Mechanism Of TP53INP2 Gene Regulating Qinchuan Beef Cattle Adipocytes Differentiation

Adipose tissue is a vital endocrine organ that maintains energy homeostasis and is critical for mammalian development.In addition,the quality of beef is closely related to the intramuscular fat mass.However,the content of intramuscular fat at the molecular and cellular level depends on the proliferation and differentiation of adipocytes.Tumor protein p53 inducible nuclear protein 2(TP53INP2),is a dual regulator of transcription and autophagy,some results have reported that TP53INP2 could negatively regulate adipogenesis in human and mouse preadipocytes.However,the molecular mechanism of whether TP53INP2 can regulate bovine adipocyte differentiation through autophagy has not been clarified.We focus on a new Qinchuan beef strain(referred to as "Qinchuan beef cattle")in our research,the mechanism of TP53INP2 gene on bovine adipocyte differentiation was systematically explored.In this study,bovine pre-adipocytes were isolated from a one-day-old healthy calf.We investigate whether TP53INP2 is involved in the regulation of autophagy during bovine adipocyte differentiation and how TP53INP2 affects the differentiation of bovine adipocytes in molecular and cellular level by constructing overexpression vectors and synthesis of si RNA.The obtained experimental results are as follows:1.TP53INP2 gene is highly abundantly expressed in the muscle tissues of Qinchuan beef cattle(p<0.01),and is expressed in the subcutaneous adipose tissue of Qinchuan beef cattle,RT-q PCR results showed that the m RNA expression of TP53INP2 gene reached its peak on the second day after induction(p<0.01),then the expression level was decreased,indicating that this gene may be involved in the early differentiation of preadipocytes.2.PCR amplified bovine TP53INP2 gene CDS region,and obtained a total length of 675 bp CDS region;successfully constructed pc DNA3.1(+)-TP53INP2 overexpression vector,overexpression efficiency of 24 h,48h,72 h and 150 h were about 400 times,500 times,35 times and 14 times,both significantly higher than the control group(p<0.01);among the three synthesized si RNAs,one si RNA with the best interference efficiency was selected,and the interference efficiency of 24 h,48h,72 h and 150 h were about 84%,83%,70%,and 40%,and all are significantly higher than the control group(p<0.01).3.Interfering with the expression of TP53INP2 can significantly inhibit the formation of lipid droplets during adipocyte differentiation,and can inhibit the expression of adipogenesis marker genes PPAR?,PLIN2,FASN protein and m RNA level(p<0.05);on the contrary,overexpression of TP53INP2 promotes the process of adipocyte differentiation Promote the formation of medium lipid droplets and the expression of adipogenic differentiation marker genes expression during differentiation(p<0.05).It was revealed that the TP53INP2 gene promotes the differentiation of bovine preadipocytes.4.Knockdown of the TP53INP2 can significantly inhibit the degree of autophagy during the differentiation of adipocytes,and can significantly inhibit the m RNA expression of autophagy marker genes ATG7 and BECN1 and the expression of LC3 and p62 protein levels(p<0.05);on the contrary,overexpression of this gene can promote the occurrence of autophagy and the expression of autophagic marker proteins during adipocyte differentiation(p<0.05).It was revealed that TP53INP2 gene promotes the occurrence of autophagy during early differentiation and may regulate bovine preadipocyte differentiation through autophagy.5.When the TP53INP2 gene was overexpressed and interfered,the m RNA and protein expression levels of the differentiation marker gene PPAR? were significantly increased or decreased(p<0.05).Results of in vitro rescue test of PPAR? agonist rosiglitazone showed that rosiglitazone can significantly alleviate the phenomenon of reduced lipid droplets and down-regulation of adipogenic differentiation genes caused by interference with TP53INP2 gene(p<0.05).The results showed that the TP53INP2 gene may affect bovine precursor adipocyte differentiation through the PPAR? pathway.Overall,this results suggested that TP53INP2 can activate autophagy during the early stage of differentiation in Qinchuan beef bovine adipocytes and positively regulate adipocyte differentiation by affecting autophagy.Additionally,peroxisome proliferator-activated receptor gamma(PPAR?)also contributed to the function of TP53INP2 in modulating Qinchuan beef bovine adipocyte differentiation.