肾透明细胞癌中分泌型卷曲相关蛋白1基因甲基化及其表达与亚甲基四氢叶酸还原酶C677T基因多态性的关系

目的探讨人肾透明细胞癌组织中分泌型卷曲相关蛋白1基因（SFRP 1）的甲基化状态及其mRNA表达与亚甲基四氢叶酸还原酶C677T（MTHFR C677T）基因多态性的关系。方法采用PCR-RFLP技术分别检测38例手术切除的肾透明细胞癌区和外周正常肾脏组织标本MTHFR基因677（C→T）多态,并分别以Real-time PCR和和甲基化特异性PCR（MSP）技术检测SFRP 1基因的表达和甲基化状态。结果 SFRP 1基因失表达和/或低表达的肾透明细胞癌组织其启动子区处于高甲基化状态,MTHFR 677CC基因型的肾透明细胞癌组织SFRP 1甲基化升高且其表达降低,SFRP 1的甲基化及其表达与MTHFRC677T基因多态性有明显相关性。结论 MTHFR基因多态性通过影响SFRP 1基因的甲基化状态而调控其表达,可能在肾癌的发生、发展中具有重要作用。     

MTHFR、MS基因多态及NF2基因甲基化与乳腺癌发病关系的研究

目的：本研究探讨MTHFR、MS基因多态性与抑癌基因NF2甲基化及乳腺癌发病的关系。方法：选取原发性乳腺癌患者47例,健康人52例及乳腺良性病变患者15例,采用RT-PCR、DNA甲基化特异性PCR（methylation-sPecific PCR,MSP）聚合酶链反应-限制性片段长度多态性（PCR-RFLP）技术检测NF2的基因表达、甲基化水平及MTHFR、MS基因多态性。结果：NF2基因的表达量在乳腺癌组织（0.16±0.11）及癌旁组织（0.27±0.14）明显低于乳腺良性病变组织（0.64±0.17,P〈0.05）。NF2基因甲基化发生率在乳腺癌组织和癌旁组织中分别为57.4%（27/47）和23.4%（11/47）,两组差异有统计学意义（P〈0.05）。MTHFR677等位基因T型较C等位基因T型在病例组和对照组分布有显著差异（P〈0.05）。携带MTHFR677TT基因型与携带MTHFRCC基因型比较,前者使乳腺癌患病风险增加5.44倍（95%CI,1.91~15.48）。MS2756等位基因G型较等位基因A型在病例组和对照组分布有显著差异（P〈0.05）。携带MS 2756AG基因型与携带MS 2756AA基因型比较,前者使乳腺癌患病风险增加2.73倍（95%CI,1.42~5.24）。MTHFR677多态性与NF2基因甲基化有相关性（χ2=7.29,P〈0.05）。MS2756多态性与NF2基因甲基化无显著关联。结论：NF2甲基化与乳腺癌发生密切相关,MTHFR677、MS2756多态性可以增加乳腺癌的患病风险,MTHFR677多态性可能通过影响部分肿瘤相关基因发生甲基化而调控其表达。     

