MiR-122-5p Regulates The Differentiation Of Abdominal Preadipocytes In Gushi Hicken By Targeting FABP5

Gushi chicken is a famous local chicken type of meat and eggs in China.It is famous for its delicious meat quality,and the formation mechanism of meat traits and genetic evaluation are increasingly valued.The development status of chicken abdominal fat is closely related to the meat quality traits,and it affects the economic benefits.The development of abdominal fat is related to the proliferation and hypertrophy of adipocytes,involving the proliferation and differentiation of preadipocytes and the accumulation of lipid droplets in adipocytes,which are regulated by genes and other regulatory factors.Studies have shown that post transcriptional regulation of genes and miRNAs is closely related to adipocyte development.However,the understand of the key genes and their posttranscriptional regulation mechanism related to abdominal fat development in Gushi chickens is not yet thorough.Therefore,12 cDNA libraries were constructed from abdominal adipose tissue of Gushi chickens at the age of 6,14,22 and 30 weeks.The mRNA expression profiles of four development stages were detected by RNA-seq,and the differentially expressed mRNAs(DE-mRNAs)related to abdominal adipose development were identified.Based on the previous miRNA sequencing data,the regulatory networks of miRNA-mRNA related to abdominal fat development in Gushi chicken were constructed.In addition,the role of miR-122-5p and FABP5 in the differentiation of abdominal preadipocytes in Gushi chicken was further studied by overexpression and interference.The targeted interaction between mir-122-5p and FABP5 was proved by double fluorescence reporting system and remedial test.The main research results are as follows:(1)Transcriptional profile characteristics and Regulatory Network Construction of abdominal Fat Development in Gushi ChickenIn this study,1893 DE-mRNAs were screened by RNA-seq in abdominal adipose tissue at four developmental stages.Cluster analysis of DE-mRNA time series showed that the expression patterns of these mRNAs were divided into four clusters.Among them,cluster 1 genes were significantly enriched in TGF-β signaling pathway,PPAR signaling pathway,WNT signaling pathway and p53 signaling pathway;Cluster4 genes are significantly enriched in cytokine-cytokine-receptor interaction,apoptosis,PPAR signaling pathway,NOD-like receptor signaling pathway and fatty acid biosynthesis.In addition,we constructed miRNA-mRNA interaction networks related to abdominal fat development.The results showed that miR-122-5p could target many genes,including FABP5,ACSBG2 and TGFB3.These results may provide new insights for a better understanding of the potential post-transcriptional regulation mechanism of abdominal fat development in chickens.(2)MiR-122-5p inhibits the proliferation and promotes the differentiation of chicken abdominal preadipocytesTissue expression profile analysis showed that miR-122-5p was highly expressed in liver and abdominal fat tissues of 14 week old Gushi chickens;Temporal expression analysis showed that the expression level of miR-122-5p increased gradually with the differentiation of preadipocytes,and reached the peak on the 5th day after differentiation.These results suggest that miR-122-5p may play an important role in fat deposition.After overexpression of miR-122-5p,the proliferation marker gene p21 was significantly up-regulated(P < 0.01),and CDK1,PCNA and CCND1 were significantly down regulated(P < 0.05);The results of CCK-8 and flow cytometry showed that the number of preadipocytes decreased significantly and the cell cycle was blocked after overexpression of miR-122-5p.Inhibition of miR-122-5p resulted in the opposite result.After the differentiation of chicken preadipocytes was induced,the overexpression of miR-122-5p could up regulate the differentiation marker gene PPAR γ And C / EBP α Expression at mRNA level increased the number of lipid droplets and triglyceride content;Inhibition of miR-122-5p yielded the opposite result.(3)FABP5 inhibits the differentiation of chicken abdominal preadipocytesqPCR analysis showed that the expression of FABP5 decreased continuously with the differentiation of chicken abdominal preadipocytes,and reached the lowest point on the fifth day of differentiation.We believe that FABP5 may be involved in the formation of chicken abdominal fat.Therefore,we constructed FABP5 overexpression vector and interference fragment.Overexpression of FABP5 in preadipocytes can inhibit the differentiation marker gene PPAR γ and C/EBP α Expression at mRNA level decreased the number of lipid droplets and triglyceride content;Interference with FABP5 results in the opposite.(4)MiR-122-5p promotes the differentiation of chicken abdominal preadipocytes by targeting FABP5In order to further reveal the mechanism of miR-122-5p in the process of abdominal fat development and deposition,QRT-PCR showed that there was a significant negative correlation between the expression level of miR-122-5p and FABP5 during the induction and differentiation of preadipocytes;Mirdb and rnahybrid bioinformatics analysis showed that miR-122-5p could target FABP5;Secondly,overexpression of miR-122-5p can significantly reduce the expression of FABP5 at the mRNA level,while interference with miR-122-5p can up regulate the expression of FABP5.Therefore,we constructed the wild-type and mutant vectors of FABP5 3’UTR.The double fluorescence test showed that there was a direct targeting relationship between miR-122-5p and FABP5.In addition,through remedial experiments,we confirmed that miR-122-5p promoted the differentiation of abdominal preadipocytes and the production of lipid droplets by directly targeting FABP5.