pig ACSLs showed that ACSL1 was mainly expressed in the liver,ACSL3 was mainly expressed in liver and muscle tissues,ACSL4 was mainly expressed in liver and spleen,and both ACSL5 and ACSL6 were expressed in various tissues.Lower in the muscle tissues of the high and low fat groups,ACSL1,ACSL3,and ACSL4 were differentially expressed and reached significant levels(P<0.01).ACSL5 and ACSL6 were different but not significant.(2)miR-26a-5p and miR-223-3p were differentially expressed in the liver tissues of pigs with different intramuscular fat content and reached extremely significant levels,consistent with high-throughput sequencing results.In the muscle tissues of the high and low fat groups,miR-26a-5p and miR-223-3p and the gene ACSL3 were differentially expressed and reached significant levels.(3)Dual luciferase system reporter gene test results: when wild-type ACSL3 3? UTRand miR-26 a mimic were transfected into PK15 cells,the ratio of firefly luciferase signal to Renilla luciferase signal detected was compared with other control groups.Compared with the significant decrease(P<0.01)and no significant difference between the other control groups,the results indicated that the seed sequence of ACSL3 3? UTR can bind to the seed sequence of miR-26a-5p to quench the firefly luciferase.The firefly signal reduces the ratio of the signal to the Renilla luciferase signal,thereby determining the direct targeting relationship between ACSL3 and miR-26a-5p;the ACSL3 3? UTR wild type dual luciferase reporter vector and miR-223-3p When mimic was transfected into PK15 cells,the ratio of firefly luciferase signal to Renilla luciferase signal detected was not significantly different from that of other controls,indicating that there was no direct targeting of ACSL3 and miR-223-3p.relationship.(4)The mRNA level of ACSL3 increased gradually in the early stage of IMF cell differentiation,reached the peak at the 2nd day of differentiation(P<0.05),and then gradually decreased,and entered the plateau after 6d.(5)transfection of miR-26a-5p mimic / inhibitor can affect the expression of ACSL3 mRNA in primary intramuscular fat cells: inhibition of miR-26 a can significantly up-regulate the expression of ACSL3 mRNA(P <0.01),overexpression of miR-26 a can The expression level of ACSL3 mRNA was down-regulated.(6)Overexpression of miR-26a-5p can inhibit the differentiation of porcine intramuscular fat cells: overexpression of miR-26 a can significantly down-regulate the expression of PPAR? and SRREBP-1c in adipocyte differentiation markers(P<0.01),indicating overexpression of miR.-26a-5p can inhibit the differentiation of porcine intramuscular fat cells.(7)Overexpression of miR-26a-5p can inhibit lipid synthesis in pig intramuscular fat cells: After overexpression of miR-26 a,the results of oil red O staining and TG content showed significant reduction of synthetic lipid droplets,indicating overexpression of miR-26a-5p can inhibit lipid synthesis in pig intramuscular fat cells.The above results show that miR-26 a reduces intracellular TG synthesis by targeting the binding gene ACSL3;ssc-miR-26 a can inhibit the differentiation of porcine intramuscular fat cells.The results provide a basis for further study of the mechanism of miRNA effects onintramuscular fat deposition in pigs.