口腔鳞状细胞癌中凋亡蛋白酶活化因子甲基化的研究

目的：探讨凋亡蛋白酶活化因子1（APAF1）基因的启动子区甲基化状态与口腔鳞状细胞癌（OSCC）的关系。方法：采用甲基特异性PCR的方法分别检测23例正常口腔黏膜组织和53例OSCC组织中APAF1基因的启动子区甲基化情况。结果：23例正常口腔黏膜组织中无1例检测到APAF1基因启动子区的甲基化,53例OSCC组织中有41例（77．36％）APAF1基因启动子区完全甲基化,5例（9．43％）APAF1基因部分甲基化,总甲基化率为86．79％（46／53）,与正常口腔黏膜组织甲基化率比较差异有显著性（P〈0．01）。APAF1基因的甲基化与病理分级、年龄、性别无关。结论：APAF1基因启动子区的甲基化是该基因在OSCC组织中表达降低的机制之一,基因甲基化引起的基因功能静默可能与OSCC的发生有关。     

人成釉细胞瘤中端粒酶反转录酶启动子区的DNA甲基化

目的研究成釉细胞瘤（ameloblastoma，AB）中hTERT启动子区的DNA甲基化，并探讨其生物学意义。方法选取新鲜标本AB12例，同时用11例正常黏膜做对照观察，用甲基化特异PCR检测上述组织中人类端粒酶反转录酶（human telomerase reverse transcripase，hTERT）启动子区的DNA甲基化。结果AB、正常黏膜中hTERT启动子区的DNA非甲基化阳性分别为4例（4／12）和6例（6／11）。AB、正常黏膜中hTERT启动子区的DNA甲基化阳性分别为11例（11／12）和3例（3／11），其中4例AB和1例正常黏膜同时表现hTERT启动子区DNA的甲基化和非甲基化。结论AB的hTERT启动子区的DNA甲基化比正常黏膜常见且有意义；hTERT启动子的甲基化可能对hTERT基因起调节作用。     

Hypermethylation of Death-Associated Protein Kinase (DAPK1) and its association with oral carcinogenesis - An experimental and meta-analysis study

ObjectivesThe value of abnormal DNA methylation of DAPK1 promoter and its association with various cancers have been suggested in the literature. To establish the significance of DNA methylation of DAPK1 promoter in oral squamous cell carcinoma (OSCC), we a) performed a case-control study, b) evaluated published data for its utility in the diagnosis and prognosis of OSCC and c) identified the association of DAPK1 gene expression with promoter DNA methylation status.DesignBisulfite gene sequencing of DAPK1 promoter region was performed on non-malignant and malignant oral samples. Further, using a systematic search, 330 publications were retrieved from PubMed, Scopus, and Google Scholar and 11 relevant articles were identified.ResultsSignificant association of DAPK1 promoter methylation with OSCC (p < 0.0001) was observed in the case-control study. The studies chosen for meta-analysis showed prognostic and predictive significance of DAPK1 gene promoter, despite defined inconsistencies in few studies. Overall, we obtained a statistically significant (p-value < 0.001) association for both sensitivity and specificity of DAPK1 DNA promoter methylation in oral cancer cases, without publication bias.ConclusionDNA hypermethylation of DAPK1 gene promoter is a promising biomarker for OSCC prediction/prognostics and suggests further validation in large distinct cohorts to facilitate translation to clinics.     

