Mechanism Of Circ-FoxO3 Regulating High-temperature Induced Apoptosis And Autophagy In Bovine Hepatocytes

Circ-Fox O3 is a novel class of non-coding RNA,a covalently closed loop formed by reverse shearing of m RNA precursors,which plays an important role in cell differentiation,metabolism,embryonic development,neurodevelopment and the development of human diseases.Circ-Fox O3 is derived from a product formed by the cyclization of the second exon of the parental gene Fox O3.Studies have shown that Circ-Fox O3 plays an important role in cell differentiation.In this study,the effects of high temperature on the proliferation,apoptosis and autophagy of bovine hepatocytes and the expression of Circ-Fox O3 gene were investigated using isolated and cultured bovine hepatocytes as experimental materials.We report a systematic investigation of the mechanism of Circ-Fox O3 on the proliferation apoptosis and autophagy of bovine hepatocytes under high temperature conditions to provide a theoretical basis for alleviating heat stress in beef cattle.The main findings of this experiment are as follows.1.HS affects apoptosis and autophagy in bovine hepatocytes and the expression of Circ-Fox O3The autophagy and apoptosis of bovine hepatocytes were examined by applying HS(0h,2h,6h,12 h and 24h)for different periods of time,and it was found that the degree of apoptosis and autophagy of bovine hepatocytes were positively correlated with the time of heat stress.The results of q RT-PCR showed that the expression of Circ-Fox O3 increased with the time of heat stress treatment.2.Circ-Fox O3 is involved in the regulation of apoptosis and autophagy in bovine hepatocytes under heat stress3.Circ-Fox O3 overexpression plasmid was constructed and Circ-Fox O3 si RNA was synthesized.p LV-ci R and NC-si RNA were transfected into bovine hepatocytes,and Circ-Fox O3 overexpression significantly increased the expression of apoptosis and apoptosis-related genes caspase3 and Ba X at the m RNA level,and inhibited the expression of Bcl-2 at the m RNA level.Besides,it also decreased the expression of autophagy and autophagy-related genes P62,Beclin1,ULK1 and LC3 at the m RNA level.mi R-153 can regulate apoptosis and autophagy in bovine hepatocytes under HS through mi R-153.Bioinformatic analysis predicted that there are potential binding sites for Circ-Fox O3 and mi R-153,and that mi R-153 can regulate the expression of ULK1 genes involved in autophagy and apoptosis.Bovine hepatocytes were transfected with mi R-153 mimics and NC-mi R-153,respectively,and the results showed that overexpression of mi R-153 inhibited apoptosis and promoted autophagy in bovine hepatocytes,and overexpression of mi R-153 downregulated the expression of Circ-Fox O3 and promoted the expression of ULK1.The target fragment containing the specific binding site of Circ-Fox O3 and mi R-153 was recombinantly incorporated into the psi-CHECK reporter vector,and the binding site was targeted for mutation.The wild-type and mutant psi-CHECK reporter vectors were transfected with mi R-153 mimics and NC-mi R-153 into bovine hepatocytes,respectively.The dual luciferase activity assay showed that compared with the control group,the wild-type group showed a significant decrease in dual luciferase activity,while the mutant vector showed no significant change.This suggests that Circ-Fox O3 deregulates its promotion of ULK1 through sponge adsorption of mi R-153.In conclusion,Circ-Fox O3 expression was elevated in bovine hepatocytes under HS conditions,and HS promoted apoptosis and inhibited cell proliferation and autophagy.Circ-Fox O3 promoted apoptosis and inhibited cell proliferation and autophagy in bovine hepatocytes under HS.mi R-153 can be involved in the onset of autophagy and apoptosis caused by HS through the Circ-Fox O3/mi R-153/ ULK1 axis by targeting binding to Circ-Fox O3.