胰腺癌中P16、PTEN、XAF1基因的转录表达及甲基化修饰的研究
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摘要
目的：本实验的目的是研究胰腺癌中P16、PTEN、XAF1基因的转录表达及甲基化修饰和肿瘤发生发展的关系。
方法：实验收集了30例胰腺癌组织、癌旁组织、正常胰腺组织和3株胰腺癌细胞(AsPC1、BxPC-3、PANC-1)作为研究对象。实验利用RT-PCR、免疫组化、免疫荧光、Western Blot蛋白印迹、甲基化特异性PCR、细胞周期检测等方法，检测胰腺癌中P16、PTEN、XAF1基因的转录表达及甲基化修饰的情况，将胰腺癌的临床资料与检测结果进行统计分析，明确抑癌基因P16、PTEN、XAF1的转录表达甲基化修饰与肿瘤发生发展的可能关系。再利用去甲基化药物5-aza-2′-deoxycytidine诱导胰腺癌细胞，检测药物对癌细胞的P16、PTEN、XAF1基因的表达和甲基化修饰以及细胞增殖的影响。
结果：胰腺癌中P16、PTEN、XAF1基因的转录表达较癌旁及正常组织下调，且和甲基化修饰水平呈负相关(p＜0．05)。P16、PTEN、XAF1基因的转录表达及甲基化修饰与肿瘤的分化程度、淋巴结转移情况、临床分期有关(p＜0．05)。胰腺癌P16、PTEN、XAF1基因的甲基化修饰水平和胰腺肿瘤标志物CA19-9水平相关(p＜0．05)。利用5-aza-2′-deoxycytidine诱导胰腺癌细胞后，P16、PTEN、XAF1基因的表达增加，其甲基化被逆转，并且使胰腺癌细胞株的增殖受阻于G1／S。
结论：胰腺癌中P16、PTEN、XAF1基因的转录表达下调和基因甲基化修饰水平增高是胰腺癌区别于良性正常组织的生物学标志。胰腺癌中P16、PTEN、XAF1基因甲基化修饰是一种普遍现象。胰腺癌P16、PTEN、XAF1基因的转录表达及甲基化修饰水平和胰腺癌的分化程度、淋巴结转移情况、临床分期有关。胰腺癌P16、PTEN、XAF1基因甲基化修饰水平可望成为胰腺癌的早期诊断指标。药物5-aza-2′-deoxycytidine能够逆转AsPC1、BxPC-3、PANC-1细胞中P16、XAF1基因异常甲基化，诱导P16、PTEN、XAF1基因的转录表达上调，并能抑制肿瘤细胞的增殖。
Objectives:To observe the transcription,expression and methylation status of gene P16,PTEN and XAF1 in pancreatic cancer tissues and cell lines.
Methods:Collect pancreatic tumor(n=30),ajacent(n=10),normal(n=10) tissues andthree pancreatic cancer cell lines.Add in demethylation drug 5-aza-2′-deoxycytidine toact on cell lines.Perform RT-PCR,WesternBlot,immunohistochemistry andimmunofluorescence tests to detect the expression of mRNA and protein in pancreatictissues and cell lines.The MSP method is used to investigate the methylation status intissues and cell lines.Flow cytometry is carried out to analyse the cell cycle.
Results:The expressions of gene P16,PTEN and XAF1 are down-regulated in tumortissues while the methylation status of these genes are up-regulated,which showscertain statistical signification(p＜0.05).The expression and methylation status of P16,PTEN and XAF1 genes are closely related with tumor differentiation,lymph nodemetastasis and clinical stages(p＜0.05).The methylation status of P16,PTEN andXAF1 are related with the pancreatic tumor marker CA19-9 with greatsignification(p＜0.05).Through adding 5-aza-2′-deoxycytidine to induce the pancreaticcell lines,improvement of expression of gene P16,PTEN and XAF1 can be acquired,with the demethylation status and blocked proliferation in G1 phase.
Conclusions:The reduction of expressions of P16,PTEN and XAF1 and increase ofmethylation status of these genes in pancreatic cancer can be considered as the biological marker to distinguish it from normal pancreas.The transcription,expressionand methylation status of gene P16,PTEN and XAF1 are closely related with severalclinical factors,such as the differentiation,lymph node metastasis and clinical stages.Thus the methylation level of P16,PTEN and XAF1 could be expected as an indicatorof early diagnosis of pancreatic cancer.5-aza-2′-deoxycytidine can reverse abnormalgene methylation,as well as induce the transcription and expression of gene P16,PTENand XAF1.Moreover,this drug can inhibit tumor cell proliferation.It implies the5-aza-2′-deoxycytidine can be used as a antineoplastic drug.
