摘要
口腔鳞状细胞癌(Oral Squamous Cell Carcinoma, OSCC)是头颈部最常见的恶性肿瘤。尽管传统的手术、放疗、化疗及其综合治疗对早期OSCC可获得较好的治疗效果,但对于晚期或复发的病人难以达到满意的疗效。因此,研究OSCC发病的分子机制,寻找其早期诊断和治疗的分子标志物,将有助于发现新的治疗方法和提高口腔肿瘤病人的生存率和生存质量。
越来越多的研究提示,恶性肿瘤实质上是一种基因疾病,其发生、发展涉及到多种基因的改变。抑瘤基因(tumor suppressor gene, TSG)在人体内往往发挥非常重要的生理功能,其失活、缺失与肿瘤的发生发展密切相关。许多抑瘤基因的发现都是通过研究等位基因杂合性丢失或纯合性丢失(loss of heterozygosity/homozygosity, LOH)而实现的。研究表明,染色体3p21.3区域在肺癌、鼻咽癌等多种肿瘤组织中表现为高频缺失,并存在多个与肿瘤发生密切相关的候选抑瘤基因。多个研究小组发现,该区域在OSCC中同样存在不同程度高频缺失(23%-50%)。目前,该区域内候选抑瘤基因在OSCC发生发展中的作用还鲜少有人研究,大量的信息还有待进一步挖掘。
基于以上原因,本研究拟在文献复习及生物信息学方法分析的基础上,选择该区域内鲜有研究的候选抑瘤基因作为研究对象。筛选在OSCC中表达下调的候选基因并进一步分析其表达失活的可能原因及其在OSCC中的生物学功能。
[3p21.3区候选抑瘤基因在口腔鳞状细胞癌中的筛选]
首先,通过文献复习,生物信息学的方法分析基因的可能功能,确定11个基因(AUXD1、BAP1、FUS1、GNAT1、LARS2、LTF, NPRL2、RASSF1A、SEMA3F、SEMA3B和ZNF35)为初步研究对象。采用RT-PCR方法分析11个基因在12例OSCC癌组织及其旁侧正常对照组织中的表达情况。结果发现,LTF, GNAT1、SEMA3B、AUXD1基因分别在50%(6／12),58.3%(7／12),41.7%(5／12),41.7%(5／12)的口腔鳞癌组织中表达下调或缺失,与在对应正常口腔组织中的表达存在明显差异(P<0.05)。SEMA3F、RASSFIA基因在口腔鳞癌组织中均表现为25%(3／12)的表达下调或缺失。而ZNF35和LARS2基因在口腔鳞癌组织中表现为33.3%(4／12)和25%(3／12)上调表达。其它3个基因(NPRL2、BAP1、FUS1)在口腔鳞癌组织及其对侧正常对照组织中未见明显表达差异(P>0.05)。初步筛选的结果提示,LTF、GNAT1、EMA3B、AUXD14个基因在OSCC组织中呈现较高频率的下调表达,可能与OSCC发生发展密切相关。为此,我们选择这4个基因作为进一步研究的对象。
[启动区异常甲基化在基因表达中的作用]
为能较为准确地判断4个基因在OSCC组织中的表达情况,我们采用RT-PCR方法,分析了4个基因在舌癌细胞系TCA8113细胞和36对OSCC组织标本中的mRNA表达水平。结果发现,除AUXD1基因在TCA8113细胞中表达外,LTF、GNAT1，SEMA3B均表达下调或缺失。与癌旁对照口腔组织相比,LTF, GNAT1、SEMA3B、AUXD1基因分别在61.1%(22／36),44.4%(16／36),50%(18／36),47.2%(17／36)的口腔鳞癌组织中表达下调或缺失,统计学分析,4个基因在两组间的表达具有显著差异(P<0.05)。
为考察4个基因的异常表达与临床相关因素的关系。采用χ2检验对数据进行统计分析,结果提示,LTF, AXUD1、GNAT1和SEMA3B的表达均与患者性别无相关性；而仅LTF基因的表达下调与OSCC发生转移有关,有转移的患者,其LTF的表达明显低于没有转移的患者(P<0.05),其它三个基因均与患者肿瘤转移无明显相关性(P>0.05)。
为进一步探讨基因启动子区甲基化对于基因在OSCC组织或细胞中表达异常的影响,课题采用甲基化特异性PCR (methylation specific PCR, MSP)分析了LTF, GNAT1、SEMA3B在其表达下调的OSCC组织中各自启动子区甲基化的情况。结果发现,LTF, GNATl和SEMA3B在OSCC组织中启动子区均有高甲基化,甲基化率分别为72.7%(16／22)、75%(12／16)、77.8%(14／18),而在癌旁对照组织中三个基因也存在不同程度的的甲基化,其频率也分别为22.7%(5／22)、56.25%(9／16)和38.9%(8／18)。χ2分析表明,LTF和SEMA3B在两种组织中存在启动子区甲基化差异(P=-0.000<0.01),但GNAT1基因启动子区的甲基化在两组间不具有显著差异(P=0.264>0.01)。
甲基转移酶抑制剂5-杂氮-2-脱氧胞苷(5-aza-2'-deoxycytidine, 5-Aza-Cdc)处理舌癌细胞株TCA8113,通过RT-PCR和甲基化特异性PCR分别检测了处理前和用不同浓度的药物处理细胞后基因mRNA表达水平和其启动子区甲基化的情况。结果提示：高甲基化能明显抑制舌癌细胞株TCA8113中LTF基因和SEMA3B基因的mRNA表达,而去甲基化后能部分逆转细胞中两个基因的启动子甲基化状态,重新激活或上调该基因的表达。这些结果均提示启动子区高甲基化对于LTF基因和SEMA3B基因表达失活具有重要的作用。
[LTF基因在舌癌细胞中的功能研究]
相对RT-PCR方法,Real-time PCR能更为准确地检测基因mRNA表达水平,为此,我们在前期分析的基础上,采用Real-time PCR方法进一步验证了LTF基因在OSCC及其癌旁正常对照组织中的表达情况。结果LTF基因表达在OSCC组织中的平均Ct值(9.490±1.675)明显大于癌旁对照组(6.212±4.00),说明LTF基因在OSCC组织中的表达明显下调(P<0.01),其下调频率为63.3%(19／30),并与RT-PCR初筛结果相似。
蛋白质是基因功能的执行者,蛋白质的表达水平与基因功能的发挥密切相关。因此,在检测了LTF mRNA表达水平之后,我们进一步采用免疫组化方法考察了30例OSCC患者石蜡切片中LTF蛋白表达分布的情况。结果显示,LTF蛋白主要表达在细胞胞浆中,并主要分布在鳞状上皮中。30例OSCC石蜡切片中,除4例表现较弱信号外,其余26例癌旁组织上皮均可检测到较强的阳性信号(++)或(+++),而在癌组织中,除12例检测到阳性信号外,其余18例表现为阴性信号(一),或较弱信号(+),其下调频率为60%(18／30),两组间存在明显差异(P<0.01)。
为了进一步探讨LTF在舌癌细胞中的作用,我们从购买的pCMV6-XL5-LTF载体中,采用酶切方法分离获得包含LTF开放阅读框序列在内的2.8kb cDNA片段,经测序证实LTF cDNA存在三个插入或错义SNP位点(23R, A29T, K47R),并经生物信息学分析推测这些位点的多态性可能与LTF的生物学功能无关。将LTF基因的开放阅读框序列克隆到pcDNA3.1(-)载体中,构建了LTF基因的真核表达载体LTF/pcDNA3.1(-),并重新命名为pcLTFo利用脂质体转染技术将pcLTF导入TCA8113细胞,G418筛选获得抗性克隆,进一步采用RT-PCR检测LTF基因的mRNA表达,采用Western Blotting检测LTF蛋白的表达,结果表明我们已成功地建立稳定转染pcLTF的TCA8113细胞系TCA-LTF。免疫细胞化学实验显示表达的LTF蛋白主要定位于胞浆。
随后我们检测了LTF基因稳定表达后对TCA8113细胞的生物学特性的影响。利用MTT法和平板克隆形成实验观察LTF基因稳定表达后对LTF细胞增殖能力和克隆形成能力的影响。MTT的结果表明TCA-LTF细胞的增殖速度明显低于对照组(P<0.05)；平板克隆形成实验结果显示TCA-LTF细胞的克隆形成率明显低于对照组(27.7%vs 51.1%／47.7%)。流式细胞分析结果表明,与对照组相比,转染pcLTF后可以使TCA8113细胞停滞于G0-G1期,G0-G1期细胞比例增加(66.37%vs 53.7%),S期(24.6%vs 32.83%)和G2-M期细胞比例减少(9.05%vs 13.47%)。提示LTF基因稳定表达后,阻止细胞周期进程,导致TCA8113细胞的增殖能力和体外克隆形成能力降低。
总之,本课题以染色体3p21.3缺失区域为切入点,在oscc组织及其癌旁对照组织中分析了该区域内11个候选抑瘤基因的表达情况。结果提示,在oscc组织中,LTF、GNAT1、SEMA3B和AUXD14个基因的mRNA表达呈较高水平下调或缺失,并且启动子甲基化可能是影响LTF和SEMA3B表达下调的重要机制之一,但与GNAT1基因表达下调无关。恢复LTF基因的表达,改善了舌癌细胞TCA8113的恶性生物学特性。这些研究结果将有助于了解口腔黏膜癌变的分子机制,也为寻找新的治疗方法和用于早期诊断、治疗的靶标分子提供一定的理论和实验依据。