SFRP1基因启动子甲基化在甲状腺乳头状癌中的表达及意义

目的检测甲状腺乳头状癌(papillary thyroid cancer,PTC)组织中SFRP1基因启动子DNA甲基化状态,分析其与临床特征和生化指标的相关性及对mRNA表达的影响,探究其在临床应用中的价值。方法选择2017年10月至2019年10月在烟台市烟台山医院行手术治疗的97例初发PTC患者作为研究对象,取患者的PTC组织(观察组)和癌旁组织(对照组),采用甲基化特异性PCR(methylation special PCR,MSR)检测组织中SFRP1基因启动子区甲基化状态,采用荧光定量PCR法检测SFRP1基因mRNA表达量,使用甲基转移酶抑制剂5-Azacytidine处理PTC细胞株,且进行生化指标的检测和临床特征的收集。结果PTC组织的SFRP1基因启动子区甲基化阳性率(70.1%)显著高于癌旁组织的甲基化阳性率(33.0%)(χ2=26.747,P<0.001);PTC组织的SFRP1基因mRNA表达量显著低于癌旁组织的mRNA表达量(t=2.886,P=0.007),甲基化阳性PTC组织的mRNA表达量也显著低于甲基化阴性PTC组织mRNA的表达量(t=2.065,P=0.038)。随着5-Azacytidine浓度的增加,CGTHW-3细胞SFRP1基因mRNA表达量也逐渐上升。此外,PTC组织中甲基化阳性率与TNM分期(χ2=5.487,P=0.019)和是否有淋巴结转移(χ2=4.659,P=0.031)有关,且甲基化组的血清TSH和TGAb水平均高于非甲基化组(TSH:t=2.234,P=0.023;TGAb:t=2.312,P=0.021)。结论SFRP1基因启动子区高DNA甲基化可通过下调该基因mRNA表达,参与到PTC的发生、发展中。     

膀胱癌组织中Wnt拮抗因子分泌型卷曲相关蛋白1甲基化和异常表达的意义

目的 探讨Wnt拮抗因子分泌型卷曲相关蛋白1(SFRP1)甲基化和表达在膀胱癌发病机制中的作用. 方法 采用甲基化特异性聚合酶链反应检测人膀胱癌细胞株T24、5637和SCaBER及45例膀胱癌组织和对应癌旁正常组织的SFRP1基因甲基化状态,蛋白质印迹方法检测SFRP1蛋白表达. 结果 SFRP1在膀胱癌细胞株T24和5637中呈现甲基化状态,而SCaBER细胞未检出SFRP1甲基化.将去甲基化试剂5’-氮杂-脱氧胞苷酸(1μg/ml)加入发生甲基化的膀胱癌细胞株T24和5637后,细胞SFRP1 mRNA和蛋白出现表达.45例膀胱癌组织中SFRP1甲基化表型28例(62.2％),癌旁正常组织6例(13.3％),组间频率差异有统计学意义(P＜0.01). 结论 SFRP1甲基化及表达下调可能参与了膀胱癌的发病过程.     

肝细胞癌中SFRP1和APC基因启动子甲基化与其mRNA的表达

目的探讨肝细胞癌（HCC）中SFRP1和APC基因启动子甲基化状态及其mRNA表达的关系。方法应用甲基化特异性聚合酶链反应（MSP）和逆转录-聚合酶链反应（RT—PCR）技术，检测30例肝细胞癌（HCC组）及相应的癌旁组织（癌旁组）和10例正常肝组织（正常对照组）中SFRP1及APC基因启动子甲基化状态和mRNA的表达水平，分析甲基化与某些临床参数与mRNA表达的关系。结果HCC组、癌旁组及正常对照组中的SFRP1和APC基因启动子甲基化率分别是11／30，4／30，0／10和14／30，5／30，0／10；HCC组明显高于其余两组（P〈0．05）。SFRP1基因启动子甲基化与临床资料无关（P〉0．05）；APC基因启动子甲基化与年龄（〈60）岁和癌肿无假包膜有关（P〈0．05）。HCC组SFRP1基因mRNA表达明显低于其余两组（P〈0．05），各组APC基因mRNA表达差异无显著性。两基因甲基化之间无相关性。结论SFRP1和APC基因启动子甲基化与HCC的形成和进展有关，但HCC组织中基因甲基化与mRNA表达的关系尚不明确。     

SPINT2基因启动子甲基化与肾癌预后关系的研究

目的 检测肾癌中SPINT2基因启动子甲基化状态,探讨其与肿瘤特征及患者预后的关系.方法 选取2002～2004年本我院就诊的肾透明细胞癌石蜡组织,用甲基化特异性PCR检测其中SPINT2基因启动子甲基化状态,分析其与肿瘤特征及患者预后的关系.结果 共42例肾透明细胞癌患者密切随访,平均随访39个月(3 ～78个月).其中完全甲基化19例,不完全甲基化13例,无甲基化10例.不同甲基化状态的患者预后存在显著性差异(P=0.042),甲基化与肿瘤分期显著相关(P=0.007).结论 SPINT2基因启动子甲基化在肾透明细胞癌组织中广泛存在,其甲基化状态与肾癌的肿瘤分期和预后密切相关.     

