| The Fibroblast Growth Factors(FGFs)family consists of 22 structurally related proteins in mammals.These proteins have about 120 amino acid sequence homology.Most FGF ligands transmit signals into the cell by binding and activating the tyrosine kinase receptor FGFR family on the cell surface,regulating cell proliferation,survival,migration,differentiation,apoptosis,and metabolism.Because of the important function of FGFs,its role in adipose tissue development and fat metabolism has also attracted much attention from scholars.FGF9 is one of the important members of FGFs,and is widely expressed in various tissues and cells.It participates in various physiological processes such as angiogenesis,embryo development,and damage repair through binding with the receptors FGFR2 and FGFR3.At present,FGF9 related research mostly focuses on skeletal development,gonadal differentiation,and cancer development.There are some reports that FGF9 m RNA is highly expressed in adipose tissue of humans and mice,and the role of FGF9 in the differentiation of goat intramuscular adipocytes is rare.It has been reported that only the predicted sequence of goat FGF9 gene is found on NCBI,which is difficult to provide reliable basic data for animal husbandry research.Therefore,the purpose of this study was to clone the goat FGF9 gene sequence using RT-PCR technology,and to explore the effect of FGF9 on goat intramuscular adipocytes differentiation through cell culture,interference,and overexpression.The main findings are as follows:(1)The cloned goat FGF9 gene sequence was 818 bp,including a 627 bp CDS region,encoding 208 amino acids.Get Gen Bank accession number: MH358358.FGF9 is widely expressed in goat tissues,and its relative expression in kidney is the highest,which is extremely significantly higher than other tested tissues(P<0.01),followed by intestinal tissues,and liver and spleen are the lowest.(2)With the differentiation of goat intramuscular adipocytes,the expression of FGF9 increased gradually from 0 to 5 d,and the expression level increased extremely significantly on the 6th day after induction of differentiation(P<0.01).(3)The interference efficiency of FGF9-si RNA2 reached 64%(P<0.01).After interfering with FGF9,the accumulation of lipid droplets in goat intramuscular adipocytes increased,and triglyceride content also increased significantly(P<0.05).At the same time,interfering with FGF9 up-regulated the expression of adipocyte differentiation marker genes PPARγ,C/EBPα,C/EBPβ,and SREBP1.Among them,the expression of PPARγ reached a very significant level(P<0.01),while the gene that inhibited differentiation Pref1 was significantly down-regulated(P<0.01);ACACA and FASN related to the de novo synthesis of fatty acids were significantly increased up to 30 times(P<0.01)and 50 times(P<0.01),and FABP3 related to fatty acid transport was also significantly increased up to 62 times(P<0.01),DGAT1 related to triglyceride synthesis was slightly up-regulated and GAP2 was significantly increased approximately 5 times(P<0.01);LPL related to fatty acid absorption and hydrolysis was slightly up-regulated,and HSL related to lipolysis was not significantly changed while ATGL was significantly increased about 3 times(P<0.01)(compared with the control group).(4)The overexpression vector of FGF9 adenovirus was constructed,and the overexpression efficiency was about 2749 times(P<0.01).After over-expression of FGF9,the accumulation of lipid droplets in intramuscular adipocytes was reduced,and triglyceride content was significantly reduced(P<0.05).Overexpression of FGF9 down-regulated the adipocyte differentiation marker genes C/EBPα,C/EBPβ,and SREBP1,and the expression of PPARγ was significantly down-regulated(P<0.05),while the differentiation inhibition gene Pref1 was significantly up-regulated(P<0.05);at the same time,ACACA related to the de novo synthesis of fatty acids was significantly up-regulated 4 times(P<0.01),while FASN was not significantly changed,FABP3 related to fatty acid transport was slightly down-regulated,DGAT1 related to triglyceride synthesis was not significantly changed,and GAP2 was significantly down-regulated(P<0.05),LPL related to fatty acid absorption and hydrolysis did not change significantly,while HSL and ATGL related to lipolysis were significantly increased by about 2.5 times(P<0.01)and 2 times(P<0.01)(compared with the control group).(5)The expression pattern of the receptor FGFRs gene during the differentiation of goat intramuscular adipocytes showed that FGFR1,FGFR3,and FGFR4 expression were similar,and all showed a significant downward trend.However,only FGFR2 showed a slowly increasing trend when the intramuscular precursor adipocytes started to differentiate.By the 6th day of differentiation,the expression level increased significantly to the highest level(P<0.01),which was the same as the expression pattern of FGF9 in adipocyte differentiation.(6)After interfering with FGF9,FGFR2 and FGFR3 receptor gene expression levels were down-regulated,and FGFR2 reached a very significant level(P<0.01).si RNA was used to interfere with the expression of FGFR1-4 in goat intramuscular adipocytes respectively.It was found that the expression levels of PPARγ and C/EBPβ were extremely significantly increased after interference with FGFR2(P<0.01).C/EBPα and SREBP1 did not increase significantly,and Pref1 expression decreased(compared with the control group),which was consistent with the change in the expression of adipose differentiation marker genes after interference with FGF9.(7)In goat intramuscular preadipocytes co-transfected with FGF9 adenovirus overexpression vector and different concentrations of FGFR2-si RNA(0.08,0.10,0.12,0.14,0.16 μmol/L),the accumulation of lipid droplets showed a significant decrease and a slight increase with increasing interference si RNA concentration.The adipocyte differentiation marker genes PPARγ,C/EBPα,and C/EBPβ also showed a trend of first decline and then increase,SREBP1 showed a downward trend,and Pref1 kept a high expression with the increase of interference concentration(compared to the group infected with FGF9 adenovirus only).The above results indicate that FGF9 interacts with FGFR2 and inhibits the differentiation of goat intramuscular adipocytes by regulating the expression of PPARγ and Prefl.This provides necessary data for the further study of the signaling pathway mechanism of FGF9 regulating goat intramuscular adipocyte differentiation. |