Oral squamous cell carcinoma (OSCC) remains to be the major malignant tumor in head and neck tumor. Although the traditional surgery, radiotherapy, chemotherapy and combined therapy have a great therapeutic efficacy to early oral cancer, a satisfied effect can not be achieved to those patients who were in advanced stage or recurrence stage. Therefore, studying the molecular mechanism of carcinogenesis of oral mucous membrane and finding the molecular marker of early diagnosis and treatment can attribute to acquire new therapy and raise survival rate and quality.
Increasing study suggested that malignant tumor is actually a gene disease and the occurrence and development involved the change of several genes. Tumor suppressor gene (TSG) always plays an important role in physiological function and its inactivation and deletion are closely related to the occurrence and development of tumor. Many tumor suppressor genes are found by studying the loss of heterozygosity or homozygosity of allele. Study showed high frequency deletion of chromosome 3p21.3 was existed in several tumors such as lung cancer, nasopharyngeal carcinoma (NPC), and so on. Several groups find that the region of chromosome 3p21.3 also exist different degree of high frequency deletion in OSCC. Currently, little study has done to the role of TSGs at this region in the occurrence and development of OSCC and amount of information remain to further be mined.
Based on the reasons above, in this study, we prepare to choose the candidate TSGs little reported as the research object. Screening the candidate genes down-expression in OSCC and further analyze the possible reason of expression inactivation and the biological function in OSCC.
[The screening of candidate TSGs located at 3p21.3 in OSCC]
We preliminarily determined 11 genes as the research object, such as AUXD1、BAP1、FUS1、GNAT1、LARS2、LTF、NPRL2、RASSF1A、SEMA3B、SEMA3F and ZNF1, by literature review and analyzing the possible function of genes with bioinformatics. RT-PCR was performed to analyze the expression of 11 genes at the transcription level in primary OSCC biopsies and matched tumor-adjacent normal tissues. The results showed that LTF, GNAT1, SEMA3B, AUXD1 were down-regulation or deletion in 50%(6/12),58.3%(7/12),41.7%(5/12),41.7%(5/12) respectively in OSCC and there were significant difference compared to matched tumor-adjacent normal tissues (P<0.05). SEMA3F, RASSF1A were down-regulated or deletion in 25%(3/12), while ZNF35, LARS2 were up-regulated in 33.3%(4/12) and 25%(3/12) in OSCC. No significant difference in transcriptional levels of other 3 genes (NPRL2、BAP1、FUS1) was found between OSCC and matched tumor-adjacent normal tissues (P>0.05). Results of preliminary screening suggested LTF, GNAT1, SEMA3B, AUXD1 were highly down-regulated in OSCC. Therefore, we choose these 4 gens as the object for further research.
[The role of aberrant promoter methylation in gene expression]
To understand exactly the expression statues of 4 genes (LTF, GNAT1, SEMA3B, AUXD1) in OSCC, we analyzed the expression levels of 4 genes in tongue cancer cell line-TCA8113 and 36 OSCC and matched tumor-adjacent normal tissues by RT-PCR. Results revealed LTF, GNAT1, SEMA3B were all down-regulated or deleted except AUXD1 expressed in TCA8113. Compared to matched tumor-adjacent normal tissues, LTF, GNAT1, SEMA3B, AUXD1 were down-regulated or deleted in 61.1%(22/36),44.4%(16/36),50%(18/36),47.2%(17/36) in OSCC biopsies. Statistical analysis showed that the 4 genes have statistical significance between the two groups (P<0.05).
These data were further analyzed to determine the relationship between the aberrant expression of 4 genes and clinical related factor withχ2 test.Statistical analysis indicated that no any significant correlation was found between gender and expression of LTF、AXUD1、GNAT1 and SEMA3B (P>0.05); Only the under-expression of LTF but not other three genes was significantly correlated with metastasis. The expression level of LTF in patients with metastasis was significantly lower than those with no metastasis (P<0.05).
