AIM:To investigate the expression and methylation status of the secreted frizzled-related protein 2(SFRP2) in esophageal squamous cell carcinoma(ESCC) and explore its role in ESCC carcinogenesis.METHODS:Seven ESCC cel...AIM:To investigate the expression and methylation status of the secreted frizzled-related protein 2(SFRP2) in esophageal squamous cell carcinoma(ESCC) and explore its role in ESCC carcinogenesis.METHODS:Seven ESCC cell lines(KYSE 30,KYSE150,KYSE410,KYSE510,EC109,EC9706 and TE-1) and one immortalized human esophageal epithelial cell line(Het1A),20 ESCC tissue samples and 20 paired adjacent non-tumor esophageal epithelial tissues were analyzed in this study.Reverse-transcription polymerase chain reaction(RT-PCR) was employed to investigate the expression of SFRP2 in cell lines,primary ESCC tumor tissue,and paired adjacent normal tissue.Methylation status was evaluated by methylation-specific PCR and bisulfite sequencing.The correlation between expression and promoter methylation of the SFRP2 gene was confirmed with treatment of 5-aza-2'-deoxycytidine.To assess the potential role of SFRP2 in ESCC,we es-tablished stable SFRP2-transfected cells and examined them with regard to cell proliferation,colony formation,apoptosis and cell cycle in vivo and in vitro.RESULTS:SFRP2 mRNA was expressed in the immortalized normal esophageal epithelial cell line but not in seven ESCC cell lines.By methylation-specific PCR,complete methylation was detected in three cell lines with silenced SFRP2 expression,and extensive methylation was observed in the other four ESCC cell lines.5-aza-2'-deoxycytidine could restore the expression of SFRP2 mRNA in the three ESCC cell lines lacking SFRP2 expression.SFRP2 mRNA expression was obviously lower in primary ESCC tissue than in adjacent normal tissue(0.939 ± 0.398 vs 1.51 ± 0.399,P < 0.01).SFRP2 methylation was higher in tumor tissue than in paired normal tissue(95% vs 65%,P < 0.05).The DNA methylation status of the SFRP2 correlated inversely with the SFRP2 expression.To assess the potential role of SFRP2 in ESCC,we established stable SFRP2 transfectants and control counterparts by introducing pcDNA3.1/v5 hisA-SFRP2 or pcDNA3.1/v5 hisA-empty vector into KYSE30 cells lacking SFRP2 expression.After transfection,the forced-expression of SFRP2 was confirmed by the RT-PCR.In comparison with the control groups,stably-expressed SFRP2 in KYSE 30 cells significantly reduced colony formation in vitro(47.17% ± 15.61% vs 17% ± 3.6%,P = 0.031) and tumor growth in nude mice(917.86 ± 249.35 mm 3 vs 337.23 ± 124.43 mm 3,P < 0.05).Using flow cytometry analysis,we found a significantly higher number of early apoptotic cells in SFRP2-transfected cells than in the control cells(P = 0.025).The mean cell number in the S and G2-M phases of the cell cycle was also significantly lower in SFRP2-transfected KYSE30 cells compared with mock transfected counterparts.CONCLUSION:Silencing of SFRP2 expression through promoter hypermethylation may be a factor in ESCC carcinogenesis through loss of its tumor-suppressive activity.展开更多
Objective：To investigate the feasibility of the combination of detecting hypermethylated secreted frizzled-related protein 1 （SFRP1） and secreted frizzled-related protein 2（SFRP2） in feces as a panel of biomarker...Objective：To investigate the feasibility of the combination of detecting hypermethylated secreted frizzled-related protein 1 （SFRP1） and secreted frizzled-related protein 2（SFRP2） in feces as a panel of biomarkers for colorectal cancer（CRC） screening. Methods： Methylation-specific PCR（MSP） was performed to analyze methylation status of SFRP1 and SFRP2 in a blinded fashion in tumor tissues and in matched stool samples from 39 patients with primary CRC, 34 patients with adenomas, 17 patients with hyperplastic polyps and 20 endoscopically normal subjects as normal controls. Simultaneously we analyzed the correlation of hypermethylated SFRP1 and SFRP2 with the clinicopathological features of CRC. Results：Hypermethylated SFRP1 was detected in 92.3%, 76.5%, 47.1% of tissue samples and in 89.7%, 64.7%, 35.3% of matched fecal samples from CRC, adenoma and hyperplastic polyp, respectively. Hypermethylated SFRP2 was detected in 87.2%, 67.6%, 35.3% of tissue samples and in 82.1%, 55.9%, 29.4% of matched fecal samples from CRC, adenoma and hyperplastic polyp, respectively. Of these two genes, at least one hypermethylated was 94.9%, 82.4%, 52.9% in tissue samples and 92.3%,73.5%, 47.1% in