促甲状腺激素受体胞内环基因突变与乳头状甲状腺癌发病的相关性研究

目的探讨乳头状甲状腺癌的发病与促甲状腺激素受体(TSHR)基因突变的相关性。方法采用巢氏-多聚酶联反应-单链构象多态性分析(NEST-PCR-SSCP)和DNA测序方法,对65例乳头状甲状腺癌和44例正常甲状腺组织TSHR3个胞内环基因进行检测。结果NEST-PCR-SSCP检测乳头状甲状腺癌促甲状腺激素受体(TSHR)3个胞内环未发现明显带型异常;DNA测序后,检测的第3胞内环2例对照组织和3例甲状腺癌组织TSHR2000位点碱基均由C→T,使得所编码的601位氨基酸由组氨酸(CAT)→酪氨酸(TAT)(His→Tyr),余胞内环未发现基因突变。结论乳头状甲状腺癌TSHR3个胞内环未发现基因突变,提示乳头状甲状腺癌发病与TSHR胞内环基因突变无关;5例标本601位氨基酸均为酪氨酸,考虑中国人TSHR基因可能与国外人群存在差异,TSHR2000位点碱基存在基因多态性。     

抑癌基因p16和RASSF1A启动子甲基化与乳头状甲状腺癌的相关性研究

乳头状甲状腺癌(PTC)是甲状腺最常见的恶性肿瘤,目前国外有少量研究报道甲状腺癌的发生与p16和RASSF1A两种抑癌基因启动子的高甲基化密切相关[1-2],本研究采用RT-PCR和甲基化特异性PCR(methylation-specific PCR,MSP)法对PTC及对照组织进行研究,以探讨这两种抑癌基因启动子的高甲基化与PTC的发生是否有关.     

甲状腺乳头状癌组织中TSHR基因启动子甲基化研究

目的探讨甲状腺乳头状癌组织中促甲状腺激素受体基因（Thyroid Stimulating Hormone Receptor,TSHR）基因启动子区5端CpG岛甲基化改变的特点与临床特征的关系。方法采用甲基化特异性PCR（MSP,methylation-specific PCR）方法检测TSHR基因启动子甲基化情况。结果（1）22／34例甲状腺乳头状癌组织中检测到TSHR基因启动子甲基化,9／34例甲状腺乳头状癌癌旁组织中检测到TSHR基因启动子甲基化,癌组织中TSHR基因启动子甲基化率显著增高（χ^2=10．019,P=0．002〈0．05）;（2）有淋巴结转移的甲状腺乳头状癌组织（15／18例）TSHR基因启动子的甲基化显著高于无淋巴结转移组（7／16例）（χ^2=5．812,P=0．016〈0．05）。结论TSHR基因启动子异常甲基化是甲状腺乳头状癌发展过程中的分子事件之一,可能影响了甲状腺乳头状癌细胞的摄碘的功能。     

胃癌形成过程中p16IN4a、Runx3和O-6-甲基鸟嘌呤-DNA甲基转移酶基因甲基化及其表达研究

目的 研究胃癌形成过程中p16INK4a、Runx3和O-6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)基因启动子区的高甲基化状态,同时检测MGMT的蛋白表达情况.探讨抑癌基因启动子区高甲基化与胃癌发生的关系.方法 选择经透明帽法进行首次黏膜病变切除者43例,其中异型增生27例,早期胃癌16例.选择胃镜活检证实为慢性萎缩性胃炎伴肠上皮化生者14例.另取20例正常胃黏膜活检组织作为对照.采用甲基化特异聚合酶链反应(MSP)检测每例组织中p16INK4a、Runx3和MGMT基因启动子区的甲基化状态,对所有甲基化p16INK4a产物进行测序,免疫组化检测MGMT蛋白表达情况.结果 肠上皮化生、异型增生和早期胃癌中p16INK4a基因甲基化率依次为14.3%(2/14)、22.2%(6/27)和37.5%(6/16);Runx3基因甲基化率依次为14.3%(2/14)、48.1%(13/27)和50.0%(8/16);MGMT基因甲基化率依次为7.1%(1/14)、48.1%(13/27)和50.0%(8/16).20名正常对照均未检出基因甲基化,与异型增生和早期胃癌相比差异有统计学意义(P＜0.05).Runx3和MGMT两种基因在异型增生和早期胃癌中的甲基化率显著高于肠上皮化生组(P＜0.05).各组病变中三种基因甲基化联合分析发现,异型增生和早期胃癌中甲基化的基因种类高于肠上皮化生组.差异有统计学意义(P＜0.01).基因甲基化与息者年龄、性别、幽门螺杆菌感染以及病变部位无相关性,但p16INK4a和MGMT基因甲基化与血清癌胚抗原水平升高显著相关(P值分别为0.003和0.039).MGMT基因启动子区高甲基化与其蛋白失表达密切相关(χ2=12.821,P=0.001).结论 抑癌基因启动子区高甲基化是基因失活的主要机制,可能是胃癌发生的早期分子事件.p16INK4a、Runx3和MGMT基因启动子区高甲基化在胃癌形成过程中起着重要的作用.     

