DNMT1 and MBD2 expression determined by realtime-PCR (A,B) and Western blot (C,D).

<p>Data are representative of at least 3 independent experiments. Early effects mean 2(A,C); Delay effects mean 1 month postirradiation (B,D). PBMC, peripheral blood mononuclear cell. 1, control; 2, acute exposed to 0.5 Gy; 3, chronic fractionated exposure. ^*<i>P</i><0.05 versus control.</p

Rad23b and Ddit3 methylation determined by BSP.

<p>Bisulfite treated DNA was amplified by PCR with BSP primers. PCR products were cloned into the pUC57 vector, and five clones selected and sequenced from each sample. Methylation level was defined as the ratio of methylated CpG sites in all clones. (A) Typical sequencing results; (B) early effects, tissues were collected 2 h postirradiation; (C) delay effects, 1 month postirradiation. ^*<i>P</i><0.05 versus control.</p

Genome-Wide Screen of DNA Methylation Changes Induced by Low Dose X-Ray Radiation in Mice

<div><p>Epigenetic mechanisms play a key role in non-targeted effects of radiation. The purpose of this study was to investigate global hypomethylation and promoter hypermethylation of particular genes induced by low dose radiation (LDR). Thirty male BALB/c mice were divided into 3 groups: control, acutely exposed (0.5Gy X-rays), and chronic exposure for 10 days (0.05Gy/d×10d). High-performance liquid chromatography (HPLC) and MeDIP-quantitative polymerase chain reaction (qPCR) were used to study methylation profiles. DNMT1 and MBD2 expression was determined by qPCR and western blot assays. Methylation and expression of Rad23b and Ddit3 were determined by bisulfate sequencing primers (BSP) and qPCR, respectively. The results show that LDR induced genomic hypomethylation in blood 2 h postirraditaion, but was not retained at 1-month. DNMT1 and MBD2 were downregulated in a tissue-specific manner but did not persist. Specific hypermethylation was observed for 811 regions in the group receiving chronic exposure, which covered almost all key biological processes as indicated by GO and KEGG pathway analysis. Eight hypermethylated genes (Rad23b, Tdg, Ccnd1, Ddit3, Llgl1, Rasl11a, Tbx2, Scl6a15) were verified by MeDIP-qPCR. Among them, Rad23b and Ddit3 gene displayed tissue-specific methylation and downregulation, which persisted for 1-month postirradiation. Thus, LDR induced global hypomethylation and tissue-specific promoter hypermethylation of particular genes. Promoter hypermethylation, rather than global hypomethylation, was relatively stable. Dysregulation of methylation might be correlated with down-regulation of DNMT1 and MBD2, but much better understanding the molecular mechanisms involved in this process will require further study.</p></div

Functional and pathway analysis of the 811 genes with hypermethylated promoter identified by MeDIP-chip.

<p>Gene ontology (GO) analysis by three domains: Biological Process (A), Cellular Component (B) and Molecular Function (C). (D) KEGG Pathway analysis.</p

Rad23b and Ddit3 expression determined by realtime-PCR. Data analysis was done using the 2^-ΔΔCT method for relative quantification.

<p>The expression data was normalized to blood sample in control group. (A) early effects; (B) delay effects. ^*<i>P</i><0.05 versus control.</p

Effects of low dose X-rays on global methylation levels in mouse blood.

<p>Whole blood was sampled 2(dC and 5-mdC) detected by HPLC. Standard curve of dC(A) and 5mdC (B). DNA hydrolyzate compared with standard by liquid chromatography, early effects (C) and delay effects (D). Global methylation represented by 5-mdC(%), early effects (E) and delay effects (F). *<i>P</i><0.05 versus control. ^△<i>P</i>>0.05 versus control.</p

Real-time PCR on MeDIP-enriched DNA^a.

a<p>The data were the mean ratios of the signals in the immunoprecipitated DNA <i>vs</i> input DNA.</p><p>* <i>P</i><0.05 versus control and acute exposure group.</p><p>NA, no amplification.</p

Additional file 1: Figure S1. of Downregulation of MiR-31 stimulates expression of LATS2 via the hippo pathway and promotes epithelial-mesenchymal transition in esophageal squamous cell carcinoma

Survival analysis was displayed using TCGA expression data. There were no statistical differences in the analysis results. A. Survival analysis on 164 patients’ specimens was performed to investigate the effect of LATS2 and TAZ expression on prognosis using TCGA expression data. B. Survival analysis on patients with metastasis from 164 specimens was performed to investigate the effect of LATS2 and TAZ expression on metastasis using TCGA expression data. (TIFF 179 kb