肾透明细胞癌中VHL基因失活的检测及意义

目的 探讨肾透明细胞癌中Von Hippel-Lindau(VHL)基因突变和超甲基化情况及其在肾癌发生发展中的作用及临床意义.方法 采用聚合酶链反应-单链构象多态性分析(PCRSSCP)银染法,甲基化特异性PCR(MSP)及测序等方法对33例肾透明细胞癌和相应远离肿瘤正常肾组织VHL基因突变和超甲基化情况进行检测.结果 19/33(57.6%)例肾透明细胞癌中出现VHL基因突变;6/33(18.2%)例肾透明细胞癌中出现VHL基因超甲基化;VHL基因突变和超甲基化与肾癌临床分期和淋巴结转移相关.结论 肾癌中存在VHL基因突变和超甲基化,且与临床分期、淋巴结转移相关;VHL基因失活的检测可作为肾透明细胞癌的诊断指标,VHL基因可望成为肾透明细胞癌基因治疗的重要目的 基因.     

膀胱癌细胞中DAPK基因启动子甲基化状态分析

目的 分析DAPK抑癌基因在T24、5637、ScaBER三种膀胱癌细胞中的表达情况及该基因启动子区的甲基化状态。方法 采用半定量RT-PCR和Westernblot分别检测三种膀胱癌细胞中DAPK基因的表达，用甲基化特异PCR（MSP）方法检测三种细胞中的DAPK基因启动子甲基化状态。观察甲基转移酶（DMNT）抑制剂5-aza-2’-deoxy．cytidine（5-aza-CdR）对各细胞中的基因表达和甲基化状态的影响。结果 T24细胞中未检测到DAPK基因的表达，也未检测到启动子甲基化；5637细胞中该基因表达水平极低，在SeaBER细胞中该基因的表达较前两者要高。5637细胞和ScaBER细胞中均有甲基化改变。2．5μmol／l浓度的5-aza-CdR可以有效上调5637和ScaBER细胞的DAPK基因的表达。结论 抑癌基因DAPK的表达异常与移行细胞癌的发生发展有重要联系，且基因启动子区CpG岛的异常甲基化在调节DAPK基因的表达中发挥重要作用。     

肾脏特异性高表达基因SLC12A1在肾透明细胞癌中低表达的研究

目的验证肾脏特异性高表达基因SLC12A1在肾透明细胞癌中的低表达,初步研究该基因的表达与肾透明细胞癌发生发展的相关性并探讨其临床意义。方法临床收集23例肾透明细胞癌患者癌组织和癌旁正常组织样本,采用荧光定量PCR、免疫组织化学和Western blot的方法分别检测肾透明细胞癌组织和癌旁正常组织中的SLC12A1基因mRNA和蛋白质的表达水平差异。结果肾透明细胞癌组织中SLC12A1的mRNA和蛋白质表达水平明显低于癌旁正常组织(P0.01)。结论肾透明细胞癌组织中SLC12A1表达量明显降低。该结果对进一步阐明肾透明细胞癌发生、发展的分子机理具有一定的学术意义,可能为肾透明细胞癌的诊断和治疗提供一种新的思路。     

