The Role Of MiR-215-5p In Regulating The Proliferation And Differentiation Of Gushi Chicken Abdominal Preadipocytes And The Identification Of CeRNA Mediated By It

Chicken is an important agricultural economic animal.Its abdominal fat development is closely related to meat quality.Studies have shown that non-coding RNAs such as miRNA and circRNA are closely related to the regulation of adipose tissue development,and miRNA and circRNA can jointly regulate fat formation in the form of ceRNA.However,there is still a lack of research on the functions of specific miRNAs and their ceRNA interaction networks during the development of chicken belly fat.Our previous studies have shown that miR-215-5p is differentially expressed in high abundance during the development of abdominal fat in Gushi chickens,but its functional mechanism is still unclear.This study verified the effect of miR-215-5p and its predicted target gene NCOA3 on Gushi chicken abdominal pre-adipocytes.Illumina Sequencing technology was used to detect the circRNA expression profile of the abdominal adipose tissue of the Chinese local breed Gushi chicken at the age of 6,14,22,and 30 weeks.The circRNAs related to the development of abdominal fat were identified,and the potential functions of these circRNAs A comprehensive analysis.In addition,we further combined the transcriptome and circular RNA sequencing data for joint analysis,constructed a comprehensive interaction network of circ RNA,miRNA,and mRNAs and completed miR-215-5p-mediated ceRNA identification,to understand fat deposition more systematically Molecular mechanism.The main results obtained are as follows:（1）Study on the role of miR-215-5p in the proliferation and differentiation of Gushi chicken abdominal preadipocytesIn order to reveal the mechanism of miR-215-5p in Gushi chicken abdominal fat deposition,this study used real-time fluorescent quantitative PCR to analyze the differentiation of miR-215-5p in 8 tissues and abdominal pre-adipocytes in 14-week-old Gushi chickens.Expressive characteristics of the process.Through miR-215-5p mimics and inhibitors transfection experiments,using CCK-8,EdU,flow cytometry,oil red O staining,triglyceride content determination and other methods,the effects of miR-215-5p on Gushi chickens were evaluated.The proliferation and differentiation of abdominal pre-adipocytes.The results show that miR-215-5p is highly expressed in Gushi chicken abdominal fat and sebum tissues,and has obvious sequential expression characteristics during the differentiation of preadipocytes.When miR-215-5p mimics was transfected,the expression levels of proliferation marker genes CDK1 and PCNA in preadipocytes were significantly deereased（P<0.01）,and the expression levels of cell cycle arrest factor p21 were significantly increased（P<0.01）,EdU The proportion of positive cells and the total number of pre-adipocytes were significantly reduced,and the results of flow cytometry showed that the cell cycle was significantly blocked.At the same time,it can significantly inhibit the expression of adipocyte differentiation marker genes PPARG,CEBPA,FABP4 and LPL,and reduce lipid droplet production and triglyceride content in adipocytes.In contrast,after miR-215-5p inhibitors transfected pre-adipocytes,the proliferation and differentiation of pre-adipocytes were reversed to those after miR-215-5p mimics transfection.Therefore,miR-215-5p is a negative regulator of abdominal fat formation in Gushi chicken.（2）Identification of target genes of miR-215-5p in Gushi chicken abdominal preadipocytesIn order to further reveal the mechanism of miR-215-5p in the development and deposition of abdominal fat,the target gene of miR-215-5p was predicted using an online website.KEGG enrichment analysis was performed on the obtained target genes,and the genes related to lipid metabolism（NCOA3,TGFBR3 and PRKCI）were screened out to verify the target relationship.The results showed that overexpression of miR-215-5p can inhibit the fluorescence activity of the 3’-UTR region of the NCOA3 gene,and the binding site of the mutant 3’-UTR region of the NCOA3 gene and the miR-215-5p seed sequence can relieve its inhibitory effect.Neither TGFBR3 nor PRKCI has this effect,that is,only NCOA3 is a potential target gene of miR-215-5p.QPCR analysis showed that NCOA3 was negatively correlated with the expression level of miR-215-5p during the differentiation process of Gushi chicken preadipocytes.The above results prove that miR-215-5p can inhibit the proliferation and differentiation of Gushi chicken abdominal preadipocytes and directly target NCOA3 gene during adipocyte differentiation.Therefore,miR-215-5p may regulate the development and deposition of abdominal fat in Gushi chickens by targeting the NCOA3 gene.（3）Gushi chicken abdominal fat development-related circular RNA expression profile and miR-215-5p-mediated ceRNA identificationIn order to reveal the characteristics of the expression profile of the cyclic RNA related to the development of abdominal fat,and to identify the circRNA that interacts with miR-215-5p,this study completed the sequencing of the four developmental stages of abdominal fat circRNA.A total of 1,579 circular RNAs derived from known genes and 187 circular RNAs with no clear gene origin were identified.Among them,275 kinds of circular RNAs are significantly differentially expressed,and their expression patterns can be divided into 4 types.Bioinformatics analysis shows that these differential circRNAs are mainly involved in fat cell differentiation regulation,lipid metabolism regulation,fatty acid metabolism regulation,unsaturated fatty acid metabolism,etc.in the biological processes related to lipid metabolism or deposition.In addition,we also constructed a differentially expressed circRNA-miRNA-mRNA interaction network related to the development of abdominal fat.The results show that circRNA mainly affects the metabolism or deposition of abdominal fat,the proliferation and differentiation of adipocytes,matrix interactions and cell-cell connections in Gushi chickens through a complex interaction network.In addition,it was found that multiple circular RNAs and genes and miR-215-5p together constitute a ceRNA network.These results provide new insights for a better understanding of the post-transcriptional regulatory mechanism of chicken abdominal fat development.（4）gga_circ₀002520-miR-215-5p-NCOA3 ceRNA verificationIn order to verify the relationship between gga_circ₀002520,miR-215-5p and NCOA3 genes,we used bioinformatics methods to make predictions.The analysis showed that gga_circ₀002520 and miR-215-5p have binding sites.QPCR analysis showed that overexpression of miR-215-5p can significantly reduce the expression level of gga_circ₀002520.Therefore,we constructed the wild-type and mutant vectors of gga_circ₀002520,and the dual-luciferase report test showed that there is a direct targeting relationship between gga_circ₀002520 and miR-215-5p.These results indicate that miR-215-5p may interact with gga_circ₀002520 and NCOA3 genes,and together play an important role in the early differentiation of preadipocytes.In summary,the above results prove that miR-215-5p can inhibit the proliferation and differentiation of chicken abdominal preadipocytes,at least through direct targeting of NCOA3.In addition,Illumina Sequencing technology was used to identify a large number of circRNAs related to adipogenesis from the abdominal adipose tissues of the four developmental stages of Gushi chickens,and found multiple ceRNA networks that have a targeting relationship with miR-215-5p,dual fluorescent reporters enzyme reporter system experiments confirmed that miR-215-5p may regulate the formation and development of abdominal fat in Gushi chickens through the gga_circ₀002520-miR-215-5p-NCOA 3 ceRNA network.