膀胱癌中XPC基因表达缺陷及其分子调控机制研究
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摘要
着色性干皮病基因C(Xeroderma pigmentosum group C,XPC)核苷酸切除修复(Nucleotied Excision Repair,NER)中最早的DNA损伤识别蛋白,XPC蛋白可与HHR23B蛋白紧密结合形成一个异二聚体,研究表明XPC-HR23B复合体是第一个识别并结合在损伤位点的蛋白成分。XPC蛋白在DNA损伤诱导的其他细胞反应中也扮演重要角色,包括细胞周期检查点的调节、细胞凋亡等等。XPC在膀胱癌中的表达明显明显低于正常组织和细胞,这提示XPC的转录抑制与膀胱肿瘤的发生、发展相关。
目前关于XPC的转录调控机制尚不十分清楚,为了研究XPC基因的转录调控机制及其转录抑制与膀胱癌的发生、发展的相关性,我们进行了如下的研究:
1.XPC蛋白水平减低预示膀胱癌患者预后不良
用石蜡包埋的膀胱癌全切术后标本做免疫组织化学的方法检测XPC水平。K Kaplan-Meier方法分析证实XPC低水平表达的膀胱癌患者生存时间明显低于XPC高表达的患者。同时Cox分析进一步得出,XPC蛋白水平可以作为膀胱癌患者的一个独立的预后因素。
2.膀胱癌组织中人XPC基因启动子CpG岛甲基化状态的研究
运用“MethPrimer”软件对XPC启动子区进行分析,预测CpG岛,通过硫化测序PCR (Bisulfite sequencing PCR, BSP),检测XPC启动子区CpG岛的甲基化状况,结果显示:膀胱癌组织中XPC基因启动子区存在CpG岛的甲基化,这可能是其在膀胱癌中表达下降的原因之一。
3.人XPC基因启动子的活性分析
通过生物信息学预测软件(http://www.fruitfly.org/ cgi-bin/seq_tools/promoter.pl.)对人XPC基因的5′侧翼区约2000bp的序列进行分析,预测5′侧翼区内具有启动子功能的区域。应用TransFac professional 8.1(http://jupiter.coh.org/ cgi-bin/transfac/bin/start.cgi)软件对该区域进行分析,获得可能的转录因子结合位点。通过构建包含XPC基因5′上游序列的荧光素酶报告质粒,利用脂质体瞬时转染,检测荧光素酶活性,对XPC启动子区进行分析,结果显示,启动子活性最小区域位于-43 ~ -27bp,预测结果表明,在这一区域可能存在着CREB1转录因子结合位点。
4.细胞内、细胞外鉴定CREB1与XPC基因启动子活性区域的结合
染色质免疫沉淀(chromatin immunoprecipitation assay, ChIP)及电泳迁移率变动实验(electrophoresis mobility shift and supershift assay, EMSA)证明:CREB1可在细胞内、细胞外与XPC启动子区域相结合。
5.CREB1对XPC基因启动子活性的调控
CREB1真核表达质粒与XPC启动子重组报告基因质粒或与突变报告基因质粒(含转录因子结合位点突变)的共转染实验表明:CREB1通过与XPC启动子-43 ~ -27bp区域结合,调节XPC启动子的活性, Western blot进一步证明:PKA抑制剂H89可以抑制UV-C诱导的HEK293细胞中XPC的表达。
6.XPC启动子报告质粒经SssI甲基化处理后极大的降低了它的启动子活性,
EMSA检测结果表明位点特异性甲基化会影响其CREB1与XPC启动子上的顺式作用元件的结合。
本研究对人XPC基因5′侧翼区序列进行了一系列缺失分析,确定了启动子的最小活性区域-43 ~ -27bp,并鉴定了与之相结合的转录因子CREB1对XPC启动子的调节作用。此外,我们还从表观遗传学调控方面研究了XPC基因启动子CpG岛的甲基化状态,发现膀胱癌组织中XPC基因启动子区存在CpG岛的甲基化,证明了XPC在膀胱癌中的转录抑制与XPC启动子CpG岛的甲基化相关,而XPC低表达则预示着膀胱癌患者的预后不良。本研究结果为阐明XPC基因自身转录调控机制以及XPC的转录抑制与肿瘤的相关性奠定了基础。
Xeroderma pigmentosum group C (XPC) is a DNA damage recognition protein that plays an important role in the nucleotide excision repair (NER) process. The XPC protein binds tightly with an HR23B protein to form a stable XPC-HR23B complex . Studies indicate that the XPC-HR23B complex is the first protein component that recognizes and binds to the damaged sites . The XPC protein might also play an important role in other DNA damage-induced cellular responses, including cell cycle checkpoint regulation and apoptosis. It has been found that XPC expression is significantly attenuated in bladder cancer. Therefore, the characterization of the elements that regulate the XPC promoter is of crucial interest to a better understanding the mechanisms of XPC gene expression. However, very little is known about the regulation of the expression of XPC gene itself. To better understand the molecular mechanisms that regulate the expression of XPC gene, we carried out this study and the major results are summarized below:
