喉癌细胞系中RASSF1A基因启动子去甲基化与基因表达的研究

目的:探讨甲基化转移酶抑制剂[5-氮杂-2′-脱氧胞苷(5-Aza-dC)]及组蛋白去乙酰化转移酶抑制剂[曲古抑菌素A (TSA)]对喉癌细胞系中抑癌基因RASSF1A甲基化水平及基因表达的影响.方法:5-Aza-dC及TSA处理体外培养的Hep-2细胞,应用Realtime PCR方法及甲基化特异性PCR(MSP)方法分别检测药物干预前后细胞中抑癌基因RASSF1A表达及甲基化情况.结果:①在Hep-2细胞中未经药物干预前抑癌基因RASSF1A表现为甲基化,抑癌基因RASSF1A弱表达.②在5-Aza-dC及TSA的作用下,Hep-2细胞系中RASSF1A基因的甲基化状态得到了逆转.其中5-Aza-dC及TSA联合应用效果与单独应用5-Aza-dC效果相似,单独应用TSA无明显效果.③在5-Aza-dC及TSA的作用下,Hep-2细胞系中RASSF1A基因表达上调,其中5-Aza-dC作用较TSA作用强,2种药物联合应用起协同增效作用.结论:喉癌细胞系中抑癌基因RASSF1A启动子甲基化可能是导致其基因失活的主要原因但并不是惟一原因.应用5-Aza-dC及TSA能够通过逆转抑癌基因RASSF1A的DNA甲基化水平和组蛋白去乙酰化水平从而使其表达得到了增强.     

5-Aza-dC和TSA对人喉癌细胞系中CHFR基因启动子甲基化及mRNA表达水平的影响

目的:探讨5-杂氮-2′-脱氧胞苷(5-Aza-dC)和制霉菌素A(TSA)对人喉癌细胞系中CHFR基因启动子区甲基化与mRNA表达水平的影响。方法:采用荧光定量PCR技术和甲基化特异性PCR技术检测5-Aza-dC和TSA处理前后的Hep-2人喉癌细胞系基因CHFR启动子区甲基化与mRNA表达水平的变化。结果:在对照组Hep-2人喉癌细胞系中CHFR基因启动子区显示DNA甲基化,用5-Aza-dC作用后DNA甲基化程度明显减弱,mRNA表达量上调(1.75±0.21);用TSA作用后DNA甲基化程度和mRNA表达量(1.05±0.13)无明显变化。联合使用5-Aza-dC和TSA后DNA甲基化程度明显减弱,mRNA表达量明显上调(2.15±0.18)。结论:CHFR基因启动子区DNA甲基化在喉癌的发生、发展中是一个频发事件,是导致其mRNA表达下调的主要原因,5-Aza-dC单独应用和联合TSA能够逆转DNA甲基化状态,从而调控其基因表达,为临床使用药物治疗喉癌提供了新思路。     

5-氮杂-2'-脱氧胞苷对喉癌细胞中MGMT基因表达水平的影响

目的 探讨5-氮杂-2'-脱氧胞苷(5-aza-dC)对喉癌细胞系抑癌基因MGMT表达活性的影响.方法 体外培养Hep-2喉癌细胞,以不同药物浓度的5-aza-dC进行干预处理,应用逆转录PCR方法检测药物干预前后MGMT的表达活性,比较分析不同药物浓度的干预效应.结果 Hep-2喉癌细胞系经不同药物浓度的5-aza-dC作用后,能够诱导靶细胞中受抑制而处于不表达状态的抑癌基因MGMT重新恢复表达活性,尤以7μmol/L浓度时效应明显.结论 MGMT基因启动子甲基化可能是导致该基因失活的主要原因,5-aza-dC能显著增强Hep-2喉癌细胞肿瘤抑制基因的表达活性.     

5-氮杂-2＇-脱氧胞苷对喉癌细胞中MGMT基因表达水平的影响

目的探讨5-氮杂-2＇-脱氧胞苷（5-aza—dC）对喉癌细胞系抑癌基因MGMT表达活性的影响。方法体外培养Hep-2喉癌细胞,以不同药物浓度的5-aza—dC进行干预处理,应用逆转录PCR方法检测药物干预前后MGMT的表达活性,比较分析不同药物浓度的干预效应。结果Hep-2喉癌细胞系经不同药物浓度的5-aza—dC作用后,能够诱导靶细胞中受抑制而处于不表达状态的抑癌基因MGMT重新恢复表达活性,尤以7μmol／L浓度时效应明显。结论MGMT基因启动子甲基化可能是导致该基因失活的主要原因,5-aza—dCN能显著增强Hep-2喉癌细胞肿瘤抑制基因的表达活性。     

