Role And Mechanism Studies Of MiR-503 And MiR-135a In Adipogenesis By Targeting Zfp217

Adipocytes are differentiated and developed from multipotent mesenchymal stem cells in mesoderm.The development process from mesenchymal stem cells to mature adipocytes includes two parts: adipogenic commitment and differentiation,which are regulated by the highly coordinated and complex network.Adipogenesis is a process that lasts for the whole development of the embryo and still has effects in adult animals.In recent years,it is reported that zinc finger protein gene could function as key transcriptional regulators involved in adipogenesis.Zinc Finger proteins can not only regulate the adipogenic commitment but also affect adipogenic differentiation and fat deposition.It is worth noting that more and more reports that the gene of zinc finger protein related to lipid metabolism processes is influenced by the miRNA regulation and then influence the differentiation process.In our early research,we have screened a conserved differentially expressed gene which is related to adipogenic differentiation in early porcine SV cells with transcriptome sequencing and real-time quantitative PCR.Then the research preliminarily determined its role in the development process from adipose stromal cells to adipose cells through the RNAi technology.However,the functions of Zfp217 in adipogenic commitment and differentiation,and the mechanism of the regulation by miRNAs remain unclear.The main results of this study are as follows:1.Bioinformatic analysis of zinc finger protein 217The multilevel structure and sequence properties of Zfp217 were analyzed with bioinformatics technology.The results showed that Zfp217 contained 8 zinc finger motifs among the three species(hsa,mmu and ssc),and the sixth and seventh zinc finger had the highest sequence similarity.The constructed 3D structure model of Zfp217 could meet the stereochemical standards using the method of homology modeling.The physicochemical property analysis of Zfp217 amino acid sequence showed that it was a hydrophilic,low thermal stability and unstable protein among the three species.Analysis of transmembrane domain showed that the transmembrane domains only existed in mice and pigs.Post-transcriptional modifications showed that O-Gal NAc protein glycosylation sites and phosphorylation sites were predicted all in humans,mice and pigs.The NCBI database was used to search the protein sequence of Zfp217 in eukaryote,and then the homologous phylogenetic tree was constructed.The zinc finger motifs of those protein sequences were also analyzed.The results showed that mammalian was the largest one from the perspective of the phylogenetic tree,and the phylogenetic relationships among all classes or orders in the phylogenetic tree were quite clear.The C2H2 type zinc finger motifs were widely distributed in the sequences of various species,and the number of this motif ranged from 5 to 8.The majority of the C2H2 number among the species was 8.The microarray datasets which related to the gain or loss of Zfp217's function obtained from GEO Database.It outputted 4 genes which were consistent with Zfp217,and 33 genes in opposite via R project.Those differentially expressed genes were analyzed by functional enrichment,and found that they had high biological activity,and enriched in the biological process of tumor and cancer.One hundred and twenty-one differentially expressed miRNAs were obtained using GEO data mining of breast cancer,19 of them were predicted to target Zfp217 both in human and mouse.Under the strict screening standard,a total of 164 conserved miRNAs were predicted to target Zfp217.Further literature discovery revealed that 102 miRNAs were associated with cancer,and the most associated cancer was breast cancer.In addition,data mining also showed that Zfp217 was highly correlated with obesity and adipogenic marker genes.It is speculated that Zfp217 might be involved in cell adipogenic differentiation and was targeted by miRNAs.2.The expression and function of Zfp217The expression patterns of Zfp217 in C3H10T1/2 cells,different tissues of pig and mouse,as well as adipose tissue of obese mice were analyzed by QPCR or Western blot.The results showed that up-regulated expression of Zfp217 was consistent with that of adipogenic differentiation core transcription factor gene Pparg2 during the adipogenic differentiation of C3H10T1/2 cells.In visceral adipose tissue,the expression trend of Zfp217 and Pparg2 was consistent,and showed a similar significance.The expression pattern of Zfp217 was also consistent with some of the early marker genes that pro-adipogenic differentiation(Cebpb,KLF4,Ebf1 and ZNF395).The murine CDS of Zfp217 was cloned to explore its effect in adipogenesis under the gain or loss of this gene's function.The results showed that the expression of Zfp217 was positively correlated with the lipid drop formation and triglyceride content of C3H10T1/2 cells.The mechanism of Zfp217 regulating cell adipogenic differentiation was analyzed by QPCR,flow cytometry,mammalian two-hybrid assay and so on.The results showed that overexpression of Zfp217 could significantly inhibit the m RNA level of Wnt signaling genes.In addition,adipogenic marker genes were also significantly changed in different treatment groups of Zfp217 after 4 days of adipogenic induction.There was a protein interaction between Zfp217 and Ezh2 in the murine background by two-hybrid experiments.All the results showed that Zfp217 could promote adipogenic differentiation by inhibiting cell DNA synthesis,affecting cell cycle,and interacting with Ezh2 to inhibit Wnt signaling gene expression.3.The screening,validation and functional studies of the miRNA which targeting Zfp217Using various bioinformatics websites to predict miRNAs which can target Zfp217.A total of 176 miRNAs targeting Zfp217 were predicted in human while 213 miRNAs were predicted in mouse,and 42 miRNAs were coexisting in both human and mouse.Furthermore,we obtained 5 candidate miRNAs: miR-503,miR-135 a,miR-26 a,miR-19a/b and miR-1a by functional annotation analysis and evolutionary analysis.With the help of dual-luciferase assay,QPCR and Western blot,it was verified that miR-503,miR-135 a and miR-19 a could reduce the expression of Zfp217 in the cell by targeting the 3'untranslated region(3'UTR).The expression patterns of candidate miRNAs in C3H10T1/2 cells,different tissues of pig and mouse,as well as adipose tissue of obese mice were tested,and then the effects of miR-503 and miR-135 a on cell proliferation,cycle and DNA synthesis were analyzed.The results showed that miR-503,miR-135 a and miR-19 a targeted Zfp217 and inhibited adipogenic differentiation.