To further explore the role of promoter methylation in abnormal expression of genes in OSCC biopsies or cell lines, we analyzed the promoter methylation statues of LTF, GNAT and SEMA3B in OSCC patients with down-regulated expression of each 3 genes. MSPCR analysis revealed LTF, GNAT, SEMA3B promoter hypermethylation in 72.7%(16/22),75%(12/16),77.8%(14/18) primary OSCCs respectively, and in 22.7%(5/22)、56.25%(9/16) and 38.9%(8/18) matched tumor-adjacent normal tissues respectively.χ2 analysis indicated that there was a significant difference in methylation frequency of LTF (P=0.000<0.01) between OSCCs and tumor-adjacent normal tissues, while no difference in that of GNAT1 between this two groups (P=0.264>0.01). In the other hand, restoration of LTF or SEMA3B expression along with decrease in methylated allele of LTF or SEMA3B could be achieved in TCA8113 cell line after the treatment with 5-aza-2'-deoxycytidine (Aza), a DNA methyltransferase inhibitor. It suggested that promoter hypermethylation should be the important mechanism responsible for inactivation of LTF or SEMA3B in OSCC.
[Investigation of LTF functions in tongue cancer cells]
Real-time PCR can detect more exactly than RT-PCR in mRNA expression levels. Based on previous analysis, we further determined the expression levels of LTF in OSCC biopsies and matched tumor-adjacent normal tissues using real-time PCR method. Results showed the average of Ct value (9.490±1.675) denoting the expression level of LTF in OSCC biopsies was higher than that in matched tumor-adjacent normal tissues (6.212±4.00). It is similar to previous results of RT-PCR that LTF expression was obviously down-regulation (P<0.01) and the ratio of down-regulation was 63.3%(19/30).
To better understand the role of LTF protein in OSCC, immunohistochemical method was also used to detect the protein levels and location of LTF in 30 OSCC specimens. The results showed LTF protein was mainly located in the cytoplasm and stratified squamous epithelial cell. Additionally, positive strong signal (++)or(+++) can be detected in 26 tumor-adjacent normal tissues besides 4 expressed weak signal, while 60%(18/30) of the OSCC tissues showed negative (-) or very low expression level (+) of LTF protein.
In order to investigate the functions of LTF in tongue cancer cells, using enzyme digestion methods, we obtained-2.8kb cDNA containing the open reading frame (ORF) sequence of LTF gene from bought pCMV6-XL5-LTF. Sequencing results showed that LTF cDNA sequence contained three missense SNP site (23R, T29A, R47K), but these had no influence on LTF function according to bioinformatic analysis. Subsequently, the eukaryotic expression vector of LTF (named pcLTF) was constructed by inserting the LTF ORF into the pcDNA3.1(-) vector. Then the pcLTF vector was transferred into TCA8113 cells by Lipofectamine transfection technology. After G418 selection, several G418-resistant cell clones were obtained. It was demonstrated that LTF expressed stably both at mRNA and protein level in the G418-resistant cell clones detected by RT-PCR and western blotting, respectively, showing that the TCA8113 cell line stably expressing LTF gene (named TCA-LTF) was successfully established. The recombinant LTF protein was detected mainly in the cytoplasm of TCA-LTF by immunocytochemistry.
Subsequently, we examined the changes in biological characteristics TCA-LTF cells. The proliferative activity and clonality ability of TCA-LTF were measured by MTT analysis and colony formation assay, respectively. Compared with TCA8113 cells transfected with blank vector (named TCA-pc3.1) or untransfected TCA8113 cells, TCA-LTF cells showed especial proliferation inhibition (P<0.05) and reduction in colony formation efficiency (27.7% vs 51.1%/47.7%). Flow cytometry (FCM) analysis showed that TCA-LTF could block the cell cycle progression of TCA8113 cells in G0-G1 phase, as the pencentage of G0-G1 phase cells were increased (66.37% vs 53.7%) while S phase (24.6% vs 32.83%) and G2-M phase cells (9.05%vs 13.47%) were reduced. These results indicated that LTF overexpression blocked TCA8113 cells at G0-G1 phase resulting in attenuated proliferation and clony forming ability in vitro.
In summary, we elected the deletion region of chromosome 3p21.3 as a breakthrough point to find other TSG candidates closely related OSCC, expression of 11 genes from which in OSCC biopsies and matched tumor-adjacent normal tissues have been done. Our results suggest that 4 genes, such as LTF, GNAT1, SEMA3B, AUXD1, expresses is significantly down-regulated or absent in OSCC tissues. Promoter hypermethylation plays a key role in inactivating LTF and SEMA3B in OSCC, but not in down-regulation expression of GNAT1. Re-expression of LTF in TCA8113 cells can confer partial reversion of the malignant phenotype of tongue cance cells. These results will help us comprehensively understand molecular mechanism underlying OSCC carcinogenesis, and provide the theoretical and experimental evidence for finding new therapy method and biomarker used in early diagnosis or therapy.
引文
[1]Massano J, Regateiro FS, Januario G. Oral squamous cell carcinoma:review of prognostic and predictive factors [J]. Oral Surg Oral Med Oral Pathol Oral Radiol Endod,2006,102(1):67-76.
[2]Parkin DM, Bray F, Ferlay J, et al. Global cancer statistics,2002[J]. CA Cancer J Clin,2005,55(2):74-108.
[3]Vokes EE, Weichselbaum RR, Lippman SM, Hong WK. Head and neck cancer[J]. N Engl J Med,1993,328(3):184-94.
[4]Lippman SM, Hong WK. Molecular markers of the risk of oral cancer [J]. N Engl J Med,2001,344(17):1323-6.
[5]Friend SH, Bernards R, Rogelj S, et al. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma[J]. Nature, 1986,323:643-6.
[6]Chiba I, Shindoh M, Yasuda M, et al. Mutations in the p53 gene and human papillomavirus infection as significant prognosis factors in squamous cell carcinomas of the oral cavity[J]. Oncogene,1996,18:1663-8.
[7]Call KM, Glaser T, Ito CY, et al. Isolation and characterization of a zinc finger polypeptide gene at the human chromosome 11 Wilms'tumor locus[J]. Cell, 1990,60:509-20.
[8]Kamb A, Shattuck-Eidens D, Eeles R, et al. Analysis of the p16 gene (CDKN2) as a candidate for the chromosome 9p melanoma susceptibilitylocus[J]. Nat Genet 1994,8:23-6.
[9]Aoki K, Taketo MM. Adenomatous polyposis coli (APC):a multi-functional tumor suppressor gene[J]. J Cell Sci,2007,120(Pt 19):3327-35.
[10]Hahn SA, Schutte M, Hoque AT, et al. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1[J]. Science,1996,271:350-3.
