miR-25 is upregulated before the occurrence of esophageal squamous cell carcinoma

Abstract

MicroRNAs (miRNAs) are potential biomarkers for cancer detection including esophageal squamous cell carcinoma (ESCC); however, little is known about their expression profile and diagnostic impact in esophageal squamous cell intraepithelial neoplasia, the pathological precancerous lesion of ESCC. In this study, we examined the expression levels of eight miRNAs that were reported to be deregulated in ESCC, including miR-25, let-7a, miR-100, miR-133a, miR-223, miR-375, miR-483-5p and miR-1322, in 30 pairs of esophageal squamous cell neoplasia lesion tissues and corresponding adjacent normal tissues using quantitative real-time PCR (qRT-PCR). Differential expression of miRNAs was further examined by in situ hybridization. Furthermore, the deregulated miRNAs were also measured in serum and serum exosome samples of these patients. miR-25, an oncomir that had been reported to be upregulated in ESCC tissues, were found to be overexpressed in esophageal squamous cell intraepithelial neoplasia lesions (66.7%, 20/30) compared to adjacent normal tissues (P < 0.05), while the other seven miRNAs did not show a significant difference between the lesions and controls. The miR-25 signal was stronger in lesion tissues than in normal tissues according to in situ hybridization. The concentrations of miR-25 in both serum and exosome samples of patients were not significantly different from those of healthy individuals. These findings suggested that the overexpression of miR-25 in esophageal squamous cell intraepithelial neoplasia lesions might be a promising early biomarker candidate for the prediction of ESCC.

Keywords: Esophageal squamous cell carcinoma; esophageal squamous cell intraepithelial neoplasia; in situ hybridization; miR-25; miRNA; qRT-PCR.

Figure 1

Routine endoscopy and narrow band imaging (NBI) images of representative cases of HGIN patients and LGIN patients. Conventional endoscopic view: a slightly depressed lesion with iodine unstained areas. Resected specimens with H&E (×100) stain were shown. A. Specimens were diagnosed as HGIN if stained with neoplastic cells > 50% in the thickness of the epithelium. B. Specimens were diagnosed as LGIN if stained with neoplastic cells < 50% in the thickness of the epithelium.

Figure 2

Expression levels of the eight miRNAs examined in esophageal squamous cell intraepithelial neoplasia (IN) plaque tissues (n = 30) and the matched adjacent normal control (NC). A-H. Only miR-25 was up regulated with a significant difference. The relative expression levels of miRNAs were normalized to U6 snRNA. I. Expression levels of miR-25 were ranged from the largest to the smallest. Relative expression was calculated using the 2^-Δcq method, and N refers to the normal control. *P < 0.05.

Figure 3

Expression levels of miR-25 in LGIN and HGIN tissues analyzed using qRT-PCR.

Figure 4

Representative photographs of miR-25 ISH results in esophageal squamous cell intraepithelial neoplasia. A. The levels of miR-25 (green signal) were higher in the plaques of intraepithelial neoplasia (IN) tissues than that in matched adjacent normal controls (NC). B. As negative control, a scrambled probe was used instead of the miR-25 specific probe. Original magnification: 20×objective.

Figure 5

Expression levels of miR-25 in serum and isolated exosomes in esophageal squamous cell intraepithelial neoplasia (IN) and healthy normal controls (NC). A. Expression levels of miR-25 in serum samples. B. Expression levels of miR-25 in exosome samples. The relative levels of miR-25 were normalized to miR-2911 and calculated using the 2^-Δcq method.