1. The paraffin wax-embedded tissues of bladder papillary urothelial carcinoma were collected from patients who underwent transurethral resection of bladder tumor,and XPC protein levels were evaluated by immunohistochemistry. Kaplan-Meier analysis demonstrated that the median survival of patients with lower XPC protein levels was shorter compared with patients with higher XPC levels . Cox regression analysis further indicated that XPC protein level may act as an independent prognostic factor for bladder cancer patients (P=0.043).
2. After prediction of CpG island in promoter of XPC gene using“MethPrimer”software, the status of methylation of CpG island in promoter of XPC gene was analyzed using bisulfite sequencing PCR (BSP) and sequence assay. The results revealed that the CpG island in promoter of XPC was partly methylated only in bladder cancer but not in normal bladder tissues.
3. The minimal promoter region of the human XPC gene were identified in this study.The 5′flanking region of the human XPC gene was amplified and analyzed in detail for minimal promoter localization utilizing a series of 5′and 3′deletions of the XPC 5′flanking region fused upstream of the firefly luciferase reporter gene. These experiment identified the minimal promoter region, -43/-27bp, that is necessary for the expression of human XPC gene.
4. Chromatin immunoprecipitation (ChIP) analysis and electrophoretic mobility shift assay (EMSA) revealed that transcription factor CREB interacted with the–43 ~ -27bp promoter region of the human XPC gene in vivo and in vitro.
5. The results of the co-transfection experiments showed that the transcriptional activity of human XPC promoter was found to be regulated negatively by transcription factors CREB, through binding the putative binding sites within XPC promoter region. Western blot further demonstrated that PKA inhibitor H89 decreases DNA damage-inducible expression of XPC following UV-C irradiation in HEK293 cells.
6. The SssI methylation of XPC promoter-luciferase plasmid extemely decreased the luciferase activities ,indicating that Site specific DNA methylation may play a role in interfering with the binding of CREB1 to DNA and silencing transcription. It was found CREB 1 is sensitive to the methylation status of its cognate binding sites in vitro.
In summary, the minimal promoter region of the humanXPC gene was mapped in the region from nucleotides -43 to -27bp. The transcriptional activity of human XPC promoter was found to be regulated by transcription factor CREB, through binding the CREB putative binding sites within XPC promoter region. In addition, we also investigated the epigenetic regulation of XPC gene. The results from this study suggest that hypermethylation of CpG island in promoter of XPC is probably responsible for XPC expression silencing in bladder cancer, and the methyltransferase inhibitor such as 5-Aza-dc can effectively inactivate the expression of XPC. The expression of XPC was reduced with increasing invasive potential in bladder cancer patients. Reduced XPC expression was correlated with tumor aggressiveness and poor patient survival.The information generated from this study provides a solid base for further study of the transcriptional regulation of XPC gene and a novel insight in bladder cancer diagnosis and treatment.