p16基因启动子区甲基化在人嗜铬细胞瘤和副神经节瘤中的变化

目的 检测嗜铬细胞瘤(PHEO)和副神经节瘤(PGL)中p16基因突变和启动子区DNA甲基化改变,分析其与患者临床特征之间的关系.方法收集34例(PHEO 20例、PGL14例)组织标本及患者临床资料,通过甲基化特异性PCR(MSP)测定p16基因启动子区甲基化状态,DNA测序检测基因序列以及RT-PCR方法测定其mRNA表达.结果 (1)34例肿瘤组织中未发现p16基因纯合缺失及点突变;(2)35.3%(12/34)的患者存在p16基因高甲基化,p16基因甲基化阳性标本中,PHEO和PGL分别占25%和75%,两者差异有统计学意义(P=0.005);p16基因甲基化在恶性、单发肿瘤、发病年龄早的亚组中有增高趋势(P＞0.05);(3)甲基化与非甲基化肿瘤组织之间p16 mRNA表达无统计学差异;不同特点的肿瘤中其mRNA表达亦无统计学差异,但恶性肿瘤p16 mRNA表达与良性肿瘤相比有下降的趋势(0.83±0.65对1.12±0.81,P=0.278).结论人PHEO和PGL中,p16基因纯合缺失和突变并不常见,但p16基因启动子区甲基化是其失活的主要形式.     

卵巢癌组织中肺癌抑癌基因1基因启动子甲基化状态及其临床意义

目的探讨肺癌抑癌基因1(TSLC1)在卵巢癌中基因启动子甲基化状态及临床意义。方法应用甲基化特异性PCR检测TSLC1基因启动子在50例卵巢癌组织和10例正常卵巢组织中的甲基化状态并结合临床病理资料进行分析。结果在卵巢癌组织中TSLC1甲基化阳性率(36%,18/50),正常卵巢组织未出现TSLC1甲基化。TSLC1甲基化与肿瘤淋巴结转移与Silverberg分期密切相关(P0.05)。结论 TSLC1甲基化可能是参与卵巢癌发展的重要分子事件。     

乳头状甲状腺癌与相关基因DNA甲基化

DNA甲基化是重要的表观遗传修饰方式之一,在肿瘤的发病、诊断及预后判断中有重要的临床意义.近年的研究发现甲状腺特异基因[促甲状腺激素受体(TSHR)、钠-碘转运体(NIS)]与肿瘤抑制基因(RASSF1A、P16、ECAD、ATM、SLC5A8、Rap1 GAP、TIMP3、DAPK、RARβ2、PTEN、MLH1)等的DNA甲基化在乳头状甲状腺癌的发生及发展过程中起重要作用,并与BRAF突变、RET/PTC重排及RAS/RAF/丝裂原活化蛋白激酶激酶/细胞外信号调节激酶/丝裂原活化蛋白激酶(RAS/RAF/MEK/ERK/MAPK)通路等密切相关.临床上,对乳头状甲状腺癌相关基因的DNA甲基化进行检测,可提供一种新的诊断及判断预后的方法,有望成为精确诊断肿瘤的早期指标,并从表观遗传学角度提供治疗疾病的新方法.     