[11]Ohta M, Inoue H, Cotticelli MG, et al. The FHIT gene, spanning the chromosome 3pl4.2 fragile site and renal carcinoma-associated t(3;8) breakpoint, is abnormal in digestive tract cancers[J]. Cell,1996,84:587-97.
[12]Li J, Yen C, Liaw D, et al. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer[J]. Science,1997, 275:1943-7.
[13]Steck PA, Pershouse MA, Jasser SA, et al. Identification of a candidate tumor suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers[J]. Nat Genet,1997,15:356-62.
[14]Miyakawa A, Wang XL, Nakanishi H, et al. Allelic loss on chromosome 22 in oral cancer:possibility of the existence of a tumor suppressor gene on 22q13[J]. Int J Oncol,1998,13(4):705-9.
[15]Imai FL, Uzawa K, Miyakawa A, et al. A detailed deletion map of chromosome 20 in human oral squamous cell carcinoma[J]. Int J Mol Med,2001,7(1):43-7.
[16]Yamamoto N, Mizoe J, Numasawa H, et al. Allelic loss on chromosomes 2q,3p and 21q:possibly a poor prognostic factor in oral squamous cell carcinoma[J]. Oral Oncol,2003,39(8):796-805.
[17]Sparano A, Quesnelle KM, Kumar MS, et al. Genome-wide profiling of oral squamous cell carcinoma by array-based comparative genomic hybridization[J]. Laryngoscope,2006,116(5):735-41
[18]Nakanishi H, Wang XL, Imai FL, et al. Localization of a novel tumor suppressor gene loci on chromosome 9p21-22 in oral cancer[J]. Anticancer Res, 1999,19(1A):29-34.
[19]Roz L, Wu CL, Porter S, et al. Allelic imbalance on chromosome 3p in oral dysplastic lesions:an early event in oral carcinogenesis[J]. Cancer Res,1996, 56(6):1228-31.
[20]Yamamoto N, Kuroiwa T, Katakura A, et al. Loss of heterozygosity (LOH) on chromosomes 2q,3p and 21q in Indian oral squamous cell carcinoma[J]. Bull Tokyo Dent Coll,2007,48(3):109-17.
[21]Van den Berg A, Dijkhuizen T, Draaijers TG, et al. Analysis of multiple renal cell adenomas and carcinomas suggests allelic loss at 3p21 to be a prerequisite for malignant development[J]. Genes Chromosomes Cancer,1997,19(4): 228-32.
[22]Hung J, Kishimoto Y, Sugio K, et al. Allele-specific chromosome 3p deletions occur at an early stage in the pathogenesis of lung carcinoma[J]. JAMA,1995, 273(7):2511-5.
[23]Zhu Y, Spitz MR, Zheng YL, et al. BPDE-induced lymphocytic 3p21.3 aberrations may predict head and neck carcinoma risk[J]. Cancer,2002,95(3): 563-8.
[24]Senchenko V, Liu J, Braga E, et al. Deletion mapping using quantitative
real-time PCR identifies two distinct 3p21.3 regions affected in most cervical carcinomas[J]. Oncogene,2003,22(19):2984-92.
[25]Senchenko VN, Liu J, Loginov W, et al. Discovery of frequent homozygous deletions in chromosome 3p21.3 LUC A and AP20 regions in renal, lung and breast carcinomas[J]. Oncogene,2004,23(34):5719-28.
[26]Ito M, Ito G, Kondo M, et al. Frequent inactivation of RASSF1 A, BLU, and SEMA3B on 3p21.3 by promoter hypermethylation and allele loss in non-small cell lung cancer[J]. Cancer Lett,2005,225(1):131-9.
[27]Chow LS, Lo KW, Kwong J, et al. RASSF1 A is a target tumor suppressor from 3p21.3 in nasopharyngeal carcinoma[J]. Int J Cancer,2004,109(6):839-47.
[28]Liu XQ, Sun M, Chen HK, et al. Mutation and expression of SEMA3B and SEMA3F gene in nasopharyngeal carcinoma[J]. Ai Zheng,2003,22(1):16-20.
[29]Agathanggelou A, Dallol A, Zochbauer-Muller S, et al. Epigenetic inactivation of the candidate 3p21.3 suppressor gene BLU in human cancers[J]. Oncogene, 2003,22(10):1580-8.
[30]Lai HC, Lin YW, Chang CC, et al. Hypermethylation of two consecutive tumor suppressor genes, BLU and RASSF1 A, located at 3p21.3 in cervical neoplasias[J]. Gynecol Oncol,2007,104(3):629-35.
[31]Tran TN, Liu Y, Takagi M, et al. Frequent promoter hypermethylation of RASSF1 A and pl6INK4a and infrequent allelic loss other than 9p21 in betel-associated oral carcinoma in a Vietnamese non-smoking/non-drinking female population[J]. J Oral Pathol Med,2005,34(3):150-6.
[32]万扬,高文信,陈英新,崔玉波,刘永茂.口腔粘膜癌前病变及鳞癌中RASSF1A基因甲基化与表达的研究[J].现代口腔医学杂志,2007,21(1)：31-33.
[33]Ghosh S, Ghosh A, Maiti GP, et al. Alterations of 3p21.31 tumor suppressor genes in head and neck squamous cell carcinoma:Correlation with progression and prognosis[J]. Int J Cancer,2008,123(11):2594-604.
[34]Esteller M. Epigenetics in cancer[J]. N Engl J Med,2008,358(11):1148-1159.
[35]Baylin SB, Esteller M, Rountree MR, et al. Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer[J]. Hum Mol Genet,2001,10(7):687-692.
[36]Bird A. DNA methylation patterns and epigenetic memory[J]. Genes Dev,2002, 16(1):6-21.
[37]Bird AP. CpG-rich islands and the function of DNA methylation[J]. Nature, 1986,321:209-213.
[38]Merlo A, Herman JG, Mao L, et al.5'CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers[J]. Nat Med,1995,1:686-692
[39]Baylin SB, Esteller M, Rountree MR, et al. Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer[J]. Hum Mol Genet,2001,10(7):687-692.
[40]Steinmann K, Sandner A, Schagdarsurengin U, et al. Frequent promoter hypermethylation of tumor-related genes in head and neck squamous cell carcinoma[J]. Oncol Rep.2009 Dec;22(6):1519-26
[41]Shintani S, Nakahara Y, Mihara M, et al. Inactivation of the p14(ARF), p15(INK4B) and pl6(INK4A) genes is a frequent event in human oral squamous cell carcinomas [J]. Oral Oncol,2001,37(6):498-504.
[42]Nakayama S, Sasaki A, Mese H, et al. The E-cadherin gene is silenced by CpG methylation in human oral squamous cell carcinomasj[J]. Int J Cancer,2001, 93(5):667-73.
