MicroRNAs are a new class of non-proteincoding, small RNAs that function as tumor suppressors or oncogenes. They participate in diverse biological pathways and function as gene regulators. A G>C polymorphism (rs2910164), which is located in the sequence of miR-146a precursor, results in a change from G:U to C:U in its stem region. However, it remains largely unknown whether this single nucleotide polymorphism (SNP) may alter esophageal squamous cell carcinoma (ESCC) susceptibility. In the current study, we evaluated association between rs2910164 and ESCC susceptibility in a case-control study of 444 sporadic ESCC patients and 468 matched cancer-free controls in a Chinese Han population. Compared with rs2910164 variant genotype CC, genotype GG was associated with increased risk of ESCC (Odds Ratio, 2.39, 95% Confidence Interval, 1.36-4.20). In the smokers, the risk of rs2910164 GG genotype was more notable (Odds Ratio, 3.17, 95% Confidence Interval, 1.71-4.46). In the stratification analyses, we also found there was a strong correlation between rs2910164 C/G variant and the clinical TNM stage (P < 0.01). These findings suggest that this functional SNP in pre-miR-146a could contribute to ESCC susceptibility and clinical outcome.

Invasion and metastasis are the major cause of deaths in patients with esophageal cancer. In this study, we isolated cancer stem-like cells from an esophageal squamous cell carcinoma cell line EC109 based on aldehyde dehydrogenase 1A1 (ALDH1A1), and found that ALDH1A1 high cells possessed the capacities of self-renewal, differentiation and tumor initiation, indications of stem cell properties. To support their stemness, ALDH1A1 high cells exhibited increased potential of invasion and metastasis as compared with ALDH1A1 low cells. ALDH1A1 high esophageal squamous cell carcinoma cells expressed increased levels of mRNA for vimentin, matrix metalloproteinase 2, 7 and 9 (MMP2, MMP7 and MMP9), but decreased the level of E-cadherin mRNA, suggesting that epithelial-mesenchymal transition and secretary MMPs may be attributed to the high invasive and metastatic capabilities of ALDH1A1 high cells. Furthermore, we examined esophageal squamous cell carcinoma specimens from 165 patients and found that ALDH1A1 high cells were associated with esophageal squamous dysplasia and the grades, differentiation and invasion depth, lymph node metastasis and UICC stage of esophageal squamous cell carcinoma, as well as poor prognosis of patients. Our results provide the strong evidence that ALDH1A1 high cancer stem-like cells contribute to the invasion, metastasis and poor outcome of human esophageal squamous cell carcinoma. Modern Pathology (2014) 27, 775-783; doi:10.1038/modpathol.2013.189; published online 8 November 2013Keywords: aldehyde dehydrogenase 1A1; cancer stem-like cells; esophageal squamous carcinoma; invasion; metastasis; prognosis Esophageal squamous cell carcinoma is one of the most frequent fatal malignancies in the area from northern Iran to north-central China ('Asian esophageal cancer belt'). 1,2 The 5-year survival rate of esophageal squamous cell carcinoma patients after surgery and chemotherapy remains low owing to highly invasive and metastatic nature of esophageal squamous cell carcinoma. Cancer stem-like cells are a small subpopulation within tumors with the capacities for self-renewal and generating heterogeneous tumor cell lineages. 3 Recent studies suggest that cancer stem-like cells are responsible for invasion and metastasis of many tumor types. We have reported that cancer stem-like cells possess higher capability of invasion and metastasis in solid tumors. [4][5][6] However, the biomarkers related to invasion and metastasis of cancer stem-like cells

Previous studies on esophageal squamous cell carcinoma (ESCC) indicated that it contains much dysregulation of microRNAs (miRNAs). DNA hypermethylation in the miRNA 5 0 regulatory region is a mechanism that can account for the downregulation of miRNA in tumors (Esteller, N Engl J Med 2008;358:1148-59). Among those dysregulated miRNAs, miR-203, miR-34b/c, miR-424 and miR-129-2 are embedded in CpG islands, as is the promoter of miR-34a. We investigated their methylation status in ESCC by bisulfite sequencing PCR (BSP) and methylation specific PCR (MSP). The methylation frequency of miR-203 and miR-424 is the same in carcinoma and in the corresponding non-tumor tissues. The methylation ratio of miR-34a, miR-34b/c and miR-129-2 is 66.7% (36/54), 40.7% (22/54) and 96.3% (52/54), respectively in ESCC, which are significantly higher than that in the corresponding non-tumor tissues(p < 0.01). Quantitative RT-PCR analysis in clinical samples suggested that CpG island methylation is significantly correlated with their low expression in ESCC, 5-aza-2 0 -deoxycytidine (DAC) treatment partly recovered their expression in EC9706 cell line. We conclude that CpG island methylation of miR-34a, miR-34b/c and miR-129-2 are frequent events and important mechanism for their low expression in ESCC. DNA methylation changes have been reported to occur early in carcinogenesis and are potentially good early indicators of carcinoma (Laird, Nat Rev Cancer 2003;3:253-66). The high methylation ratio of miR-129-2 indicated its potential as a methylation biomarker in early diagnosis of ESCC.

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