AIM： To explore epigenetic changes in the gene encod- ing X chromosome-linked inhibitor of apoptosis-associ- ated factor 1 （XAF1） during esophageal carcinogenesis. METHODS： Methylation status of XAF1 was detected ...AIM： To explore epigenetic changes in the gene encod- ing X chromosome-linked inhibitor of apoptosis-associ- ated factor 1 （XAF1） during esophageal carcinogenesis. METHODS： Methylation status of XAF1 was detected by methylation-specific polymerase chain reaction （MSP） in four esophageal cancer cell lines （KYSE30, KYSE70, BICl and partially methylated in TE3 cell lines）, nine cases of normal mucosa, 72 cases of pri- mary esophageal cancer and matched adjacent tissue. XAF1 expression was examined by semi-quantitative reverse transcriptional polymerase chain reaction and Western blotting before and after treatment with 5-aza- deoxycytidine （5-aza-dc）, a demethylating agent. To investigate the correlation of XAF1 expression and methylation status in primary esophageal cancer, immu- nohistochemistry for XAF1 expression was performed in 32 cases of esophageal cancer and matched adjacent tissue. The association of methylation status and clini-copathological data was analyzed by logistic regression. RESULTS： MSP results were as follows： loss of XAF1 expression was found in three of four esophageal cell lines with promoter region hypermethylation （com- pletely methylated in KYSE30, KYSE70 and BIC1 cell lines and partially in TE3 cells）; all nine cases of normal esophageal mucosa were unmethylated; and 54/72 （75.00%） samples from patients with esophageal can- cer were methylated, and 25/72 （34.70%） matched adjacent tissues were methylated （75.00% vs 34,70%, z2 = 23.5840, P = 0.000）. mRNA level of XAF1 mea- sured with semi-quantitative reverse transcription poly- merase chain reaction was detectable only in TE3 cells, and no expression was detected in KYSE30, KYSE70 or BIC1 cells. Protein expression was not observed in KYSE30 cells by Western blotting before treatment with 5-aza-dc. After treatment, mRNA level of XAF1 was detectable in KYSE30, KYSE70 and BIC1 cells. Protein expression was detected in KYSE30 after treatment with 5-aza-dc. Immunohistochemistry was performed on 32 cases of esophageal cancer and adjacent tissue, and demonstrated XAF1 in the nucleus and cytoplasm. XAF1 staining was found in 20/32 samples of adjacent normal tissue but was present in only 8/32 samples of esophageal cancer tissue （Z2= 9.143, P = 0.002）. XAF1 expression was decreased in cancer samples compared with adjacent tissues. In 32 cases of esophageal can- cer, 24/32 samples were methylated, and 8/32 esopha- geal cancer tissues were unmethylated. XAF1 staining was found in 6/8 samples of unmethylated esophageal cancer and 2/24 samples of methylated esophageal cancer tissue. XAF1 staining was inversely correlated with XAF1 promoter region methylation （Fisher＇s exact test, P = 0.004）. Regarding methylation status and clinicopathological data, no significant differences were found in sex, age, tumor size, tumor stage, or metas- tasis with respect to methylation of XAF1 for the 72 tis- sue samples from patients with esophageal cancer. CONCLUSION： XAF1 is frequently methylated in eso- phageal cancer, and XAF1 expression is regulated by promoter region hypermethylation.展开更多
基金Supported by Grants from the National Basic Research Program(973 Program), No. 2012CB934002, 2010CB912802National Key Scientific Instrument Special Programme of China, No.2011YQ03013405National Science Foundation of China,No. 81121004, 81071953 and 81161120432
文摘AIM： To explore epigenetic changes in the gene encod- ing X chromosome-linked inhibitor of apoptosis-associ- ated factor 1 （XAF1） during esophageal carcinogenesis. METHODS： Methylation status of XAF1 was detected by methylation-specific polymerase chain reaction （MSP） in four esophageal cancer cell lines （KYSE30, KYSE70, BICl and partially methylated in TE3 cell lines）, nine cases of normal mucosa, 72 cases of pri- mary esophageal cancer and matched adjacent tissue. XAF1 expression was examined by semi-quantitative reverse transcriptional polymerase chain reaction and Western blotting before and after treatment with 5-aza- deoxycytidine （5-aza-dc）, a demethylating agent. To investigate the correlation of XAF1 expression and methylation status in primary esophageal cancer, immu- nohistochemistry for XAF1 expression was performed in 32 cases of esophageal cancer and matched adjacent tissue. The association of methylation status and clini-copathological data was analyzed by logistic regression. RESULTS： MSP results were as follows： loss of XAF1 expression was found in three of four esophageal cell lines with promoter region hypermethylation （com- pletely methylated in KYSE30, KYSE70 and BIC1 cell lines and partially in TE3 cells）; all nine cases of normal esophageal mucosa were unmethylated; and 54/72 （75.00%） samples from patients with esophageal can- cer were methylated, and 25/72 （34.70%） matched adjacent tissues were methylated （75.00% vs 34,70%, z2 = 23.5840, P = 0.000）. mRNA level of XAF1 mea- sured with semi-quantitative reverse transcription poly- merase chain reaction was detectable only in TE3 cells, and no expression was detected in KYSE30, KYSE70 or BIC1 cells. Protein expression was not observed in KYSE30 cells by Western blotting before treatment with 5-aza-dc. After treatment, mRNA level of XAF1 was detectable in KYSE30, KYSE70 and BIC1 cells. Protein expression was detected in KYSE30 after treatment with 5-aza-dc. Immunohistochemistry was performed on 32 cases of esophageal cancer and adjacent tissue, and demonstrated XAF1 in the nucleus and cytoplasm. XAF1 staining was found in 20/32 samples of adjacent normal tissue but was present in only 8/32 samples of esophageal cancer tissue （Z2= 9.143, P = 0.002）. XAF1 expression was decreased in cancer samples compared with adjacent tissues. In 32 cases of esophageal can- cer, 24/32 samples were methylated, and 8/32 esopha- geal cancer tissues were unmethylated. XAF1 staining was found in 6/8 samples of unmethylated esophageal cancer and 2/24 samples of methylated esophageal cancer tissue. XAF1 staining was inversely correlated with XAF1 promoter region methylation （Fisher＇s exact test, P = 0.004）. Regarding methylation status and clinicopathological data, no significant differences were found in sex, age, tumor size, tumor stage, or metas- tasis with respect to methylation of XAF1 for the 72 tis- sue samples from patients with esophageal cancer. CONCLUSION： XAF1 is frequently methylated in eso- phageal cancer, and XAF1 expression is regulated by promoter region hypermethylation.
