Exploring The Mechanism Of CircADAMTS16 Regulating Bovine Adipocyte Proliferation And Differentiation

Beef is loved by consumers because of its high nutritional value,and the intramuscular fat content affects the juiciness,tenderness and flavor of beef.Adipose deposition is regulated by a variety of hormones,enzymes,transcription factors,RNA(non-coding RNA,ncRNA).RNA(circular RNAs,circRNA)is a covalently closed ncRNA molecule that exists in eukaryotes.Studies have shown that circRNA is involved in the regulation of bovine adipocyte development.CircADAMTS16 is derive from that coding region of AD AMTS 16 gene and has a total length of 550 bp.Analysis of previous sequencing data revealed a high expression of circADAMTS16 in adipose tissues,so we explored its molecular mechanism in adipose development.The main results of this study are as follows:(1)The real existence of circADAMTS16 was identified by RNase R digestion.Cytoplasm was separated from adipocytes,and semiquantitative localization of nuclear matter was performed on circADAMTS16,which was mainly in the cytoplasm and a small number in the nucleus.Using qPCR to construct tissue expression pattern and sequential expression pattern,and using the heart as the internal control,circADAMTS16 was expressed in the highest amount in the lung,followed by fat,and the lowest in the kidney.During adipogenic differentiation,circADAMTS16 was significantly overexpressed on day 2 and then gradually decreased to its lowest level on day 8.(2)pCD2.1-circADAMTS16 and si-circADAMTS 16 were transfected into bovine adipose cells.The results of qPCR and western blot showed that circADAMTS16 inhibited the expression of adipogenic marker genes,promoted the expression of proliferation marker genes and inhibited the expression of apoptotic genes.Oil red O phenotypic test showed significant inhibition of lipid droplet formation;The CCK-8 and EdU flow cytometry tests showed that circADAMTS16 promoted the transformation of Gl-phase cells to S-phase and inhibited the apoptosis of adipocytes.Interference with the circADAMTS16 produces the opposite result.(3)miRnada and RNAhybird were predicted and screened,and the double luciferase report test verified that miR-10167-3p targeted to circADAMTS16.(4)MiR-10167-3pagomir/miR-10167-3pagomir was transfected into bovine adipocytes.Detection by qPCR and western blot showed that miR-10167-3p promoted the expression of lipid-forming marker genes,inhibited the expression of proliferation marker genes,and promoted the expression of apoptotic genes.The O-phenotype test of oil red showed that it significantly promoted lipid droplet generation,inhibited the transformation of G1 phase cells to S phase,and promoted the apoptosis of adipocytes.After interfering with miR-10167-3p,the results were the opposite.After co-transfection with circADAMTS16,the miR-10167-3p was rescued from inhibiting the proliferation of adipocytes and alleviating its promoting effects on paper synthesis and apoptosis of adipocytes.After the disturbance,the result is the opposite.(5)RNA-seq was used to analyze the effect of overexpression of miR-10167-3p on the transcriptome of bovine adipocytes.The results of sequencing showed that 573 differentially expressed genes were obtained,including 330 up-regulated genes and 243 down-regulated genes.The results of GO classification showed that DEGs was mainly enriched in cell process,organelles and catalytic activity.According to KEGG analysis,DEGs is enriched in cancer pathway genes,accounting for 8.87%at most,and in pathways related to fat deposition and lipid metabolism,accounting for 15.19%,namely MAPK signaling pathway,PI3K-Akt signaling pathway,Wnt signaling pathway and FoxO signaling pathway.(6)The results of the double luciferase reporting test showed that miR-10167-3p targeted the TCF7L1 gene located in the Wnt signaling pathway.In summary,our data indicate that circADAMTS16 inhibits the differentiation of primary bovine adipocytes,promotes the proliferation of primary bovine adipocytes and blocks the apoptosis of adipocytes by competitively binding to miR-10167-3p.The results of this study will provide basic data for further exploration of the mechanism of circADAMTS16 in the process of bovine lipogenesis,provide a theoretical basis for the regulation of fat metabolism interaction network and provide basic data for later genetic improvement and molecular breeding of beef cattle.