[43]Araki D, Uzawa K, Watanabe T, et al. Frequent allelic losses on the short arm of chromosome 1 and decreased expression of the p73 gene at 1p36.3 in squamous cell carcinoma of the oral cavity[J]. Int J Oncol,2002,20(2):355-60.
[44]Chang KW, Kao SY, Tzeng RJ, et al. Multiple molecular alterations of FHIT in betel-associated oral carcinoma[J]. J Pathol,2002,196(3):300-6.
[45]Uzawa K, Ono K, Suzuki H, et al. High prevalence of decreased expression of KAI1 metastasis suppressor in human oral carcinogenesis[J]. Clin Cancer Res, 2002,8(3):828-35.
[46]Gasco M, Bell AK, Heath V, et al. Epigenetic inactivation of 14-3-3 sigma in oral carcinoma:association with p16(INK4a) silencing and human papillomavirus negativity [J]. Cancer Res,2002,62(7):2072-6.
[47]Ogi K, Toyota M, Ohe-Toyota M, et al. Aberrant methylation of multiple genes and clinicopathological features in oral squamous cell carcinoma[J]. Clin Cancer Res,2002,8(10):3164-71.
[48]Ono K, Uzawa K, Nakatsuru M, et al. Down-regulation of FEZ1/LZTS1 gene with frequent loss of heterozygosity in oral squamous cell carcinomas [J]. Int J Oncol.2003,23(2):297-302.
[49]Marsit CJ, Liu M, Nelson HH, et al. Inactivation of the Fanconi anemia/BRCA pathway in lung and oral cancers:implications for treatment and survival[J]. Oncogene,2004,23(4):1000-4.
[50]Kulkarni V, Saranath D. Concurrent hypermethylation of multiple regulatory genes in chewing tobacco associated oral squamous cell carcinomas and adjacent normal tissues[J]. Oral Oncol,2004,40(2):145-53.
[51]Gao S, Worm J, Guldberg P, et al. Genetic and epigenetic alterations of the blood group ABO gene in oral squamous cell carcinoma[J]. Int J Cancer,2004, 109(2):230-7.
[52]Youssef EM, Lotan D, Issa JP, et al. Hypermethylation of the retinoic acid receptor-beta(2) gene in head and neck carcinogenesis[J]. Clin Cancer Res, 2004,10(5):1733-42.
[53]Endo Y, Uzawa K, Mochida Y, et al. Sarcoendoplasmic reticulum Ca(2+) ATPase type 2 downregulated in human oral squamous cell carcinoma[J]. Int J Cancer,2004,110(2):225-31.
[54]Gao S, Worm J, Guldberg P, et al. Loss of heterozygosity at 9q33 and hypermethylation of the DBCCR1 gene in oral squamous cell carcinoma[J]. Br J Cancer,2004,91(4):760-4.
[55]Noguchi T, Tanimoto K, Shimokuni T, et al. Aberrant methylation of DP YD promoter, DP YD expression, and cellular sensitivity to 5-fluorouracil in cancer cells[J]. Clin Cancer Res,2004,10(20):7100-7.
[56]Imai MA, Moriya T, Imai FL, et al. Down-regulation of DMBT1 gene expression in human oral squamous cell carcinoma[J]. Int J Mol Med,2005, 15(4):585-9.
[57]Uesugi H, Uzawa K, Kawasaki K, et al. Status of reduced expression and hypermethylation of the APC tumor suppressor gene in human oral squamous cell carcinoma[J]. Int J Mol Med,2005,15(4):597-602.
[58]Gao S, Skeldal S, Krogdahl A, et al. CpG methylation of the PAI-1 gene 5'-flanking region is inversely correlated with PAI-1 mRNA levels in human cell lines[J]. Thromb Haemost,2005,94(3):651-60.
[59]Estecio MR, Youssef EM, Rahal P, et al. LHX6 is a sensitive methylation marker in head and neck carcinomas[J]. Oncogene,2006,25(36):5018-26.
[60]Nakagawa T, Pimkhaokham A, Suzuki E, et al. Genetic or epigenetic silencing of low density lipoprotein receptor-related protein 1B expression in oral squamous cell carcinoma[J]. Cancer Sci,2006,97(10):1070-4.
[61]Nakaya K, Yamagata HD, Arita N, et al. Identification of homozygous deletions of tumor suppressor gene FAT in oral cancer using CGH-array[J]. Oncogene, 2007,26(36):5300-8.
[62]Suzuki E, Imoto I, Pimkhaokham A, et al. PRTFDC1, a possible tumor-suppressor gene, is frequently silenced in oral squamous-cell carcinomas by aberrant promoter hypermethylation[J]. Oncogene,2007,26(57):7921-32.
[63]Nakamura E, Kozaki K, Tsuda H, et al. Frequent silencing of a putative tumor suppressor gene melatonin receptor 1 A (MTNR1A) in oral squamous-cell carcinoma[J]. Cancer Sci,2008,99(7):1390-400.
[64]Shiah SG, Chang LC, Tai KY, et al. The involvement of promoter methylation and DNA methyltransferase-1 in the regulation of EpCAM expression in oral squamous cell carcinoma[J]. Oral Oncol,2009,45(1):el-8.
[65]Kurasawa Y, Shiiba M, Nakamura M, et al. PTEN expression and methylation status in oral squamous cell carcinoma[J]. Oncol Rep,2008,19(6):1429-34.
[66]Sogabe Y, Suzuki H, Toyota M, et al. Epigenetic inactivation of SFRP genes in oral squamous cell carcinoma[J]. Int J Oncol,2008,32(6):1253-61.
[67]Shaw RJ, Omar MM, Rokadiya S, et al. Cytoglobin is upregulated by tumour hypoxia and silenced by promoter hypermethylation in head and neck cancer [J]. Br J Cancer,2009,101(1):139-44.
[68]曾益新.肿瘤学(第2版)[M].北京：人民卫生出版社,2003；97.
[69]Kuroki T, Trapasso F, Yendamuri S, et al. Allelic Loss on Chromosome 3p21.3 and Promoter Hypermethylation of Semaphorin3B in Non-Small Cell Lung Cancer[J]. Cancer Res,2003,63(12):3352-5.
[70]Partridge M, Kiguwa S, Langdon JD. Frequent deletion of chromosome 3p in oral squamous cell carcinoma[J]. Eur J Cancer B Oral Oncol,1994,30B (4): 248-51.
[71]Partridge M, Emilion G, Langdon JD. LOH at 3p correlates with a poor survival in oral squamous cell carcinoma[J]. Br J Cancer,1996,73(3):366-71.
[72]Hart LM, Hansen T, Rietveld I, et al. Evidence that the mitochondrial leucyl tRNA synthetase (LARS2) gene represents a novel type 2 diabetes susceptibility gene [J]. Diabetes,2005,54(6):1892-5.
