新疆哈萨克族食管鳞癌miR-203基因甲基化改变及其意义
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摘要
目的：检测新疆哈萨克族食管鳞癌、癌旁非癌组织和正常食管粘膜中miR-203基因的甲基化状态,探讨miR-203基因甲基化与食管鳞癌之间的关系及其意义。
方法：运用基质辅助激光解析电离时间飞行质谱仪(MALDI-TOF MS)检测99例新疆哈萨克族食管鳞癌、46例癌旁非癌组织和46例正常食管粘膜中miR-203基因甲基化状态。运用Cluster 3.0软件、TreeView软件对三组样本miR-203基因甲基化数据进行分层聚类分析。运用GraphPad Prism 5软件对三组样本miR-203基因甲基化数据进行分布状况分析。运用SPSS16.0软件对食管鳞癌/癌旁非癌组织/正常食管粘膜三组样本、ESCC临床病理学特征、HPV16感染间miR-203基因甲基化数据进行非参数秩和检验统计学分析。
结果：1、食管鳞癌、癌旁非癌组织和正常食管粘膜三组miR-203_15甲基化数据比较结果：新疆哈萨克族鳞癌、癌旁非癌组织中miR-203_15呈高甲基化改变,且miR-203_15有10个CpG单位(CpG_7.8、CpG_10、CpG_11、CpG_12、CpG_14、CpG_15.16、CpG_17.18.19.20、CpG_22.23、CpG_24、CpG_31.32)的高甲基化改变与新疆哈萨克族食管鳞癌的发生相关(p<0.05)；而miR-203_15有两个CpG单位(CpG_10、CpG_11)在食管鳞癌与癌旁非癌组织之间存在甲基化水平的差异(p<0.017),且这两个CpG单位的甲基化水平在癌旁非癌组织中较高。2、miR-203_15甲基化与食管鳞癌临床病理学特征比较结果：与性别比较,miR-203_15各CpG单位甲基化水平在两组间无差异性(p>0.05)；与年龄相关性分析显示,CpG_22.23甲基化水平与年龄呈止相关(p<0.05)：与肿瘤发生部位比较,CpG 31.32甲基化水平在不同部位间存在差异性(p<0.05),且甲基化率比较：下段>中段>上段；与肿瘤大体类型比较,CpG_24甲基化水平在不同类型间存在差异性(p<0.05)；与肿瘤分化程度比较,CpG_17.18.19.20甲基化水平在高、中、低分化程度间存在差异性(p<0.05)且甲基化率比较：中分化组>高分化组>低分化组；与淋巴结转移、TNM分期(Ⅰ期+Ⅱ期和Ⅲ期+Ⅳ期)比较,均无甲基化水平的差异性(p>0.05)。3、miR-203_15甲基化与食管鳞癌HPV16感染比较结果：CpG_15.16、CpG_17.18.19.20在HPV16(+)组与HPV16(-)组之间存在甲基化水平差异性(p<0.05),且甲基化率比较：感染组>非感染组。
结论：1、新疆哈萨克族食管鳞状细胞癌与癌旁非癌组织miR-203_15甲基化水平显著高于正常食管粘膜。2、新疆哈萨克族食管鳞状细胞癌miR-203_15有10个CpG单位(CpG_7.8、CpG_10、CpG_11、CpG_12、CpG_14、CpG_15.16、CpG_17.18.19.20、CpG_22.23、CpG_24、CpG_31.32)呈高甲基化改变,这种改变可能是新疆哈萨克族食管鳞癌发生的一个早期事件。3、新疆哈萨克族食管鳞状细胞癌miR-203_15有两个CpG单位(CpG_15.16、CpG_17.18.19.20)高甲基化改变与HPV16感染相关,两者可能协同作用共同参与新疆哈萨克族食管鳞状细胞癌的发生、发展。
Objective:To detect the methylation status of miR-203 gene of the Hazakh's esophageal squamous cell carcinoma, non-cancerous adjacent tissue and normal esophageal squamous epithelium, and explore the correlation and significance between the methylation of CpG islands of miR-203 and ESCC.
Methods:The genomic DNA of 191 samples, including 99 cases of esophageal squamous cell carcinoma (ESCC),46 cases of non-cancerous adjacent tissue (NCAT) and 46 cases of normal esophageal squamous epithelium (NESE), were modified by the EZ DNA MethylationTM Kit. The CpG islands methylation status of miR-203 gene of 191 cases was detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). All of the methylation data were analyzed by the software of Cluster3.0, GraphPad Prism 5 and SPSS 16.0.
