Differential gene expression profiling of gastric intraepithelial neoplasia and early-stage adenocarcinoma

Abstract

Aim: To investigate the differentiated whole genome expression profiling of gastric high- and low-grade intraepithelial neoplasia and early-stage adenocarcinoma.

Methods: Gastric specimens from an upper magnifying chromoendoscopic targeted biopsy were collected from March 2010 to May 2013. Whole genome expression profiling was performed on 19 low-grade intraepithelial neoplasia (LGIN), 20 high-grade intraepithelial neoplasia (HGIN), 19 early-stage adenocarcinoma (EGC), and 19 chronic gastritis tissue samples using Agilent 4 × 44K Whole Human Genome microarrays. Differentially expressed genes between different types of lesions were identified using an unpaired t-test and corrected with the Benjamini and Hochberg false discovery rate algorithm. A gene ontology (GO) enrichment analysis was performed using the GeneSpring software GX 12.6. The differentially expressed gene was verified using a real-time TaqMan® PCR assay with independent tissue samples, including 26 LGIN, 15 HGIN, 14 EGC, and 20 chronic gastritis. The expression of G0S2 were further validated by immunohistochemical staining (IHC) in 24 LGIN, 40 HGIN, 30 EGC and 61 chronic gastritis specimens.

Results: The gene expression patterns of LGIN and HGIN tissues were distinct. There were 2521 significantly differentially expressed transcripts in HGIN, with 951 upregulated and 1570 downregulated. A GO enrichment analysis demonstrated that the most striking overexpressed transcripts in HGIN compared with LGIN were in the category of metabolism, defense response, and nuclear factor κB (NF-κB) cascade. While the vast majority of transcripts had barely altered expression in HGIN and EGC tissues, only 38 transcripts were upregulated in EGC. A GO enrichment analysis revealed that the alterations of the immune response were most prominent in the progression from HGIN to EGC. It is worth noting that, compared with LGIN, 289 transcripts were expressed at higher levels both in HGIN and EGC. A characteristic gene, G0/G1 switch 2 (G0S2) was one of the 289 transcripts and related to metabolism, the immune response, and the NF-κB cascade, and its expression was validated in independent samples through real-time TaqMan® PCR and immunohistochemical staining. In real-time PCR analysis, the expression of G0S2 was elevated both in HGIN and EGC compared with that in LGIN (P < 0.01 and P < 0.001, respectively). In IHC analysis, G0S2 immunoreactivity was detected in the cytoplasmic of neoplastic cells, but was undetectable in chronic gastritis cells. The G0S2 expression in HGIN was higher than that of LGIN (P = 0.012, χ (2) = 6.28) and EGC (P = 0.008, χ (2) = 6.94).

Conclusion: A clear biological distinction between gastric high- and low-grade intraepithelial neoplasia was identified, and provides molecular evidence for clinical application.

Keywords: G0/G1 switch 2; Gastric early-stage adenocarcinoma; High-and low-grade intraepithelial neoplasia; Immunohistochemical staining; Quantitative real-time PCR; Whole genome expression microarray.

Figure 1

All of the transcripts that were statistically significant both in gastric early-stage adenocarcinoma vs low-grade intraepithelial neoplasia and high-grade intraepithelial neoplasia vs low-grade intraepithelial neoplasia and were involved in the Gene Ontology enrichment analysis. LGIN: Low-grade intraepithelial neoplasia; EGC: Early-stage adenocarcinoma; HGIN: High-grade intraepithelial neoplasia.

Figure 2

Unsupervised cluster analysis of gastric low-grade intraepithelial neoplasia and early-stage adenocarcinoma (A), low-grade intraepithelial neoplasia and high-grade intraepithelial neoplasia (B) tissue samples with the 289 differentially expressed transcripts. The transverse blue bar indicates the histopathological type of the tissue samples: A: Light blue, low-grade intraepithelial neoplasia (LGIN); Dark blue, early-stage adenocarcinoma (EGC); B: Light blue, LGIN; Dark blue, high-grade intraepithelial neoplasia (HGIN). Each column represents a sample, and each row represents a transcript. The expression abundance from low levels to high levels is shown in colors from blue to red.

Figure 3

G₀S₂ expression is increased in gastric high-grade intraepithelial neoplasia and early-stage adenocarcinoma in the microarray. The G₀S₂ mRNA expression in gastric high-grade intraepithelial neoplasia (HGIN), tissue shows a statistically significant elevation compared with that of low-grade intraepithelial neoplasia (LGIN). No statistically significant difference in G₀S₂ expression was found between HGIN and early-stage adenocarcinoma (EGC). The results are expressed as the mean ± SE.

Figure 4

G₀S₂ expression was validated by quantitative polymerase chain reaction in the independent samples. The results are shown as the mean ± SE. -ΔCTG₀S₂ represents the expression level of G₀S₂. The differences of -ΔCTG₀S₂ in the 4 groups [low-grade intraepithelial neoplasia (LGIN), high-grade intraepithelial neoplasia (HGIN), early-stage adenocarcinoma (EGC) and gastritis] were compared using an analysis of variance (one-way ANOVA). A test for the homogeneity of variances shows P = 0.105, indicating that the variances are equal. The mean difference between LGIN and HGIN was statistically significant (P = 0.004). The mean difference between LGIN and EGC was statistically significant (P < 0.001).

Figure 5

Representative immunohistochemistry for G₀S₂ in gastric samples. Representative samples are shown from G₀S₂ (A) chronic gastritis tissues with negative expression, and G₀S₂ (B) low-grade intraepithelial neoplasia tissues with low expression, G₀S₂ (C) high-grade intraepithelial neoplasia tissues with medium expression, and G₀S₂ (D) early-stage adenocarcinoma tissues with high expression (bar represents 100 μm).