[73]Munakata K, Iwamoto K, Bundo M, et al. Mitochondrial DNA 3243A>G mutation and increased expression of LARS2 gene in the brains of patients with
bipolar disorder and schizophrenia [J]. Biol Psychiatry,2005,57(5):525-32.
[74]Zhou W, Feng X, Li H, et al. Inactivation of LARS2, located at the commonly deleted region 3p21.3, by both epigenetic and genetic mechanisms in nasopharyngeal carcinoma[J]. Acta Biochim Biophys Sin,2009,41(1):54-62.
[75]Frankel AD, Pabo CO. Fingering too many proteins [J]. Cell,1988,53(6):675.
[76]Giesecke AV, Fang R, Joung JK, et al. Synthetic protein-protein interaction domains created by shuffling Cys2His2 zinc-fingers [J]. Mol Syst Bio,2006,2: 2006-2011.
[77]Gao L, Sun C, Qiu HL, et al. Cloning an characterization of a novel human zinc finger gene, hkid3, from a C2H2-ZNF enchiched human embryonic cDNA library[J]. Biochem Biophys Res Commun,2004,325(4):1145-1152.
[78]Huang CX, Jia YC, Yang SL, et al. Characterization of ZNF23, a KRAB-containing protein that is downregulated in human cancers and inhibits cell cycle progression[J]. Experimental Cell Research,2007,313(2):254-263.
[79]Donti E, Lanfrancone L, Huebner K, et al. Localization of the human HF.10 finger gene on a chromosome region (3p21-22) frequently deleted in human cancers[J]. Hum Genet,1990,84(5):391-5.
[80]Jensen DE, Proctor M, Marquis ST, et al. BAP1:a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression[J]. Oncogene,1998,16(9):1097-1112.
[81]Ventii KH, Devi NS, Friedrich KL, et al. BRCA1-associated protein-1 is a tumor suppressor that requires deubiquitinating activity and nuclear localization[J]. Cancer Res,2008,68(17):6953-62.
[82]Ji L, Roth JA. Tumor suppressor FUS1 signaling pathway[J]. J Thorac Oncol, 2008,3(4):327-30.
[83]Prudkin L, Behrens C, Liu DD, et al. Loss and reduction of FUS1 protein expression is a frequent phenomenon in the pathogenesis of lung cancer[J]. Clin Cancer Res,2008,14(1):41-7.
[84]Uno F, Sasaki J, Nishizaki M, et al. Myristoylation of the fusl protein is required for tumor suppression in human lung cancer cells[J]. Cancer Res,2004, 64(9):2969-76.
[85]Lerman MI, Minna JD. The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3:identification and evaluation of the resident candidate tumor suppressor genes. The International Lung Cancer Chromosome 3p21.3 Tumor Suppressor Gene Consortium[J]. Cancer Res,2000,60(21): 6116-33.
[86]Li J, Wang F, Haraldson K, et al. Functional characterization of the candidate tumor suppressor gene NPRL2/G21 located in 3p21.3C[J]. Cancer Res,2004, 64(18):6438-43.
[87]Kurata A, Katayama R, Watanabe T, et al. TUSC4/NPRL2, a novel PDK1-interacting protein, inhibits PDK1 tyrosine phosphorylation and its downstream signaling[J]. Cancer Sci,2008,99(9):1827-34.
[88]Otani S, Takeda S, Yamada S, et al. The tumor suppressor NPRL2 in hepatocellular carcinoma plays an important role in progression and can be served as an independent prognostic factor[J]. J Surg Oncol,2009,100(5): 358-63.
[89]Donninger H, Vos MD, Clark GJ. The RASSF1A tumor suppressor[J]. J Cell Sci, 2007,120(Pt 18):3163-72.
[90]Hesson LB, Cooper WN, Latif F. The role of RASSF1A methylation in cancer[J]. Dis Markers,2007,23(1-2):73-87.
[91]Agathanggelou A, Cooper WN, Latif F. Role of the Ras-association domain family 1 tumor suppressor gene in human cancers[J]. Cancer Res,2005,65(9): 3497-508.
[92]Tomizawa Y, SekidoY, Kondo M, et al. Inhibition of lung cancer cell growth and induction of apoptosis after reexpression of 3p21.3 candidate tumor suppressor gene SEMA3B[J]. Proc. Natl.Acad. Sci. USA,2001,98(24): 13954-13959.
[93]Tse C, Xiang RH, Bracht T, et al. Human Semaphorin 3B (SEMA3B) located at chromosome 3p21.3 suppresses tumor formation in an adenocarcinoma cell line[J]. Cancer Res,2002,62(2):542-546.
[94]Ochi K, Mori T, Toyama Y, et al. Identification of semaphorin3B as a direct target of p53[J]. Neoplasia,2002,4(1):82-87.
[95]Riquelme E, Tang M, Baez S, et al. Frequent epigenetic inactivation of chromosome 3p candidate tumor suppressor genes in gallbladder carcinoma[J]. Cancer Lett,2007,250(1):100-6.
[96]Brambiila E, Constantin B, Drabkin H, et al. Semaphorin SEMA3F localization in malignant Human lung and cell lines:A suggested role in cell adhesion and cell migration[J]. Am J Pathol,2000,156(3):939-950.
[97]Ji L, Minna JD, Roth JA.3p21.3 tumor suppressor cluster:prospects for translational applications[J]. Future Oncol,2005,1(1):79-92.
[98]Ishiguro H, Tsunoda T, Tanaka T, et al. Identification of AXUD1, a novel human gene induced by AXIN1 and its reduced expression in human carcinomas of the lung, liver, colon and kidney[J]. Oncogene,2001,20(36): 5062-6.
[99]Ngo JT, Bateman JB, Klisak I, et al. Regional mapping of a human rod alpha-transducin (GNAT1) gene to chromosome 3p22[J]. Genomics,1993, 18(3):724-5.
[100]易红梅,任彩萍,彭丹,周亮,李辉,姚开泰.鼻咽癌组织中GNAT1基因的表达、杂合性丢失及甲基化分析[J].癌症,2007,26(1)：9-14.
[101]Rey MW, Woloshuk SL, deBoer HA, et al. Complete nucleotide sequence of human mammary gland lactoferrin[J]. Nucleic Acids Res,1990,18(17):5288.
[102]Anderson BF, Baker HM, Norris GE, et al. Apolactoferrin structure demonstrates ligand-induced conformational change in transferrins[J]. Nature, 1990,344(6268):784-7.
[103]Ward PP, Paz E, Conneely OM. Multifunctional roles of lactoferrin:a critical overview[J]. Cell Mol Life Sc,2005,62(22):2540-8.
[104]Finkbeiner WE, Carrier SD, Teresi CE. Reverse transcription-polymerase chain reaction (RT-PCR) phenotypic analysis of cell cultures of human tracheal epithelium, tracheobronchial glands, and lung carcinomas[J]. Am J Respir Cell Mol Biol,1993,9(5):547-56.