matched fecal samples from CRC, adenoma and hyperplastic polyp, respectively. In contrast, no hypermethylated SFRP1 and SFRP2 were detected in mucosa tissues of normal controls, only 2 cases of fecal samples was detected with hypermethylated SFRP1 and another 1 case was detected with hypermethylated SFRP2. Moreover, no significant associations were observed between hypermethylated SFRP1,SFRP2 and clinicopathological features of CRC. Conclusion： Hypermethylation of SFRP1 and SFRP2 in feces are novel epigenetic biomarkers of CRC and carded high potential for the remote detection of CRC as non-invasive screening method, and combined analysis of hypermethylated SFRP1 and SFRP2 in fecal could further increase the detection rate of CRC and premalignant lesions.展开更多
目的:探讨粪便中分泌型卷曲相关蛋白2(secreted frizzled-related protein 2,SFRP2)基因超甲基化作为筛查大肠癌的非侵袭性工具的可行性。方法:用甲基化特异性PCR(MSP)技术分析69例大肠癌、34例腺瘤和26例增生性息肉患者的瘤组织和粪便...目的:探讨粪便中分泌型卷曲相关蛋白2(secreted frizzled-related protein 2,SFRP2)基因超甲基化作为筛查大肠癌的非侵袭性工具的可行性。方法:用甲基化特异性PCR(MSP)技术分析69例大肠癌、34例腺瘤和26例增生性息肉患者的瘤组织和粪便中SFRP2基因启动子甲基化状态,30例内镜下正常的健康者作为对照。其中大肠癌患者的粪便分别于术前及术后第9天采集。同时分析SFRP2基因甲基化与肿瘤临床病理因素之间的关系。结果:SFRP2基因在91.3%(63/69)的大肠癌组织、79.4%(27/34)的腺瘤组织和53.8%(14/26)的增生性息肉组织中发生超甲基化,并在同一患者所对应的粪便中有87.0%(60/69)、61.8%(21/34)和42.3%(11/26)检测到发生超甲基化。在正常对照的结肠黏膜中没有检测到SFRP2基因甲基化,但在粪便中有2例发生了SFRP2基因甲基化。在大肠癌患者术前和术后第9天的粪便中SFRP2基因超甲基化比较差异有统计学意义(P<0.001)。此外,SFRP2超甲基化与肿瘤的临床特征包括性别、年龄、肿瘤分期、位置及病理分级等无显著相关性。结论:粪便中SFRP2基因超甲基化是大肠癌的一种新的分子标志,对于非侵袭性检测大肠癌具有高度的潜力。展开更多
基金Supported by National Natural Science Foundation of China,No. 81050016Research Fund for the Doctoral Program of Higher Education of China,No. 200800250003
文摘AIM:To investigate the expression and methylation status of the secreted frizzled-related protein 2(SFRP2) in esophageal squamous cell carcinoma(ESCC) and explore its role in ESCC carcinogenesis.METHODS:Seven ESCC cell lines(KYSE 30,KYSE150,KYSE410,KYSE510,EC109,EC9706 and TE-1) and one immortalized human esophageal epithelial cell line(Het1A),20 ESCC tissue samples and 20 paired adjacent non-tumor esophageal epithelial tissues were analyzed in this study.Reverse-transcription polymerase chain reaction(RT-PCR) was employed to investigate the expression of SFRP2 in cell lines,primary ESCC tumor tissue,and paired adjacent normal tissue.Methylation status was evaluated by methylation-specific PCR and bisulfite sequencing.The correlation between expression and promoter methylation of the SFRP2 gene was confirmed with treatment of 5-aza-2'-deoxycytidine.To assess the potential role of SFRP2 in ESCC,we es-tablished stable SFRP2-transfected cells and examined them with regard to cell proliferation,colony formation,apoptosis and cell cycle in vivo and in vitro.RESULTS:SFRP2 mRNA was expressed in the immortalized normal esophageal epithelial cell line but not in seven ESCC cell lines.By methylation-specific PCR,complete methylation was detected in three cell lines with silenced SFRP2 expression,and extensive methylation was observed in the other four ESCC cell lines.5-aza-2'-deoxycytidine could restore the expression of SFRP2 mRNA in the three ESCC cell lines lacking SFRP2 expression.SFRP2 mRNA expression was obviously lower in primary ESCC tissue than in adjacent normal tissue(0.939 ± 0.398 vs 1.51 ± 0.399,P < 0.01).SFRP2 methylation was higher in tumor tissue than in paired normal tissue(95% vs 65%,P < 0.05).The DNA methylation status of the SFRP2 correlated inversely with the SFRP2 expression.To assess the potential role of SFRP2 in ESCC,we established stable SFRP2 transfectants and control counterparts by introducing pcDNA3.1/v5 hisA-SFRP2 or pcDNA3.1/v5 hisA-empty vector into KYSE30 cells lacking SFRP2 expression.After transfection,the forced-expression of SFRP2 was confirmed by the RT-PCR.In comparison with the control groups,stably-expressed SFRP2 in KYSE 30 cells significantly reduced colony formation in vitro(47.17% ± 15.61% vs 17% ± 3.6%,P = 0.031) and tumor growth in nude mice(917.86 ± 249.35 mm 3 vs 337.23 ± 124.43 mm 3,P < 0.05).Using flow cytometry analysis,we found a significantly higher number of early apoptotic cells in SFRP2-transfected cells than in the control cells(P = 0.025).The mean cell number in the S and G2-M phases of the cell cycle was also significantly lower in SFRP2-transfected KYSE30 cells compared with mock transfected counterparts.CONCLUSION:Silencing of SFRP2 expression through promoter hypermethylation may be a factor in ESCC carcinogenesis through loss of its tumor-suppressive activity.
