Regulatory Mechanism Of Circ＿015343 On Milk Fat Synthesis Of Ovine Mammary Epithelial Cells

circular RNA(circRNA)is a class of non-coding RNA,and it can increase the m RNAs expression by act as micro RNA(miRNA)sponges.They have important molecular functions in mammalian mammary gland development and lactation.In our previous study,circ＿015343 was found to be highly expressed in the ovine mammary gland tissue at lactation period by using RNA sequencing(RNA-seq),but the molecular mechanism of regulating lactation in sheep is still unknown.Therefore,the reverse transcription-quantitative polymerase chain reaction(RT-q PCR),CCK-8 and Edu assay were used to study the effect of circ＿015343 and miR-25 on ovine mammary epithelial cells,the targeting relationship between circ＿015343 and miR-25,between miR-25 and INSIG1 were verified using dual-luciferase reporter assay.The main results are as follows:1.The circ＿015343 was derived from complete ten exons between exon 2 and exon 11 of AASS,it mainly expressed in cytoplasm of ovine mammary epithelial cells,and there is also low expression in nucleus.circ＿015343 and AASS was found to be expressed in eight ovine tissues,with the highest expression level in the mammary gland,showing obvious tissue-specific expression.The circ＿015343 differentially expressed in mammary gland of two sheep breeds,and its expression in mammary gland tissues of Small Tail Han ewes was lower than that of Gansu Alpine Merino ewes.2.The si-circ＿015343 increased the expression levels of fatty acid-binding protein 4(FABP4),acetyl-coenzyme A carboxylase alpha(ACACA)and peroxisome proliferator activated receptor gamma(PPARG)(P < 0.05),but decreased the expression level of its parent gene AASS(P < 0.05).The si-circ＿015343 also increased the viability and proliferation of ovine mammary epithelial cells.This indicated that circ＿015343 inhibited the milk fat synthesis,cell viability and proliferation of ovine mammary epithelial cells.3.The results of the activity determination of double luciferase showed that miR-25 mimic significantly reduced the activity of luciferase in circ＿015343 for wild-type vector(P < 0.05),but it had no significant effect on activity of luciferase in the mutant vector,this indicates that there is a targeting relationship between circ＿015343 and miR-25.RT-q PCR results showed that circ＿015343 decreased the expression of miR-25(P < 0.01).4.RT-qPCR results showed that miR-25 mimic increased the expression levels of FABP4,stearoyl-Co A desaturase(SCD),sterol regulatory element-binding protein 1(SREBP1),ACACA and content of triglyceride in ovine mammary epithelial cells(P <0.05).However,miR-25 inhibitor decreased the expression levels of FABP4,fatty acid synthase(FASN)and content of triglyceride(P < 0.05).miR-25 mimic significantly increased the activity and proliferation of ovine mammary epithelial cells(P < 0.05),while miR-25 inhibitor showed the opposite results.Therefore,miR-25 can promote milk fat synthesis,the activity and proliferation of ovine mammary epithelial cells.5.miR-25 mimic significantly reduced the activity of luciferase of INSIG1 in wild-type vector(P < 0.05),but it had no significant effect on the luciferase activity of INSIG1 in mutant vector,this indicates that INSIG1 is a target gene of miR-25.RT-q PCR results showed that miR-25 decreased the expression of INSIG1(P < 0.01).si-circ＿015343 reduced the expression of INSIG1(P < 0.05).The above results showed that there is a regulatory pathway of circ＿015343-miR-25-INSIG1 in ovine mammary epithelial cells.circ＿015343 can inhibit the milk fat synthesis of ovine mammary epithelial cells by regulating the expression of miR-25,and miR-25 can promote the milk fat synthesis of ovine mammary epithelial cells by down-regulating the expression of INSIG1.