Mechanisms By Which CircSSBP2 Regulates The Proliferation Of Intramuscular Adipocytes In Qinchuan Cattle

Intramuscular fat(IMF)is a crucial factor in beef quality.IMF is primarily distributed between muscle fibers,and its accumulation can have an impact on the marbling shape and meat quality of beef.IMF formation and deposition is a complex process,and in recent years,a group of non-coding RNAs(nc RNAs)known as circ RNAs have been discovered to play an important role in regulating intramuscular fat deposition.Circ RNAs form a covalent loop structure after reverse splicing of precursor m RNAs.They can work by adsorbing mi RNAs and thus reducing their repressive effects on downstream target genes.Based on highthroughput sequencing of circ RNAs in intramuscular fat of Qinchuan and Wagyu cattle,we identified a new circ SSBP2 that is differentially expressed between the two species and associated with adipogenesis.We used RNA interference,overexpression,a dual-luciferase reporter,and a sensor to investigate the role of circ SSBP2 in intramuscular fat deposition in cattle.By competitively binding mi R-2400 to the NDRG1 gene,circ SSBP2 inhibited the proliferation of bovine intramuscular adipocytes.The purpose of this study is to explain the molecular mechanism of circ SSBP2 regulation of intramuscular fat deposition in Qinchuan cattle in order to lay the groundwork for the production of high quality Qinchuan beef rich in marbling and the selection of new strains of Qinchuan beef with excellent meat quality.The following are the key findings:1.circ SSBP2 inhibits the proliferation of bovine intramuscular adipocytesSanger sequencing confirmed the authenticity of circ SSBP2 and was supported by g DNA and c DNA experiments,as well as an actinomycin D assay.RT-q PCR revealed that it was significantly higher in Qinchuan cattle than in Japanese Black cattle,and that it was significantly increased during the proliferation of bovine intramuscular adipocytes,but that its content did not change significantly during cell differentiation,implying that it plays an important role in the development of intramuscular fat in Qinchuan cattle.Disruption of circ SSBP2 and overexpression of circ SSBP2 followed by EDU,flow cytocycling,CCK8,RTq PCR of proliferation marker genes,and Western blot assays revealed that disruption of circ SSBP2 promoted proliferation of bovine intramuscular adipocytes while overexpression of circ SSBP2 inhibited proliferation.2.circ SSBP2 inhibits bovine intramuscular preadipocyte proliferation by targeting mi R-2400The Bi Bi Serv online website was used to predict a binding site between circ SSBP2 and mi R-2400.Mi R-2400 overexpression significantly reduced the activity of the circ SSBP2 wildtype vector compared to the control,but had no effect on the activity of the mutant vector,demonstrating that the two bind to each other.The mi R-2400 Sensor vector was then constructed,and the assay results showed that mi R-2400 overexpression significantly reduced the luciferase activity of the mi R-2400 Sensor vector,but when circ SSBP2 was also overexpressed,the luciferase activity of the mi R-2400 Sensor vector was partially restored,and the extent of this restoration was positively correlated with the dose of circ SSBP2.Overexpression of mi R-2400 was shown to increase proliferation of bovine intramyocardial preadipocytes by EDU,flow cytometry,CCK8,RT-q PCR and Western blot assays of proliferation marker genes.Bovine intramuscular adipocytes co-transfected with the mi R-2400 mimic and/or the p CD5-circ SSBP2 overexpression vector demonstrated the combination of both.3.circ SSBP2 suppresses the proliferation of bovine intramuscular adipocytes by competitively binding mi R-2400 to the NDRG1 geneRNA-seq and online site prediction were used to identify the downstream gene NDRG1.The findings of RT-q PCR revealed a significant decrease in NDRG1 expression after mi R-2400 overexpression.The dual luciferase reporter method confirmed that NDRG1 is a mi R-2400 target gene.Overexpression of mi R-2400 significantly reduced the activity of the NDRG1 wild-type vector but had no impact on the activity of the mutant vector.NDRG1 interference substantially reduced NDRG1 m RNA and protein expression.EDU,flow cytometry,CCK8,RT-q PCR of proliferation marker genes,and Western blot assays all showed that inhibiting NDRG1 increased proliferation of bovine intramuscular adipocytes.The simultaneous co-transfection of circ SSBP2 reversed NDRG1 expression.Circ SSBP2 clearly suppresses bovine intramuscular preadipocyte proliferation by competitively binding mi R-2400 to the NDRG1 gene.By constructing a ce RNA network,this study clarified the functional role of circ SSBP2 in inhibiting the proliferation of bovine intramuscular adipocytes and revealed the mechanism of the circ SSBP2/mi R-2400/NDRG1 axis in intramuscular adipogenesis in Qinchuan cattle,providing a certain reference for molecular breeding and breed improvement work in Qinchuan cattle.