| MicroRNAs(miRNAs)play an important role in the transcription regulation of gene expression.The absence of miRNAs also affects the shape,structure and size of adipose tissue,which can also lead to damage of basic function.BCAA consists of leucine,isoleucine and valine.And excessive intake of BCAA will be converted into stored fat,adipose tissue effectively to the carbon skeleton of BCAA transformed into new fatty acids,as adipocyte differentiation and fat synthesis of carbon source,so the intake of BCAA is conducive to adipogenic function.BCAT2 is the first catalytic enzyme in the metabolism of BCAA,which catalyzes the decomposition of BCAA into the reversible reaction of branched chain keto acids.In this process,BCAT2 is involved in the metabolism of BCAA.During the subsequent decomposition process,branched chain keto acids can only be decomposed under the action of the speed limiting BCKDH.BCKDHA and BCKDHB are the alpha and beta subunits of BCKDH E1 structure.The abnormal expression of BCAT2,BCKDHA and BCKDHB can lead to the disorder of BCAA metabolism,the accumulation of metabolites,and the obstacle of the three carboxylic acid cycle.To a certain extent,it will affect the supply of energy materials and even cause some metabolic diseases to some extent.In this study,the ovine preadipocytes were used as experimental samples.In order to increase the reliability of all the results,preadipocytes were first tested.During the different stages of culture and differentiation,cells were photographed and oil red O stained and the markers of adipocyte differentiation were detected.Then the relationship between BCAT2 and miR-330-5p,BCKDHB and miR-433-3p is revealed by theoretical prediction and test verification.Bioinformatics software is used to predict miRNAs that can be combined with BCAT2 and BCKDHB.Construction of BCAT2-3'-UTR and BCKDHB-3'-UTR double luciferase reporter PmirGLO-BCAT2-3'-UTR and PmirGLO-BCKDHB-3'-UTR,and purchased the overexpressed carrier mimics of miR-433-3p and miR-330-5p.HEK-293T cells were cultured and cells were collected after 72 h.The fluorescence activity of the cells was detected by the double luciferase reporter system to verify the relationship between BCAT2 and miR-330-5p,BCKDHB and miR-433-3p.Overexpression of miR-330-5p explored the regulation of miR-330-5p on BCAT2 in ovine preadipocytes,overexpressed miR-433-3p,and explored the regulation of miR-433-3p on BCKDHB in ovine preadipocytes,each of which was divided into overexpression group and negative control group.RT-qPCR was used to detect the time sequence of BCAT2 and miR-330-5p,BCKDHB and miR-433-3p in the differentiation process of preadipocytes,respectively.At the same time,the overexpression of BCAT2 by adenovirus vector pMSCV,using BCAT2-siRNA knockout BCAT2 that were used to detect the BCAT2,BCKDHA and BCKDHB mRNA and protein.And RT-qPCR was used to detect the markers of fatty differentiation gene PPAR?,C/EBPa,FABP4 and ADIPOQ,and detected before and after transfection the change of lipid droplets,which shows that the effect of BCAT2 on preadipocyte differentiation process.The main results are as follows:1 The cultured preadipocytes with spindle or triangle shape.There were no lipid droplets in the cells.As the differentiation process proceeds,fat droplets increase from small to small,fat droplets gradually aggregate into large fat drops,and lipid droplets become more and more transparent.ORO staining shows that the area of coloured lipid droplets is bigger and bigger.During differentiation,the RT-qPCR results of differentiation markers PPARy,C/EBP?,FABP4 and ADIPOQ showed an upward trend,indicating that cultured preadipocytes could serve as samples.2 Dual luciferase reporter vector BCAT2-3'-UTR and BCKDHB-3'-UTR was successfully constructed,showing the group transfected fluorescence activity of dual luciferase reporter assay:total fluorescence activity transfected with PmirGLO-BCAT2-3'-UTR and mimics miR-330-5p were significantly lower than the fluorescence activity(P<0.05)were transfected into PmirGLO-BCAT2-3'-UTR and miR-330-5p NC,also significantly lower than(P<0.05)fluorescence activity in the control group,indicating that BCAT2 and miR-330-5p have a relationship between the target binding sites.Co transfection of PmirGLO-BCKDHB-3 '-UTR and miR-433-3p mimics were significantly lower than the fluorescence activity(P<0.05)were transfected into PmirGLO-BCKDHB-3'-UTR and miR-433-3p NC(P<0.05),also significantly lower than the fluorescence activity of blank control group,indicating that BCKDHB and miR-433-3p have target relationship.3 Overexpression of miR-330-5p in ovine preadipocytes after the comparison of relative expression of mRNA and protein expression in BCAT2 group and negative control group in the amount found overexpression group of BCAT2 mRNA and protein expression was significantly lower than the negative control group(P<0.05),indicating that miR-330-5p not only reduced BCAT2 expression of mRNA,also lowered the expression of its protein.After overexpression of miR-433-3p,by comparing the relative expression of mRNA and protein expression in BCKDHB group and negative control group in the amount found over expression group of BCKDHB mRNA and protein expression was significantly lower than the negative control group(P<0.05),miR-433-3p in ovine preadipocyte of BCKDHB have a negative regulation.4 During the differentiation,there was a negative correlation between the mRNA expression of miR-330-5p and BCAT2,and the negative correlation between the expression of miR-433-3p and BCKDHB.5 After overexpressing BCAT2,the expression of mRNA and protein in BCKDHA and BCKDHB decreased significantly.The expression levels of PPARy,C/EBPa,FABP4 and ADIPOQ were significantly increased(P<0.05).The results of oil red O staining showed that lipid droplets also increased significantly(P<0.05).After silencing BCAT2,the expression of mRNA and protein in BCKDHA and BCKDHB increased significantly(P<0.05).The expression of PPARy,C/EBPa,FABP4 and ADIPOQ in markers genes and the fat drop results of ORO staining were opposite to those of overexpressed BCAT2.These results suggest that BCAT2 has a negative regulatory effect on the expression of BCKDHA and BCKDHB,and that BCAT2 promotes the differentiation of adipocytes.These results fully demonstrate that miR-330-5p can regulate the expression of target genes and their encoded proteins by binding to BCAT2-3'-UTR and miR-433-3p through the combination of BCKDHB-3'-UTR,and inhibits the differentiation of sheep precursor adipocytes.Meanwhile,it is also indicated that there is a certain negative relationship between BCAT2 and downstream genes in the BCAA pathway.It provides a scientific basis for further study of the related genes in miRNA and BCAA pathway,and the molecular mechanism of gene interaction between genes in the BCAA pathway to regulate the metabolism of sheep fat. |
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