Effect Of Bta-miR-novel3 On Lipid Deposition In Cow Brown Adipocytes Under High NEFAs Concentration

Cows in the perinatal period have a negative energy balance because of the increase in energy demand and the decrease in dry matter intake.At this time,fat mobilization rapidly aggravate and further develop into lipid metabolism disorders in adipocytes,resulting in high non-esterified fatty acids(NEFAs)hyperlipidemia,which is a major clinical pathological feature of perinatal cows.The release of excessive amounts of NEFAs can cause various diseases in dairy cows,such as cow’s ketoacidosis,fatty liver,and oxidative stress,etc.At the same time,the lipid toxicity of high NEFAs can seriously damage the health of dairy cows.Brown adipocytes play an important role in mobilizing fat and producing calories.Therefore,understanding the lipid metabolism of dairy brown adipocytes in this state is critical for the study of high NEFAs hyperlipidemia in cows.MicroRNA(miRNA)is a type of non-coding small RNA molecule(about 22 nucleotides)that has been studied popularly in recent years.It is mainly through the combination with the non-coding region of the 3’-end of the target gene mRNA,thereby inhibiting the target gene’s expression to regulate the physiological functions of cells.The role of miRNA is very extensive,and studies have found that about 30% of mammalian genes are regulated by miRNA.However,the mechanism of miRNA regulation in dairy cattle brown adipocytes under high NEFAs has rarely been reported.Therefore,the purpose of this dissertation is to study the regulation of miRNA on brown adipocytes in dairy cows under high NEFAs status,and to provide new ideas and strategies for the prevention and treatment of high NEFAs hyperlipemia in dairy cows.In our study,NEFAs(0 mmol/L,0.3 mmol/L,0.6 mmol/L,1.2 mmol/L,2.4 mmol/L)were directly added to brown adipocytes(11 days of fully differentiated and mature cells)to simulate the cellular status of dairy cows with high NEFAs hyperlipidemia.The results showed that when the concentration of NEFAs was higher than 1.2 mmol/L,the content of intracellular triacylglycerol(TG)was significantly reduced,and the glycerol content in the culture fluid increased significantly.To explore its intrinsic regulatory mechanisms,this model was applied to detect changes in miRNA expression profiles in brown adipocytes under the action of high NEFAs(1.2 mmol/L)using second-generation sequencing technology.Sequencing results showed that the expression of bta-miRnovel3 was significantly increased compared to the blank control group,and glycerol-3-phosphate acyltransferases1(GPAT1)expression was significantly reduced,and was verified by qRT-PCR and Western Blot.In addition,we also confirmed the targeting relationship between bta-miR-novel3 and GPAT1 through dual luciferase reporter assays.Further studies revealed that bta-miR-novel3 was over-expressed in brown adipocytes of dairy cows by transfection with the bta-miR-novel3 mimics.The results showed that GPAT1 expression and intracellular TG content decreased significantly in the adipocytes of the mimics group;the glycerol content increased significantly in the culture medium.When the bta-miR-novel3 inhibitor(inhibitor)was transfected to inhibit the expression of bta-miR-novel3,the results showed that GPAT1 expression and intracellular TG content were significantly increased in the adipocytes of the inhibitor group;the glycerol content declined significantly in the culture medium.In addition,bta-miR-novel3 inhibitor was used to inhibit bta-miR-novel3 expression when adipocytes were treated with high concentrations of NEFAs(1.2 mmol/L).The results showed that GPAT1 expression,intracellular TG content and lipid droplets(LDs)levels in bta-miR-novel3 inhibitor group were increased,while the glycerol content in culture medium was decreased compared with NEFAs group.The above results show that it is possible to ameliorate the impairment of fat accumulation capacity of brown adipocytes by inhibiting the expression of bta-miR-novel3 under high concentrations of NEFAs(1.2 mmol/L).In summary,high concentrations of NEFAs cause the up-regulation of bta-miRnovel3 expression in primary brown adipocytes,and bta-mi R-novel3 inhibits TG synthesis by inhibiting the expression of the target gene GPAT1,which aggravates the production of NEFAs and high NEFAs hyperlipemia.