目前关于XPC的转录调控机制尚不十分清楚,为了研究XPC基因的转录调控机制及其转录抑制与膀胱癌的发生、发展的相关性,我们进行了如下的研究:
1.XPC蛋白水平减低预示膀胱癌患者预后不良
用石蜡包埋的膀胱癌全切术后标本做免疫组织化学的方法检测XPC水平。K Kaplan-Meier方法分析证实XPC低水平表达的膀胱癌患者生存时间明显低于XPC高表达的患者。同时Cox分析进一步得出,XPC蛋白水平可以作为膀胱癌患者的一个独立的预后因素。
2.膀胱癌组织中人XPC基因启动子CpG岛甲基化状态的研究
运用“MethPrimer”软件对XPC启动子区进行分析,预测CpG岛,通过硫化测序PCR (Bisulfite sequencing PCR, BSP),检测XPC启动子区CpG岛的甲基化状况,结果显示:膀胱癌组织中XPC基因启动子区存在CpG岛的甲基化,这可能是其在膀胱癌中表达下降的原因之一。
3.人XPC基因启动子的活性分析
通过生物信息学预测软件(http://www.fruitfly.org/ cgi-bin/seq_tools/promoter.pl.)对人XPC基因的5′侧翼区约2000bp的序列进行分析,预测5′侧翼区内具有启动子功能的区域。应用TransFac professional 8.1(http://jupiter.coh.org/ cgi-bin/transfac/bin/start.cgi)软件对该区域进行分析,获得可能的转录因子结合位点。通过构建包含XPC基因5′上游序列的荧光素酶报告质粒,利用脂质体瞬时转染,检测荧光素酶活性,对XPC启动子区进行分析,结果显示,启动子活性最小区域位于-43 ~ -27bp,预测结果表明,在这一区域可能存在着CREB1转录因子结合位点。
4.细胞内、细胞外鉴定CREB1与XPC基因启动子活性区域的结合
染色质免疫沉淀(chromatin immunoprecipitation assay, ChIP)及电泳迁移率变动实验(electrophoresis mobility shift and supershift assay, EMSA)证明:CREB1可在细胞内、细胞外与XPC启动子区域相结合。
5.CREB1对XPC基因启动子活性的调控
CREB1真核表达质粒与XPC启动子重组报告基因质粒或与突变报告基因质粒(含转录因子结合位点突变)的共转染实验表明:CREB1通过与XPC启动子-43 ~ -27bp区域结合,调节XPC启动子的活性, Western blot进一步证明:PKA抑制剂H89可以抑制UV-C诱导的HEK293细胞中XPC的表达。
6.XPC启动子报告质粒经SssI甲基化处理后极大的降低了它的启动子活性,
EMSA检测结果表明位点特异性甲基化会影响其CREB1与XPC启动子上的顺式作用元件的结合。
本研究对人XPC基因5′侧翼区序列进行了一系列缺失分析,确定了启动子的最小活性区域-43 ~ -27bp,并鉴定了与之相结合的转录因子CREB1对XPC启动子的调节作用。此外,我们还从表观遗传学调控方面研究了XPC基因启动子CpG岛的甲基化状态,发现膀胱癌组织中XPC基因启动子区存在CpG岛的甲基化,证明了XPC在膀胱癌中的转录抑制与XPC启动子CpG岛的甲基化相关,而XPC低表达则预示着膀胱癌患者的预后不良。本研究结果为阐明XPC基因自身转录调控机制以及XPC的转录抑制与肿瘤的相关性奠定了基础。
Xeroderma pigmentosum group C (XPC) is a DNA damage recognition protein that plays an important role in the nucleotide excision repair (NER) process. The XPC protein binds tightly with an HR23B protein to form a stable XPC-HR23B complex . Studies indicate that the XPC-HR23B complex is the first protein component that recognizes and binds to the damaged sites . The XPC protein might also play an important role in other DNA damage-induced cellular responses, including cell cycle checkpoint regulation and apoptosis. It has been found that XPC expression is significantly attenuated in bladder cancer. Therefore, the characterization of the elements that regulate the XPC promoter is of crucial interest to a better understanding the mechanisms of XPC gene expression. However, very little is known about the regulation of the expression of XPC gene itself. To better understand the molecular mechanisms that regulate the expression of XPC gene, we carried out this study and the major results are summarized below:
1. The paraffin wax-embedded tissues of bladder papillary urothelial carcinoma were collected from patients who underwent transurethral resection of bladder tumor,and XPC protein levels were evaluated by immunohistochemistry. Kaplan-Meier analysis demonstrated that the median survival of patients with lower XPC protein levels was shorter compared with patients with higher XPC levels . Cox regression analysis further indicated that XPC protein level may act as an independent prognostic factor for bladder cancer patients (P=0.043).
