The Effect Of Wnt Signaling Pathway And TCF7L2 On Type 2 Diabetes

Objective:To investigate the change of gene expression profile in pancreas of patients with type 2 diabetes mellitus (T2DM); to screen differentially expressed genes in Wnt signaling pathway; and to validate the expression level of genes involved in transcription factor 7-like 2 (TCF7L2)-dependent Wnt signaling pathway.Methods:Samples of Pancreas were obtained from the tissue bank of pathology department, Shanghai Medical College, Fudan University. We used microarray to detect 2 samples with T2DM (average age 55 years) and one without T2DM (50 years old), and used PCR to validate gene expressions for four cases with T2DM (average age 72±3.72 years) and five without T2DM (average age 52±11.1 years). Pancreas samples were isolated from each donor after death and extracted for total RNA. The cDNA microarray was hybridized using hybond membrane with 18,000 clones. After the raw array data were normalized, the differential expression of genes between diabetic and control groups was evaluated using fold change (cutoff=1.5). We performed functional enrichment analysis of KEGG (Kyoto Encyclopedia of Genes and Genomes) for these differential expressional genes with DAVID (http://david.abcc.ncifcrf.gov/) online tools. The expression level of selected genes from the Wnt signaling pathway was validated by real time quantitive PCR, and the nucleoprotein TCF7L2 was detected by the Western Blot. The statistical test of non-parametric Mann-Whiteney was used for testing the difference between the result of microarray and that of PCR.Results:Compared with the control, there were 4200 differentially expressed genes in the gene chip of pancreas of patients with T2DM. The analysis of KEGG pathway showed that these differential expression genes were mainly enriched in more than 40 pathway including Wnt signaling pathway, focal adhesion, insulin signaling pathway, ubiquitin mediated proteolysis, oxidative phosphorylation; and there were 41 differentially expressed genes in Wnt signaling pathway, which were distributed in various branches of Wnt signaling pathway. Using quantitative PCR, in pancreas of patients with T2DM compared with non-diabetic patients, relative mRNA expression of wingless-type MMTV integration site family, member 4 (Wnt4) was 1.691, frizzled homolog 4 (FZD4) 1.158, glycogen synthase kinase 3 beta (GSK 3β) 2.273 platelet-activating factor acetylhydrolase, isoformⅠb, alpha subunit (PAFAHB1) 1.889, protein phosphatase 2, regulatory subunit B (B56), gamma isoform(PPP2R5C) 1.892, dishevelled 1(DVL1)1.603, Catenin, beta-1 (CTNNB1) 1.190 TCF7L2 1.929, which corresponded with the expression level of gene chips. In addition, the protein of TCF7L2 was decreased in pancreas of patients with T2DM.Conclusion:Islet P-cell dysfunction in T2DM may be associated with Wnt signaling pathway, focal adhesion, insulin signaling pathway, ubiquitination-mediated protein degradation pathway, and oxidative phosphorylation et.al. Based on the expression of Wnt signaling pathway genes in microarray data and PCR results, it was supposed that the Wnt signaling was dysregulated in diabetic pancreas, and it may be inhibited or not activited. TCF7L2 is a key Wnt signaling pathway transcription factors, and the change of TCF7L2 expression may have important impact on its downstream target genes. Therefore, the effect of TCF7L2 on the whole genome transcription need to be further studied. Objective:TCF7L2 is the key transcription factor of classical Wnt signaling pathway. Recent studies have confirmed that TCF7L2 is strongly associated with T2DM. In the study, the new chromatin immunoprecipitation sequence (ChIP-Seq) technique was used to investigate the whole genome binding sites of TCF7L2 in mouse pancreatic isletβcell in order to seek out the potential target genes of TCF7L2.Methods:The TCF7L2 protein level was detected by Western Blot in mouse pancreaticβcell line MIN6 cell. MIN6 cell was cultured in medium with lithium chloride for 24 hours. The binding fragment of the whole genome of TCF7L2 protein was obtained by chromatin immunoprecipitation technique (ChIP). Using the new generation of sequencing technology based on Solexa, the DNA fragments of TCF7L2 obtained and Input control were sequenced on the Illumina Genome Analyzer II X platform. Then the sequenced fragments were mapped to the reference mouse genome(mm9) by Eland algorithm; the candidate binding sites of TCF7L2 in the whole genome were obtained by MACS packages; The motifs of TCF7L2 were calculated using meme package; the TCF7L2 binding sites in the genome-wide distribution were counted by package developed by our group; based on the Gene Expression Omnibus (GEO) from National Center for Biotechnology information (NCBI), the effect of the binding between TCF7L2 and DNA on the gene transcription was analyzed; these genes, whose promoter region were bound by TCF7L2 protein, were performed functional enrichment analysis using online tools of DAVID.Results:Using ChIP-Seq,24.18 million and 34 million of the raw sequence cluster were respectively obtained in the sample immunoprecipited by TCF7L2 and Input sample; after mapping to mm9,4.5 million and 12.2 million of Unique Reads were respectively obtained, their sequencing error rates were both less than 0.5%; 17,347 binding sites of TCF7L2 protein in the whole-genome were obtained by bioinformatics analysis; 17.3%of those binding sites was distributed in promoter regions,23.7% in introns,2.1% in exons,57.0% in intergenic; The meme, a motif finding package, defined the in vivo-occupied TCF7L2-binding site as GAGA and TCTC repeat sequence; combined with gene expression data, it showed that peaks of binding sites of TCF7L2 were lower near the transcription start sites (TSS) of genes whose gene expression were higher, the peaks were high near the low expression genes; there were 3600 candidate target genes of TCF7L2 within TSS±10kb of annotated genes,15 of which were known as Wnt targeted genes, and 12 known as susceptibility gene of T2DM; there were 703 candidate target genes of TCF7L2 within TSS±2kb of annotated genes, KEGG pathway analysis revealed that those candidate target genes were enriched in more than 20 pathway, including mitogen-activated protein kinase signaling pathway (MAPK), Toll-like receptor signaling pathway, Janus kinase-signal transducer activator of transcription (JAK-STAT), insulin signaling pathway, cell cycle, apoptosis; Gene Ontology (GO) analysis showed that these genes were involved in many biological processes, such as cell differentiation, meiotic cell cycle, cell maturation, cell cycle stages.Conclusion:In MIN6 cell, TCF7L2 protein mostly bound to intergenic, but had higher affinity with promoter and intron; it was supposed that TCF7L2 protein could inhibit and activate the gene transcription, but transcriptional inhibition may be dominant. The binding sites of TCF7L2 protein in the whole-genome included known Wnt targeted genes and diabetic susceptibility gene. The candidate target genes of TCF7L2 mostly were involved in cell growth, development and inflammation-related metabolic pathways, such as MAPK, JAK-STAT, Toll-like receptors pathway. By the analysis of candidate target gene of TCF7L2 in mouse pancreaticβcell, it was speculated that TCF7L2 may participate in the development of diabetes through those known diabetic genes, genes regulating pancreaticβcell proliferation and apoptosis, genes involved in inflammation. In order to identify true target TCF7L2 gene, further studies are needed to investigate the effect of TCF7L2 on transcription of gene expression in pancreatic islet (3 cell.