Experimental Study On Promoter Hypermethylation And Expression Silencing Of The RUNX3 Gene In Esophageal Squamous Cell Carcinoma

Esophageal carcinoma(EC) is one of the most common malignant tumors in human, it is the sixth most frequent cancer incidence and its morbidity has risen to the fourth cause in recent years in China, esophageal squamous cell carcinoma(ESCC) accounts for 90％of the esophageal carcinoma in Asian countries. Previous studies showed that multiple genetic alterations involved in carcinogenesis of esophagus, but its molecular mechanisms are poorly understood. From the normal esophageal epithelial cell to esophageal cancer must undergo a series of process which including hyperplasia, in situ carcinoma and metastasis carcinoma, in which involves activation of oncogenes and inactivation of tumor suppressor genes. However, these changes of oncogenes and tumor suppressor genes could not completely explain the activation/inactivation by the classical regulatory control(two-hit hypothesis). Recent data suggests that ESCC appears to be a process that is fulled by genetic alterations and by epigenetic mechanism. It mainly involves DNA methylation and histone deacetylation. DNA methylation is the most important mechanism in epigenetic, DNA hypermethylation is established as the third mechanism by which tumor suppressor genes are inactivated. Although multiple genetic and epigenetic changes have been detected in ESCC, the precise molecular mechanisms governing the development and/or progression of ESCC still remain unknown.RUNX3 has been recognized as a putative tumor suppressor gene in human gastric cancer. Loss of RUNX3 expression through high frequency hemizygous deletion and hypermethylation was found in 25-75％of gastric, colorectal and hepatocellular cancers. Comparative genomic hybridization of ESCC has revealed the frequent occurrence of multiple chromosomal deletions at 1p, 3p, 4p, 18q, 19p, 19q, etc. RUNX3 locus 1p36 is commonly deleted in a variety of human cancers, including ESCC. Taken all together, there is a strong indication that RUNX3 gene is a novel tumor suppressor gene, and epigenetic alterations in RUNX3 gene may play an important role in ESCC. The aim of this study is to evaluate potential clinical implications of promoter hypermethylation of the RUNX3 gene in ESCC.PartⅠStudy of expression silencing of the RUNX3 gene and clinical implications in ESCCObjective: To evaluate expression of the RUNX3 gene and potential clinical implications in Eca-109cell line, 42 ESCC tissues and matched 42 adjacent normal tissues.Methods: To detect expression of RUNX3 mRNA by RT-PCR in Eca-109 cell line, 42 primary ESCC tissues and corresponding normal tissues, Western blotting was evaluated for protein expression in the same specimens, the results were compared with the clinicopathological data.Results: Loss or down regulation expression of RUNX3 mRNA expression was detected in the Eca-109 cell line and 23(54.8％) of the 42 ESCC tissues by RT-PCR, whereas RUNX3 mRNA was detectable in all matched 42 adjacent normal tissues. There was significant difference of loss of expression of RUNX3 mRNA between ESCC tissues and matched adjacent normal tissues(P＜0.001). Loss or down regulation of RUNX3 mRNA was significantly correlated with lymph node metastasis and tumor TNM stage(P＜0.05). No relationship was observed between expression of RUNX3 mRNA and sex, age, tumor size, tumor differentiation or drinking(P＞0.05). Western blotting analysis revealed loss or down regulation of RUNX3 protein expression was detected in the Eca-109 cell line and 20 (47.6％) of the 42 ESCC tissues, whereas RUNX3 protein expression was detectable in all corresponding normal tissues. There was significant difference of loss or down regulation of the expression of RUNX3 protein between ESCC tissues and corresponding normal tissues(P＜0.001). Loss or down regulation of RUNX3 protein expression was significantly correlated with lymph node metastasis and TNM stage (P=0.047 and 0.026 respectively). No relationship was observed between the expression of RUNX3 protein and sex, age, tumor size, tumor differentiation or drinking (P＞0.05). Conclusion: Loss or down regulation expression of RUNX3 gene is frequent in ESCC, which is closely associated with lymph node metastasis and TNM stage, RUNX3 is a putative tumor suppressor gene in ESCC. PartⅡStudy of promoter hypermethylation of the RUNX3 gene and demethylating treatment in ESCCPart A Study of promoter hypermethylation of the RUNX3 gene and clinical implications in ESCCObjective: To evaluate promoter hypermethylation of the RUNX3 gene and potential clinical implications in Eca109 cell line, 42 ESCC tissues and corresponding normal tissues.Methods: Methylation-specific PCR (MSP) was evaluated for promoter region methylation status of the RUNX3 gene in ESCC, the results were compared with the clinicopathological data.Results: MSP analysis demonstrated that 64.3% (27/42) of primary ESCC but none of adjacent normal tissues was hypermethylated in the promoter of the RUNX3 gene. There was significant difference of promoter hypermethylation of the RUNX3 gene between ESCC tissues and adjacent normal tissues (P＜0.001). The clinicopathological analysis showed that the RUNX3 gene promoter hypermethylation was closely associated with lymph node metastasis and TNM stage (P=0.029 and 0.008, respectively). Compared with 43.8% of hypermethylation in group without lymph node metastasis, it was significantly higher in those with lymph node metastasis (76.9%). The frequency of promoter hypermethy lation in stageⅠ-Ⅱand stageⅢ-Ⅳwas 38.9% and 83.3%, respectively. Among 27 of RUNX3 gene hypermethylated in the promoter, 85.2% (23/27) RUNX3 mRNA and 74.1% (20/27) RUNX3 protein loss or down regulation of primary ESCC. Promoter hypermethylation of the RUNX3 gene was statistically associated with loss or down regulation of mRNA and protein expression in ESCC (P＜0.001).Conclusion: Our findings suggest that epigenetic silencing of RUNX3 gene expression by promoter hypermethylation could play an important role in ESCC. The detection of RUNX3 hypermethylation by MSP could be a new useful molecular marker of ESCC.Part B Effect of 5-azaC on growth inhibition of Eca109 cell line and expression of the RUNX3 geneObjective: To explore the transcription and protein expression effects of promoter demethylation on RUNX3 gene in Eca109 cell line in vitro exposed to the specific demethylation agent--5-azaC, and evaluate the biological behaviors in Eca109 cell line.Methods: Eca109 cell line was treated by 10μmol/L, 20μmol/L, and 50μmol/L 5-azaC. MSP analysis was evaluated for the promoter region methylation status of the RUNX3 gene in Eca109 cell line, RT-PCR, Western blotting and flow cytometer were used to determine the mRNA expression of RUNX3 gene, protein expression and cell apoptosis.Results: RUNX3 gene promoter was hypermethylated in untreated Eca109 cell, methylated RUNX3 gene was demethylated partly after treated with 5-azaC. No expression of RUNX3 mRNA or protein was found in RUNX3 gene which promoter hypermethylated in Eca109 cell without treated by 5-azaC. On the contrary, expression of RUNX3 mRNA or protein was found after Ecal09 cell treated by 5-azaC, which had a dose-effect relationship. The rate of apoptosis was increased in Eca109 cell after treated by 5-azaC. After 5-azaC treatment, RUNX3 restoration was accompanied by growth inhibition and cell apoptosis, which was partly mediated through up regulation of RUNX3 expression and this may be one of the mechanisms for the tumor cell growth inhibition by 5-azaC.Conclusion: 5-azaC can reactivate the RUNX3 gene transcription and protein expression by demethylation, slow the growth of Eca109 cell and induce cell apoptosis.