Study On YaK Preadipocyte Differentiation Regulated By MiRNA,CircRNA And M~6A Methylation

The yak is a unique livestock breed on the Qinghai-Tibet Plateau,and its adipose tissue not only affects meat-producing and reproductive traits but also plays an important role in adapting to the harsh living environment.Studies have shown that epigenetics plays an important role in adipose tissue development by affecting the transcriptional regulation of genes.In this study,preadipocytes were isolated from Datong yak adipose tissue and induced into mature adipocytes.By using RNA-seq and Me RIP-seq,the epigenetic regulators（miRNA,circRNA and m~6A methylation modification）were identified and their genomic features were analyzed during yak preadipocyte differentiation.The differentially expressed epigenetic regulatory factors were screened and the biological functional prediction was performed.The mechanism of bta-miR-181a promoting the yak preadipocyte differentiation was elucidated at the cellular level.It provides a theoretical basis and new research ideas for further insight into the molecular mechanisms of yak adipose development.The main findings of the study are as follows.（1）Totally,707 miRNAs were identified in three stages during yak preadipocyte differentiation,and the length of miRNAs is mainly distributed at 21～23 nt.There were 45 differentially expressed miRNAs in the three stages.Among them,bta-miR-181a was highly expressed and had a more fold difference,suggesting that it has an important function during preadipocyte differentiation.The target genes of differentially expressed miRNAs were enriched in biological pathways related to transcriptional regulation and lipid metabolism,indicating that miRNAs may participate in biological processes associated with adipocyte differentiation by affecting the expression of target genes.（2）Bta-miR-181a is highly expressed in yak adipose tissue.SIRT1 is a direct target gene of bta-miR-181a.Bta-miR-181a promotes the differentiation and the lipid deposition of yak preadipocytes by inhibiting the expression of SIRT1.（3）In total,7203 circRNA were identified and derived from 2909 parental genes.Prediction of miRNA binding sites on circRNA revealed that adipocyte differentiation-specific miRNAs targeted multiple circRNAs.Novel＿circ＿0000628and novel＿circ＿0006981 may regulate the yak preadipocytes differentiation through adsorbing bta-miR-200a and bta-miR-181a with competitive endogenous RNAs.（4）Compared with preadipocytes,the expressions of methyltransferases（METTL14,WTAP,METTL3）and demethylase ALKBH5 were significantly up-regulated（P<0.05）,and the expression of FTO was extremely significantly down-regulated（P<0.01）,the expression of m~6A-binding proteins（YTHDC1,YTHDC2）was significantly up-regulated（P<0.01）,and the m RNA m~6A content was significantly increased in adipocytes（P<0.05）.It showed that the m~6A methylation modification was significantly different between preadipocytes and adipocytes.（5）A total of 14,710 m~6A peaks were detected in adipocytes,derived from 9,633transcripts,and 13,388 m~6A peaks were detected in preadipocytes,corresponding to9,142 transcripts.The results of the enrichment analysis in both groups showed that the consensus motifs of m~6A RRACH were GGACU.The m~6A peaks were predominantly enriched in the CDS region near the start and stop codons and approach the 3’UTR.The relationship of differentially methylated m~6A peaks and gene expression patterns in preadipocytes and adipocytes through cross-analysis of the m~6A-seq and RNA-seq results,in which a positive correlation existed in differentially methylated m~6A peaks and gene expression levels.Differentially methylated genes KLF9,FOXO1 and UHRF1 were involved in lipid metabolism-related signaling pathways.Studying m~6A-modified sites and relative expression level of genes revealed that the genes have 3 or 4 modified sites appear to be more abundant in contrast with other m~6A-modified sites.It was revealed that m~6A methylation regulated yak preadipocyte differentiation by affecting gene expression levels.In conclusion,bta-miR-181a promotes yak preadipocyte differentiation and lipid deposition in cells.Novel＿circ＿0006981 and novel＿circ＿0000628 are candidate competitive endogenous RNAs for regulating preadipocyte differentiation.The genes of KLF9,FOXO1 and UHRF1 may be key potential candidate methylated genes that regulate preadipocyte differentiation.