Studies On The Regulation Mechanism Of MiR-194-5p By Targeting BCKDHA Gene In Ovine Adipogenesis

Branched chain amino acid(BCAA)is not only raw material for synthesis of proteins,but also an important energy substances.They were oxidatived decarboxylation to provide energy for the body.In addition,BCAA may also serve as important signaling molecules involved in glucose and lipid metabolism.BCKDHA,as a subunit of branched-chain α-ketoacid dehydrogenase complex(BCKDH),its abnormal can lead to the dysfunction of BCKDH complex,which can lead to the aggregating of BCAA and its metabolism in vivo,further reducing the transport and utilization of pyruvate and fatty acid.Finally,the energy metabolism can be interfered in vivo.MiRNAs(microRNAs)play an important role in post-transcriptional regulation of gene by completely or incompletely combining with mRNAs of target genes.They mainly combined with target gene’s 3’-untranslated region(3’ UTR)to inhibit the translation of the target gene,or to degrade the mRNA.However,the regulation mechanism of miRNAs for BCKDHA has not been reported.In order to better understand the functional mechanism of BCKDHA gene,miRNA and their crosstalk in regulating adipocyte differentiation.In this study,coding sequence(CDS)region and amino acid sequence of BCKDHA gene was analyzed using bioinformatics.Three online softwares were used to predict the miRNAs that may target BCKDHA.Double fluorescent reporter vector of BCKDHA 3’ UTR was constructed and transfected with miR-194-5p mimic into HEK293T cells.Target relationship between them was validated by detecting the fluorescence activity of dual-luciferase with.After overexpressing and inhibitting miR-194-5p in ovine SVF differentiation process,we observed the change of SVF during adipocyte differentiation process.The expression of miR-194-5p,BCKDHA mRNA and protein during ovine SVF adipocyte differentiation or transfected with miR-194-5p was detected using quantitative real-time PCR(qPCR)and Western blotting.The relationship and mechanism between miR-194-5p and BCKDHA in the pre-adipocyte differentiation and metabolism was analyzed from the RNA and protein level.The results of studies were as follows:(1)The full length of CDS sequence of ovine BCKDHA gene was 1344 bp and encoding 447 amino acids with a molecular mass at 50.7 kDa.The predicted BCKDHA is a basic instable and basic protein with isoelectric point of 8.62 and instability of 41.68.BCKDHA does not contain signal peptides and transmembrane domains.Ten O-glycosylation,3 N-glycosylation sites and 24 phosphorylation sites were predicted in the protein.The target gene prediction results showed that miR-194-5p was a key miRNA that may regulate BCKDHA expression with fully complementation between its seed sequence(2-8)and the 190～196th bases of BCKDHA 3’ UTR.(2)We successfully constructed BCKDHA 3’ UTR double fluorescent reporter vectors.Dual-luciferase activity testing results showed that fluorescence activity for groups co-transfected with pmir-BCKDHA and of miR-194-5p mimic was significantly(P<0.001)lower than that in the control group,suggesting that miR-194-5p inhibits fluorescence signal by combining with BCKDHA 3’ UTR.Therefore,the mechanism that miR-194-5p regulating BCKDHA was:miR-194-5p inhibits the expression of BCKDHA by fully combining between its seed sequence and the 190～196th bases of BCKDHA 3’ UTR.(3)After overexpressing and inhibitting miR-194-5p during ovine SVF differentiation process,the changes of ovine SVF differentiation were observed by oil red O stain.Results showed that lipid droplets of experimental group that transfected with miR-194-5p mimic was significantly more than that in miR-194-5p NC group,miR-194-5p inhibitor group and control group.There was big difference between group transfected with miR-194-5p mimic and group transfected with miR-194-5p inhibitor.However,group of transfected with miR-194-5p NC was slightly less than that in control group.Results suggests that miR-194-5p can suppress the SVF differentiation and decrease the formation of lipid droplet.qPCR results showed opposite trends for the expression of miR-194-5p and BCKDHA in SVF differentiation process.It can be suggested that the high expression of miR-194-5p is closely related to the low expression of BCKDHA,and miR-194-5p negatively regulates BCKDHA gene.(4)After overexpressing miR-194-5p in ovine SVF,the expression of miR-194-5p was increased but the expression of BCKDHA mRNA was relatively lower,and BCKDHA protein expression was also reduced.However,the results after transfected with miR-194-5p inhibitor in ovine SVF were contrary.Which further demonstrated that miR-194-5p inhibits the expression of BCKDHA by targeting its 3’ UTR.