The Effects Of FNDC5 On Proliferation And Differentiation Of Adipocytes In Arbas Cashmere Goat Adipose-derived Stem Cells

FNDC5(Fibronectin type III domain-containing protein 5)is a single-transmembrane protein.Irisin is the post-translation processing products of FNDC5.As a kind of myokine,irisin plays an important roles in fat and energy metabolism,which can promote browning of the white adipocytes and increase thermogenic.FNDC5 also participated in the development of neurons.However,the mechanism of irisin in proliferation and differentiation into adipocytes is still in its infancy.In present study,over-expression and the interference vectors of FNDC5 were constructed to explore the effects of FNDC5 on proliferation and differentiation of adipocytes in arbas cashmere goat adipose-derived stem cells.(1)The construction of FNDC5 over-expression and the interference vectorsSequences of Arbas cashmere goats FNDC5 cDNA was cloned from the myoblast,the cloned FNDC5 fragment was connected with the pIRES2-EGFP vector.The vector was verified by restriction enzyme digestion,sequencing and fluorescent quantitative PCR,and was transferred into adipose-derived stem cells to detect the expression efficiency.The result indicates that the expression level of a FNDC5 gene is improved by 27.5 times.At the same time,2 FNDC5 siRNA sequences according to the homologous region of goat and other transcripts were designed and connected with the pSIREN-RetroQ-ZsGreen vector.The FNDC5 interference vector was constructed successfully and transfected into the ADSCs.The results showed that the expression level of FNDC5 gene could be reduced by 34%.The results indicates that the over-expression vector pIRES2-EGFP-FNDC5 and the interference vector CTC0398-1-6 were successfully constructed.(2)The effects of FNDC5 on proliferation of adipose-derived stem cells in arbas cashmere goat.The FNDC5 overexpression vector was transfected into adipose-derived stem cells by liposome transfection.The changes of ERK,p38 and AKT proteins and their phosphorylation levels were detected by Western blotting at 24h and 48h after transfection.The results showed that over expression of Fndc5 gene could increase the phosphorylation level of ERK and p38 in adipose-derived stem cells,and had no effect on the phosphorylation of AKT.The results showed that the FNDC5 gene influenced the physiological activity of the cell by the phosphorylation level of ERK1/2 and p38 in the MAPK signaling pathway,promoting cell proliferation.(3)The effects of FNDC5 on differentiation of adipose-derived stem cells in arbas cashmere goat.over-expression and the interference vectors of FNDC5 were transfected into adipose-derived stem cells by liposome transfection.The cells were then induced to differentiate into adipocytes and the mRNA and the protein levels of PPARy?SREBP1?ACC and FABP4,and the protein levels of white adipocyte signature gene ASC1 and brown adipocyte signature gene UCP1 were detected.Results show that the mRNA levels of PPAR??SREBP1?ACC and FABP4 were promoted by overexpression of FNDC5,the protein level of ASC1 was inhibited by overexpression of FNDC5,but the protein levels of PPAR??SREBP1?ACC and FABP4 had no significant change from the variation of FNDC5.The stainning of the oil red O indicates that FNDC5 has an inhibitory effect on the adipogenesis process of ADSCs.