胃癌3号染色体短臂抑癌基因CpG岛甲基化表型研究

背景:3号染色体短臂(3p)抑癌基因CpG岛甲基化表型(CIMP)涉及的甲基化异常见于多种类型的癌症中,但与胃癌的关系迄今仍未阐明。目的:研究胃癌中3p抑癌基因启动子区CIMP的临床意义。方法:采用甲基化特异性PCR(MSP)法检测100例胃癌组织及其对应癌旁组织中hOGG1、VHL、RAR-B、hMLH1、SEMA3B、RASSF1A、BLU和FHIT共8个抑癌基因启动子区CpG岛甲基化状态。以每份样本中含有4个及以上甲基化基因定义为高CIMP水平(CIMP-H)。分析CIMP-H与胃癌临床病理特征的关系。结果:胃癌组织中VHL(P=0.030)、hMLH1(P0.001)、SEMA3B(P=0.003)、RASSF1A(P0.001)和FHIT(P0.001)基因甲基化阳性率显著高于癌旁组织。胃癌组织和对应癌旁组织中CIMP-H发生率分别为44.0%和4.0%,差异有统计学意义(P0.001)。胃癌组织CIMP-H与肿瘤分化程度呈显著负相关(P=0.004),与淋巴结转移呈显著正相关(P=0.005),而与性别、年龄、肿瘤部位、肿瘤直径、浸润深度和TNM分期均无关(P0.05)。结论:3p CIMP异常状态可能在胃癌形成早期已发生,并影响肿瘤分化,导致淋巴结转移。     

结肠癌组织中RASSF1A基因启动子甲基化状态、mRNA表达变化及意义

目的观察结肠癌组织中RAS相关区域家族1A（RASSF1A）基因启动子甲基化状态、mRNA表达变化,并探讨其意义。方法分别采用甲基化特异性PCR（MSPCR）和RT-PCR技术检测80例结肠癌及癌旁正常结肠组织中RASSF1A基因启动子甲基化、mRNA。结果 80例正常结肠组织中RASSF1A基因启动子未见甲基化,80例结肠癌组织中RASSF1A基因启动子出现甲基化者58例,两者相比,P〈0.05。正常结肠组织均可检测到RASSF1A基因mRNA,结肠癌组织中RASSF1A基因启动子甲基化者未检测到RASSF1A基因mRNA、未甲基化者可检测到RASSF1A基因mRNA。RASSF1A基因启动子甲基化与结肠癌病理分型、淋巴结转移、远处转移和Dukes分期有关（P均〈0.05）。RASSF1A基因mRNA的表达与结肠癌Dukes分期有关（P〈0.05）。结论结肠癌组织中RASSF1A基因启动子出现甲基化,RASSF1A基因启动子甲基化者RASSF1A基因mRNA表达缺失,这可能与结肠癌的发生发展及转移有关。     

RASAL1基因启动子甲基化及RAS活性与结肠癌临床病理的关系

目的:检测RASAL1(ras GTPase-activatinglike protein 1)基因甲基化率及RAS活性在人结肠癌组织中的表达,探讨其与临床病理资料间的关系.方法:以40例结肠癌标本为研究对象,相应的正常组织标本为对照.甲基化特异性PCR(methylation-specific PCR,MSP)检测RASAL1启动子CpG岛甲基化状态,免疫共沉淀法检测RAS活性,分析肿瘤组织中RASAL1基因甲基化率及RAS活性与临床病理参数间的关系.结果:40例肿瘤组织中有26例检测出RASAL1基因甲基化(26/40,67.5%),对照组40例中有12例出现甲基化(12/40,30%),肿瘤组织甲基化率明显高于正常组织(P=0.0017).肿瘤组织中RAS活性的中位数为1.07,正常组织中RAS活性的中位数为0.52,P<0.001,差异有统计学意义.结肠癌组织中RASAL1基因的甲基化率、RAS活性与患者肿瘤分化程度、侵袭深度、淋巴结转移、TNM分期有统计学差异(均P<0.05).结论:RASAL1甲基化状态与RAS活性增加有关,可能在结肠癌的发生发展中起重要作用.抑制RASAL1甲基化及RAS活性,可能成为治疗结肠癌的新靶点.     