方法：实验收集了30例胰腺癌组织、癌旁组织、正常胰腺组织和3株胰腺癌细胞(AsPC1、BxPC-3、PANC-1)作为研究对象。实验利用RT-PCR、免疫组化、免疫荧光、Western Blot蛋白印迹、甲基化特异性PCR、细胞周期检测等方法，检测胰腺癌中P16、PTEN、XAF1基因的转录表达及甲基化修饰的情况，将胰腺癌的临床资料与检测结果进行统计分析，明确抑癌基因P16、PTEN、XAF1的转录表达甲基化修饰与肿瘤发生发展的可能关系。再利用去甲基化药物5-aza-2′-deoxycytidine诱导胰腺癌细胞，检测药物对癌细胞的P16、PTEN、XAF1基因的表达和甲基化修饰以及细胞增殖的影响。
结果：胰腺癌中P16、PTEN、XAF1基因的转录表达较癌旁及正常组织下调，且和甲基化修饰水平呈负相关(p＜0．05)。P16、PTEN、XAF1基因的转录表达及甲基化修饰与肿瘤的分化程度、淋巴结转移情况、临床分期有关(p＜0．05)。胰腺癌P16、PTEN、XAF1基因的甲基化修饰水平和胰腺肿瘤标志物CA19-9水平相关(p＜0．05)。利用5-aza-2′-deoxycytidine诱导胰腺癌细胞后，P16、PTEN、XAF1基因的表达增加，其甲基化被逆转，并且使胰腺癌细胞株的增殖受阻于G1／S。
结论：胰腺癌中P16、PTEN、XAF1基因的转录表达下调和基因甲基化修饰水平增高是胰腺癌区别于良性正常组织的生物学标志。胰腺癌中P16、PTEN、XAF1基因甲基化修饰是一种普遍现象。胰腺癌P16、PTEN、XAF1基因的转录表达及甲基化修饰水平和胰腺癌的分化程度、淋巴结转移情况、临床分期有关。胰腺癌P16、PTEN、XAF1基因甲基化修饰水平可望成为胰腺癌的早期诊断指标。药物5-aza-2′-deoxycytidine能够逆转AsPC1、BxPC-3、PANC-1细胞中P16、XAF1基因异常甲基化，诱导P16、PTEN、XAF1基因的转录表达上调，并能抑制肿瘤细胞的增殖。
Objectives:To observe the transcription,expression and methylation status of gene P16,PTEN and XAF1 in pancreatic cancer tissues and cell lines.
Methods:Collect pancreatic tumor(n=30),ajacent(n=10),normal(n=10) tissues andthree pancreatic cancer cell lines.Add in demethylation drug 5-aza-2′-deoxycytidine toact on cell lines.Perform RT-PCR,WesternBlot,immunohistochemistry andimmunofluorescence tests to detect the expression of mRNA and protein in pancreatictissues and cell lines.The MSP method is used to investigate the methylation status intissues and cell lines.Flow cytometry is carried out to analyse the cell cycle.
Results:The expressions of gene P16,PTEN and XAF1 are down-regulated in tumortissues while the methylation status of these genes are up-regulated,which showscertain statistical signification(p＜0.05).The expression and methylation status of P16,PTEN and XAF1 genes are closely related with tumor differentiation,lymph nodemetastasis and clinical stages(p＜0.05).The methylation status of P16,PTEN andXAF1 are related with the pancreatic tumor marker CA19-9 with greatsignification(p＜0.05).Through adding 5-aza-2′-deoxycytidine to induce the pancreaticcell lines,improvement of expression of gene P16,PTEN and XAF1 can be acquired,with the demethylation status and blocked proliferation in G1 phase.
Conclusions:The reduction of expressions of P16,PTEN and XAF1 and increase ofmethylation status of these genes in pancreatic cancer can be considered as the biological marker to distinguish it from normal pancreas.The transcription,expressionand methylation status of gene P16,PTEN and XAF1 are closely related with severalclinical factors,such as the differentiation,lymph node metastasis and clinical stages.Thus the methylation level of P16,PTEN and XAF1 could be expected as an indicatorof early diagnosis of pancreatic cancer.5-aza-2′-deoxycytidine can reverse abnormalgene methylation,as well as induce the transcription and expression of gene P16,PTENand XAF1.Moreover,this drug can inhibit tumor cell proliferation.It implies the5-aza-2′-deoxycytidine can be used as a antineoplastic drug.
引文
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