2. After prediction of CpG island in promoter of XPC gene using“MethPrimer”software, the status of methylation of CpG island in promoter of XPC gene was analyzed using bisulfite sequencing PCR (BSP) and sequence assay. The results revealed that the CpG island in promoter of XPC was partly methylated only in bladder cancer but not in normal bladder tissues.
3. The minimal promoter region of the human XPC gene were identified in this study.The 5′flanking region of the human XPC gene was amplified and analyzed in detail for minimal promoter localization utilizing a series of 5′and 3′deletions of the XPC 5′flanking region fused upstream of the firefly luciferase reporter gene. These experiment identified the minimal promoter region, -43/-27bp, that is necessary for the expression of human XPC gene.
4. Chromatin immunoprecipitation (ChIP) analysis and electrophoretic mobility shift assay (EMSA) revealed that transcription factor CREB interacted with the–43 ~ -27bp promoter region of the human XPC gene in vivo and in vitro.
5. The results of the co-transfection experiments showed that the transcriptional activity of human XPC promoter was found to be regulated negatively by transcription factors CREB, through binding the putative binding sites within XPC promoter region. Western blot further demonstrated that PKA inhibitor H89 decreases DNA damage-inducible expression of XPC following UV-C irradiation in HEK293 cells.
6. The SssI methylation of XPC promoter-luciferase plasmid extemely decreased the luciferase activities ,indicating that Site specific DNA methylation may play a role in interfering with the binding of CREB1 to DNA and silencing transcription. It was found CREB 1 is sensitive to the methylation status of its cognate binding sites in vitro.
In summary, the minimal promoter region of the humanXPC gene was mapped in the region from nucleotides -43 to -27bp. The transcriptional activity of human XPC promoter was found to be regulated by transcription factor CREB, through binding the CREB putative binding sites within XPC promoter region. In addition, we also investigated the epigenetic regulation of XPC gene. The results from this study suggest that hypermethylation of CpG island in promoter of XPC is probably responsible for XPC expression silencing in bladder cancer, and the methyltransferase inhibitor such as 5-Aza-dc can effectively inactivate the expression of XPC. The expression of XPC was reduced with increasing invasive potential in bladder cancer patients. Reduced XPC expression was correlated with tumor aggressiveness and poor patient survival.The information generated from this study provides a solid base for further study of the transcriptional regulation of XPC gene and a novel insight in bladder cancer diagnosis and treatment.
引文
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62. Ogishima T, Shiina H, Breault JE, et al. (2005) Promoter CpG hypomethylation and transcription factor EGR1 hyperactivate heparanase expression in bladder cancer. Oncogene 24:6765–6772
63. Issa JP (2007) DNA methylation as a therapeutic target in cancer.Clin Cancer Res 13:1634–1637
64. Cheng JC, Weisenberger DJ, Gonzales FA, et al. (2004) Continuous zebularine treatment effectively sustains demethylation in human bladder cancer cells. Mol Cell Biol 24:1270–1278
65. Ben-Kasus T, Ben-Zvi Z, Marquez VE, et al. (2005) Metabolic activation of zebularine, a novel DNA methylation inhibitor, in human bladder carcinoma cells. Biochem Pharmacol 70:121–133
66. Christoph F, Kempkensteffen C, Weikert S, et al. (2006) Methylation of tumour suppressor genes APAF-1 and DAPK-1 and in vitro effects of demethylating agents in bladder and kidney cancer. Br J Cancer 95:1701–1707
67. Velicescu M, Weisenberger DJ, Gonzales FA, et al. (2002) Cell division is required for de novo methylation of CpG islands in bladder cancer cells. Cancer Res 62:2378–2384
