MBD1/mdr1信号转录调控胰腺癌多药耐药性的实验研究及其临床意义
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摘要
在胰腺癌的综合治疗过程中，化疗效果始终不尽人意。多药耐药性MDR(multidrug resistance)及mdr1基因(multidrug resistance gene 1)被认为是临床化疗失败的主要原因之一。检测胰腺癌中mdr1基因的甲基化改变并试图应用甲基化CpG结合域蛋白1(methyl-CpG binding domain protein 1, MBD1)进行mdr1基因的甲基化转录调控，从而达到改变胰腺癌多药耐药性的目的。
实验研究mdr1基因5′端的主要转录区域上游具有启动子活性，富有CAAT-盒和GC-盒，缺乏TATA-盒，其中-110GC盒和-50GC盒分别是转录抑制子和增强子的结合位点，结合位点的甲基化程度与肿瘤多药耐药基因表达有着密切的联系。通过荧光定量聚合酶链反应(FQ-PCR)技术和甲基化特异性PCR(MSP)方法检测mdr1基因在胰腺癌肿瘤组织细胞中的表达及其甲基化水平，发现胰腺癌肿瘤组织细胞中mdr1mRNA与P-gp的表达存在显著相关性，同时mdr1基因转录区域的-110GC盒、-50GC盒结合位点甲基化程度与mdr1mRNA表达也存在显著相关性。所以我们认为mdr1基因转录区域结合位点甲基化可能是一种影响基因表达的重要机制，DNA甲基化状态改变可能是调控肿瘤多药耐药性的重要手段之一。
我们利用基因芯片技术分析胰腺癌基因表达谱时，发现在差异表达的基因中，多个甲基化相关基因间的异常转录调控机制有重要的研究价值。其中甲基化结合域蛋白MBD1基因在胰腺癌中表达明显上调。作为一个重要的转录调控因子，胰腺癌中MBD1介导的甲基化转录调控作用可能是造成多药耐药基因转录表达增加／下降，以致胰腺癌多药耐药性发生改变的重要原因。
BxPC-3为原发癌细胞株，且其原始mdr1基因和MBD1基因表达程度均高，因此选择BxPC-3细胞株作为进一步的研究对象。我们利用RNA干扰技术，设计合成了针对MBD1基因的siRNAs，成功构建MBD1siRNAs真核表达质粒Rotro Super-MBD1siRNAs，并成功载入质粒。采用脂质体介导的方法将MBD1siRNAs表达质粒转染胰腺癌细胞系BxPC-3，成功下调了BxPC-3细胞株MBD1基因表达水平。应用免疫组化定量分析法、FQ-PCR技术、MSP方法及MTT比色法检测BxPC-3细胞在调控前后mdr1／P-gp在蛋白水平、基因表达水平、基因甲基化程度以及肿瘤细胞增殖能力变化。研究结果显示MBD1siRNA表达质粒能显著抑制胰腺癌细胞BxPC-3中MBD1的表达，同时P-gp、mdr1基因表达随之下调，转录区域结合位点甲基化表达呈上调，细胞株对于药物的耐药性受到相应的抑制，即药物敏感性提高。
临床研究应用免疫组织化学法和FQ-PCR技术、MSP方法分别从蛋白和基因水平检测胰腺癌临床组织标本和对应外周血标本中的mdr1基因的表达及转录区域结合位点甲基化水平。研究外周血有核细胞与肿瘤组织细胞的多药耐药基因及其甲基化表达的相关性及一致性。研究结果表明，外周血与肿瘤组织细胞中的mdr1基因及其甲基化表达存在显著的相关性及一致性。推想临床上可根据外周血测定mdr1基因及其甲基化表达水平来预测胰腺癌患者化疗前后多药耐药性的变化及影响。
结论：研究结果表明，胰腺癌中P-gp与mdr1 mRNA表达呈显著一致，与mdr1基因转录区域结合位点甲基化表达程度也呈显著相关，提示mdr1基因转录区域结合位点甲基化对于胰腺癌的多药耐药性有着重要的意义。当胰腺癌细胞株BxPC-3通过脂质体介导方法转染MBD1siRNAs表达质粒后，MBD1的表达受到有效抑制，改变了mdr1／P-gp在蛋白水平、mRNA水平和基因甲基化的表达，影响了肿瘤细胞对药物的耐药能力，间接调控了胰腺癌的多药耐药性；外周血与肿瘤组织细胞中mdr1基因及其甲基化表达存在相关性及一致性。临床上可根据外周血测定mdr1基因及其甲基化变化程度来预测胰腺癌患者化疗前后多药耐药性的变化，从而指导临床。
In case of pancreatic cancer's combined therapy, the chemo-therapeutic effectwas not satisfactory all the time. We know that MDR (multidrug resistance) and mdr1gene (multidrug resistance gene 1) were the main cause of clinical chemotherapyfailure. In order to alter the multidrug resistance of pancreatic cancer, we tried todetect the alteration of mdr1 gene methylation in pancreatic cancer and to control thetranscription of methylation by using action of the MBD1(methyl-CpG bindingdomain protein 1)
Experimental study: The upper stream in the main transcription domain of mdr15'-flanking sequence had promoter activity. It was rich of CAAT-box, C-C-box andlack of TATA-box. The -110GC-box and -50GC-box among them were methylatedbinding sites and were bind of transcriptional repressor and enhanser respectively. Wedetected mdr1 and its transcriptional domain binding site methylated expression levelfrom pancreatic cancer tissues by the techniques of FQ-PCR (Real-time fluorescentquantitative PCR)and MSP (methylation—specific PCR). The results showed thatexpressions of mdr1 and P-gp were closely correlated, the methylated expressions of-110GC-box and -50GC-box were correlated significantly also. So we thought thatDNA's methylation was one kind of important mechanism, to influence the geneticexpression. We also supposed that altering the status of DNA's methylation could bethe one of important method to regulate the tumor's multidrug resistance.