Smad分子在骨形成蛋白2调控成牙本质细胞Ⅰ型胶原基因表达中的作用

目的　研究Smad蛋白在骨形成蛋白 2 (bonemorphogeneticprotein 2 ,BMP 2 )调控小鼠成牙本质细胞系MDPC 2 3内Ⅰ型胶原α2链 [collagenα2 (Ⅰ ) ,COL1A2 ]表达中的作用。方法　细胞免疫组化观察MDPC 2 3细胞内BMP 2细胞内信号分子Smad1、Smad5和Smad6的表达。瞬时转染和报告基因检测观察Smad1、Smad5和Smad6在BMP 2调控COL1A2基因转录中的作用。结果　MDPC 2 3细胞表达Smad1、Smad5和Smad6。BMP 2能诱导含COL1A2基因启动子的荧光素酶报告基因活性。Smad1或Smad5过表达增强BMP 2诱导的COLIA2基因启动子活性 ,而Smad6过表达抑制BMP 2诱导的COL1A2基因启动子活性。过表达Smad1或Smad5突变型载体可以阻断BMP 2的诱导能力。结论　在MDPC 2 3细胞内 ,Smad信号途径存在并发挥功能 ,参与调控BMP 2对COL1A2基因的转录。Smad信号途径可能在BMP 2调控成牙本质细胞分化和牙本质形成过程中发挥重要作用     

涎腺腺样囊性癌细胞系中DNA甲基化研究

目的探讨涎腺腺样囊性癌（adenoidcysticcarcinoma，ACC）细胞系中抑癌基因甲基化状况及其与mRNA、蛋白表达之间的关系。方法甲基化特异性PCR法检测ACC细胞系ACC-2、ACC-3、ACC-M中E-钙黏着蛋白（E-cadherin，E-cad）、p16、RASSFlA、DAPK、MGMT基因启动子区的甲基化状况。应用RT-PCR方法和免疫组织化学方法检测E-cad、p16在mRNA和蛋白水平的表达。结果3个ACC细胞系中均检测到E-cad、p16基因的甲基化，而没有RASSFlA、DAPK、MGMT基因的甲基化；mRNA和蛋白水平均未检测到E-cad的表达，均检测到p16的表达。结论ACC细胞系中，E-cad、p16基因启动子区的甲基化是常见事件，甲基化可能是E-cad基因失活的主要机制之一。     

CDH1基因在SD大鼠舌黏膜癌变过程中甲基化和突变的研究

目的:通过甲基化和外显子突变研究探索CDH1基因在舌鳞状细胞癌发生过程中的作用。方法:采用质量分数为0.004%的4-硝基喹啉-1-氧化物(4-nitroquinoline-1-oxide,4NQO)饮水饲养90只无特定病原体(specific pathogen free,SPF)SD大鼠以诱发舌黏膜癌变全过程,分别于第10、14、18、22、24周分批处死大鼠,取舌黏膜组织行病理分级,并提取基因组DNA。利用甲基化特异性PCR(methylation-specific PCR,MS-PCR)检测CDH1启动子甲基化水平;利用聚合酶链式反应(polymerase chain reaction,PCR)扩增CDH1外显子1-16,提纯后测序以检测突变。结果:病变各阶段均未检测出CDH1启动子甲基化产物,CDH1 mRNA第2106位发生碱基G→T错义突变。结论:4NQO饮水诱导SD大鼠舌黏膜癌变的发生、发展可能与CDH1外显子突变有关而与CDH1启动子甲基化无关。     

唾液腺腺样囊性癌中RASSF1A表达及与启动子甲基化之间的关系

目的：研究唾液腺腺样囊性癌（adenoid cystic carcinoma,ACC）中RASSF1A表达及其与启动子区甲基化之间的关系。方法：收集167例原发性唾液腺ACC,亚硫酸盐测序聚合酶链反应（bisulfite sequencing polymerase chain reaction,BSP）和甲基化特异性聚合酶链反应（methylation-specific polymerase chain reaction,MSP）方法检测RASSF1A基因启动子区甲基化状况,免疫组织化学方法检测RASSF1A蛋白表达情况。去甲基化药物decitabine处理ACC细胞系SACC-83后,检测处理前、后RASSF1A基因甲基化及表达情况。应用SPSS18.0软件包对数据进行统计学分析。结果：59/167（35.3%）例病例中检测到RASSF1A基因启动子区甲基化。101/167（60.5%）例病例中RASSF1A蛋白呈低或不表达,66/167（39.5）病例中RASSF1A蛋白呈高表达。存在RASSF1A基因甲基化组,RASSF1A蛋白表达显著低于不存在甲基化组（P=0.012）。去甲基化药物decitabine处理ACC细胞系后,RASSF1A mRNA及蛋白水平表达均升高。结论：唾液腺ACC中,启动子区甲基化是RASSF1A基因失活的主要原因,可作为该肿瘤的潜在治疗靶点。     

