Preliminary Study On The Effect Of Bta-miR6517 In The Differentiation Of Cattle Adipocytes

In this study,the mechanism of bta-miR-6517 in bovine adipocytes was investigated.The main experimental methods are as follows:bioinformatics was used to analyze the location of CpG island in the upstream region of bta-miR-6517 precursor,the transcription factors bound to the promoter region and to screen the target genes related to adipose differentiation.Fluorescence quantitative PCR was used to detect the expression of bta-miR-6517 in heart,liver,spleen,lung,kidney,fat and muscle of Qinchuan cattle and Wagyu cattle.According to the prediction results of target genes,the construction of dual luciferase reporter test was used to verify whether there is a targeted relationship between bta-miR-6517 and candidate target genes.Bovine preadipocytes were isolated and cultured by tissue mass method and digestion method.Fluorescence quantitative PCR was used to detect the expression of adipogenic marker genes PPARγ and bta-miR-6517 in different time(0d,2d,4d,6d,10d)of adipogenic differentiation,and oil red O staining was performed on the cells induced for Od and 10d.The results are as follows:(1)Biological analysis of bta-miR-6517Using PROMO,it was predicted that its promoter region had five binding sites with adipocyte proliferation and differentiation and lipid metabolism regulation,which were C/EBPα,C/EBPβ,HSF1,AP-2α and C/EBP.Bioinformatics software was used to obtain that MAT1A may be the target gene of bta-miR-6517,and targetscan prediction showed that there were two possible binding sites between MAT1A 3’UTR and bta-miR-6517,which were located at 67nt-74nt and 288nt-295nt,respectively.(2)The expression of bta-miR-6517 in tissuesIn Qinchuan cattle,the expression of bta-miR-6517 in lung,back fat and muscle was extremely significantly increased(P<0.01)than that in heart,while the expression of bta-miR-6517 in spleen was significantly lower than that in heart(P<0.05).In Wagyu cattle,the expression in backfat,abdominal fat and inguinal fat was significantly(P<0.05)higher than pericardial fat,and extremely significantly(P<0.01)higher than heart,liver,spleen and lung.(3)Identification of bta-miR-6517 target geneThe dual luciferase reporter vector was verified by PCR and sequencing and was co-transfected with bta-miR-6517 mimics/mimics NC into 293T cells.After 48h,the dual luciferase reporter test results showed that the relative fluorescence activity of wt-MAT1A-3’UTR2 mimics group was significantly lower than that of wt-MAT1A-3’UTR2 mimics NC group(P<0.01)and There was no significant difference between wt-MAT1A-3’UTR1 mimics group and wt-MAT1A-3’UTR1 mimics NC group(P>0.05).It shows that bta-miR-6517 combines with MAT1A-3’UTR2 but not with MATIA-3’UTR1.(4)The expression of bta-miR-6517 in cellsBovine primary adipocytes were successfully isolated by tissue mass and digestion method.After induction.The results showed that PPARγ and bta-miR-6517 were up-regulated with the increase of induction time,and reached the maximum on the 10d.Compared with Od,the expression of PPARγincreased significantly from the 4d(P<0.01),the expression of bta-miR-6517 increased significantly from the 2d(P<0.05),and it increased significantly from the 4d(P<0.01).The results of this study showed that there were five transcription factor binding sites,C/EBPa,C/EBPP,HSF1,AP-2α and C/EBP,related to adipocyte proliferation and differentiation and lipid metabolism regulation in the upstream promoter region of bta-miR-6517 precursor.bta-miR-6517 was highly expressed in adipose tissue and muscle tissue,and could bind with MAT1A 3’UTR2.The expression of bta-miR-6517 in the process of adipogenic differentiation showed an upward-regulated trend.Therefore,bta-miR-6517 may target MAT1A and play a role in adipocyte differentiation.