老年肺癌患者血清RASSF1A基因启动子异常甲基化检测及其临床意义

目的探讨老年肺癌患者血清RASSF1A基因启动子区域的甲基化状态及其临床意义。方法采用甲基化特异性聚合酶链反应（MSP）技术，检测45例老年肺癌患者、20例肺部良性病变患者及15例健康志愿者血清RASSF1A基因启动子区域的甲基化状态，并分析其与临床病理参数之间的相关性。结果45例老年肺癌患者血清RASSF1A基因启动子区域异常甲基化15例，检出率为33．3％，而20例肺部良性疾病患者及15例健康志愿者中检出率均为0，差异有统计学意义（均为P〈0．01）。RASSF1A启动子异常甲基化在小细胞肺癌（SCLC）中有更高的检出率，但不同病理类型的非小细胞肺癌（NSCLC）异常甲基化的频率无明显差异。RASSF1A启动子异常甲基化与患者的性别、年龄、分化、分期和治疗干预无显著相关。结论RASSF1A启动子异常甲基化在老年肺癌患者血清中有着较高的检出率，可能成为肺癌诊断的分子标记。     

前列腺癌组织中多基因异常甲基化检测及临床意义

目的 探讨前列腺癌组织中RARβ2、GSTP1和DAPK基因异常甲基化,与前列腺临床病理特征间的关系,及其在前列腺癌诊断中价值.方法 采用巢式甲基化特异性PCR(nestedmethylationspecificpolymerasechainreaction,NMSP)法对57例前列腺痛组织和35例良性前列腺增生(BPH)组织进行甲基化检测;分析其甲基化的发生与前列腺癌临床病理特征间的关系,以及在前列腺癌诊断中价值.结果 前列腺癌组织中RARβ2、GSTPI和DAPK基因甲基化检出率显著高于BPH组织(RARβ32:52.6%对0%;GSTPl:61.4%对2.9%;DAPK:43.9%对8.6%;均P<0.01).RARβ2基因甲基化率在前列腺癌不同Gleason评分和不同临床分期之间差异有统计学意义(4～7分对8～10分:34.8%对64.7%,B、C期对D期:37.0%对66.7%,x2=4.927和x2=5.004,P=0.026和P=0.025);GSTP1基因甲基化率在不同Gleason评分间差异有统计学意义(4～7分对8～10分:43.5%对73.5%,x2=11.530,P=0.001),而在不同临床分期之间差异无统计学意义(B、C期对D期:51.9%对70.0%,x2=1.975,P=0.16);DAPK基因甲基化率在不同Gleason评分和不同临床分期之间差异均无统计学意义(4～7分对8～lO分:39.1%对50.0%,B、C期对D期:33.3%对53.3%,x2=1.290和x2=2.309,均P>0.05).GSTP1基因诊断敏感度最高为61.4%(35/57),特异度为97.1%(34/35);RARβ2基因特异度最高为100%(35/35),敏感度为52.6%(30/57);DAPK基因的诊断敏感度和特异度分别为43.9%和91.4%(25/57和32/35).而RARβ2、GSTP1和DAPK基因甲基化联合检测能明显提高前列腺癌诊断敏感度,但诊断特异度却有所下降.结论 RARβ2、GSTP1和DAPK基因异常甲基化与前列腺癌的发生与发展相关,可作为前列腺癌的诊断有效指标之一.     

APC基因启动子区甲基化及环境因素与前列腺癌关系研究

目的探讨腺瘤样结肠息肉易感基因(APC)启动子区CpG甲基化及环境危险因素与中国人群中前列腺癌(PCa)发病之间的关系。方法收集60例PCa和40例前列腺增生(BPH)患者的组织标本。用亚硫酸盐修饰后测序法检测PCa组织及BPH组织中APC基因启动子区CpG甲基化情况。同时收集人口学资料以及体质量指数(BMI)、饮酒、饮茶、吸烟、体育运动等环境危险因素资料,用单因素和多因素分析法研究APC基因甲基化及环境危险因素与PCa患病之间的关系。结果 APC基因在PCa及BPH组织中CG位点甲基化率为14.00%、1.19%;基因甲基化率与前列腺特异性抗原(PSA)、Gleason评分、病理分期和PCa临床分期之间关系密切;PCa发病与饮酒、饮茶有关,比值比分别为2.46、0.29。结论 APC基因启动子区CpG甲基化与PCa发生及发展有关,其甲基化率的变化与PCa的临床分期关系密切。饮酒、饮茶是导致PCa的环境危险因素。     