Promoter methylation of MGMT in oral carcinoma: A population-based study and meta-analysis

IntroductionThe relevance of DNA methylation of O⁶-methylguanine-DNA methyltransferase (MGMT) in relation to several cancers and other disorders has been extensively explored in several cancer types.AimsTo ascertain the significance of DNA methylation of MGMT promoter in oral squamous cell carcinoma (OSCC), we undertook a study to a) analyse the methylation patterns of MGMT gene promoter in afflicted and normal population of coastal Karnataka, b) determine the expression status of MGMT in oral cancer cell lines (CAL-27 and SCC-4) and its relationship to DNA methylation and c) performed a meta-analysis of the published data.MethodsBisulfite sequencing of MGMT promoter region was performed on non-malignant/malignant oral samples, and oral cancer cell lines, followed by gene expression studies. Further, using a systematic search, 1024 publications were retrieved from PubMed, Scopus, Google Scholar and Web of Science and 23 relevant articles were reviewed.ResultsSignificant association of MGMT promoter methylation with OSCC (p < 0.0001) was observed in the case-control study. The studies chosen for meta-analysis showed predictive significance of MGMT gene promoter. Overall, we obtained a statistically significant (p < 0.0001) association for both sensitivity and specificity of MGMT DNA promoter methylation in oral cancer cases without publication bias. Gene expression was significantly elevated in both oral cancer cell lines (p < 0.03) after treatment with a demethylating agent (5-Aza-2′-deoxycytidine).ConclusionDNA promoter hypermethylation and gene expression of MGMT may associate with recursive mutagenesis and is a promising biomarker for OSCC prediction. Studies suggest further validation in large distinct cohorts to facilitate translation to clinics.     

LOH at chromosome 9q34.3 and the Notch1 gene methylation are less involved in oral squamous cell carcinomas

BACKGROUND: Previous studies of oral carcinomas have shown that both loss of heterozygosity (LOH) and hypermethylation at chromosome 9q33 to 9q34.2 are frequent. The present study investigates the frequency of Notch1 gene methylation and LOH at 9q34.3 region. METHODS: Gene promoter hypermethylation of the Notch1 gene was analysed by methylation-specific PCR and LOH was analysed using microsatellite markers. RESULTS: We found LOH at 9q34.3 in three patients and methylation of the Notch1 gene only in two patients with oral carcinoma. CONCLUSION: Comparing with the alterations at 9q33 to 34.2 regions, LOH at 9q34.3 and methylation of the Notch1 gene was less involved in oral squamous cell carcinomas.     

唾液腺恶性多形性腺瘤中CDH1基因启动子甲基化与E-cadherin表达的相关性分析

目的: 分析唾液腺恶性多形性腺瘤（malignant pleomorphic adenoma,MPA）中CDH1启动子甲基化程度与E-cadherin表达的相关性,评估CDH1启动子甲基化在E-cadherin沉默中的作用,并探讨其与MPA临床病理指标之间的相关性,对患者预后的潜在评估价值。 方法: 采用免疫组织化学法、BSP法分别检测37例MPA临床标本中E-cadherin的表达及CDH1启动子甲基化程度,进行相关性分析。收集患者资料,进行临床随访,分析CDH1甲基化与患者临床病理指标及生存率之间的相关性。采用SPSS 16.0软件包进行统计学分析。 结果: CDH1高甲基化与E-cadherin沉默表达呈显著正相关。CDH1甲基化程度与患者性别、肿瘤分化类型、组织学分级、淋巴结转移及TNM分期显著相关。CDH1高甲基化者生存率差,淋巴结转移为MPA患者预后的独立预测因子。 结论: CDH1基因高甲基化是MPA中E-cadherin沉默表达的重要调控机制之一,CDH1甲基化有望作为评估MPA患者临床预后的预测因子之一。     

Interleukin-6 gene promoter methylation in rheumatoid arthritis and chronic periodontitis

