Effects Of Ssc-miR-34a On Proliferation And Differentiation Of Intramuscular Adipocyte In Pigs

MicroRNAs(miRNAs)are small non-coding RNA molecules(20–25 nucleotides in length)which found in eukaryotic organisms that regulate gene expression at the post-transcriptional level,including development,organ formation,cell proliferation and apoptosis,cancer incidence and fat metabolism,etc.Lipid metabolism is a complex biological metabolic process involving many tissues and organs.Adipose tissue is the largest energy repository of mammals,mainly stored in adipocyte in the form of triglycerides,while the liver is an important lipid metabolism organ.In the early stage of this laboratory,miRNAs with fat depositions were selected through high-throughput sequencing,and further study was carried out through preliminary verification and selection of miR-34 a.miRNAs is conservative strictly,we assume that miR-34 a might with the proliferation differentiation of porcine intramuscular adipocyte(IA),therefore design experiment for validation.This experiment adopts the target gene prediction software to predict miR-34 a candidate target genes of acetyl-co A synthetase 4(ACSL4),293 T cell line as the experimental material,by building ACSL4 3? UTR sequence(containing seeds of wild type and mutant type of carrier),the system adopts double luciferase reporter gene test firstly verify miR-34 a relation to ACSL4 targeting,and then research the role of the miR-34 a and the candidate target genes ACSL4 by IA in swine proliferation differentiation.The results as follows:1、The initial verification of miRNAs,such as miR-31,mi R-127,miR-96-5p,miR-345-5p,miR-34 a,related to fat deposition through RT-qPCR was significant and consistent with the results of high-throughput sequencing.2、The wild type ACSL4 3?UTR with miR-34 a mimic analog transfected 293 T cells,then detect the firefly luciferase signals and sea renal luciferase ratio compared with other control group were significantly lower(P< 0.01),and there is no obvious difference between the other control groups.The results show that ACSL4 3?UTR sequence of seeds can be combined with miR-34 a,so as to the firefly luciferase signal with sea kidney luciferase signal ratio is reduced,thus we determine that there is a direct targeting between ACSL4 mRNA and miR-34 a.3、According to the growth characteristic of the IA differentiation in different periods,namely,0 d,2 d,4 d,6 d,8 d cells,collected respectively,for the extraction of RNA,the RT-qPCR of the candidate target gene ACSL4 and other marker genes such as the production of fat marker gene PPARγ,aP2,SREBP-1c and adipose decompose key ATGL,Sirt1 gene.It was found that ACSL4 mRNA level gradually increased in the early stage of the differentiation of IMF cells and reached a peak(P<0.001)in the differentiation of 4d,followed by a gradual decline with a higher level of expression,so as the PPARγ and aP2 gene,but the gene of ATGL and Sirt1 with different.4、miR-34 a mimic/NC and miR-34 a inhibitor/NC was transfected into the porcine IA and induced after 2d,take 0 d,4 d,8 d after induction IA to ACSL4 gene RT-qPCR,found that miR-34 a mimic group than the control group ACSL4 gene expression by reducing,after the induction of 0d(namely after transfection 2d)reached the significant level(P<0.05),at the day of 4 and 8,the relative expression quantity is not very significant difference,especially to the 8d relative expression returns to normal.Transfection miR-34 a inhibitor from its control ACSL4 gene increases,the relative expression of the same after the induction of 0d(namely after transfection 2d)reached the significant level(P<0.05),at the 4d and 8d,the relative expression quantity is not very significant difference,especially to the 8d relative expression returns to normal.After selecting the induced after 0d(namely after transfection 2d)cells do other marker genes quantitative research,found that PPARγ,aP2,SREBP-1c gene expression and the trend of ACSL4 gene were positively correlated,ATGL and Sirt1 gene were negatively correlated(P<0.05).Conclusion: The relative expression of miR-34 a in the liver of pigs is significant differences,and down-regulated in the high fat groups;miR-34 a directly target ACSL4 gene and suppress the mRNA expression,so as to increase the accumulation of fat droplets in adipocyte.