| Esophageal cancer is one of the most common cancers in the world. Esophageal squamous cell carcinoma(ESCC) and esophageal adenocarcinoma are the two most common esophageal cancers worldwide. Esophageal adenocarcinoma is most common in the western world. In China, ESCC is one of the most common histologic types. Despite the great advances in surgery, radiotherapy, and chemotherapy in ESCC treatment, the overall 5-year survival rate for ESCC patients is still unfavorable. Advanced ESCC patients have a poor prognosis and low quality of life. Therefore, there is a pressing need for reliable biomarkers to identify the subset of patients with a high risk of poor survival outcomes and guide treatment.It is known that epigenetic changes, including DNA methylation and covalent histone modification, are involved in tumorigenesis and tumor progression of human cancers. One of the most important mechanisms is that many cancer-related genes are silenced by the enhancer of zeste homolog 2(EZH2), which can specially trimethylate lysine 27 on histone H3(H3K27) of the target gene promoters. In the present study, the expression of EZH2 and H3K27me3 protein in esophageal squamous cell carcinomas and their corresponding normal tissues was detected by immunohistochemisty. The relationship between the expression of EZH2 and H3K27me3 protein and their clinical biological was analyzed, especially the prognostic factors. We use real-time quantitative PCR and Western blot to detect the espression of EZH2 and H3K27me3 in esophageal cancer cells. The relationship between EZH2 and invasion and migration of esophageal cancer cells were studied by Transwell invasion and migration assays. In order to provide a theoretical basis for the EZH2 and H3K27me3 as indicators of poor prognosis in patients. The main research contents and results were shown as follows:Part I The expression of EZH2 and H3K27me3 in esophageal squamous cellcarcinoma and its prognostic valueObjective: To investigate the expression of EZH2 and H3K27me3 in esophageal squamous cell carcinomas and corresponding normal tissues, and analyze the relationship between the expression of EZH2 and H3K27me3 protein and their clinical biological indicators, especially the prognostic factors.Methods:1 To investigate the expression pattern of EZH2 in ESCC patients, quantitative real-time PCR was carried out in 96 paired of ESCC specimens and the corresponding pericarcinoma esophageal specimens.2 Immunohistochemical staining was used to detect the protein expression of EZH2 and H3K27me3 protein in esophageal squamous cell carcinomas and the corresponding normal tissue and the clinical/biological factors were analyzed.Results:1 The expression level of EZH2 mRNA in ESCC specimens was significantly higher than the pericarcinoma esophageal specimens. High-expression of EZH2 was found in 84.4%(81 out of 96) of ESCC specimens and 51.0%(49 out of 96) of pericarcinoma esophageal specimens, respectively.2 The expression rate of both EZH2 and H3K27me3 in ESCC specimens was significantly higher than the pericarcinoma esophageal specimens. In ESCC cells, EZH2 was seen in both cytoplasm and the nucleus, however, H3K27me3 was only seen in cell nucleus. Both EZH2 and H3K27me3 showed weak expression in the pericarcinoma esophageal specimens, and the expression rate was 44.8%(43 out of 96) and 26.0%(25 out of 96), respectively. 68 out of 96(70.8%) and 46 out of 96(47.9%) ESCC specimens were found positive with EZH2 and H3K27me3, respectively.3 Association between EZH2 and H3K27me3 expression and the clinicopathological features of ESCC patients. EZH2 protein expression was not correlated with the patients' age, gender, tumor location and tumor status(P?0.05). EZH2 protein expression was positively correlated with the histological grade, lymph node metastasis and distant lymph node metastasis of ESCC patients(P<0.05). H3K27me3 protein expression was not correlated with the patients' age, gender, tumor location, and tumor status(P?0.05). H3K27me3 protein expression was positively correlated with the histological grade, the lymph node metastasis and the distant lymph node metastasis of ESCC patients(P<0.05).4 Association between EZH2 and H3K27me3 expression and the overall survival of ESCC patients. Kaplan-Meier survival data showed that the average overall survival of patients with positive EZH2 expression was 41.175 months, which