实时荧光定量PCR和甲基化特异性PCR检测结直肠癌APC、MLH1基因甲基化状态

背景：启动子区甲基化致肿瘤抑制基因失活在结直肠癌的发生、发展中起重要作用,检测肿瘤相关基因的甲基化状态,可能为寻找新的结直肠癌诊断、预后相关标记物提供依据.目的：比较实时荧光定量PCR（FO-PCR）与甲基化特异性PCR（MSP）检测基因甲基化状态的差异.方法：以FQ-PCR检测66例结直肠癌组织和20例癌旁组织中与结直肠癌的发生、发展相关的抑癌基因APC和错配修复基因MLH1的甲基化状态,同时以MSP检测结直肠癌组织中APC基因的甲基化状态.结果：根据FQ-PCR结果,结直肠癌组织中APC、MLH1基因甲基化阳性率显著高于癌旁组织（48.5%和54.5%对0%和0%,P=0.000）.FQ-PCR和MSP可检出的甲基化阳性对照DNA最低浓度分别为0.015 ng/μl和1.5 ng/μl,两者对APC基因甲基化状态的检测结果差异有统计学意义（P＜0.05）.结论：在DNA甲基化的检测手段中,FQ-PCR的敏感度优于MSP.     

APC基因与胃癌

ＡＰＣ基因是近年来发现的抑癌基因，有关该基因的结构、功能及与胃癌关系的研究不断得到进展。多数研究提示，该基因与细胞增殖、分化、粘附等均有一定关系，并在胃癌的早期发生及发展中可能起重要作用。因此，研究它的结构、蛋白产物及功能、致癌机制，探讨其在胃癌中的变化规律，对于胃癌的预防、诊断和治疗均具有重要意义。     

抑癌基因APC、RASSFlA、WIF-1启动子区域甲基化与消化系肿瘤

消化系统肿瘤是常见的人类恶性肿瘤,目前研究认为抑癌基因启动子区域的异常甲基化与肿瘤的形成有关.在此综述APC、RASSFIA、WIF-13种抑癌基因启动子区域的异常甲基化与消化系统肿瘤的关系.     

胃癌组织Runx3基因甲基化的检测及意义

目的探讨胃癌组织中Runx3基因甲基化情况及其意义。方法采用DNA甲基化特异性PCR技术（MSP）对35例胃癌患者手术切除的肿瘤组织及癌旁组织Runx3基因启动子区域甲基化进行检测,同时采用RT—PCR检测Runx3mRNA的表达。结果胃癌及癌旁组织Runx3基因的甲基化发生率分别为40．0％和8．5％;胃癌组织Runx3mRNA表达较癌旁正常组织下调（P〈0．05）;Runx3mRNA的表达与胃癌分化程度及淋巴结转移有关。结论Runx3基因甲基化是导致Runx3基因失活的主要原因之一,与胃癌的发生发展密切相关。     

抑癌基因RASSF1启动子甲基化在肿瘤中的研究进展

肺癌和其它一些肿瘤中普遍存在人3号染色体短臂的杂合型丢失（loss of heterozygosity,LOH）,2000年Dammann等通过酵母双杂交技术从3p21.3分离鉴定出抑癌基因RASSF1A（rasassociation domain family 1A）。在许多人类肿瘤中存在RASSF1A启动子区域高甲基化,导致基因表观遗传失活。RASSF1A启动子区高甲基化可以作为肿瘤早期诊断和评价预后的一个候选分子标志。     

多种抑癌基因在食管鳞状细胞癌中的甲基化状态及其临床意义

抑癌基因甲基化与食管癌相关,目前多种抑癌基因与肿瘤家族史相关性的报道少见。目的：研究多种抑癌基因在食管鳞状细胞癌（ESCC）中的甲基化状态及其临床意义。方法：选取2010年2～7月浙江省肿瘤医院76例ESCC患者。应用MSP技术检测肿瘤组织和相应癌旁正常组织中APC、RARl32、CDHl、p16…、RASSFlA等5个抑癌基因的甲基化状态,并分析抑癌基因甲基化状态与肿瘤家族史的关系及其对预后的影响。结果：ESCC组织APC、RARe2、CDHl、p16慨、RASSFlA的甲基化率均显著高于相应癌旁正常组织（P〈0．05）。ESCC组织中APC、RARl32、CDHl、RASSFlA甲基化与肿瘤家族史相关（P〈0．05）：CDHl、RASSFlA甲基化患者的生存期明显低于非甲基化患者（P-0．015、P=0．016）。结论：ESCC患者存在抑癌基因APC、RARe2、CDHl、p16№、RASSFlA高甲基化;且APC、RARe2、CDHl、RASSFlA甲基化与肿瘤家族史显著相关,CDHl、RASSFlA甲基化患者的预后可能较差。     