By using the technique of cDNA microarray to analyze the pancreatic cancergenetic expression spectra, we found that the abnormal transcription controlmechanism between numerous methylation related gene took very important role inthe genetic expression difference. Among them, the expression of MBD1 wasobviously up-regulated in pancreatic cancer. As a important transcriptional controlfactor, MBD1 mediated methylated transcription control in pancreatic cancer might bethe related reason that made the expression and transcription of multidrug resistancegene up/down, and changed the pancreatic cancer's multidrug resistance.
BxPC-3 was cultured from pancreatic cancer in situ, and the expression of mdr1 and MBD1 were higher. So BxPC-3 was chosen as a further research platform. ByRNA interference (RNAi) technology, the siRNA was designed and synthesied whichaimed at the MBD1 gene. MBD1 siRNAs eukaryotic expression vector RotroSuper-MBD 1 was constructed and had been successfully integrated into the plasmid.MBD1 siRNAs vector was transfected into BxPC-3 pancreatic cancer cell byliposome and had regulated the level of MBD1 gene expression down ward.Techniques of quantitative immunohistochemical analysis, FQ-PCR, MSP and MTTcolormetric assay was used to detect the difference of mdr1/P-gp protein level, geneticexpression level, genetic methylation level and tumour cell mukiplication activitybefore and after transcription control. It was demonstrated that MBD 1 siRNAs couldsignificantly suppress the expression of MBD 1 mRNA in BxPC-3, which induced thedown-regulation of mdr1 mRNA and P-gp and up-regulation of -110GC-box and-50GC-box's methylation. Among those changes, the MDR (multidrug resistance) ofBxPC-3 were also downgraded and drug sensitivity were upgraded.
Clinical study: Utilizing methods including quantitative immunohistochemicalanalysis, FQ-PCR and MSP to detect the protein, genetic expression and methylationlevel in pancreatic carcinoma tissue sample and peripheral blood sample accordingly.After investigation, it was proved that the revariation of expression and methylation inmultidrug resistance gene were dependable concordant between tumor tissue sampleand blood sample of pancreatic carcinoma patients. We could predict the effect andchange of multidrug resistance in pancreatic cancer especially after chemotherapythrough the detection of expression and methylation of multidrug resistance genemerely from peripheral blood sample.
Conclusion: Our experimental result indicated that the expressions of mdr1 andP-gp were closely correlated, the methylated expressions of -110GC-box and-50GC-box were correlated significantly also. So the methylation of mdr1 played avery important role in the process of pancreatic cancer's multidrug resistance. Theexpression of mdr1 and MBD1 in BxPC-3 pancreatic cancer cell were high. Theexpression of MBD1 was down-regulated by transfecting MBD1 siRNAs vector intoBxPC-3 mediated with liposome. The level of mdr1/P-gp protein, genetic expression,genetic methylation and tumour cell multiplication activity had been altered, and eventually also changed pancreatic cancer's multidrug resistance indirectly. Thevariation of expression and methylation in multidrug resistance gene were dependableconcordant between tumor tissue sample and blood sample of pancreatic carcinomapatients. We could predict the effect and change of multidrug resistance in pancreaticcancer especially after chemotherapy through the detection of expression andmethylation of multidrug resistance gene merely from peripheral blood sample.