68. Sachs MD, Ramamurthy M, Poel H, et al. (2004) Histone deacetylase inhibitors upregulate expression of the coxsackie adenovirus receptor (CAR) preferentially in bladder cancer cells. Cancer Gene Ther 11:477–486
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74. Karam JA, Fan J, Stanfi eld J, et al. (2007) The use of histone deacetylase inhibitor FK228 and DNA hypomethylation agent 5-azacytidine in human bladder cancer therapy. Int J Cancer 120:1795–1802
75. Ransohoff DF (2003) Cancer. Developing molecular biomarkers for cancer. Science 299:1679–1680
76. Herman JG, Baylin SB (2003) Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 349:2042–2054
77. Herman JG, Graff JR, Myohanen S, et al. (1996) Methylation specifi c PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 93:9821–9826
78. Clark SJ, Harrison J, Paul CL, et al. (1994) High sensitivity mapping of methylated cytosines. Nucleic Acids Res 22:2990–2997
79. Costello JF, Plass C, Cavenee WK (2000) Aberrant methylation of genes in low-grade astrocytomas. Brain Tumor Pathol 17:49–56
80. Santourlidis S, Florl A, Ackermann R, et al. (1999) High frequency of alterations in DNA methylation in adenocarcinoma of the prostate. Prostate 39:166–174
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82. Herman JG, Latif F, Weng Y, et al. (1994) Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma.Proc Natl Acad Sci U S A 91:9700–9704
83. Battagli C, Uzzo RG, Dulaimi E, et al. (2003) Promoter hypermethylation of tumor suppressor genes in urine from kidney cancer patients. Cancer Res 63:8695–8699
84. Ricciardiello L, Goel A, Mantovani V, et al. (2003) Frequent loss of hMLH1 by promoter hypermethylation leads to microsatellite instability in adenomatous polyps of patients with a single fi rstdegree member affected by colon cancer. Cancer Res 63:787–792
85. Kawakami K, Brabender J, Lord RV, et al. (2000) Hypermethylated APC DNA in plasma and prognosis of patients with esophageal adenocarcinoma. J Natl Cancer Inst 92:1805–1811
86. Sathyanarayana UG, Maruyama R, Padar A, et al. (2004) Molecular detection of noninvasive and invasive bladder tumor tissues and exfoliated cells by aberrant promoter methylation of laminin-5 encoding genes. Cancer Res 64:1425–1430
87. Yates DR, Rehman I, Meuth M, et al. (2006) Methylational urinalysis: a prospective study of bladder cancer patients and age stratifi ed benign controls. Oncogene 25:1984–1988
88. Liang G, Gonzales FA, Jones PA, et al. (2002) Analysis of gene induction in human fi broblasts and bladder cancer cells exposed to the methylation inhibitor 5-aza-2′-deoxycytidine. Cancer Res 62:961–966
89. Aleman A, Adrien L, Lopez-Serra L, et al. (2007) Identifi cation of DNA hypermethylation of SOX9 in association with bladder cancer progression using CpG microarrays. Br J Cancer 98:466–473
90. Sasaki M, Anast J, Bassett W, et al. (2003) Bisulfi te conversion specific and methylation-specific PCR: a sensitive technique for accurate evaluation of CpG methylation. Biochem Biophys Res Commun 309:305–309
91. Ehrich M, Nelson MR, Stanssens P, et al. (2005) Quantitative high-throughput analysis of DNA methylation patterns by basespecific cleavage and mass spectrometry. Proc Natl Acad Sci USA 102:15785–15790
92. Esquela-Kerscher A, Slack FJ (2006) Oncomirs– microRNAs with a role in cancer. Nat Rev Cancer. 6:259–269
93. Lagos-Quintana M, Rauhut R, Lendeckel W, et al. (2001) Identification of novel genes coding for small expressed RNAs. Science 294:853–858
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97. Hoque MO, Begum S, Brait M, et al. (2008) Tissue inhibitor of metalloproteinases-3 promoter methylation is an independent prognostic factor for bladder cancer. J Urol 179:743–747