乳腺癌亚甲基四氢叶酸还原酶基因多态性的临床意义

目的：探讨乳腺癌亚甲基四氢叶酸还原酶（MTHFR）基因多态性的临床意义。方法：收集54例乳腺癌女性患者血液样本（研究组）和93例正常女性血液样本（对照组）,均进行DNA提取、PCR扩增、DNA限制性片段长度多态性分析。分析MTHFR C677T、A1298C基因型在乳腺癌女性和正常女性中的分布差异。结果：PCR-RFLP法检测显示,MTHFR基因野生型纯合子CC(198 bp)只有1条带,MTHFR杂合子CT(198 bp、175 bp)产生2条带,MTHFR变异型纯合子TT(175 bp)只有1条带。研究组MTHFR 677CC、677CT和677TT基因型频率分别为37.04%、51.58%和11.11%,677C、677T等位基因频率分别为62.96%、21.51%;对照组MTHFR 677CC、CT和TT基因型频率分别为34.41%、48.39%和17.20%,677C、677T等位基因频率分别为37.04%、41.40%。两组MTHFR C677T基因型频率和等位基因频率比较差异均无统计学意义（P均>0.05）。研究组MTHFR 1298AA、AC、CC基因型频率分别为...     

膀胱癌和肾癌组织中RASSF1A基因的甲基化改变

目的研究RASSF1A基因启动子的甲基化状态及在膀胱癌和肾癌发生中的作用。方法采用甲基化特异性PCR(MSP)技术检测45例手术切除的膀胱癌组织和相应癌旁正常组织标本、9例电切膀胱癌组织、3例非肿瘤患者正常膀胱组织、12例肾癌组织和相应癌旁正常组织标本中RASSF1A基因的甲基化情况。结果膀胱癌组织中RASSF1A基因异常甲基化55.6%(30/54),其中手术切除组57.8%(26/45),电切癌组织异常甲基化4例,癌旁正常组织基因异常甲基化1例;肾癌组织中基因异常甲基化66.7%(8/12),相应癌旁正常组织中未发现基因甲基化。3例正常膀胱组织标本中未发现基因异常甲基化。膀胱癌组织中RASSF1A基因甲基化率有随组织分期升高的趋势,但与临床病理参数间无明显相关性。结论膀胱癌和肾癌组织中RASSF1A基因启动子高度甲基化,表明该基因甲基化可能与2种肿瘤的发生相关。     

Combination of angiotensin-converting enzyme and methylenetetrahydrofolate reductase gene polymorphisms as determinant risk factors for chronic allograft dysfunction

OBJECTIVE: The aim of this study was to investigate the frequency of gene angiotensin-converting enzyme insertion/deletion (ACE I/D) and methylenetetrahydrofolate reductase (MTHFR C677T and A1298C) variants, as well as to evaluate the plasma homocysteine concentrations in 217 patients who underwent renal transplantation at least 12 months prior to define risk factors for chronic allograft dysfunction. METHODS: The presence of the polymorphism ACE deletion was assessed by polymerase chain reaction (PCR) analysis. MTHFR polymorphisms were determined by PCR and restriction fragment length polymorphism (RFPL) techniques. The restriction enzymes were Hinf I and Mbo II for MTHFR variants C677T and A1298C, respectively. Plasma homocysteine concentrations were measured by liquid chromatography-tandem mass spectrometry (LS-MS/MS). RESULTS: Hyperhomocysteinemias were more common in patients with chronic allograft dysfunction (P = .004). No statistically significant differences were observed between the allelic and genotypic distributions of MTHFR and ACE polymorphisms. An effective risk factor was found when the polymorphisms of the ACE and MTHFR genes and hyperhomocysteinemia were associated (odds ratio 2.51; 95% confidence interval 1.19-5.28). In conclusion, our study identified that the presence of hyperhomocysteinemia in combination with unfavorable genotypes contributes to an increased risk for development of chronic allograft dysfunction.     

Effects of the C677T and A1298C polymorphisms of the MTHFR gene on the genetic predisposition for diabetic nephropathy.

