Follistatin supplementation induces changes in CDX2 CpG methylation and improves in vitro development of bovine SCNT preimplantation embryos.

Caudal Type Homeobox 2 (CDX2) is a key regulator of trophectoderm formation and maintenance in preimplantation embryos. We previously demonstrated that supplementation of exogenous follistatin, during in vitro culture of bovine IVF embryos, upregulates CDX2 expression, possibly, via alteration of the methylation status of CDX2 gene. Here, we further investigated the effects of exogenous follistatin supplementation on developmental competence and CDX2 methylation in bovine somatic cell nuclear transfer (SCNT) embryos. SCNT embryos were cultured with or without follistatin for 72h, then transferred into follistatin free media until d7 when blastocysts were collected and subjected to CDX2 gene expression and DNA methylation analysis for CDX2 regulatory regions by bisulfite sequencing. Follistatin supplementation significantly increased both blastocyst development as well as blastocyst CDX2 mRNA expression on d7. Three different CpG rich fragments within the CDX2 regulatory elements; proximal promoter (fragment P1, -1644 to -1180; P2, -305 to +126) and intron 1 (fragment I, + 3030 to + 3710) were identified and selected for bisulfite sequencing analysis. This analysis showed that follistatin treatment induced differential methylation (DM) at specific CpG sites within the analyzed fragments. Follistatin treatment elicited hypomethylation at six CpG sites at positions -1374, -279, -163, -23, +122 and +3558 and hypermethylation at two CpG sites at positions -243 and +20 in promoter region and first intron of CDX2 gene. Motif analysis using MatInspector revealed that differentially methylated CpG sites are putative binding sites for key transcription factors (TFs) known to regulate Cdx2 expression in mouse embryos and embryonic stem cells including OCT1, AP2F, KLF and P53, or TFs that have indirect link to CDX2 regulation including HAND and NRSF. Collectively, results of the present study together with our previous findings in IVF embryos support the hypothesis that alteration of CDX2 methylation is one of the epigenetic mechanisms by which follistatin may regulates CDX2 expression in preimplantation bovine embryos.

Reduced Caudal Type Homeobox 2 (CDX2) Promoter Methylation Is Associated with Curcumin's Suppressive Effects on Epithelial-Mesenchymal Transition in Colorectal Cancer Cells.

BACKGROUND Curcumin is a component of Curcuma longa with various biological activities. The present study aimed to investigate curcumin's inhibitory effects on epithelial-mesenchymal transition (EMT) in colorectal cancer (CRC) cells and possible mechanisms of action underlying these effects. MATERIAL AND METHODS Human SW480 CRC cells were incubated with curcumin at 0.1, 0.2, 0.4, 0.8, or 1.6 µmol/L. The 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay was utilized to evaluate cell viabilities. The DNA methylation levels of the cdx2 promoter were assessed by bisulfite sequencing polymerase chain reaction (BSP). Real-time quantitative PCR was used to measure the mRNA expression levels. Protein expression levels were evaluated with western blotting. Immunofluorescence staining was used to evaluate the nuclear translocation of ß-catenin. RESULTS Curcumin concentrations of 0.1, 0.2, and 0.4 µmol/L showed no significant association with the viability of SW480 cells, which were chosen for subsequent experiments. Curcumin incubation significantly downregulated expression levels of DNA methyltransferase1 (DNMT1), DNMT3a, and the methylation levels of the cdx2 promoter in a concentration-dependent manner. The expression levels of N-cadherin, Vimentin, Wnt3a, Snail1, and Twist, as well as the nuclear translocation levels of ß-catenin, were reduced in a curcumin concentration-dependent manner. The expression levels of E-cadherin were increased in a curcumin concentration-dependent manner. CONCLUSIONS Curcumin negatively regulated transcription factors promoting EMT in CRC cells by decreasing cdx2 promoter DNA methylation and consequently suppressing the CDX2/Wnt3a/ß-catenin signaling pathway.

Aspirin prevents NF-κB activation and CDX2 expression stimulated by acid and bile salts in oesophageal squamous cells of patients with Barrett's oesophagus.

OBJECTIVE: In previous studies using oesophageal squamous cells from patients with Barrett's oesophagus (normal oesophageal squamous (NES)-B cells) and from patients without Barrett's oesophagus (NES-G cells), we showed that acid and bile salts induced caudal-related homeobox transcription factor 2 (CDX2) expression only in NES-B cells. CDX2, a transcription factor required to form intestinal epithelium, is a target of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling, which can be inhibited by aspirin. We explored mechanisms underlying differences between NES-B and NES-G cells in CDX2 expression and effects of aspirin on that CDX2 expression. DESIGN: We exposed NES-B and NES-G cells to acid and bile salts, with and without aspirin, and evaluated effects on IκB-NF-κB-PKAc complex activation, p65 NF-κB subunit function, and CDX2 expression. RESULTS: In both NES-B and NES-G cells, acid and bile salts activated nicotinamide adenine dinucleotide phosphate oxidase to generate H2O2, which activated the IκB-NF-κB-PKAc complex. NES-B cells exhibited higher levels of phosphorylated IκB and p65 and greater NF-κB transcriptional activity than NES-G cells, indicating greater IκB-NF-κB-PKAc complex activation by acid and bile salts in NES-B cells, and p65 siRNA prevented their increased expression of CDX2. Aspirin blocked IκB phosphorylation, p65 nuclear translocation, CDX2 promoter activation and CDX2 expression induced by acid and bile salts in NES-B cells. CONCLUSIONS: Differences between NES-B and NES-G cells in NF-κB activation by acid and bile salts can account for their differences in CDX2 expression, and their CDX2 expression can be blocked by aspirin. These findings might explain why some patients with GORD develop Barrett's oesophagus while others do not, and why aspirin might protect against development of Barrett's oesophagus.