[105]Penco S, Caligo MA, Cipollini G, et al. Lactoferrin expression in human breast cancer[J]. Cancer Biochem Biophys,1999,17(1-2):163-78.
[106]Tuccari G, Giuffre G, Crisafulli C, et al. Immunohistochemical detection of lactoferrin in human astrocytomas and multiforme glioblastomas[J]. Eur J Histochem,1999,43(4):317-22.
[107]Lee JY, Eom EM, Kim DS, et al. Analysis of gene expression profiles of gastric normal and cancer tissues by SAGE[J]. Genomics,2003,82(1):78-85.
[108]Christina Teng, Wesley Gladwell, Ibrahim Raphiou, et al. Methylation and expression of the lactoferrin gene in human tissues and cancer cells[J]. BioMetals,2004,17(3):317-323.
[109]Yi HM, Li H, Peng D, et al. Genetic and epigenetic alterations of LTF at 3p21.3 in nasopharyngeal carcinoma[J]. Oncol Res,2006,16(6):261-72.
[110]Iijima H, Tomizawa Y, Iwasaki Y, et al. Genetic and epigenetic inactivation of LTF gene at 3p21.3 in lung cancers[J]. Int J Cancer,2006,118(4):797-801.
[111]Bezault J, Bhimani R, Wiprovnick J, et al. Human lactoferrin inhibits growth of solid tumors and development of experimental metastases in mice[J]. Cancer Res,1994,54(9):2310-2.
[112]Damiens E, El Yazidi I, Mazurier J, et al. Lactoferrin inhibits G1 cyclin-dependent kinases during growth arrest of human breast carcinoma cells[J]. J Cell Biochem,1999,74(3):486-98.
[113]Xiao Y, Monitto CL, Minhas KM, Sidransky D. Lactoferrin down-regulates G1 cyclin-dependent kinases during growth arrest of head and neck cancer cells[J]. Clin Cancer Res,2004,10(24):8683-6.
[114]Zhou Y, Zeng Z, Zhang W, et al. Lactotransferrin:a candidate tumor suppressor-Deficient expression in human nasopharyngeal carcinoma and inhibition of NPC cell proliferation by modulating the mitogen-activated protein kinase pathway[J]. Int J Cancer,2008,123(9):2065-72.
[115]Ehrlich M. Cancer-linked DNA hypomethylation and its relationship to hypermethylation[J]. Curr Top Microbiol Immunol,2006,310:251-74.
[116]Kanai Y. Genome-wide DNA methylation profiles in precancerous conditions and cancers[J]. Cancer Sci,2009,101(1):36-45.
[117]Costello JF, Fruhwald MC, Smiraglia DJ, et al. Aberrant CpG island methylation has non-random and tumour-type-specific patterns[J]. Nat Genet, 2000,24 (2):132-138.
[118]Herman J G, Graff J R, Myohanen S, et al. Methylation-specific PCR:a novel PCR assay for methylation status of CpG islands [J]. Proc Natl Acad Sci USA,1996,93(18):9821-9826.
[119]Tischoff I, Markwarth A, Witzigmann H, et al. Allele loss and epigenetic inactivation of 3p21.3 in malignant liver tumors[J]. Int J Cancer,2005,115(5): 684-9.
[120]Nair PN, McArdle L, Cornell J, et al. High-resolution analysis of 3p deletion in neuroblastoma and differential methylation of the SEMA3B tumor suppressor gene[J]. Cancer Genet Cytogenet,2007,174(2):100-10.
[121]Hesson L, Bieche I, Krex D, et al. Frequent epigenetic inactivation of RASSF1A and BLU genes located within the critical 3p21.3 region in gliomas[J]. Oncogene,2004,23(13):2408-19.
[122]易红梅,任彩萍,彭丹,et al. SEMA3B基因在鼻咽癌组织中的表达、杂合性丢失和甲基化分析[J].生命科学研究,2006,10(2)：183-188.
[123]Grote HJ, Schmiemann V, Geddert H, et al. Aberrant promoter methylation of p16(INK4a), RARB2 and SEMA3B in bronchial aspirates from patients with suspected lung cancer[J]. Int J Cancer,2005,116(5):720-5.
[124]Honorio S, Agathanggelou A, Schuermann M, et al. Detection of RASSF1A aberrant promoter hypermethylation in sputum from chronic smokers and ductal carcinoma in situ from breast cancer patients[J]. Oncogene 2003,22: 147-150.
[125]Herman JG, Baylin SB. Gene Silencing in Cancer in Association with Promoter Hypermethylation[J]. N Engl J Med,2003,349(21):2042-2054.
[126]李江.口腔癌的临床病理特征与预后[J].中国口腔颌面外科杂志,2008,6(1)：17-21.
[127]Son HJ, Lee SH, Choi SY. Human lactoferrin controls the level of retinoblastoma protein and its activity[J]. Biochem Cell Biol,2006,84(3): 345-50.
[128]Oh SM, Pyo CW, Kim Y, et al. Oncogene. Neutrophil lactoferrin upregulates the human p53 gene through induction of NF-kappaB activation cascade[J]. Oncogene,2004,23(50):8282-91.
[129]Wu M, Li X, Li X, et al. Signaling Transduction Network Mediated by Tumor Suppressor/Susceptibility Genes in NPC[J]. Current Genomics,2009, 10(4):216-222.