BACKGROUND: Methylenetetrahydrofolate reductase (MTHFR) is a regulatory enzyme of homocysteine metabolism. The C677T polymorphism of the MTHFR gene has been reported to be associated with elevated plasma homocysteine in patients with low folic acid intake. A recently reported second common polymorphism, A1298C, may increase homocysteine, but only in individuals carrying the T677 allele. This study aimed to investigate the influence of the C677T and A1298C polymorphisms of the MTHFR gene on the development of diabetic nephropathy in Caucasian patients with type 2 diabetes. METHODS: We genotyped 429 type 2 diabetic patients for the C677T and A1298C polymorphisms using standard PCR-based protocols, and divided them into three groups based on renal status: 159 patients with normoalbuminuria, 149 with microalbuminuria, and 121 with persistent proteinuria and chronic renal failure (CRF). The C677T and A1298C genotype frequencies were compared among the three groups. RESULTS: Although the frequencies of the CT and TT genotypes of the C677T polymorphism tended to increase with each stage of diabetic nephropathy (53, 56 and 63% in normoalbuminuria, microalbuminuria and proteinuria/CRF, respectively), these differences were not significant. When male and female patients were analysed separately, the effect was seen only in males. The CT + TT genotype was present in 46% of male patients with normoalbuminuria, in 57% with microalbuminuria and in 68% with proteinuria/CRF (OR = 2.46; 95% CI 1.13-5.38). There were no differ-ences in the A1298C polymorphism among the three groups. CONCLUSIONS: These findings indicate that the C677T polymorphism is a risk factor for diabetic nephrop-athy in male patients with type 2 diabetes.     

Association of methylenetetrahydrofolate reductase polymorphism and the risk of squamous cell carcinoma in renal transplant patients

The relative risk of developing cutaneous squamous cell carcinoma (SCC) is significantly increased after organ transplantation. We investigated the genetic association of SCC in two pathways associated with cancer risks, with the potential for modification by vitamin supplementation. A total of 367 renal transplant recipients (117 with SCC and 250 without any skin cancer) were genotyped for key polymorphisms in the folate pathway (methylene tetrahydrofolate reductase; MTHFR:C677T), and the vitamin D pathway (vitamin D receptor: Intron8G/T;). Individuals carrying the MTHFR 677T allele had a marked increase in risk of SCC (adjusted odds ratio=2.54, P=0.002, after adjustment for age, ender, skin type, sun exposure score, and immunosuppression duration; lower 95% confidence boundary odds ratio of 1.41). In contrast, vitamin D receptor polymorphisms were not significantly associated. Folate-sensitive pathways may play a critical role in the elevated rate of SCC in renal transplant recipients.     

5-杂氮脱氧胞苷逆转膀胱癌细胞WIF-1基因转录表达的研究

目的 探讨去甲基化药物5-杂氮脱氧胞苷对膀胱癌WIF-1基因表达的影响及其生物学效应.方法 用5-杂氮脱氧胞苷处理膀胱癌细胞株T24和BIU87,RT-PCR检测WIF-1 mRNA表达情况,免疫细胞化学和Western blot检测WIF-1蛋白表达变化,甲基化特异性PCR检测WIF-1启动子甲基化状态变化,MTT法检测5-杂氮脱氧胞苷对膀胱癌细胞的增殖抑制作用,TUNEL法和流式细胞仪检测细胞凋亡率.结果 5-杂氮脱氧胞苷处理72 h后,WIF-1 mRNA及蛋白表达明显增加,而且恢复为启动子未甲基化状态,膀胱癌细胞增殖明显抑制,FCM法检测T24和BIU87的凋亡率分别为(18.2±2.6)%和(17.3±2.7)%.结论 DNA甲基化是导致WIF-1不表达的重要原因,5-杂氮脱氧胞苷可以通过逆转WIF-1甲基化状态而恢复WIF-1表达,从而抑制膀胱癌细胞的生长并诱导细胞凋亡.5-杂氮脱氧胞苷有望成为膀胱癌的有效辅助化疗药.     