喉癌及癌前病变中细胞周期调控基因甲基化的研究

目的探讨细胞周期调控基因P14,P15及P16在喉癌发生发展中的作用。方法选取20例喉癌癌前病变（包括喉角化8例、白斑4例和成人复发性喉乳头状瘤8例）、30例喉鳞状细胞癌（声门型）及10例癌旁组织。采用甲基特异性聚合酶链反应（methylationspecific PCR,MSP）检测P14,P15及P16基因启动子区过甲基化。结果P14,P15及P16基因启动子过甲基化发生率在癌前病变中分别为10％（2／20）、15％（3／20）、20％（4／20）;在喉癌中分别为30．0％（9／30）、40．0％（12／30）、36．7％（11／30）;在癌旁组织中未检测到过甲基化事件。结论细胞周期调控基因p14,p15及p16启动子区过甲基化发生于喉癌早期病变中,可作为喉癌前病变及早期喉癌筛查的指标之一。     

声门上型喉癌组织中的BRMS1蛋白表达及甲基化的研究

目的:探讨声门上型喉鳞状细胞癌组织中乳腺癌转移抑制基因(BRMS1)的蛋白表达以及BRMS1基因启动子区域甲基化情况及其临床意义。方法:采用Western blotting法检测70例声门上型喉癌组织原发灶、60例癌旁正常喉黏膜组织和44例颈部淋巴结转移灶中BRMS1蛋白的表达情况;甲基化特异聚合酶链反应(MSP)法检测上述组织中的BRMS1基因启动子区域甲基化情况,探讨BRMS1基因启动子甲基化与其基因表达的相关性;分析BRMS1蛋白表达及BRMS1基因启动子甲基化情况与患者的临床分期、病理分级、颈部淋巴结转移等方面的相关分析。结果:Western blotting检测发现声门上型喉癌原发灶、癌旁正常喉黏膜组织和颈部淋巴结转移灶均有BRMS1蛋白表达,在癌组织及转移淋巴结中BRMS1蛋白表达下调(P<0.05)。MSP法检测发现BRMS1基因启动子区甲基化,在BRMS1蛋白低表达患者的原发灶中检测到34例BRMS1基因启动子区甲基化,44例BRMS1蛋白低表达的颈部淋巴结转移灶中检测到32例BRMS1基因启动子区甲基化,60例癌旁正常喉黏膜组织中均未检测到BRMS1基因启动子区甲基化,统计学分析结果表明在声门上型喉癌中BRMS1基因启动子甲基化与其基因表达下调密切相关(rs=0.66,P<0.05)。结论:声门上型喉癌组织中BRMS1蛋白表达下调。BRMS1蛋白表达下调与声门上型喉癌的P-TNM分期、病理分化程度和颈部淋巴结转移密切相关。BRMS1基因启动子甲基化与声门上型喉癌组织中BRMS1基因表达下调相关,也可能是声门上型喉癌组织中BRMS1基因表达下调的原因之一。     

喉癌组织中IGFBP-rP1基因的甲基化及表达

目的：探讨喉癌中胰岛素样生长因子结合蛋白一相关蛋白1（IGFBP—rP1）基因启动子区甲基化与蛋白表达之间的关系。方法：采用甲基化特异性PCR方法和免疫组织化学的方法分别检测45例喉癌组织及18例癌旁组织中IGFBP-rP1基因的甲基化状态和蛋白表达情况。结果：喉癌组织IGFBP—rP1启动子区甲基化率为33．3％（15／45）,相应癌旁组织甲基化率为5．6％（1／18）,喉癌组织中IGFBP—rP1基因甲基化率明显高于癌旁组织（P〈0．05）。喉癌组织中IGFBP—rP1蛋白表达显著低于癌旁正常组织（P％0．05）,且与其启动子区甲基化状态呈负相关。结论：IGFBP—rP1基因启动子区甲基化导致基因沉默可能是喉癌发生的机制之一。     

RASSF2A基因甲基化在喉癌组织中的表达及意义

目的 探讨抑癌基因RASSF2A表达水平及其基因异常甲基化与喉癌的关系及意义。 方法 采用RT-PCR及甲基化特异性PCR(MSP)检测50例喉癌组织(实验组)和10例癌旁正常黏膜组织(对照组)中RASSF2A基因mRNA的表达水平及其启动子甲基化状态。 结果 喉癌组织中有54%(27/50)存在RASSF2A基因启动子,而对照组正常黏膜组织中未发现RASSF2A基因甲基化现象,差异有统计学意义(χ2=9.818, P=0.002)。RASSF2A甲基化水平程度与喉癌患者临床病理特征无明显相关性。喉癌组织中RASSF2A基因mRNA的表达水平明显低于正常黏膜组织(χ2=9.818, P=0.002)。RASSF2A基因甲基化的喉癌组织中其mRNA的表达减少,反之亦然。 结论 喉癌组织中RASSF2A基因CpG岛甲基化与其基因表达缺失有关,可能参与了喉癌的发生发展过程。     

DNA甲基化在头颈肿瘤中的研究进展[综述]

头颈肿瘤是近年来最常见的恶性肿瘤之一,预后较差,其确切的分子机制和预后因素尚不清楚。识别特异性的生物标志物可使有效的靶向治疗策略应用到头颈肿瘤诊断治疗中,从而改善患者预后。越来越多的证据证实DNA甲基化在肿瘤的起始和进展中起重要作用。本文 就DNA甲基化与头颈肿瘤的关系及研究进展做一综述。     

