Mechanism Of CircBTBD7 Inhibition Of Adipogenesis In Bovine Adipocytes Via Bta-miR-183/SMAD4 Axis

Fat has a great influence on the quality of livestock meat,and appropriate levels of intramuscular fat and subcutaneous fat can improve the flavor,tenderness and taste of meat products.CircRNAs play an important role in the growth and development of mammals.However,the regulation mechanism of circRNAs on fat deposition is still unclear.In this study,we investigated the differentially expressed circRNAs in bovine longest dorsal muscle,perirenal adipose and subcutaneous adipose tissue,screened and identified a new circRNA,named circBTBD7,and further investigated the function and regulatory mechanism of circBTBD7 in bovine precursor adipocytes.The main findings are as follows:1.Differential expression analysis of circRNAs in longissimus dorsi,perirenal fat and subcutaneous fat tissue and screening of candidate circRNAsA total of 27,054 circRNAs were detected in the longest dorsal muscle,perirenal adipose and subcutaneous adipose tissues of Qinchuan cattle.According to log₂|Fold Change|>1 and P<0.05,there were 796 differentially expressed circRNAs in the longest dorsal muscle and perirenal adipose tissues,631 differentially expressed circRNAs in the longest dorsal muscle and subcutaneous adipose tissues There were 98 circRNAs differentially expressed in perirenal adipose and subcutaneous adipose tissues.Among them,the expression of differentially expressed circRNAs in both perirenal and subcutaneous adipose tissues was too low to perform subsequent functional validation tests,so the identification of adipogenesis-related circRNAs was changed.10 candidate circRNAs potentially involved in adipogenesis were identified in the longest dorsal muscle and both adipose tissues,and the circRNA with the highest expression level was explored-circBTBD7,the most highly expressed circRNA,was investigated for its function and regulatory mechanism in bovine preadipocytes.2.circBTBD7 suppresses adipogenesisCircRNA4807 was formed by reverse splicing of the second and third exons of the bovine BTBD7 gene,which was named circBTBD7.circBTBD7 was disrupted,lipid droplet and triglyceride content increased significantly at day 6 of induction of differentiation.The expression levels of adipose differentiation marker genes（PPARγand C/EBPα）were significantly upregulated at day 2 of induction of differentiation,and the expression levels of adipogenesis-related genes（FABP4,FASN and ACCα）regulated by PPARγand C/EBPαwere also significantly upregulated at day 6 of differentiation.This suggests that circBTBD7inhibited adipogenesis in bovine preadipocytes.3.circBTBD7 inhibited adipogenesis through the bta-miR-183/SMAD4 axisCircBTBD7 was mainly expressed in the cytoplasm,and 23 miRNAs were predicted to bind to circBTBD7 by Miranda and Targetscan software.Among them,bta-miR-183 was shown to bind to circBTBD7.In addition,SMAD4 in bta-miR-183 predicted target genes has been reported to be involved in adipogenesis.Dual luciferase reporter vector and rescue assays showed that bta-miR-183 was able to target and bind SMAD4 and reduce its expression level,while circBTBD7 was able to sponge bta-miR-183 and attenuate the repressive effect of bta-miR-183 on SMAD4.Meanwhile,bta-miR-183 and SMAD4 were also shown to promote and inhibit adipogenesis in bovine precursor fat,respectively.This suggests that circBTBD7inhibits adipogenesis through the bta-miR-183/SMAD4 axis.In this study,we analyzed the differentially expressed circRNAs in the longest dorsal muscle,perirenal adipose and subcutaneous adipose tissue of Qinchuan cattle,and identified a new circRNA,circBTBD7,and further explored the function and mechanism of action of circBTBD7 in bovine adipocytes,which provides a molecular theoretical basis for the next step of beef cattle breeding.