[1]Shahnavaz SA,Regezi JA,Bradley G,et al. p53 gene mutations in sequential oral epithelial dysplasias and squamous cell carcinomas [J]. J Pathol,2000,190(4):417-422
[2]Jang SJ,Chiba I,Hirai A,et al.Multiple oral squamous epithelial lesions:are they genetically related? [J].Oncogene,2001,20(18):2235-2242
[3]Razin A,Cedar H.DNA methylation and embryogenesis [J]. EXS,1993,64:343-357
[4]方练,凌玲.DNA甲基化与头颈部肿瘤[J].实用肿瘤杂志,2003,18(5)：425-427
[5]Bestor TH.Gene silencing.Methylation meets acetylation [J].Nature,1998,393(6683):311-312
[6]Nan X,Ng HH,Johnson CA, et al.Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase comples [J]. Nature,1998,393(6683):386-389
[7]Jones PL,Veenstra GJ,Wade PA,et al.Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription [J].Nat Genet,1998,19(2):187-191
[8]Summer Student小组,程斌.DNA甲基化与口腔鳞癌的发生发展[J].国外医学口腔医学分册,2001,28(3)：139-141
[9]李新,罗鹏,刘莉.DNA甲基化与结肠癌[J].国际消化病杂,2009,29(1)：29-32
[10]Robertson KD,Jones PA.DNA methylation:past, present and future directions [J]. Carcinogenesis,2000,21(3):461-467
[11]Costello JF,Fruhwald Mc,Smiraglia DJ,el al.Aberrant CpG-island methylation has non-random and tumour-type-specific patterns[J].Nat Genet,2000,24(2):132-138
[12]陈勇,陈双郧,冯景.DNA甲基化与宫颈癌关系的研究进展[J].郧阳医学院学报,2009,28(1)：102-104
[13]Attwood JT,Yung RL,Richardson BC.DNA methylation and the regulation of gene transcription [J].Cell Mol Life Sci,2002,59(2):241-257
[14]Matsuda Y,Ichida T,Matsuzawa J,et al.p16(INK4) is inactivated by extensive CpG methylation in human hepatocellular carcinoma [J]. Gastroenterology,1999,116(2):394-400
[15]Shintani S,Nakahara Y,Mihara M,et al.Inactivation of the p14(ARF), p15(INK4B) and p16(INK4A) genes is a frequent event in human oral squamous cell carcinomas [J].Oral Oncol,2001,37(6):498-504
[16]阚卫兵,方肇勤,耿琳.CDKN2A研究进展[J].国外医学肿瘤学分册,2004,31
(7)：483-486
[17]Ogi K,Toyota M,Ohe-Toyota M,et al.Aberrant methylation of multiple genes and clinicopathological features in oral squamous cell carcinoma [J].Clin Cancer Res,2002,8(10):3164-3171
[18]Kresty LA,Mallery SR,Knobloch TJ,et al.Alterations of p16(INK4a) and p14(ARF) in patients with severe oral epithelial dysplasia[J].Cancer Res,2002,62(18):5295-5300
[19]Lopez M,Aguirre JM,Cuevas N,et al.Gene promoter hypermethylation in oral rinses of leukoplakia patients--a diagnostic and/or prognostic tool? [J]. Eur J Cancer,2003,39(16):2306-2309
[20]Dong SM,Sun DI,Benoit NE,et al.Epigenetic inactivation of RASSF1A in head and neck cancer [J].Clin Cancer Res,2003,9(10 Pt 1):3635-3640
[21]李春艳,高文信,周延民,等.口腔鳞状细胞癌中凋亡蛋白酶活化因子甲基化的研究[J].口腔医学研究,2009,25(3)：279-281
[22]Vellucci VF,Germino FJ,Reiss M.Cloning of putative growth regulatory genes from primary human keratinocytes by subtractive hybridization [J].Gene,1995,166(2):213-220
[23]Gasco M,Bell AK,Heath V,et al.Epigenetic inactivation of 14-3-3 sigma in oral carcinoma:association with p16(INK4a) silencing and human papillomavirus negativity [J].Can cer Res,2002,62(7):2072-2076
[24]Kulkarni V,Saranath D.Concurrent hypermethylation of multiple regulatory genes in chewing tobacco associated oral squamous cell carcinomas and adjacent normal tissues [J].Oral Oncol,2004,40 (2):145-153
[25]Chen Q,Lipkina G,Song Q,et al.Promoter methylation regulates cadherin switching in squamous cell carcinoma [J]. Biochem Biophys Res Commun,2004,315(4):850-856
[26]Viswanathan M,Tsuchida N,Shanmugam G,et al.Promoter hypermethylation profile of tumor-associated genes p16, p15, hMLH1, MGMT and E-cadherin in oral squamous cell carcinoma[J].Int J Cancer,2003,105(1):41-46
[27]Nakayama S,Sasaki A,Mese H,et al.The E-cadherin gene is silenced by CpG methylation in human oral squamous cell carcinomas [J]. Int J Cancer,2001,93(5):667-673
[28]沈佳尧,侯鹏,祭美菊,等.DNA甲基化方法研究现状[J].生命的化学,2003,23(2)：149-151
[29]Herman JG,Graff JR,Myohanen S,et al.Methylation-specific PCR:a novel PCR assay for methylation status of GpG islands [J].Proc Natl Acad Sci USA,1996,93(18):9821-9826
[30]Frommer M,McDonald LE,Millar DS,et al.A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands[J]. Proc Natl Acad Sci USA,1992,89(5):1827-1831
[31]Xiong Z,Laird PW.COBRA:a sensitive and quantitative DNA methylation assay[J].Nucleic Acids Res,1997,25(12):2532-2534
[32]Sadri R,Hornsby PJ.Rapid analysis of DNA methylation using new restriction enzyme sites created by bisulfite modification [J].Nucleic Acids Res,1996,24(24): 5058-5059
[33]Li Q,Ahuja N,Burger PC,et al.Methylation and silencing of the Thrombospondin-1 promoter in human cancer[J].Oncogene,1999,18(21): 3284-3289
[34]邓大君,邓国仁,吕有勇,等.变性高效液相色谱法检测CpG岛胞嘧啶甲基化[J].中华医学杂志,2001,81(3)：158-161
[35]Huang TH,Perry MR,Laux DE.Methylation profiling of CpG islands in human breast cancer cells [J].Hum Mol Genet,1999,8(3):459-470
[36]Gitan RS,Shi H,Chen CM,et al.Methylation-specific oligonucleotide microarray: a new potential for high-throughput methylation analysis [J].Genome Res,2002,12(1):158-164
[37]郭万松,杨波,丛宪玲,等.DNA甲基化与膀胱癌研究进展[J].中国实验诊断学,2009,13(8)：1123-1128
[38]朱景德.DNA甲基化及其在肿瘤分子诊断中的前景[J].中华检验医学杂志,2005,28(7)：679-683
[39]Egger G,Liang G,Aparicio A,et al.Epigenetics in human disease and prospects for epigenetic therapy [J].Nature,2004,429(6990):457-463
[40]Stresemann C,Brueckner B,Musch T,et al.Functional diversity of DNA methyltransferase inhibitors in human cancer cell lines [J].Cancer Res,2006,66(5):2794-2800
[41]Suzuki M,Sunaga N,Shames DS,et al.RNA interference-mediated knockdown of DNA methyltransferase 1 leads to promoter demethylation and gene re-expression in human lung and breast cancer cells[J].Cancer Res,2004,64(9):3137-3143
[42]Bhattacharya SK,Ramchandani S,Cervoni N,et al.A mammalian protein with specific demethylase activity for mCpG DNA[J]. Nature,1999,397(6720):579-583
[43]Hendrich B,Hardeland U,Ng HH,et al.The thymine glycosylase MBD4 can bind to the product of deamination at methylated CpG sites [J]. Nature,1999,401(6750):301-304
[44]Fang MZ,Chen D,Sun Y,et al.Reversal of hypermethylation and reactivation of p16INK4a, RARbeta,and MGMT genes by genistein and other isoflavones from soy[J].Clin Cancer Res,2005,11(19 Pt 1):7033-7041