MiRNAs Profiles And Functions Of MiR-148a And MiR-200b In Ovine Adipogenesis

Adipose tissue plays an important role in providing energy,maintaining body temperature,and protecting internal organs in sheep.Fat deposits can also affect the production performance of livestock,especially meat quality.Moderate fat deposition can improve the tenderness and flavor of mutton in sheep.Therefore,the in-depth understanding of the molecular mechanism that regulates the development of adipose tissue provides an opportunity to improve meat quality in sheep.The adipocytes cultured in vitro are the basis for in-depth research on the regulation of lipid metabolism and lipid deposition at the cellular and molecular levels.In mammalian cells,micro RNAs(miRNAs)are a class of important regulators of lipid deposition and metabolism.Up to date,numerous miRNAs have been identified in adipose tissue and adipocytes of domestic animal.However,systemic investigated of miRNAs profile and function analysis of miRNAs regulated fat metabolism and deposition still elusive.Therefore,this study isolate and culture sheep adipocytes derived from subcutaneous adipose tissue,and the RNA-Seq technology was used to analyze the differentially expressed miRNAs during the differentiation of sheep adipocytes.The regulation mechanism of miR-148 a and miR-200 b on the proliferation and differentiation of sheep preadipocytes was deeply explored using over-expression,small interference,RT-q PCR,Oil Red O staining,dual luciferase reporter detection system and Ed U labeling experiment.1.The ovine adipocytes have a strong ability to differentiate: after induction,day 0 is the preadipocyte stage,day 2 is the early differentiation stage,and day 8 is the terminal stage of differentiation,2.MiRNAs have different expression patterns in adipocyte differentiation of sheep.The high-throughput sequencing analysis results showed that the Cleans reads obtained from the three libraries were 11396070,12342040 and 12438379,respectively.127,220 and 318 differentially expressed miRNAs were identified in D2 vs.D0,D8 vs.D2 and D8 vs.D0 groups,respectively.Temporal expression trend cluster analysis showed that these differentially expressed miRNAs were enriched in 8 expression patterns.Functional enrichment analysis showed that the predicted target genes of differentially expressed miRNAs were significantly enriched to 186?197 and 201 GO items,respectively;KEGG enrichment analysis shows that the predicted target genes are all enriched in 345 signal pathways.sixteen of the differentially expressed miRNAs were randomly selected for RT-q PCR verification,and the results were consistent with the results of RNA-Seq.3.MiR-148 a and miR-200 b affect the differentiation and lipid synthesis of ovine adipocytes.Among the 8 different tissues of sheep,miR-148 a and miR-200 b were predominantly expressed in subcutaneous fat tissue and liver.During the differentiation of ovine preadipocytes,the expression of miR-148 a was continuously up-regulated while the expression of miR-200 b was decreased.Up-regulation of miR-148 a in adipocytes significantly promoted adipogenesis,while over-regulation of miR-200 b significantly inhibited adipogenic differentiation.In addition,the results of dual luciferase report detection showed that homologous protein phosphatase-tension(PTEN)and Krüppel-like factor 9(KLF9)is the target gene of miR-148 a and miR-200 b in ovine adipocytes,respectively.The target gene function analysis showed that the up-regulation of PTEN in adipocytes inhibited the differentiation of preadipocytes,but up-regulation of KLF9 significantly promoted the differentiation of adipocytes.This indicates that miR-148 a and miR-200 b regulates the differentiation of ovine adipocytes by inhibiting the expression of PTEN and KLF9.4.MiR-148 a and miR-200 b were involved in the regulation of the proliferation of ovine preadipocytes.Although the Small RNA-Seq in this study did not involve the proliferation stage of sheep preadipocytes,a large number of researches have confirmed that miRNA play a key regulator both in the proliferation and differentiation of adipocytes.In order to comprehensive understand the regulation of miR-148 a and miR-200 b on the development of sheep adipose tissue,the effect of miR-148 a and miR-200 b on the proliferation of ovine preadipocytes was further studied.Up-regulation of miR-148 a in preadipocytes significantly inhibited preadipocytes proliferation,the over-expression of miR-200 b promoted preadipocyte proliferation.We also confirmed that miR-200 b directly targets the cyclin-dependent kinase inhibitor 1B(p27)and inhibits the expression of p27 in sheep preadipocytes.The p27 function verification showed that the over-expression of p27 in sheep preadipocytes inhibited the proliferation of preadipocytes,while down-regulation of p27 expression promoted the proliferation activity of preadipocytes.In this study,an adipogenic differentiation model of ovine adipocytes was established and the expression profile of miRNAs in adipocyte differentiation was obtained.Meanwhile,the function of differentially expressed miRNAs in adipocyte differentiation and lipid deposition was explored preliminarily.This study also systematically explored the effect of the miR-148 a and miR-200 b and their target genes in the proliferation and differentiation of adipocytes.The results of the study provide basic data for further understanding of the mechanisms by which miRNAs regulate the differentiation and lipid deposition of sheep adipocytes,and lay a theoretical foundation for improving the quality of sheep meat.