Background: Methylation status of the cytokine genes may play a role in the pathogenesis of inflammatory diseases, such as rheumatoid arthritis (RA) and chronic periodontitis (CP). This study was undertaken to evaluate whether the DNA methylation profile of the interleukin‐6 (IL‐6) gene promoter was unique to individuals with RA and CP. Methods: The study participants consisted of 30 patients with RA, 30 patients with CP, and 30 age‐, sex‐, and smoking status–balanced healthy controls. Genomic DNA isolated from peripheral blood was modified by sodium bisulfite and analyzed for DNA methylation levels of IL‐6 gene with direct sequencing. Levels of IL‐6 were determined by an enzyme‐linked immunosorbent assay. Results: The region of IL‐6 gene promoter from ?1200 to +27 bp was shown to contain 19 CpG motifs. The methylation levels of the CpG motif at ?74 bp were significantly lower in patients with RA and CP than those in controls (P = 0.0001). Both levels of serum IL‐6 and IL‐6 production by mononuclear cells were significantly different between individuals with and without the methylation at ?74 bp (P = 0.03). The +19 bp motif exhibited differential levels of the methylation among the groups, which was not associated with serum levels of IL‐6. The other 17 CpG motifs exhibited comparable levels of the methylation between the groups. Conclusion: These results suggest that hypomethylated status of a single CpG in the IL‐6 promoter region may lead to increased levels of serum IL‐6, implicating a role in the pathogenesis of RA and CP.     

DNMT1干扰对唾液腺腺样囊性癌细胞系ACC-M E-cadherin表达的影响

目的：建立DNA甲基转移酶1（DNMT1）表达稳定抑制的唾液腺腺样囊性癌细胞系ACC-M,探讨DNMT1表达抑制对ACC-M细胞E-cadherin表达的影响。方法：设计靶向干扰DNMT1的shRNA序列,构建携带该序列的慢病毒载体并转导ACC-M细胞,对筛选出的抗性克隆采用RT-PCR、荧光定量PCR、免疫印迹方法分别检测DNMT1mRNA、蛋白质水平,筛选获得DNMT1表达稳定抑制的ACC-M细胞,并通过甲基化特异性PCR检测E-cadherin基因启动子的甲基化状态,荧光定量PCR检测E-cadherin的表达情况。采用SPSS11.0软件包对数据进行t检验。结果：筛选获得DNMT1稳定表达抑制的ACC-M细胞,其mRNA、蛋白相对表达水平（0.156±0.008,0.163±0.013）显著低于空白对照组和空载对照组。进一步检测发现,E-cadherin基因启动子甲基化水平明显降低,E-cadherinmRNA表达水平显著增高,P〈0.05。结论：shRNA慢病毒载体介导的RNA干扰能够有效、稳定地抑制ACC-M细胞DNMT1的表达,并降低ACC-M细胞E-cadherin基因启动子的甲基化水平,从而使E-cadherin基因表达增强。     

MMP-1 promoter polymorphism: association with chronic periodontitis severity in a Brazilian population

BACKGROUND: A single nucleotide polymorphism was described in the promoter region of the human MMP-1 gene, and this polymorphism has been associated with risk of cancer metastasis and inflammatory diseases. In this paper, we studied the possible relationship between the MMP-1 promoter polymorphism and the severity of chronic periodontitis. METHODS: Genomic DNA from oral mucosa was amplified by PCR and analyzed by restriction endonuclease. The alleles were separated by polyacrylamide gel electrophoresis. The significance of the differences in observed frequencies of polymorphism in moderate and severe disease and healthy groups was assessed by Chi-squared test. RESULTS: In the healthy group, the 2G allele was observed with a frequency of 48.7%, while in severely diseased patients the 2G allele was seen in 69.2% (P = 0.0344). The genotype 2G/2G was found in 46.15% of the group with severe periodontitis, and 24.3% and 25.0%, respectively, of the healthy and moderate groups (P = 0.0647). CONCLUSION: These results show that a polymorphism in the promoter region of MMP-1 gene is associated with the severe chronic periodontitis phenotype in non-smokers.     