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64. Cheng JC, Weisenberger DJ, Gonzales FA, et al. (2004) Continuous zebularine treatment effectively sustains demethylation in human bladder cancer cells. Mol Cell Biol 24:1270–1278
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74. Karam JA, Fan J, Stanfi eld J, et al. (2007) The use of histone deacetylase inhibitor FK228 and DNA hypomethylation agent 5-azacytidine in human bladder cancer therapy. Int J Cancer 120:1795–1802
75. Ransohoff DF (2003) Cancer. Developing molecular biomarkers for cancer. Science 299:1679–1680
76. Herman JG, Baylin SB (2003) Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 349:2042–2054
77. Herman JG, Graff JR, Myohanen S, et al. (1996) Methylation specifi c PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 93:9821–9826
78. Clark SJ, Harrison J, Paul CL, et al. (1994) High sensitivity mapping of methylated cytosines. Nucleic Acids Res 22:2990–2997
79. Costello JF, Plass C, Cavenee WK (2000) Aberrant methylation of genes in low-grade astrocytomas. Brain Tumor Pathol 17:49–56
80. Santourlidis S, Florl A, Ackermann R, et al. (1999) High frequency of alterations in DNA methylation in adenocarcinoma of the prostate. Prostate 39:166–174
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83. Battagli C, Uzzo RG, Dulaimi E, et al. (2003) Promoter hypermethylation of tumor suppressor genes in urine from kidney cancer patients. Cancer Res 63:8695–8699
84. Ricciardiello L, Goel A, Mantovani V, et al. (2003) Frequent loss of hMLH1 by promoter hypermethylation leads to microsatellite instability in adenomatous polyps of patients with a single fi rstdegree member affected by colon cancer. Cancer Res 63:787–792
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86. Sathyanarayana UG, Maruyama R, Padar A, et al. (2004) Molecular detection of noninvasive and invasive bladder tumor tissues and exfoliated cells by aberrant promoter methylation of laminin-5 encoding genes. Cancer Res 64:1425–1430
87. Yates DR, Rehman I, Meuth M, et al. (2006) Methylational urinalysis: a prospective study of bladder cancer patients and age stratifi ed benign controls. Oncogene 25:1984–1988
88. Liang G, Gonzales FA, Jones PA, et al. (2002) Analysis of gene induction in human fi broblasts and bladder cancer cells exposed to the methylation inhibitor 5-aza-2′-deoxycytidine. Cancer Res 62:961–966
89. Aleman A, Adrien L, Lopez-Serra L, et al. (2007) Identifi cation of DNA hypermethylation of SOX9 in association with bladder cancer progression using CpG microarrays. Br J Cancer 98:466–473
90. Sasaki M, Anast J, Bassett W, et al. (2003) Bisulfi te conversion specific and methylation-specific PCR: a sensitive technique for accurate evaluation of CpG methylation. Biochem Biophys Res Commun 309:305–309
91. Ehrich M, Nelson MR, Stanssens P, et al. (2005) Quantitative high-throughput analysis of DNA methylation patterns by basespecific cleavage and mass spectrometry. Proc Natl Acad Sci USA 102:15785–15790
92. Esquela-Kerscher A, Slack FJ (2006) Oncomirs– microRNAs with a role in cancer. Nat Rev Cancer. 6:259–269
93. Lagos-Quintana M, Rauhut R, Lendeckel W, et al. (2001) Identification of novel genes coding for small expressed RNAs. Science 294:853–858
94. Huang JC, Babak T, Corson TW, et al. (2007) Using expression profiling data to identify human microRNA targets. Nat Methods 4:1045–1049
95. Kim WJ, Kim EJ, Jeong P, et al. (2005) RUNX3 inactivation by point mutations and aberrant DNA methylation in bladder tumors.Cancer Res 65:9347–9354
96. Yu J, Zhu T, Wang Z, et al. (2007) A novel set of DNA methylation markers in urine sediments for sensitive/specifi c detection of bladder cancer. Clin Cancer Res 13:7296–7304
97. Hoque MO, Begum S, Brait M, et al. (2008) Tissue inhibitor of metalloproteinases-3 promoter methylation is an independent prognostic factor for bladder cancer. J Urol 179:743–747