Results:1. Comparison of the methylation rate of miR-203_15 among ESCC, NCAT and NESE: miR-203_15 were hypermethylated in ESCC and NCAT, and there were 10 CpG units, which were CpG_7.8, CpG_10, CpG_11, CpG_12, CpG_14, CpG_15.16, CpG_17.18.19.20, CpG_22.23, CpG_24 and CpG_31.32, were correlated with the occurrence of ESCC of Hazakh in Xinjiang Province (p<0.05). At the same time, the average methylation rate of the 10 CpG units in ESCC and NCAT were higher than NESE. But, the Comparison between ESCC and NCAT there were 2 CpG units among the 10 CpG units (CpG_10, CpG_11), which NCATs were higher than ESCC.2. Comparison between the methylation rate of miR-203_15 and Clinical pathology characteristic:Comparing with gender, there were no CpG unit of miR-203_15 had difference (p>0.05). Comparing with age, methylation status of CpG_22.23 was correlated positively with it. Comparing with tumor position, methylation status of CpG_31.32 had difference (p<0.05) and the average methylation rate was hypomere> midpiece> epimere. Comparing with tumor type, methylation status of CpG_24 had difference (p<0.05). Comparing with differentiation degree, methylation status of CpG_17.18.19.20 had difference (p<0.05) and the average methylation rate was moderately differentiated> well-differentiated> poorly differentiated. Comparing with metastasis of lymph node and TNM staging, there were no CpG unit of miR-203_15 had difference (p>0.05). 3. Comparison between the methylation rate of miR-203_15 and HPV16 infection:methylation status of CpG_15.16, CpG_17.18.19.20 had differences between HPV16-positive and HPV16-negative (p<0.05) and the average methylation rate was HPV16-positive> HPV16-negative.
Conclusions:1.The methylation status of miR-203_15 in ESCC and NCAT has a higher level than in NESE.2. There are 10 CpG units have been hypermethylated and it may be a molecular event in earlier Hazakh's ESCC.3. Hypermethylation of CpG_15.16, CpG_17.18.19.20 of miR-203 may correlate with the infection of HPV16 and participate the occurrence and development of Hazakh's ESCC together.
方法：运用基质辅助激光解析电离时间飞行质谱仪(MALDI-TOF MS)检测99例新疆哈萨克族食管鳞癌、46例癌旁非癌组织和46例正常食管粘膜中miR-203基因甲基化状态。运用Cluster 3.0软件、TreeView软件对三组样本miR-203基因甲基化数据进行分层聚类分析。运用GraphPad Prism 5软件对三组样本miR-203基因甲基化数据进行分布状况分析。运用SPSS16.0软件对食管鳞癌/癌旁非癌组织/正常食管粘膜三组样本、ESCC临床病理学特征、HPV16感染间miR-203基因甲基化数据进行非参数秩和检验统计学分析。
结果：1、食管鳞癌、癌旁非癌组织和正常食管粘膜三组miR-203_15甲基化数据比较结果：新疆哈萨克族鳞癌、癌旁非癌组织中miR-203_15呈高甲基化改变,且miR-203_15有10个CpG单位(CpG_7.8、CpG_10、CpG_11、CpG_12、CpG_14、CpG_15.16、CpG_17.18.19.20、CpG_22.23、CpG_24、CpG_31.32)的高甲基化改变与新疆哈萨克族食管鳞癌的发生相关(p<0.05)；而miR-203_15有两个CpG单位(CpG_10、CpG_11)在食管鳞癌与癌旁非癌组织之间存在甲基化水平的差异(p<0.017),且这两个CpG单位的甲基化水平在癌旁非癌组织中较高。2、miR-203_15甲基化与食管鳞癌临床病理学特征比较结果：与性别比较,miR-203_15各CpG单位甲基化水平在两组间无差异性(p>0.05)；与年龄相关性分析显示,CpG_22.23甲基化水平与年龄呈止相关(p<0.05)：与肿瘤发生部位比较,CpG 31.32甲基化水平在不同部位间存在差异性(p<0.05),且甲基化率比较：下段>中段>上段；与肿瘤大体类型比较,CpG_24甲基化水平在不同类型间存在差异性(p<0.05)；与肿瘤分化程度比较,CpG_17.18.19.20甲基化水平在高、中、低分化程度间存在差异性(p<0.05)且甲基化率比较：中分化组>高分化组>低分化组；与淋巴结转移、TNM分期(Ⅰ期+Ⅱ期和Ⅲ期+Ⅳ期)比较,均无甲基化水平的差异性(p>0.05)。3、miR-203_15甲基化与食管鳞癌HPV16感染比较结果：CpG_15.16、CpG_17.18.19.20在HPV16(+)组与HPV16(-)组之间存在甲基化水平差异性(p<0.05),且甲基化率比较：感染组>非感染组。
