Identification And Functional Validation Of Bta-miR-124a Target Gene In Dairy Cattle

MicroRNA(referred to as microRNA)is a single-stranded small molecule RNA with a length of about 22 nucleotides.It can complement the binding sites of the non-coding region of target gene and inhibit the expression of target gene.Studies have shown that mi R-124 can regulate the expression of a variety of genes,with cell differentiation,cell proliferation,and participate in the biological processes such as sheep fat formation and deposition,porcine adipocyte differentiation and so on.However,lipid synthesis and metabolism are important physiological processes,but studies on the regulation of milk fat metabolism by mi R-124 in dairy cows are still limited.Therefore,the aim of this study is to identify the target genes related to milk fat metabolism of bta-mi R-124a(cow mi R-124a)and to verify its biological function.This study provides a basis for further study on the molecular mechanism of mi R-124 regulating lipid metabolism,and provides an important experimental support for molecular breeding of new dairy cow lines with high quality milk quality traits.Previous research group obtained the expression profiles of mi RNA in mammary epithelial cells of high-fat and low-fat dairy cows by high-throughput sequencing.From the highly differentially expressed mi RNA,bta-mi R-124 a was selected as the research object,and the target gene of bta-mi R-124 a and its biological function were validated by using mammary epithelial cells of dairy cows as experimental materials.Firstly,the conservativeness of the mature region of bta-mi R-124 a was analyzed by bioinformatics method,and the candidate target genes were predicted.At the same time,the signal pathway of bta-mi R-124 a candidate target genes was deeply analyzed.The results showed that the mature sequence of bta-mir-124 a was highly conservative among different species.57 candidate target genes were predicted from Target Scan and Mir Walk databases.The candidate target genes could participate in the cancer genes,MAPK(mitogen-activated protein kinase),AMPK(AMP-activated protein),c AMP(Cyclic adenosine monophosphate)signal transduction,lipid metabolism and fatty acid synthesis signaling pathway.Among them,PECR(peroxisomal trans-2-enoyl-Co A reductase),SCD(stearoyl-Co A desaturase),ELOVL5(fatty acid elongase 5),CPT1A(carnitine palmitoyl transferase 1A)andother candidate genes related to lipid metabolism.Then,we selected the highly matched gene PECR to study the transcription and translation level of bta-mi R-124 a pairs of target genes by quantitative real-time polymerase chain reaction(q PCR).In this study,PECR gene was selected for further target gene identification and functional validation.Dual luciferase reporter gene and real-time fluorescence quantitative PCR(q PCR)were used to further identify the targeting relationship between bta-mi R-124 a and target gene.The results showed that the expression of percent and protein in overexpressing bta-mi R-124 a cells were significantly Lower than those in control group(p < 0.05),and the expression of percent and protein in inhibiting bta-mi R-124 a cells were significantly higher than those in control group(p < 0.05).In addition,the expression of ELOVL2(fatty acid elongase 2)downstream percent,triglycerides and free fatty acids in PECR downstream gene were further detected after transfection of bta-mi R-124 a mimics and bta-mi R-124 a inhibitor.In the overexpression group of transfected bta-mi R-124 a,bta-mi R-124 a could significantly and positively regulate the expression of ELOVL2(p < 0.05),triglyceride(p <0.01)and free fatty acid(p < 0.05)downstream gene of PECR.Compared with the control group,the expression of ELOVL2 was significantly negatively regulated by bta-mi R-124 a after transfection with bta-mi R-124 a inhibitor(p < 0.01).However,the expression of triglyceride was higher than that of control group and the content of free fatty acid was lower than that of control group.There was no significant difference between the two groups.The above results conclude that:(1)Through bioinformatics analysis,fluorescence quantitative PCR and dual-luciferase validation,PECR has a targeting relationship with bta-mi R-124,and PECR is the target gene of bta-mi R-124 a.(2)bta-mi R-124 a can regulate the expression of ELOVL2,a downstream gene of PECR,in the lipid metabolism pathway.(3)bta-mi R-124 a can effectively affect the content of triglycerides and free fatty acids in dairy cow mammary epithelial cells,thus involved in regulating the synthesis and secretion of milk.