Background: Measuring the DNA methylome may offer the opportunity to identify novel disease biomarkers and insights into disease mechanisms. Although aberrant DNA methylation has been investigated in many human cancers and precancerous lesions, the DNA methylation landscape of gastric cardiac intestinal metaplasia (IM) remains unknown. Therefore, we aimed to investigate the genome-wide DNA methylation landscape and to search for potential epigenetic biomarkers of gastric cardiac IM. Methods: Histopathologic profiling was performed on a total of 118 gastric cardiac biopsies from cancer-free individuals. Genome-wide DNA methylation analysis was performed on 11 gastric cardiac mucosal biopsies (IM = 7; normal = 4) using Illumina 850K microarrays. Transcriptional relevance of any candidate epigenetic biomarker was validated by qRT-PCR. Results: The detection rate of gastric cardiac IM was 23% (27/118) in cancer-free individuals. Genome-wide DNA methylation profiling showed a global decrease in methylation in IM compared with normal tissues (median methylation = 0.64 and 0.70 for gastric cardiac IM and normal tissues, respectively). Differential methylation analysis between gastric cardiac IM and normal tissues identified 38,237 differentially methylated probes (DMPs) with a majority of sites showing hypermethylation in IM compared with normal tissues (56.3% vs. 43.7%). Subsequent analysis revealed a significant enrichment of hypermethylated DMPs in promoter and CpG islands (p < 0.001 for both, Pearson χ 2 test). For DMPs located in promoter CpG islands showing extreme hypermethylation, the candidate gene with the largest number of DMPs (n = 7) was mapped to HOXA5. Accordingly, mRNA expression of HOXA5 was significantly reduced in IM compared to normal tissue. Conclusions: Our results suggest the implication of alterations in DNA methylation in gastric cardiac IM and highlight that HOXA5 hypermethylation may be a promising epigenetic biomarker, emphasizing the role of aberrant HOXA5 expression in the pathogenesis of gastric cardiac IM.

Background Esophageal carcinoma is a common gastrointestinal tumor in humans. Cyclopamine, a Hedgehog (Hh)-pathway-specific inhibitor, is an effective chemotherapeutic drug for suppressing tumor cell differentiation, with unclear mechanisms. We investigated glioma-associated oncogene protein-1 (Gli-1) expression in human esophageal carcinoma tissue and the inhibition of cyclopamine on EC9706 esophageal carcinoma cell growth. Material/Methods Gli-1 in tumor tissue was measured by immunohistochemistry (IHC). EC9706 cells were treated with different concentrations of cyclopamine and incubated for different times. MTT method, flow cytometry, and Acridine orange/ethidium bromide (AO/EB) double-fluorescence staining were applied to detect cell proliferation and apoptosis. Western blot (WB) analysis was performed to assess Gli-1 expression. Results Gli-1 was associated with patient age, gender, lymphatic metastasis, tumor recurrence, and stage, with significantly ( P <0.05) positive correlations with age, lymphatic metastasis, tumor recurrence, and stage. At 12 h ( F =214.57), 24 h ( F =76.832), 48 h ( F =236.90), and 72 h ( F =164.55), the higher the concentration of cyclopamine, the higher the inhibition rate of suppressing EC9706 proliferation, and this effect was significant ( P <0.05). The number of early-apoptosis cells increased as the concentration of cyclopamine increased. Morphology of EC9706 cells appeared as round with rough edges, karyopyknosis, and karyorrhexis. After 48 h, apoptosis rates of EC9706 cells treated with different concentrations of cyclopamine were (7.73±1.25)% at 2.5 μM, (13.37±1.42)% at 5.0 μM, (22.3±2.92)% at 10.0 μM, and (33.57±1.75)% at 20.0 μM, and the effect was dose-dependent. Gli-1 was obviously reduced after cyclopamine treatment and the effect was dose-dependent. Conclusions Gli-1 is highly expressed in human esophageal carcinoma, and could be a marker for use in assessing tumor stage and the deciding on treatment target.

BackgroundEsophageal squamous cell carcinoma (ESCC) is a common cancer with poor prognosis. Caveolin-1 (Cav1) and Rho/ROCK pathway play important roles in tumor metastasis, separately. However, less research was focused on the relationship between Cav1 and Rho/ROCK in ECSS metastasis. Therefore, we investigated the relationship between Cav1 and Rho/ROCK pathway in ESCC metastasis.Material/MethodsCav1 and phosphorylated Cav1 (PY14Cav1) were examined in ESCC and in adjacent and non-tumorous tissues from ESCC patients by immunohistochemistry (IHC). Small interfering RNA (siRNA) targeting Cav1 or Rho/ROCK inhibitor was used to treat EC109, Eca109, TE1, and TE13 cells. Western blotting (WB) was used to detect Cav1 and PY14Cav1 expression. The wound healing scratch test and transwell assays were used to assess migration and invasion.ResultsCav1 and PY14Cav1 were gradually expressed at higher levels in ECSS than in adjacent and non-tumor tissues as ESCC stage and lymphatic metastasis increased, and this difference was significant (P<0.05). Cav1 was expressed at higher levels in TE1 and TE13 than in EC109 and Eca109, while PY14Cav1 was enhanced in TE1 and TE13 cells but not in EC109 and Eca109, and the difference was significant (P<0.05). TE1 and TE13 had significantly (P<0.05) stronger motility, migratory, and invasion abilities than EC109 and Eca109 cells. Silencing Cav1 decreased PY14Cav1 expression in TE1 and TE13 cells, as well as suppressing the migration and invasion of all ECSS cells, and these differences were significant (P<0.05). Suppressing the Rho/ROCK pathway obviously inhibited Cav1 and PY14Cav1 expressions, as well as significantly (P<0.05) decreasing migration and invasion of ESCC cells.ConclusionsCav1 and PY14Cav1 were positively correlated with ESCC lymphatic metastasis and cancer stages. Rho/ROCK pathway activation promoted ESCC metastasis by regulating Cav1.

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