The Role Of Bta-miR-27a-3p Targeted INSR In The Development Of Fatty Liver In Perinatal Cows

Fatty liver is a frequent nutritional metabolic disorder in dairy cows during the perinatal period and its pathogenesis is mainly associated with excessive fat mobilisation and insulin resistance（IR）.Insulin exerts its biological effects by binding to the insulin receptor（INSR）on the cell membrane and then triggering a cascade reaction.It has been shown that IR may be central to the pathogenesis of fatty liver in dairy cows.Therefore,an in-depth study of the role of INSR and insulin signalling pathways in glucolipid metabolism in cows with fatty liver is particularly important for the prevention and treatment of fatty liver in dairy cows.miRNA can regulate gene expression by catabolizing the m RNA of target genes or by blocking m RNA translation.Studies in human medicine and mice have identified that miR-27a-3p is involved in glucose uptake and utilization,triglyceride（TG）synthesis and other glucolipid metabolic processes in hepatocytes.However,to date,there are few reports in the literature on the biological functions of miR-27a-3p in the glycolipid metabolism of dairy hepatocytes.The present study aims to investigate the regulatory role of miR-27a-3p and its target genes in hepatic glucose and lipid metabolism through a combination of in vivo and in vitro assays,with a view to partially revealing the molecular pathogenesis of fatty liver in dairy cows from the perspective of endogenous regulation.In vivo trial,blood biochemical parameters and TG content of liver tissue samples were measured in cows with fatty liver,and the expression of key genes of INSR and PI3K-AKT insulin signalling pathway in liver tissue was measured,as well as the expression of miRNAs closely linked to INSR genes.In vitro assay,miR-27a-3p mimics and inhibitors were transfected into cow hepatocytes to achieve overexpression and inhibition of miR-27a-3p at the cellular level.q RT-PCR and Western blot techniques were used to detect changes in the expression of INSR and PI3K-AKT insulin signaling pathway-related genes,and the targeting relationship was verified with the help of dual luciferase reporter gene analysis.A fatty liver cell model was established using sodium oleate to observe whether miR-27a-3p inhibitor could resist the damage caused by sodium oleate to hepatocytes.Finally,INSR si RNA was transfected into cow hepatocytes and the activity of the downstream signalling pathway was examined.The main test results are as follows:（1）Disturbances in glucolipid metabolism occurred in fatty liver cows,with reduced INSR expression in the liver,significantly reduced of p-PI3 K protein expression（P < 0.05）,increased activity of the glycogen synthase kinase-3β（GSK-3β）gene,which inhibits glycogen synthesis,and increased activation of the hormone-sensitive lipase（HSL）form of lipid hydrolysis.At the same time,miR-27a-3p expression in the liver of cows with fatty liver was significantly higher and statistically significant（P < 0.05）.The results suggest that PI3 KAKT insulin signalling is impaired when cows develop fatty liver and that this process may be linked to the regulation of miR-27a-3p.（2）Overexpression of miR-27a-3p significantly reduced INSR expression（P < 0.05）,inhibited the PI3K-AKT insulin signalling pathway,increased GSK-3β gene activity and increased protein abundance of p-HSL,resulting in decreased insulin sensitivity,reduced glycogen synthesis and enhanced lipolysis in hepatocytes.In contrast,inhibition of miR-27a-3p significantly increased INSR expression（P < 0.05）,improved impaired PI3K-AKT insulin signaling pathway,inhibited GSK-3β and HSL activity,promoted hepatic glycogen synthesis,reduced lipid mobilization,and contributed to the maintenance of stable hepatocyte glucolipid metabolism.The 3’UTR of INSR was found to be directly targeted and significantly regulated by miR-27a-3p by dual luciferase reporter gene analysis（P < 0.001）,indicating that INSR is a target gene for miR-27a-3p.（3）Dairy cows were treated with different concentrations of sodium oleate（0,0.25,0.50 and 1.00 mmol/L）for 24 h.The results of Oil Red O staining demonstrated that hepatocytes showed significant lipid deposition at a concentration of 0.50 mmol/L of sodium oleate and were in good cellular condition.In addition,the intracellular lipid droplets in the "sodium oleate + inhibitor" group were smaller and the extent of lipid accumulation was reduced compared to the sodium oleate alone treatment group;at the same time,INSR expression was significantly increased（P < 0.05）and PI3K-AKT insulin signalling pathway expression returned to normal levels,suggesting that miR-27a-3p inhibitors can resist hepatocyte damage by sodium oleate.（4）The si RNA sequence INSR-Bos-4156 significantly reduced INSR protein expression（P < 0.01）,and INSR knockdown resulted in decreased PI3K-AKT insulin signaling pathway activity,causing decreased glycogen synthesis and increased lipolysis in hepatocytes.The results suggest that INSR is an important mediator linking miR-27a-3p and the downstream PI3K-AKT insulin signalling pathway.In conclusion,this experiment confirmed that the expression of INSR in the liver decreased and the PI3K-AKT insulin signaling was impaired when dairy cows developed fatty liver;miR-27a-3p affected the PI3K-AKT insulin signaling pathway by targeting INSR,Then,it regulates the key genes GSK-3β and HSL of glucose and lipid metabolism,and participates in the process of glucose and lipid metabolism in dairy cow hepatocytes.This indicates that miR-27a-3p can be used as a potential biomarker and therapeutic site for the detection of fatty liver in dairy cows in the future,and has certain application value for the diagnosis and treatment of fatty liver in dairy cows.