Aberrant promoter hypermethylation of p16 and MGMT genes in oral squamous cell carcinomas and the surrounding normal mucosa

Purpose Several genetic alterations have been reported to contribute to the development of oral squamous cell carcinoma (OSCC). Recent studies have shown roles of promoter hypermethylation of tumor suppressor genes, including p16 and MGMT, in several types of cancers. The purpose of this study is to examine the hypermethylation status of p16 and MGMT genes in both oral cancers and normal mucosa, surrounding the cancers.Methods Promoter hypermethylation status of p16 and MGMT genes were examined by the methylation-specific PCR (MSP) in OSCC (n = 51), verrucous carcinoma (n = 2), and carcinoma in situ (n = 2) tissues. Moreover, normal mucosa surrounding the cancers were also examined in 22 cases out of the 51 OSCCs. As a normal control, oral mucosa from healthy volunteers (n = 18) was used.Results Aberrant promoter hypermethylation of p16 and MGMT genes was detected in 50.9% (28 of 55) and 56.4% (31 of 55) of the total malignant cases, respectively. As for the 22 OSCC cases, in which paired cancerous tissues and the surrounding normal mucosa were examined simultaneously, promoter hypermethylation of p16 and MGMT genes was confirmed in 72.73% (16 of 22) and 68.18% (15 of 22), respectively. In contrast, as for the surrounding normal mucosa, promoter hypermethylation of p16 and MGMT genes was recognized in 27.27% (6 of 22) cases and 40.91% (9 of 22), respectively. Conclusions Hypermethylation of both p16 and MGMT genes was frequently detected in not only OSCC tissues, but also the surrounding normal mucosa around the cancerous tissues. Thus promoter hypermethylation of p16 and MGMT genes are an important, probably early event in oral carcinogenesis.     

p16INK4A gene alterations are not a prognostic indicator for survival in patients with hepatocellular carcinoma undergoing curative hepatectomy

BACKGROUND AND AIM: Hepatocellular carcinoma (HCC) is a common malignancy worldwide that is highly associated with chronic hepatitis B or C infection and cirrhosis. The tumor suppressor gene p16INK4A is an important component of the cell cycle and inactivation of the gene has been found in a variety of human cancers. The present study was performed to determine genetic and epigenetic alterations in the p16INK4A tumor suppressor gene and the effect of these on HCC progression. METHODS: The status of p16INK4A was evaluated in 117 HCC tumoral nodules and 110 corresponding peritumoral tissues by loss of heterozigosity (LOH) at the 9p21-22 region, homozygous deletions, single-strand conformation polymorphism-polymerase chain reaction (PCR) mutational analysis and methylation specific PCR. RESULTS: The most frequent inactivation mechanism was hypermethylation of the promoter region, which was found in 63.2% of the tumor samples and in 28.2% of the peritumoral samples. Loss of heterozygosity at the 9p21 region was detected in 27.3% and 10% of tumor and peritumoral tissues, respectively. Homozygous deletions and mutations were less common events in hepatocarcinogenesis. The authors found 5.9% of the tumor cases with exon 2 homozygous deletions and 8.6% with mutations. Two polymorphisms were detected, one at codon 148 (GCG --> ACG, Ala --> Thr) in three cases and the other in exon 3 at 540 bp (34.2% of the samples). No association was found between inactivation of p16INK4A and clinicopathological characteristics or prognosis. CONCLUSION: p16INK4A is altered frequently and early in HCC, being the predominant mechanism of inactivation promoter hypermethylation. The present results suggest that the p16INK4A gene plays an important role in the pathogenesis of HCC.     