Aberrant methylation of the HRPT2 gene in parathyroid carcinoma

OBJECTIVES: The incidence of parathyroid carcinoma in hyperparathyroidism-jaw tumor syndrome (HPT-JT) is reported to be as high as 15%. We used a methylation-specific polymerase chain reaction (MS-PCR) technique to investigate whether hypermethylation is one mechanism of HRPT2 gene inactivation in parathyroid tumors. METHODS: DNA was extracted from samples of parathyroid tumors embedded in paraffin. MS-PCR was performed on 11 parathyroid carcinomas, 37 sporadic parathyroid adenomas from control subjects, and 6 parathyroid adenomas from 3 patients with HPT-JT. Methylated and unmethylated PCR products from 2 tumors (Nos. 2 and 6) were cloned. Clones containing inserts were sequenced. RESULTS: Two of 11 (18%) parathyroid carcinomas showed amplification patterns consistent with methylation, compared to 0 of 37 sporadic parathyroid adenomas, and 1 of 6 (17%) parathyroid tumor samples from 3 HPT-JT patients. These results were confirmed by sequencing multiple clones from each of these samples. CONCLUSIONS: There is increasing evidence that loss of HRPT2 gene expression is strongly associated with parathyroid carcinomas. Our data indicate that methylation of the HRPT2 promoter may be another mechanism by which HRPT2 gene inactivation gives rise to parathyroid carcinomas.     

喉癌组织RKIP基因甲基化表达调控研究

摘要：目的检测喉癌组织中Raf 1激酶抑制蛋白（ Raf 1 kinase inhibitor protein, RKIP ）基因启动子区域的甲基化状态及其蛋白表达情况,并探讨其临床意义。方法采用甲基化特异性PCR（methylation specific PCR,MSP ）技术及免疫组化方法检测53例喉癌组织及34例癌旁组织中RKIP基因启动子区域的甲基化状态及蛋白表达情况。结果喉癌及癌旁组织中RKIP基因启动子甲基化发生率分别为17%、3%,两者比较差异具有统计学意义（P=0.045）。结论RKIP基因启动子区高甲基化状态可能是造成喉癌组织中RKIP表达缺失的分子机制之一,RKIP蛋白表达缺失与喉癌的发生发展及转移有关。     

Comprehensive analysis of gene expression and DNA methylation for human nasopharyngeal carcinoma

European Archives of Oto-Rhino-Laryngology - Nasopharyngeal carcinoma (NPC) is one of the most malignant head and neck carcinomas with unique epidemiological features. In this study, we aimed to...     

m6A RNA methylation regulators have prognostic value in papillary thyroid carcinoma

BackgroundN⁶-Methyladenosine (m⁶A) is a ubiquitous RNA modification with vital roles in various cancers, but little is known about its role in papillary thyroid carcinoma (PTC), a common endocrine malignancy.MethodsIn this study, an m⁶A RNA methylation regulator-based biomarker signature was developed for the effective prediction of prognosis in patients with PTC. The gene expression profiles of m⁶A RNA methylation regulators and the corresponding clinical information was downloaded from The Cancer Genome Atlas (TCGA). Differentially expressed m⁶A RNA methylation regulators between tumor and normal control samples, and correlation expression levels, clinical parameters, and outcomes were evaluated. And a prognostic signature was built using a PTC cohort from TCGA.ResultsThe expression level of HNRNPC was remarkably upregulated in tumor samples, while WTAP, RBM15, YTHDC2, YTHDC1, FTO, METTL14, METTL3, ALKBH5, KIAA1429, YTHDF1, and ZC3H13 were significantly downregulated in the cancer specimens compared with those in control samples. A three-gene prognostic signature comprising RBM15, KIAA1429, and FTO could predict overall survival in patients with PTC. In addition, the prognostic signature-based risk score was identified as an independent prognostic indicator for PTC.ConclusionsWe established a robust m⁶A RNA methylation regulator-based molecular signature for predicting prognosis in patients with PTC with high accuracy; this signature might provide important guidance for therapeutic strategies.     

Study of aberrant methylation of TSG in saliva in case of upper-aerodigestive-tract cancer

CURRENT SITUATION: Early detection of the relapse in case of tumor located in the upper aerodigestive tract (UADT) is an important point for improving prognosis. Clinical control has not been efficient and, until today, no biological biomarker has been validated for surveillance of patients. In a preliminary study, we have shown the benefit of the methylation status of six tumor suppressor genes (TSG): TIMP3, ECAD, p16, MGMT, DAPK, and RASSF1A in saliva for early diagnosis of tumor relapse. The main objective of this second study is to confirm the initial results. MATERIAL AND METHODS: This is a bicenter, prospective, diagnostic, single-blind study. The study started in December 2007, running for one year. The main inclusion criterion is a patient with squamous-cell carcinoma of the oral cavity or the oropharynx, stages I to II, without previous treatment for this location. Eighty patients will be included. The data analysis will use a multivariate Cox model. EXPECTED RESULTS: If our preliminary results are confirmed, identification of methylations in saliva would be able to constitute the first usable biomarker, for the follow-up of patients with UADT cancer.