Regulatory Pathway And Effects Of Autocrine Motility Factor Receptor On Porcine Preadipocyte Differentiation

AMFR plays an important role in the Tumorigenesis and metastasis，the formation of newblood vessels，nervous development，growth and neural differentiation， cholesterol synthesis andlipid metabolism. In this study, porcine adipocyte was used to identify the role of AMFR gene onthe process of the differentiation and explore the mechanism of this gene in pig fat deposition,providing a theoretical basis for the improvement of pig meat quality.Porcine adipocyte was isolated through the method of trypsinization, and then was inducedby differentiation inducer as insulin for examination of triglyceride content and Oil red staining.The results showed that lipid droplet was increased and OD valve of triglyceride content wasup-regulated in the process of adipocyte induction. The transcriptional level of marker geneinvolved in adipose differentiation as PPARγ, C/EBPα, KLF2were examined by RT-PCR,suggesting the expression of PPARγ and C/EBPα were increased, negativeregulation gene KLF2was down-regulaed, following the change of the morphology in the process of adipocyte induction,and these show the success of the adipose differentiation.To explore the function of porcine AMFR gene, cDNA of AMFR was cloned to get its mRNAsequence. In this study, primers were designed according to the homologous fragment of AMFRgenes from human and rodent, then amplificating and sequencing to get the2150bp mRNA ofnucleotide sequence, including the CDS of1944bp. The homology of the AMFR CDS betweenporcine and rodent was87.19%, and the homology between porcine and human was89.19%.In the process of adipocyte differentiation, the transcriptional level of AMFR， Insig1，Insig2，SREBP1-a and SREBP2were examined by RT-PCR. The data showed that the expressionof AMFR and Insig1exhibit the significant change compared to the pre-differentiation, suggestingthese genes were involved in the precursor adipose differentiation.Silencing AMFR through design of siRNA according to AMFR CDS, the mRNA level ofAMFR associated genes as Insig1，SREBP1-a and SREBP2, and the maker gene C/EBPα involvedin adipose differentiation were down-regulated, while the expression of the maker gene KLF2wasincreased, and the expression of PPARγ was not significant. The above results suggested thatAMFR could be able to regulate the expression of Insig1, SREBP1-a, SREBP2, C/EBPα andKLF2.After silencing AMFR, adipocyte was induced by differentiation inducer to144h, lipiddeposition and OD value of treatment group were significantly decreased compared to the control group. The expression of KLF2, the negativeregulation gene involved in precursor adiposedifferentiation, was decreased. The results suggested that silencing AMFR led to disturbanceprecursor adipose differentiation, decrease in the synthesis of triglycerides.In conclusion, the study demonstrated that AMFR regulated the precursor adiposedifferentiation through SREBP pathway and adipose differentiation regulation of gene C/EBPαand KLF2. The conclusion provides the theoretical basis for studying the molecular mechanism ofAMFR in lipid metabolism and improvement of the pork quality traits.