基金supported by the grant from Programs of Science and Technology Commission Foundation of Jiangsu Province(NO.BS2005036)
文摘Objective：To investigate the feasibility of the combination of detecting hypermethylated secreted frizzled-related protein 1 （SFRP1） and secreted frizzled-related protein 2（SFRP2） in feces as a panel of biomarkers for colorectal cancer（CRC） screening. Methods： Methylation-specific PCR（MSP） was performed to analyze methylation status of SFRP1 and SFRP2 in a blinded fashion in tumor tissues and in matched stool samples from 39 patients with primary CRC, 34 patients with adenomas, 17 patients with hyperplastic polyps and 20 endoscopically normal subjects as normal controls. Simultaneously we analyzed the correlation of hypermethylated SFRP1 and SFRP2 with the clinicopathological features of CRC. Results：Hypermethylated SFRP1 was detected in 92.3%, 76.5%, 47.1% of tissue samples and in 89.7%, 64.7%, 35.3% of matched fecal samples from CRC, adenoma and hyperplastic polyp, respectively. Hypermethylated SFRP2 was detected in 87.2%, 67.6%, 35.3% of tissue samples and in 82.1%, 55.9%, 29.4% of matched fecal samples from CRC, adenoma and hyperplastic polyp, respectively. Of these two genes, at least one hypermethylated was 94.9%, 82.4%, 52.9% in tissue samples and 92.3%,73.5%, 47.1% in matched fecal samples from CRC, adenoma and hyperplastic polyp, respectively. In contrast, no hypermethylated SFRP1 and SFRP2 were detected in mucosa tissues of normal controls, only 2 cases of fecal samples was detected with hypermethylated SFRP1 and another 1 case was detected with hypermethylated SFRP2. Moreover, no significant associations were observed between hypermethylated SFRP1,SFRP2 and clinicopathological features of CRC. Conclusion： Hypermethylation of SFRP1 and SFRP2 in feces are novel epigenetic biomarkers of CRC and carded high potential for the remote detection of CRC as non-invasive screening method, and combined analysis of hypermethylated SFRP1 and SFRP2 in fecal could further increase the detection rate of CRC and premalignant lesions.
文摘目的:探讨粪便中分泌型卷曲相关蛋白2(secreted frizzled-related protein 2,SFRP2)基因超甲基化作为筛查大肠癌的非侵袭性工具的可行性。方法:用甲基化特异性PCR(MSP)技术分析69例大肠癌、34例腺瘤和26例增生性息肉患者的瘤组织和粪便中SFRP2基因启动子甲基化状态,30例内镜下正常的健康者作为对照。其中大肠癌患者的粪便分别于术前及术后第9天采集。同时分析SFRP2基因甲基化与肿瘤临床病理因素之间的关系。结果:SFRP2基因在91.3%(63/69)的大肠癌组织、79.4%(27/34)的腺瘤组织和53.8%(14/26)的增生性息肉组织中发生超甲基化,并在同一患者所对应的粪便中有87.0%(60/69)、61.8%(21/34)和42.3%(11/26)检测到发生超甲基化。在正常对照的结肠黏膜中没有检测到SFRP2基因甲基化,但在粪便中有2例发生了SFRP2基因甲基化。在大肠癌患者术前和术后第9天的粪便中SFRP2基因超甲基化比较差异有统计学意义(P<0.001)。此外,SFRP2超甲基化与肿瘤的临床特征包括性别、年龄、肿瘤分期、位置及病理分级等无显著相关性。结论:粪便中SFRP2基因超甲基化是大肠癌的一种新的分子标志,对于非侵袭性检测大肠癌具有高度的潜力。
