Transcriptional Regulation Mechanism Of Porcine GPR120 Gene And Studies On The Inhibition Effect Of MiR-25 On Myoblast Energy Metabolism

Energy metabolism playes a crital role in the processes of animal growth,production and other important life activities. It is also closely related to human metabolic syndrome. Therefore, the study of energy metabolism regulation is helpful to improve animal production economic benefits, it also playes a great significance to improving human health. In our previous studies, the tissue expression profile analysis of Large White tissue expression certified GPR120 is a differentially expressed gene. Reports indicate that GPR120 gene involved in multiple physiological process of energy balance, regulated systemic energy and nutrition metabolic balance. This study aimed to investigate the regulatory mechanism of GPR120 gene, revealed the effect of GPR120 gene on the regulation process of energy metabolism.And this study also aimed to investigate the effect of mi R-25 on energy metabolism and verified biology based on high expression mi RNA of muscle tissue of embryonic stage of Large White and Tongcheng pigs by Solexa sequencing. mi R-25 was subjected to sequence analysis,target prediction and function analysis. Meanwhile,we aimed to investigate the transcriptional regulation mechanism of mi R-25, explored the effect of transcription factors on regulating transcription of mi R-25. The main results as follows: 1. Study of transcriptional regulatory mechanism of pig GPR120 geneTo determine the deletion fragmentsof GPR120 promoter were constructed into luciferase reporter vector based on the predicted Cp G islands. The data of the luciferase activity assay showed that the deletion fragment P5(-323 to +165 bp) was GPR120 core promoter. We predicted one potential binding site(TGTCG) for C/EBPβ in GPR120 core promoter by the TFSEARCH ver.1.3. Further analysis of a putative C/EBPβ binding site suggested that it is highly conserved in human, mouse and pig. Site-directed mutagenesis indicated the modification of C/EBPβ binding site significantly suppressed the promoter activity. The Ch IP analysis revealed that the binding of C/EBPβ to the GPR120 core promoter in vivo. Over-expression of C/EBPβ significantly enhances endogenous GPR120 expression. Consistent with this,inhibition of C/EBPβ expression significantly suppressed the expression of GPR120. Furthermore, HFD-feeding induced the expression of C/EBPβ, and up-regulated GPR120 and PPAR-γ2 in adipose tissue, and increased the binding of C/EBPβ on GPR120 promoter. 2.The role of mi R-25 in muscle cells energy metabolismmi R-25-3p abundance decreased first and then increased during differetiation of C2C12 cells.The expression levels of metabolic maker genes(PI3K、Map2k4) were significantly decreasedin mi R-25-3p group.We used several mi RNA target prediction softwares to predicted target genes of mi R-25-3p, the result showed that there is a potential targeting site of mi R-25-3p in Akt13’UTR region, and sequence alignment analysis suggested the sequences of mi R-25-3ptargeting siteof Akt1 3’UTR are highly conservative in mammals. The result of luciferase activity assay showed that mi R-25-3p significantly suppressed the Akt1 3’UTR luciferase activity, revealed mi R-25-3p specifically targeting to Akt1 3’UTR. Over-expression of mi R-25-3p significantly suppressed the m RNA and protein levels of Akt1 and PI3 K.In conclusion,mi R-25-3p could target Akt1 3’UTR and suppressed the m RNA and protein levels of Akt1 and PI3 K,might affect the activation of PI3K/Akt signal pathway, which affect the energy metabolism process of muscle cells. 3.Study of transcriptional regulatory mechanism of mi R-25In this study, a 2034 bp 5’-flanking region sequence of mi R-25-3p was cloned from mouse genomic DNA. Then,the promoter deletion fragments of mi R-25-3p were constructed into p GL3-Basic luciferase reporter vector. The data of the luciferase activity assay showed that the deletion fragment P9(-119/+144)was mi R-25-3p promoter. We predicted one potential binding site for AP-2α in mi R-25-3pcore promoter by bioinformatics softwares. Site-directed mutagenesis indicated the modification of AP-2α binding site significantly suppressed mi R-25-3p promoter activity. The Ch IP-Q-PCR analysis revealed that the binding of AP-2α to the mi R-25-3pcore promoter in vivo. Moreover, the bingding of AP-2α to the mi R-25-3pcore promoter was decreased during C2C12 cells differentiation. Over-expression of AP-2α significantly enhances significantly suppressed the promoter activity. AP-2α significantly induced endogenous mi R-25-3pexpression. Furthermore,AP-2α suppressed the m RNA and protein levels of Akt1. Consistent with this,inhibition of AP-2α expression significantly suppressed the expression of mi R-25-3p and up-regulated the m RNA and protein levels of Akt1.The experimental results demonstrated transcription factor AP-2α playes an important role in regulating the transcription of mi R-25-3p,and identified the binding site of mi R-25-3p. Furthermore,AP-2αwas able to influence the expression of Akt1 gene.