Study On The Regulation Mechanism Of Bta-miR-2400 In Yak (Bos Grunniens) Preadipocyte Proliferation And Differentiation

Yak(Bos grunniens)is a grazing animal with important cultural and productive significance living in cold and hypoxia areas.Due to the restriction of topography,climate and other environmental factors,the growth and decline of adipose tissue fluctuates greatly.During the warm season,due to abundant forage,the adipose tissue of yaks is rapidly hoarded to maintain sufficient body temperature and energy metabolism during the long cold season.At present,many studies have found that adipose tissue development and lipid metabolism can not be affected by the molecular regulation of miRNAs,but there is a lack of research on adipogenesis and adipose deposition-related miRNAs in yaks.Therefore,the study of the molecular mechanism of miRNAs in the proliferation,differentiation and regulation of special lipid metabolism of yak adipocytes may provide a theoretical basis for the physiological study of adipogenesis and fat deposition in yaks.In this study,real-time quantitative PCR(RT-qPCR)was used to detect the expression of bta-miR-2400 in yak heart,liver,spleen,lung,kidney,dorsal maximus muscle,subcutaneous fat,perirenal fat and mesenteric fat.Yak precursor adipocytes were cultured,and the accumulation of lipid droplets during the process of induction and differentiation was observed by oil red O staining,and the expression levels of bta-miR-2400 and lipid differentiation marker genes in the process were detected by RT-qPCR.After transfecting the inhibitors of bta-miR-2400 mimics and bta-miR-2400 into yak precursor adipocytes,and using RT-qPCR and cck-8 to detect the expression changes of adipocyte proliferation and differentiation related marker genes and proliferation of yak precursor adipocytes after overexpression and inhibition of bta-miR-2400.The target genes of bta-miR-2400 were predicted by online bioinformatics prediction software,and the direct targeting relationship between bta-miR-2400 and target genes was detected by in vitro dual luciferase report.The research results are as follows:1.bta-mir-2400 was widely expressed in different tissues of yak,and there were significant differences among different tissues.In the process of induced differentiation of yak preadipocytes,the expression level of bta-miR-2400 decreased gradually with the deepening of differentiation days and degree of differentiation.2.bta-miR-2400 promoted the proliferation of yak precursor adipocytesAfter over-expression of bta-miR-2400,the expression levels of cell proliferation related genes CDK2 and CDK4 mRNA were significantly increased.After CCK-8assay,Compared with the negative control group,over-expression of bta-miR-2400 the proliferation activity of yak precursor adipocytes was significantly increased.On the contrary,after inhibiting the expression of bta-miR-2400,the expression levels of CDK2 and CDK4 were down-regulated,and the cells activity was significantly decreased.Together,bta-miR-2400 enhanced yak precursor adipocytes proliferation.3.bta-miR-2400 inhibited the differentiation of yak precursor adipocytesAfter bta-miR-2400 mimics transferred,the content of fatty acids in yak precursor adipocytes decreased and the expression of adipocyte differentiation related genes significantly decreased.On the contrary,after bta-miR-2400 inhibitors transferred,the fatty acid content in the cells increased significantly,and the expression level of adipose-cell differentiation marker genes increased significantly.4.bta-miR-2400 targets SUMO1 to regulate yak precursor adipocytesThe online prediction software found that SUMO1 is a potential target gene of bta-miR-2400.The Luciferase reporter assay verified that bta-miR-2400 directly targeted of SUMO1.The results of RT-qPCR found that bta-miR-2400 and SUMO1 showed a reverse expression trend during the differentiation of yak precursor adipocytes.In addition,compared with negative control group,after over-expression of bta-miR-2400,the expression levels of SUMO1 were significantly decreased.After inhibiting the expression of bta-miR-2400,the expression level of SUMO1 significantly increased compared with the negative control group.In summary,we speculate that bta-miR-2400 directly targeted of 3'-UTR of SUMO1 to suppress its expression,which may involved in the regulation of yak adipogenesis.