实验研究mdr1基因5′端的主要转录区域上游具有启动子活性，富有CAAT-盒和GC-盒，缺乏TATA-盒，其中-110GC盒和-50GC盒分别是转录抑制子和增强子的结合位点，结合位点的甲基化程度与肿瘤多药耐药基因表达有着密切的联系。通过荧光定量聚合酶链反应(FQ-PCR)技术和甲基化特异性PCR(MSP)方法检测mdr1基因在胰腺癌肿瘤组织细胞中的表达及其甲基化水平，发现胰腺癌肿瘤组织细胞中mdr1mRNA与P-gp的表达存在显著相关性，同时mdr1基因转录区域的-110GC盒、-50GC盒结合位点甲基化程度与mdr1mRNA表达也存在显著相关性。所以我们认为mdr1基因转录区域结合位点甲基化可能是一种影响基因表达的重要机制，DNA甲基化状态改变可能是调控肿瘤多药耐药性的重要手段之一。
我们利用基因芯片技术分析胰腺癌基因表达谱时，发现在差异表达的基因中，多个甲基化相关基因间的异常转录调控机制有重要的研究价值。其中甲基化结合域蛋白MBD1基因在胰腺癌中表达明显上调。作为一个重要的转录调控因子，胰腺癌中MBD1介导的甲基化转录调控作用可能是造成多药耐药基因转录表达增加／下降，以致胰腺癌多药耐药性发生改变的重要原因。
BxPC-3为原发癌细胞株，且其原始mdr1基因和MBD1基因表达程度均高，因此选择BxPC-3细胞株作为进一步的研究对象。我们利用RNA干扰技术，设计合成了针对MBD1基因的siRNAs，成功构建MBD1siRNAs真核表达质粒Rotro Super-MBD1siRNAs，并成功载入质粒。采用脂质体介导的方法将MBD1siRNAs表达质粒转染胰腺癌细胞系BxPC-3，成功下调了BxPC-3细胞株MBD1基因表达水平。应用免疫组化定量分析法、FQ-PCR技术、MSP方法及MTT比色法检测BxPC-3细胞在调控前后mdr1／P-gp在蛋白水平、基因表达水平、基因甲基化程度以及肿瘤细胞增殖能力变化。研究结果显示MBD1siRNA表达质粒能显著抑制胰腺癌细胞BxPC-3中MBD1的表达，同时P-gp、mdr1基因表达随之下调，转录区域结合位点甲基化表达呈上调，细胞株对于药物的耐药性受到相应的抑制，即药物敏感性提高。
临床研究应用免疫组织化学法和FQ-PCR技术、MSP方法分别从蛋白和基因水平检测胰腺癌临床组织标本和对应外周血标本中的mdr1基因的表达及转录区域结合位点甲基化水平。研究外周血有核细胞与肿瘤组织细胞的多药耐药基因及其甲基化表达的相关性及一致性。研究结果表明，外周血与肿瘤组织细胞中的mdr1基因及其甲基化表达存在显著的相关性及一致性。推想临床上可根据外周血测定mdr1基因及其甲基化表达水平来预测胰腺癌患者化疗前后多药耐药性的变化及影响。
结论：研究结果表明，胰腺癌中P-gp与mdr1 mRNA表达呈显著一致，与mdr1基因转录区域结合位点甲基化表达程度也呈显著相关，提示mdr1基因转录区域结合位点甲基化对于胰腺癌的多药耐药性有着重要的意义。当胰腺癌细胞株BxPC-3通过脂质体介导方法转染MBD1siRNAs表达质粒后，MBD1的表达受到有效抑制，改变了mdr1／P-gp在蛋白水平、mRNA水平和基因甲基化的表达，影响了肿瘤细胞对药物的耐药能力，间接调控了胰腺癌的多药耐药性；外周血与肿瘤组织细胞中mdr1基因及其甲基化表达存在相关性及一致性。临床上可根据外周血测定mdr1基因及其甲基化变化程度来预测胰腺癌患者化疗前后多药耐药性的变化，从而指导临床。
In case of pancreatic cancer's combined therapy, the chemo-therapeutic effectwas not satisfactory all the time. We know that MDR (multidrug resistance) and mdr1gene (multidrug resistance gene 1) were the main cause of clinical chemotherapyfailure. In order to alter the multidrug resistance of pancreatic cancer, we tried todetect the alteration of mdr1 gene methylation in pancreatic cancer and to control thetranscription of methylation by using action of the MBD1(methyl-CpG bindingdomain protein 1)
Experimental study: The upper stream in the main transcription domain of mdr15'-flanking sequence had promoter activity. It was rich of CAAT-box, C-C-box andlack of TATA-box. The -110GC-box and -50GC-box among them were methylatedbinding sites and were bind of transcriptional repressor and enhanser respectively. Wedetected mdr1 and its transcriptional domain binding site methylated expression levelfrom pancreatic cancer tissues by the techniques of FQ-PCR (Real-time fluorescentquantitative PCR)and MSP (methylation—specific PCR). The results showed thatexpressions of mdr1 and P-gp were closely correlated, the methylated expressions of-110GC-box and -50GC-box were correlated significantly also. So we thought thatDNA's methylation was one kind of important mechanism, to influence the geneticexpression. We also supposed that altering the status of DNA's methylation could bethe one of important method to regulate the tumor's multidrug resistance.
