Analysis of H3K27me3 expression and DNA methylation at CCGG sites in smoking and non-smoking patients with non-small cell lung cancer and their clinical significance

Abstract

Smoking frequently leads to epigenetic alterations, including DNA methylation and histone modifications. The effect that smoking has on the DNA methylation levels at CCGG sites, the expression of trimethylation of histone H3 at lysine 27 (H3K27me3) and enhancer of zeste homolog 2 (EZH2), and their interactions in patients with non-small cell lung cancer (NSCLC) were analyzed. There were a total of 42 patients with NSCLC, 22 with adenocarcinomas and 20 with squamous cell carcinomas enrolled in the present study. Expression of H3K27me3, EZH2 and proliferating cellular nuclear antigen (PCNA) were immunohistochemically detected. DNA methylation at CCGG sites was evaluated via histoendonuclease-linked detection of DNA methylation sites. The apoptotic index of cancerous tissues obtained from patients of different smoking statuses was evaluated via the terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labeling method. The association with clinicopathological data was calculated relative to different smoking statuses. Compared with the non-smokers, smokers with NSCLC exhibited a significantly lower apoptotic index (P<0.05), and frequently had a lower level of DNA methylation at CCGG sites, lower H3K27me3 expression and a higher EZH2 expression (P<0.05). DNA methylation levels at CCGG sites were negatively correlated to the Brinkman index (P=0.017). Furthermore, there was a parallel association between the H3K27me3 and EZH2 expression levels in the majority of smokers, whereas in the majority of non-smokers, there was a diverging association (P=0.015). There was a diverging association between the PCNA and EZH2 expression levels in the majority of smokers; however, in the majority of non-smokers, there was a parallel association (P=0.048). In addition, the association between the CCGG methylation ratio and immunohistochemical expression of H3K27me3 was a parallel association in the majority of smokers, while in the majority of non-smokers there was a diverging association (P=0.049). Conclusively, patients with NSCLC and different smoking statuses exhibit different epigenetic characteristics. Additionally, DNA methylation levels at the CCGG sites may have the ability to determine associations between the expression levels of H3K27me3, EZH2 and PCNA.

Keywords: DNA methylation; EZH2; non-small cell lung cancer; smoking; trimethylation of histone H3 at lysine 27.

Figure 1.

Immunostaining for H3K27me3, EZH2 and PCNA in NSCLC tissues of smoking and non-smoking patients. (A) H&E staining for NSCLC tissues of non-smoking patients. (B) Strong expression of H3K27me3 in NSCLC tissues of non-smoking patients. (C) Weak staining of EZH2 in NSCLC tissues of non-smoking patients. (D) PCNA expression in NSCLC tissues in non-smoking patients. (E) H&E staining for NSCLC tissues of smoking patients. (F) Weak expression of H3K27me3 in NSCLC tissues of smoking patients. (G) Strong staining of EZH2 in NSCLC tissue of smoking patients. (H) PCNA expression in NSCLC tissues of smoking patients. Scale bar, 20 µm. NSCLC, non-small cell lung cancer; H3K27me3, trimethylation of histone H3 at lysine 27; EZH2, enhancer of zeste homolog 2; PCNA, proliferating cellular nuclear antigen; H&E, hematoxylin and eosin.

Figure 2.

(A) Trimethylation of histone H3 at lysine 27 expression levels differed in NSCLC patients with various smoking statuses (P=0.045). (B) Enhancer of zeste homolog 2 expression levels differed in NSCLC patients with various smoking statuses (P=0.045). (C) No difference in the proliferating cellular nuclear antigen expression levels in NSCLC patients with various smoking statuses (P=0.321). (D) A notable difference in the terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labeling apoptotic index between smoking and non-smoking patients was observed (8.02±2.12 vs. 1.99±0.95; P<0.001). NSCLC, non-small cell lung cancer; H3K27me3, trimethylation of histone H3 at lysine 27; EZH2, enhancer of zeste homolog 2; PCNA, proliferating cellular nuclear antigen; TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labeling.

Figure 3.

Apoptotic tumor cells stained by (A and B) hematoxylin and eosin and (C and D) terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labeling for non-small cell lung cancer tissues of smoking and non-smoking patients. The arrows indicate apoptotic tumor cells in non-smoker and smoker characterized by chromatin condensation, formation of crescentic caps of condensed chromatin at the nuclear periphery and formation of apoptotic bodies. Scale bar, 20 µm. TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labeling; H&E, hematoxylin and eosin.

Figure 4.

Non-small cell lung cancer tissues of smoking patients stained with (A-1 and B-1) H&E; (A-2 and B-2) H3K27me3; (A-3 and B-3) EZH2; (A-4 and B-4) and PCNA. In accordance with the scoring system aforementioned, patient A exhibited strong (A-2) H3K27me3 expression, (A-3) strong EZH2 expression and (A-4) strong PCNA expression. Patient B exhibited (B-2) weak H3K27me3 expression, (B-3) weak EZH2 expression and (B-4) weak PCNA expression. Scale bar, 20 µm. NSCLC, non-small cell lung cancer; H3K27me3, trimethylation of histone H3 at lysine 27; EZH2, enhancer of zeste homolog 2; PCNA, proliferating cellular nuclear antigen; H&E, hematoxylin and eosin.

Figure 5.

Non-small cell lung cancer tissues of smoking patients stained with (A-1 and B-1), H3K27me3, EZH2(c) H&E; (A-2 and B-2) H3K27me3; (A-3 and B-3) EZH2; and (A-4 and B-4) PCNA. Patient C exhibited strong (A-2) H3K27me3 expression, weak (A-3) EZH2 expression and weak (A-4) PCNA expression. Patient D exhibited weak (B-2) H3K27me3 expression, strong (B-3) EZH2 expressionand strong (B-4) PCNA expression. Scale bar, 20 µm. NSCLC, non-small cell lung cancer; H3K27me3, trimethylation of histone H3 at lysine 27; EZH2, enhancer of zeste homolog 2; PCNA, proliferating cellular nuclear antigen; H&E, hematoxylin and eosin.