Methylation-silencing RCC1 expression is associated with tumorigenesis and depth of invasion in gastric cancer

Abstract

Introduction: Regulator of chromosome condensation 1 (RCC1) is a critical cell cycle regulator. We firstly identified RCC1 gene hypermethylation in gastric tumor tissues using the differential methylation hybridization (DMH) microarray, but the role of RCC1 in the pathogenesis of gastric carcinoma is largely unknown.

Methods: Three gastric cancer cell lines (AGS, MKN45, and TSGH9201) were used to analyze RCC1 gene methylation, mRNA and protein expressions. Furthermore, 85 pairs of matched human gastric carcinoma samples in a tissue microarray were used to analyze RCC1 expression by immunohistochemistry staining.

Results: A differential methylation pattern was found in TSGH9201 (100%), MKN45 (87%), and AGS (62%) cell lines at the 9th CpG site of RCC1 exon 1. RCC1 mRNA and protein expressions in AGS cells were significantly higher than in TSGH9201 and MKN45 cell lines (P < 0.05). Tissue array data showed that RCC1 expression was detected in 21% (18/85) of gastric carcinoma tissues and in 80% (76/95) of adjacent non-tumor tissues. The expression of RCC1 in gastric carcinoma tissues was significantly lower than in adjacent non-tumor tissues (P < 0.001). Furthermore, an association between RCC1 expression and clinicopathological features showed that RCC1 expression was closely correlated with tumor differentiation and depth of invasion (P < 0.05).

Conclusions: Our data indicate that RCC1 expression is frequently lost in poorly differentiated gastric cell lines and gastric carcinoma tissues. Loss of RCC1 expression is correlated with tumor differentiation and depth of invasion. These findings suggest that RCC1 may play a tumor suppressor role in gastric carcinoma.

Keywords: DMH microarray; DNA methylation; RCC1; gastric carcinoma; immunohistochemistry; invasion; tumor tissue array.

Figure 1

Validation of RCC1 mRNA expression in gastric cancer cell lines by quantitative reverse transcription PCR (qRT-PCR). A. Primer and probe designs of RCC1 and GAPDH genes for qRT-PCR analysis. B. Quantitative data of relative RCC1 mRNA expression levels in three different differentiation states of gastric cancer cell lines. The results are shown as the mean ± SD of three independent experiments. Each bar represents the relative RCC1 expression percentage normalized to the GAPDH housekeeping gene and compared to the AGS cell line. *P=0.002 (Kruskal Wallis test).

Figure 2

Protein expression levels of RCC1 analyzed in three different gastric cancer cell lines by western blotting. A. Protein expression levels of RCC1 and β-actin in three gastric cancer cell lines (AGS, TSGH9201, and MKN45) as determined by western blot analyses. B. Quantification of relative RCC1 protein levels in three different gastric cancer cell lines using image J. β-actin was used as an internal control. *P < 0.05 compared to AGS cells.

Figure 3

DNA methylation analysis of RCC1 promoter CpG islands by MALDI-TOF mass spectrometry. A. Schematic map of CpG island distribution in the RCC1 gene promoter to exon 1. B. Localization of the 24 total CpG sites present in the sequence of the RCC1 gene promoter to exon 1. The putative transcription factor binding motifs located in the CpG sites were plotted by a transcription factor prediction system. C. The methylation amplicons were plotted in each CpG site according to their status of DNA methylation ratio as determined by MALDI-TOF MS analysis. D. Quantification of DNA methylation ratios at the 9th CpG site in the RCC1 gene regulation region in three different gastric cancer cell lines.

Figure 4

Immunohistochemical (IHC) examination of RCC1 protein expression in gastric carcinoma tissues and adjacent non-tumor tissues (80×). Examples of IHC staining images of different grades of gastric carcinomas, including (A) Grade 1, (B) Grade 2, and (C) Grade 3. Strong RCC1 protein staining signals were detected in the nuclei and cytoplasms of adjacent normal tissues (N), and lower RCC1 expression was found in gastric carcinoma tissue (T) under 80× magnification. Higher magnification 400× images are also shown in the bottom panels corresponding to each grade of gastric carcinoma tissues.