结论：1、新疆哈萨克族食管鳞状细胞癌与癌旁非癌组织miR-203_15甲基化水平显著高于正常食管粘膜。2、新疆哈萨克族食管鳞状细胞癌miR-203_15有10个CpG单位(CpG_7.8、CpG_10、CpG_11、CpG_12、CpG_14、CpG_15.16、CpG_17.18.19.20、CpG_22.23、CpG_24、CpG_31.32)呈高甲基化改变,这种改变可能是新疆哈萨克族食管鳞癌发生的一个早期事件。3、新疆哈萨克族食管鳞状细胞癌miR-203_15有两个CpG单位(CpG_15.16、CpG_17.18.19.20)高甲基化改变与HPV16感染相关,两者可能协同作用共同参与新疆哈萨克族食管鳞状细胞癌的发生、发展。
Objective:To detect the methylation status of miR-203 gene of the Hazakh's esophageal squamous cell carcinoma, non-cancerous adjacent tissue and normal esophageal squamous epithelium, and explore the correlation and significance between the methylation of CpG islands of miR-203 and ESCC.
Methods:The genomic DNA of 191 samples, including 99 cases of esophageal squamous cell carcinoma (ESCC),46 cases of non-cancerous adjacent tissue (NCAT) and 46 cases of normal esophageal squamous epithelium (NESE), were modified by the EZ DNA MethylationTM Kit. The CpG islands methylation status of miR-203 gene of 191 cases was detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). All of the methylation data were analyzed by the software of Cluster3.0, GraphPad Prism 5 and SPSS 16.0.
Results:1. Comparison of the methylation rate of miR-203_15 among ESCC, NCAT and NESE: miR-203_15 were hypermethylated in ESCC and NCAT, and there were 10 CpG units, which were CpG_7.8, CpG_10, CpG_11, CpG_12, CpG_14, CpG_15.16, CpG_17.18.19.20, CpG_22.23, CpG_24 and CpG_31.32, were correlated with the occurrence of ESCC of Hazakh in Xinjiang Province (p<0.05). At the same time, the average methylation rate of the 10 CpG units in ESCC and NCAT were higher than NESE. But, the Comparison between ESCC and NCAT there were 2 CpG units among the 10 CpG units (CpG_10, CpG_11), which NCATs were higher than ESCC.2. Comparison between the methylation rate of miR-203_15 and Clinical pathology characteristic:Comparing with gender, there were no CpG unit of miR-203_15 had difference (p>0.05). Comparing with age, methylation status of CpG_22.23 was correlated positively with it. Comparing with tumor position, methylation status of CpG_31.32 had difference (p<0.05) and the average methylation rate was hypomere> midpiece> epimere. Comparing with tumor type, methylation status of CpG_24 had difference (p<0.05). Comparing with differentiation degree, methylation status of CpG_17.18.19.20 had difference (p<0.05) and the average methylation rate was moderately differentiated> well-differentiated> poorly differentiated. Comparing with metastasis of lymph node and TNM staging, there were no CpG unit of miR-203_15 had difference (p>0.05). 3. Comparison between the methylation rate of miR-203_15 and HPV16 infection:methylation status of CpG_15.16, CpG_17.18.19.20 had differences between HPV16-positive and HPV16-negative (p<0.05) and the average methylation rate was HPV16-positive> HPV16-negative.
Conclusions:1.The methylation status of miR-203_15 in ESCC and NCAT has a higher level than in NESE.2. There are 10 CpG units have been hypermethylated and it may be a molecular event in earlier Hazakh's ESCC.3. Hypermethylation of CpG_15.16, CpG_17.18.19.20 of miR-203 may correlate with the infection of HPV16 and participate the occurrence and development of Hazakh's ESCC together.
引文
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