胃癌维甲酸信号通路相关基因启动子区协同高甲基化的研究

背景：启动子区高甲基化与胃癌中多种抑癌基因表达沉默密切相关。目的：探讨维甲酸信号通路相关基因维甲酸受体B（RAR13）、细胞维生素A结合蛋白1（CRBP1）和他扎罗汀诱导基因1（TIG1）启动子区高甲基化与胃癌的关系。方法：以甲基化特异性聚合酶链反应（MSP）检测40例胃癌标本、10例正常胃黏膜标本和6株胃癌细胞株的RAR13、CRBPI和TIG1基因启动子区甲基化状态，分析i者甲基化状态的相关性及其与胃癌1晦床病理特征的关系。以逆转录聚合酶链反应（RT—PCR）检测胃癌细胞株RAR13、CRBP1和TIG1mRNA表达。结果：40例胃癌组织的RAR13、CRBPI和TIG1基因甲基化率分别为45．0％、32．5％和57．5％，10例正常胃黏膜组织均未检测到上述基因甲基化（P〈0．05）。胃癌组织中RAR13的甲基化状态与CRBP1和TIG1的甲基化状态显著相关（P〈0．05），但三者的甲基化状态与胃癌临床病理特征无相关性。启动子区高甲基化胃癌细胞株相应基因mRNA表达缺失或减弱。结论：胃癌组织常发生维甲酸信号通路相关基因RAR13、CRBP1和TIG1启动子区高甲基化，高甲基化可能是相应基因转录失活的重要原因。     

Hypermethylation of the p15INK4B gene promoter in B-chronic lymphocytic leukemia

The p15 gene is a putative tumor suppressor gene that encodes a member of the cyclin dependent kinase inhibitors. Inactivation of p15 by promoter hypermethylation has been postulated as a possible way by which tumor suppressor genes are inactivated in cancer. In this study, we examined the methylation status of the p15 gene promoter in 34 patients with B-Chronic Lymphocytic Leukemia (B-CLL), by the Methylation-Specific Polymerase Chain Reaction. Selective methylation of the p15 gene promoter was found in 4/34 cases (11.8%). According to Rai staging, the four patients with methylated p15 were staged on diagnosis as: 1 on Stage 0, 1 in Stage I, 1 in Stage III, and 1 in Stage IV. Our results suggest that methylation of the p15 gene promoter can be detected in a small subset of B-CLL patients, at all stages of the disease.     

p16 promoter hypermethylation： A useful serum marker for early detection of gastric cancer

AIM： TO determine p15 promoter hypermethylation in gastric tumoral tissue and serum samples, its impact on p16-protein expression, and correlation with clinical and histological features. METHODS： Samples were obtained from 52 histologically confirmed cases of gastric adenocarcinoma. Gastric tissue and serum of 50 age- and sex-matched individuals with normal gastroscopy and biopsy were obtained as control samples. Methylation-specific polymerase chain reaction （MSP） was used to evaluate methylation status of p16 promoter, p16-protein expression was analyzed by immunohistochemical staining on paraffin-embedded sections. RESULTS： Methylation was detected in 44.2% （23/52） of tumoral tissues. 60.9% of them were also methylated in serum, i.e., 26.9% of all patients （14/52）. Methylation was not detected in tissue and sera of control samples. p16-protein expression was decreased in 61.5% of cases （32/52）, and was significantly associated with promoter hypermethylation （P 〈 0.001）. Methylation was significantly more frequent in higher pathological grades （P 〈 0.05）. Methylation was not associated with other clinicopathological features and environmental factors including Hpylori infection and smoking. CONCLUSION： p16 promoter hypermethylation is an important event in gastric carcinogenesis. It is the principle mechanism of p16 gene silencing. It is related to malignant tumor behavior. Detection of DNA methylation in serum may be a biomarker for early detection of gastric cancer.     