was significantly shorter than the patients with negative EZH2 expression(54.166 months)(?2=13.218, P=0.000). Similarly, the average overall survival of patients with positive H3K27me3 expression was 38.677 months, which was significantly shorter than the patients with negative H3K27me3 expression(50.800 months)(?2=15.990, P=0.000). The average overall survival of patients with both EZH2 and H3K27me3 expression was 36.136 months, which was significantly shorter than the patients with only one protein expression(48.027 months) or with negative expression of both(55.627 months)(?2=27.517, P=0.000).5 The Cox analysis suggested that EZH2 expression was an independent prognostic factor for poor prognosis of ESCC patients(HR, 3.574; 95% CI, 1.742-7.331; P=0.001). Similar results were observed for histological grade(HR, 2.042; 95% CI, 1.200-3.475; P=0.008) and distant lymph node metastasis(HR, 8.667; 95% CI, 4.280-17.547; P?0.001). Although H3K27me3 was not shown to be an independent prognostic factor, co-expression of EZH2 and H3K27me3 was positive associated with the distant lymph node metastasis of ESCC(?2=15.681; P=0.000).Conclusions: Co-overexpression of EZH2 and H3K27me3 could serve as biomarkers in the prediction of ESCC patients' poor prognosis.Part ? Expression and clinical significance of EZH2 and H3K27me3 in esophageal cancer-derived cells and the effect of EZH2 on the invision of esophageal cancer cellsObjective:We analyzed the expression of EZH2 in esophageal carcinoma cells lines of KYSE30, KYSE170, TE-1, Eca109. They were transfected with the EZH2 over-expression or low-expression vector to investigate the relationship between EZH2 and H3K27me3,and the effect of EZH2 on the biological behavior of esophageal carcinoma cell lines such as invasion,migration in vitro.Methods:1 qRT-PCR(Quantificational real-time PCR), Western Blot were performed to evaluate the expression of EZH2 in esophageal carcinoma cells lines of KYSE30, KYSE170, TE1, Eca109.2 The expression levels of EZH2 and H3K27me3 of esophageal cancer cells with EZH2 over-expression or low-expression were detected by qPCR and Western Blot.3 The expression levels of MMP1, MMP2, MMP7 and MMP9 of esophageal cancer cells with EZH2 over-expression or low-expression were detected by qPCR.4 The cell motility and migration ability of esophageal carcinoma cells with the EZH2 over-expression or low-expression plasmid was evaluated on wound scrape assay.5 The invasion ability of esophageal carcinoma cells with the EZH2 over-expression or low-expression plasmid was revealed in vitro assay on Matrigel filters.Results:1 The expression of EZH2 and H3K27me3 in Eca109 and TE1 cells were slightly higher than its expression level in KYSE30, KYSE170 cells by qRT-PCR and Western Blot(P<0.05).2 Western blotting demonstrated that the over-expression of EZH2 promoted the expression of H3K27me3 in esophageal carcinoma cells lines of KYSE30, KYSE170(P<0.05).3 Western blotting demonstrated that the low-expression of EZH2 inhibited the expression of H3K27me3 in esophageal carcinoma cells lines of Eca-109, TE1(P<0.05).4 qRT-PCR demonstrated that the over-expression of EZH2 promoted the expression of MMP1, MMP2, MMP7 and MMP9 in esophageal carcinoma cells lines of KYSE30, KYSE170(P<0.05). Transwell invasion chamber assays demonstrated that over-expression of EZH2 promoted invasion abilities of KYSE30 and KYSE170(P<0.05). To determine whether EZH2 acts as a tumor migration regulator, we used the wound scrape assay to evaluate cell motility. It demonstrated that over-expression of EZH2 promoted migration abilities of KYSE30 and KYSE170(P<0.05).5 qRT-PCR demonstrated that the low-expression of EZH2 inhibited the expression of MMP1, MMP2, MMP7 and MMP9 in esophageal carcinoma cells lines of Eca109 and TE1(P<0.05). Transwell invasion chamber assays demonstrated that low-expression of EZH2 inhibited invasion abilities of Eca109 and TE1(P<0.05). To determine whether EZH2 acts as a tumor migration regulator, we used the wound scrape assay to evaluate cell motility. It demonstrated that low-expression of EZH2 inhibited migration abilities of Eca109 and TE1(P<0.05).Conclusion: EZH2 can specially trimethylate lysine 27 on histone H3(H3K27) of the target gene promoters, and EZH2 promoted the invasion abilities of esophageal carcinoma cells lines. |
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