The Role Of METTL14 Gene In Proliferation,Differentiation And Lipid Deposition Of Yak Preadipocytes

The regulation of lipid deposition in yak(Bos grunniens)has obvious specificity in the special ecological environment and natural selection of alpine hypoxia.The research on the correlation between m~6A methylation modification and mammalian fat deposition mainly focuses on demethylases,and there are few studies on methylases.There are fewer related studies.In this experiment,by understanding the bioinformatics characteristics and expression pattern of yak METTL14,we explored the effect of METTL14 on the proliferation,differentiation and lipid deposition of yak adipocytes,and explored the potential genes and genes involved in METTL14 affecting the process of yak adipocyte proliferation,differentiation and lipid deposition.signaling pathway,in order to provide a theoretical basis for the physiological study of yak lipid deposition.The experiment took Datong yak as the research object,took yak adipose tissue c DNA as the template,cloned the METTL14 CDS region sequence to analyze the biological properties,and used fluorescence quantitative PCR technology to analyze the 7 tissues of yak and 2 stages of 18 and 30 months of age.Expression regulation of METTL14 during cell differentiation;construct si RNA-METTL14 and METTL14 lentiviral overexpression vectors to transiently transfect yak preadipocytes,and observe cell proliferation by CCK-8staining and Ed U staining;Oil red O staining,triglyceride Ester content detection and WB detection were used to analyze cell differentiation,and RT-q PCR was used to detect cell proliferation and differentiation marker genes,as well as the m RNA expression levels of adipogenesis and lipolysis-related genes.Transcriptome sequencing of body fat cells was used to mine the potential genes and signaling pathways related to METTL14 at the transcriptome level on yak adipocyte proliferation,differentiation and lipid deposition.The following research results were obtained:(1)The CDS region of METTL14 in yak adipose tissue is 1 371 bp in length and encodes a total of 456 amino acids.The encoded protein is an unstable water-soluble protein with a conserved domain of MT-A70 located in the nucleus at 183-363 bp.METTL14 is expressed in 7 different tissues of yak.Compared with the expression level in adipose tissue,METTL14 has the lowest expression level in spleen,and the difference is extremely significant(P<0.01).The expression level in heart and longissimus dorsi is extremely high.significantly higher than that in adipose tissue(P<0.01);with the growth and development of yak,the expression level of METTL14 in adipose tissue of18-month-old yak was significantly higher than that of 30-month-old yak(P<0.05).The whole showed an upward trend,and the expression level on 12 d was significantly higher than that on 0 d(P<0.01),and the expression level on 4 and 8 d was significantly higher than that on 0 d(P<0.05).(2)After interfering with METTL14,compared with the si-Control control group,the adipocyte proliferation activity was enhanced,the total number of cells increased significantly(P<0.05),more lipid droplets formed alicyclic rings and the triglyceride content increased significantly(P<0.05).At the m RNA level,the cell proliferation-related gene CDK4 was significantly increased(P<0.01),PCNA and CCND1 were significantly increased(P<0.05),and the cell differentiation marker gene FABP4(P<0.01)was also promoted.The genes ACCαand ELOVL1 were significantly increased(P<0.01),and FAS,LPL,and SREBF1 were significantly increased(P<0.05).After overexpression of METTL14,compared with the vi-Control control group,the cell proliferation and differentiation were inhibited,and the distribution of aliphatic rings was higher.few.At the m RNA level,CDK4,C/EBPα,LPL were significantly decreased(P<0.01),and PCNA,SCD,DGAT1,ELOVL1,SREBF1 were significantly decreased(P<0.05).The expression of C/EBPαand PPARγencoded by cell differentiation marker genes was significantly decreased(P<0.01).(3)RNA-seq analysis of the transcriptome profiles of adipocytes in the si-METTL14interference group and si-Control control group obtained 195 significantly differentially expressed genes,of which 88 differentially expressed genes were significantly up-regulated and 107 differentially expressed genes were significantly down-regulated,found potential related genes GPLD1,CDCA7L,PCDHB7 and CPT1B with adipocyte proliferation and differentiation,lipid deposition,GO and KEGG database analysis,obtained two main signaling pathways related to cell proliferation,differentiation and lipid deposition:MAPK and PI3K/AKT signal path.It indicated that METTL14 gene could inhibit the proliferation,differentiation and lipid deposition of yak preadipocytes by regulating genes and signaling pathways related to adipocyte proliferation,differentiation and lipid deposition.The experimental results show that yak METTL14 is an unstable water-soluble protein located in the nucleus.METTL14 is widely expressed in different tissues of yak and in the process of growth and development,and has inhibitory effects on the proliferation,differentiation and lipid deposition of yak preadipocytes.In yak preadipocytes,METTL14may inhibit the proliferation,differentiation and lipid deposition of yak preadipocytes by regulating key genes of cell proliferation and differentiation,lipid deposition,and some potential genes and signaling pathways.