LMXlA基因启动子甲基化以及在胃癌发展中的临床意义

目的通过检测胃癌癌旁距离原发灶不同距离组织及胃癌原发灶LMXlA基因启动子区甲基化状态的差异,分析正常胃组织、癌前病变及癌组织中该基因甲基化的动态变化以及LMXlA基因甲基化与胃癌原发灶病理学的关系,探讨LMXlA基因启动子区甲基化在胃癌阶段性发生及进展中的作用及临床意义。方法采用甲基化特异性PCR法（MSP）检测距胃癌癌灶边缘1、3、5cm组织及胃癌原发灶组织中LMXlA基因甲基化状态。结果LMXlA基因启动子区甲基化发生率在胃癌组织及距胃癌灶边缘1、3、5cm组织中分别为46．0％（23／50）、22．O％（11／50）、8．0％（4／50）和4．0％（2／50）,从距癌灶边缘5cm胃组织至胃癌原发灶组织中的表达中随距癌灶边缘距离的减少呈上升趋势,原发灶中甲基化阳性率显著高于距原发灶边缘3cm和5cm组织（x^2=15．42,P〈0．05;x^2=12．63,P〈0．01）。LMXlA基因启动子区甲基化发生率在正常胃组织、癌前病变组织及胃癌原发灶中分别为O％（0／25）、16．0％（4／25）和46．O％（23／50）,三者之间的差异具有统计学意义（×^2=24．85,P〈0．01）。LMXlA基因甲基化发生率在胃癌患者透浆膜组57．6％（19／33）显著高于未透浆膜组14．8％（4／27）（X^2=16．50,P〈0．05）;在转移淋巴结数大于7枚以上组52．9％（9／17）显著高于小于7枚组37．5％（6／16）（X^2=12．74,P〈0．05）。LMXlA基因甲基化在性别、年龄、不同大体类型、生长方式及分化程度上胃癌患者间差异无统计学意义。结论LMXlA基因启动子区异常甲基化可能与胃癌的临床进展有一定的相关性。     

原发性肝细胞癌中HBV各基因片段整合与癌基因,抑癌基因表达？…

目的 研究肝细胞癌中ＨＢＶ各基因整合与癌基因、抑制基因表达的关系。方法 以随机引物法制备ＨＢＶ全基因及各基因探针,用Ｓｏｕｔｈｅｒｎ杂交测定ＨＢＶ ＤＮＡ的整合状态,以斑点杂交检查整合型ＨＣＣ中ＨＢＶ各基因的整合,以免疫组织细胞化学方法检查癌基因、抑癌基因的表达。结果 ３２例纯整合型ＨＣ中ＨＢＶ Ｘ．Ｓ．Ｐｅｒ－Ｓ和Ｃ的整合率分别为８７．５％（２８／３２）,６２．５％（２０／３２）,６２．５％（２     

DNA甲基化和胃液固有荧光光谱联合检测在胃癌诊断中的意义

目的探索在胃癌患者胃液、外周血清中检测基因甲基化的可行性，并结合胃液稀释固有荧光光谱评价二者在胃癌诊断中的作用。方法采用甲基化特异性PCR方法，检测50例胃癌患者的原发肿瘤组织、外周血清和胃液脱落细胞的死亡相关蛋白（DAP）激酶、p16基因启动子区域甲基化状态，并以胃良性溃疡和慢性浅表性胃炎各20例、慢性萎缩性胃炎30例作为对照，同时检测胃癌患者和对照者的胃液稀释固有荧光光谱。结果50例胃癌患者的肿瘤组织、外周血清和胃液脱落细胞中p16和DAP激酶基因甲基化阳性率分别为74．4％和68．1％、52．0％和58．0％、58．6％和76．0％，20例慢性浅表性胃炎患者中均未检测到基因异常甲基化；20例胃溃疡患者溃疡周边组织、胃液脱落细胞中p16和DAP激酶甲基化阳性率为10．0％和20．0％、5．0％和15．0％，在外周血清中未检测到异常甲基化；30例慢性萎缩性胃炎患者胃黏膜组织、外周血清、胃液脱落细胞p16和DAP激酶基因甲基化阳性率分别为10．0％和23．3％、3．3％和3．3％、3．3％和20．0％。胃癌患者胃液固有荧光光谱强度较对照者明显增强（P〈0．05）；以P1FI≥111．8为分界点分析胃液固有荧光光谱诊断胃癌的敏感性和特异性分别为76．1％和78．6％。p16和DAP激酶基因甲基化和胃液固有荧光光谱结合后诊断胃癌的敏感性提高到95．6％和97．8％。结论胃癌患者外周血清及胃液脱落细胞中可检测到与原发肿瘤组织一致的基因异常甲基化，胃液固有荧光光谱和DNA甲基化联合对检测胃癌有良好的临床应用价值。