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。     

Aberrant promoter hypermethylation in biliary tract carcinoma

Biliary tract carcinoma is a relatively rare tumor with a poor survival rate. The molecular biological mechanisms underlying the development of biliary tract carcinomas are not well understood. Promoter methylation is an important epigenetic mechanism for suppressing tumor-suppressor gene activity. There is limited information regarding the abnormal methylation of cancer-related genes in biliary tract carcinoma; however, a few insights have been obtained into the role of epigenetic silencing in the progression of biliary tract carcinoma. In this review, we summarize recent data on gene silencing by promoter hypermethylation, and we discuss the implications for biliary tract carcinomas.     

APC promoter methylation and protein expression in hepatocellular carcinoma

Purpose We investigated the impact of promoter methylation on APC protein expression in patients with hepatocellular carcinoma (HCC). Materials and methods 50 patients [HCC (n=19), liver metastasis (n=19), cholangiocellular cancer (n=7), and benign liver tumors (n=5)] were studied for methylation using Methylight analysis. APC mutation was investigated by protein truncation test and direct sequencing of genomic DNA. The protein expression was evaluated by immunohistochemistry and Western blot analysis. Results The APC promoter was hypermethylated in 81.8% of non-cancerous liver tissue samples. All HCC samples and ten patients with liver metastasis (52.6%) exhibited APC promoter methylation. The degree of methylation was significantly higher in samples from HCC compared to the non-cancerous liver tissue samples (63.1% vs. 24.98%; p=0.001). The level of APC protein expression was significantly reduced in HCC samples compared to that of the corresponding non-tumor liver tissue (p<0.05). Conclusions Promoter methylation of the APC gene seems to be of significance in hepatocarcinogenesis and results in reduced protein expression in HCC. Interestingly, APC promoter methylation is also present in the vast majority of non-cancerous liver tissue whose (patho)physiological function remains unresolved.     

Protein expression and gene promoter hypermethylation of CD99 in transitional cell carcinoma of urinary bladder

Purposes  

To investigate the significance of CD99 protein expression and gene promoter hypermethylation status in urinary bladder carcinoma and its correlation with the histopathologic parameters.     

Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma

Inactivation of the genes involved in DNA mismatch repair is associated with microsatellite instability (MSI) in colorectal cancer. We report that hypermethylation of the 5′ CpG island of hMLH1 is found in the majority of sporadic primary colorectal cancers with MSI, and that this methylation was often, but not invariably, associated with loss of hMLH1 protein expression. Such methylation also occurred, but was less common, in MSI− tumors, as well as in MSI+ tumors with known mutations of a mismatch repair gene (MMR). No hypermethylation of hMSH2 was found. Hypermethylation of colorectal cancer cell lines with MSI also was frequently observed, and in such cases, reversal of the methylation with 5-aza-2′-deoxycytidine not only resulted in reexpression of hMLH1 protein, but also in restoration of the MMR capacity in MMR-deficient cell lines. Our results suggest that microsatellite instability in sporadic colorectal cancer often results from epigenetic inactivation of hMLH1 in association with DNA methylation.     

Reelin promoter hypermethylation in schizophrenia

Reelin mRNA and protein levels are reduced by approximately 50% in various cortical structures of postmortem brain from patients diagnosed with schizophrenia or bipolar illness with psychosis. In addition, the mRNA encoding the methylating enzyme, DNA methyltransferase 1, is up-regulated in the same neurons that coexpress reelin and glutamic acid decarboxylase 67. We have analyzed the extent and pattern of methylation within the CpG island of the reelin promoter in genomic DNA isolated from cortices of schizophrenia patients and nonpsychiatric subjects. Ten (The Stanley Foundation Neuropathology Consortium) and five (Harvard Brain Collection) schizophrenia patients and an equal number of nonpsychiatric subjects were selected from each brain collection. Genomic DNA was isolated, amplified (from base pair -527 to base pair +322) after bisulphite treatment, and sequenced. The results show that within the promoter region there were interesting regional variations. There was increased methylation at positions -134 and 139, which is particularly important for regulation, because this portion of the promoter is functionally competent based on transient transfection assays. This promoter region binds a protein present in neuronal precursor nuclear extracts that express very low levels of reelin mRNA; i.e., an oligonucleotide corresponding to this region and that contains methylated cytosines binds more tightly to extracts from nonexpressing cells than the nonmethylated counterpart. Collectively, the data show that this promoter region has positive and negative properties and that the function of this complex cis element relates to its methylation status.     