By using the technique of cDNA microarray to analyze the pancreatic cancergenetic expression spectra, we found that the abnormal transcription controlmechanism between numerous methylation related gene took very important role inthe genetic expression difference. Among them, the expression of MBD1 wasobviously up-regulated in pancreatic cancer. As a important transcriptional controlfactor, MBD1 mediated methylated transcription control in pancreatic cancer might bethe related reason that made the expression and transcription of multidrug resistancegene up/down, and changed the pancreatic cancer's multidrug resistance.
BxPC-3 was cultured from pancreatic cancer in situ, and the expression of mdr1 and MBD1 were higher. So BxPC-3 was chosen as a further research platform. ByRNA interference (RNAi) technology, the siRNA was designed and synthesied whichaimed at the MBD1 gene. MBD1 siRNAs eukaryotic expression vector RotroSuper-MBD 1 was constructed and had been successfully integrated into the plasmid.MBD1 siRNAs vector was transfected into BxPC-3 pancreatic cancer cell byliposome and had regulated the level of MBD1 gene expression down ward.Techniques of quantitative immunohistochemical analysis, FQ-PCR, MSP and MTTcolormetric assay was used to detect the difference of mdr1/P-gp protein level, geneticexpression level, genetic methylation level and tumour cell mukiplication activitybefore and after transcription control. It was demonstrated that MBD 1 siRNAs couldsignificantly suppress the expression of MBD 1 mRNA in BxPC-3, which induced thedown-regulation of mdr1 mRNA and P-gp and up-regulation of -110GC-box and-50GC-box's methylation. Among those changes, the MDR (multidrug resistance) ofBxPC-3 were also downgraded and drug sensitivity were upgraded.
Clinical study: Utilizing methods including quantitative immunohistochemicalanalysis, FQ-PCR and MSP to detect the protein, genetic expression and methylationlevel in pancreatic carcinoma tissue sample and peripheral blood sample accordingly.After investigation, it was proved that the revariation of expression and methylation inmultidrug resistance gene were dependable concordant between tumor tissue sampleand blood sample of pancreatic carcinoma patients. We could predict the effect andchange of multidrug resistance in pancreatic cancer especially after chemotherapythrough the detection of expression and methylation of multidrug resistance genemerely from peripheral blood sample.
Conclusion: Our experimental result indicated that the expressions of mdr1 andP-gp were closely correlated, the methylated expressions of -110GC-box and-50GC-box were correlated significantly also. So the methylation of mdr1 played avery important role in the process of pancreatic cancer's multidrug resistance. Theexpression of mdr1 and MBD1 in BxPC-3 pancreatic cancer cell were high. Theexpression of MBD1 was down-regulated by transfecting MBD1 siRNAs vector intoBxPC-3 mediated with liposome. The level of mdr1/P-gp protein, genetic expression,genetic methylation and tumour cell multiplication activity had been altered, and eventually also changed pancreatic cancer's multidrug resistance indirectly. Thevariation of expression and methylation in multidrug resistance gene were dependableconcordant between tumor tissue sample and blood sample of pancreatic carcinomapatients. We could predict the effect and change of multidrug resistance in pancreaticcancer especially after chemotherapy through the detection of expression andmethylation of multidrug resistance gene merely from peripheral blood sample.
引文
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