口腔粘膜癌前病变及鳞癌中RASSF1A基因甲基化与表达的研究

目的 研究RASSF1A基因甲基化状态及其基因表达异常与口腔癌前病变及鳞癌发生发展的关系。方法 采用甲基化特异性PCR（MSPCR）技术检测了10例正常口腔粘膜、8例上皮单纯增生、20例上皮异常增生、32例鳞癌组织中RASSF1A基因甲基化状况；运用逆转录-聚合酶链反应（RT-PCR）研究口腔粘膜癌前病变及鳞癌组织中RASSF1A基因mRNA的表达水平。结果 10例正常口腔粘膜无甲基化。且RASSF1A基因mRNA呈100％表达；8例上皮单纯增生中，有1例甲基化，1例RASSF1A mRNA表达下降；20例上皮异常增生中，有3例甲基化，5例RASSF1A mRNA表达下降；32例鳞癌组织中，有13例甲基化，占40．63％，15例RASSF1A mRNA表达下降或无表达。占46．88％。结论 RASSF1A基因甲基化是口腔癌癌变的早期事件，与口腔鳞癌的发生发展有关。RASSF1A基因甲基化与RASSSF1A基因转录抑制高度相关。     

唾液腺腺样囊性癌细胞系中5个抑癌基因甲基化及其表达

目的：探讨唾液腺腺样囊性癌（adenoid cystic carcinoma,ACC）细胞系中CDH13、RASSF2A、TFPI-2、hMLH-1、MyoD1基因的甲基化及其与mRNA表达之间的关系。方法：甲基化特异性PCR（methylation-specific PCR,MSP）法检测ACC细胞系ACC-2、ACC-3、ACC—M中CDH13、RASSF2A、TFPI-2、hMLH—1、MyoD1基因的甲基化：RT—PCR及荧光实时定量PCR对存在甲基化的基因进行mRNA水平的检测。结果：3个细胞系中均存在CDH13、RASSF2A、TFPI-2基因的甲基化和未甲基化,只存在MyoD1基因的甲基化和hMLH-1基因的未甲基化：ACC-2中CDH13基因mRNA水平的表达显著高于ACC—M,ACC-3中RASSF2A的表达显著高于ACC-2、ACC—M,3个细胞系中均未检测到MyoD1基因的表达。结论：ACC细胞系中,CDH13、RASSF2A、TFPI-2、MyoD1基因的甲基化为常见事件,甲基化可能为MvoD1基因失活的主要机制．CDH13基因的表达可能与ACC的转移相关．     

DNA methylation status of the IL8 gene promoter in oral cells of smokers and non-smokers with chronic periodontitis

Aim: This study analysed the status of DNA methylation in the promoter region of the IL8 gene in oral mucosa cells from healthy, smoker and non-smoker subjects with chronic periodontitis and compared these findings among groups with mRNA levels.
Material and Methods: Genomic DNA from epithelial oral cells of 41 healthy subjects, 30 smokers with chronic periodontitis and 40 non-smokers with chronic periodontitis were purified and modified by sodium bisulphite. Genomic DNA from blood leucocytes and gingival cells from biopsies of 13 subjects of each group were also purified and modified by sodium bisulphite. Modified DNA was submitted by methylation-specific polymerase chain reaction (PCR) (MSP), electrophoresed on 10% polyacrylamide gels and stained with SYBR Gold. Total RNA from gingival cells was also isolated using the TRIzol reagent, and real-time PCR performance was used to detect the levels of interleukin-8 mRNA.
Results: Our results indicate that individuals with chronic periodontitis, independent of smoking habit, have a higher percentage of hipomethylation of the IL8 gene than those controls in epithelial oral cells ( p <0.0001), and expression of higher levels of interleukin-8 (IL-8) mRNA than controls in gingival cells ( p= 0.007). No significant differences among groups were observed in gingival cells and blood cells.
Conclusion: We conclude that inflammation in the oral mucosa might lead to changes in the DNA methylation status of the IL8 gene in epithelial oral cells.