分拣蛋白相关受体L1基因异常甲基化在晚发散发性阿尔茨海默病中的研究

目的研究分拣蛋白相关受体L1(sortilin-related receptor 1,SORL1)基因启动子区异常甲基化与阿尔茨海默病(AD)发生的相关性。方法选择晚发散发性AD(SAD)患者40例为SAD组,另选择健康体检者8例为对照组。提取外周血DNA,设计硫化测序PCR引物以及甲基化特异性PCR引物,进行PCR扩增以及测序分析。结果对照组3例和SAD组3例经硫化修饰以及PCR扩增,对照组SORL1基因甲基化率分别为1.5%、1.0%、1.0%,SAD组SORL1基因甲基化率分别为74.0%、70.5%、73.0%。甲基化特异性PCR分析结果显示,SORL1基因在8例健康人中呈完全非甲基化状态,在40例SAD患者中其甲基化阳性例数为21例,甲基化阳性率为52.5%(P<0.05)。结论 SORL1基因启动子区异常甲基化可能参与SAD的发生,为疾病的早期监测提供可能的分子理论依据。     

肺癌患者Ras相关区域家族1A基因启动子异常甲基化的检测

目的探讨肺癌组织和外周血浆、支气管肺泡灌洗液(BALF)中Ras相关区域家族1A(RASSF1A)基因启动子异常甲基化状况及其在肺癌诊断中的价值。方法用甲基化特异PCR方法对肺癌患者癌组织、癌旁组织及相应血浆、BALF进行RASSF1A异常甲基化检测。结果45例肺癌组织中,RASSF1A基因启动子异常甲基化率为53.33%(24/45),相应血浆中RASSF1A的甲基化检出率为28.89%(13/45),BALF检出率为42.22%(19/45),而癌旁组织中的RASSF1A启动子甲基化检出率为13.04%(3/23)、正常对照血浆、非肺癌患者BALF中未检出甲基化,只检出未甲基化的RASSF1A。血浆、BALF中甲基化改变与肿瘤组织甲基化状况显著相关(P<0.01);但与患者年龄、性别、肿瘤大小、恶性程度、肿瘤分类的差异无统计学意义(P>0.05)。结论血浆、BALF中RASSF1A基因异常甲基化改变的检测在肺癌的特异诊断等方面有一定的应用价值。     

Tumor suppressor gene BLU is frequently downregulated by promoter hypermethylation in myelodysplastic syndrome

Purpose  

BLU methylation status was investigated in bone marrow mononuclear cells from newly diagnosed myelodysplastic syndrome (MDS) patients and patients who received 5-aza-2′-deoxycytidine (decitabine) treatment so as to determine the effect of BLU in the pathogenesis of MDS.     

Hemizygous deletion and hypermethylation of RUNX3 gene in hepatocellular carcinoma

AIM：To analyze the genetic and epigenetic alterations of RUNX3 gene, a potential putative tumor suppressor gene,in hepatocellular carcinoma (HCC).METHODS: PCR-based loss of heterozygosity (LOH) detection, analysis of mutation with PCR-single strand conformational polymorphism (SSCP) and sequencing, and methylation study with methylation specific PCR (MSP) were performed on RUNX3 gene in a series of 62 HCCs along with their matched normal tissues.RESULTS:Mutation of RUNX3 gene was not found, but one single nucleotide polymorphism with T to A transversion at the second nucleotide of the 18th condon was found.Nine of 26 informative cases (34.6%) showed allelic loss on the polymorphic site and 30 cases (48.4%) revealed hypermethylation of RUNX3 gene in promoter CpG islands.Furthermore,of the 9 cases with LOH, 8 (88.9%) also had hypermethylation.CONCLUSION:Our findings indicate that inactivation of RUNX3 gene through allelic loss and promoter hypermethylation might be one of the major mechanisms in hepatocellualr carcinogenesis.