DNA损伤修复基因XRCC1启动子甲基化对肝癌易感性及其预后的影响

目的 探讨原发性肝癌组织和正常肝脏组织的DNA损伤修复基因XRCC1启动子甲基化状态,分析其与肝癌易感性的关系及对患者预后的影响.方法 甲基化特异性PCR检测手术切除的肝癌组织78例及正常肝组织78例的XRCC1基因甲基化情况,并随访3年以上.结果 肝癌组的XRCC1启动子甲基化率远高于对照组（P＜0.05）,肝癌组发生XRCC1启动子甲基化的危险是对照组的13倍（4.089 vs 41.332）;XRCC1启动子甲基化的个体发生肝癌的危险是非甲基化的10.36倍（3.423 vs 31.354）（P＜0.05）;XRCC1启动子甲基化可致肝癌患者无进展期生存率和总体生存率降低（P＜ 0.05）.结论 DNA损伤修复基因XRCC1启动子甲基化在肝癌发生和发展的过程中起着重要作用,并对肝癌患者较差的预后具有提示作用.     

The C677T methylenetetrahydrofolate reductase gene mutation does not influence cardiovascular risk in the dialysis population: results of a multicentre prospective study.

BACKGROUND: Although the methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism has been identified as an independent cardiovascular risk factor (CRF) in the general population and among uraemic subjects, the validity of this association remains controversial. METHODS: To verify this hypothesis, we enrolled all subjects on maintenance dialysis treatment from a specific Italian district. We also enrolled, from the same area, 1307 subject to serve as controls. Genomic DNA was obtained and MTHFR C677T gene polymorphisms were determined. After a baseline evaluation, patients were followed-up for 37+/-13 months, and all cardiovascular events and causes of mortality were recorded. RESULTS: A total of 461 patients (417 on haemodialysis and 44 on peritoneal dialysis) were investigated, and these included patients with and without cardiovascular diseases at baseline. At enrollment, mean age was 58.8+/-15.6 years and dialytic age was 82+/-69 months. Genotype frequencies were not different between controls and uraemics. During the follow-up, the mean mortality rate was 8.81%/year, with cardiovascular events as the most frequent cause of death (n = 68, 56.6%). There was no relationship between the MTHFR genotype and cardiovascular morbidity, overall mortality or cardiovascular mortality. CONCLUSIONS: In end-stage renal disease, MTHFR C677T polymorphisms were not associated with cardiovascular disease or mortality.     

组织因子途径抑制物-2在肾癌组织中的表达及甲基化

目的 探讨组织因子途径抑制物-2(TFPI-2)对肾癌侵袭性及细胞凋亡的影响,TFPI-2在肾癌组织中的表达与基因启动子甲基化关系.方法 免疫组织化学、蛋白质印迹、荧光定量RT-PCR检测37例肾癌及11例正常肾脏组织TFPI-2基因表达;TUNEL法检测细胞凋亡;荧光定量甲基化特异性PCR检测TFPI-2基因启动子甲基化.结果肾癌与正常肾脏组TFPI-2蛋白质印迹条带吸光度(A)值分别为0.92±0.36、1.61±0.13;2组TFPI-2 mRNA相对表达量为0.0019±0.0011、0.0065±0.0008.随肾癌分期增加,TFPI-2表达下降.肾癌TFPI-2表达1～4级细胞凋亡指数分别为2.41%、3.90%、6.78%、9.57%.随TFPI-2表达增加,肾癌细胞凋亡指数上升.肾癌TFPI-2基因启动子甲基化阳性组与阴性组TFPI-2 mRNA相对表达量分别为0.0015±0.0011、0.0024±0.0009,2组蛋白质印迹条带A值为0.82±0.35、1.04±0.34.基因启动子甲基化阳性组TFPI-2表达低于阴性组.结论 肾癌TFPI-2基因表达与侵袭性呈负相关,促进细胞凋亡;启动子高甲基化是肾癌TFPI-2基因低表达机制之一.     

肝细胞癌中WIF-1基因启动子甲基化状态及mRNA表达的研究

目的 探讨细胞癌中WIF-1基因启动子甲基化状态及其对mRNA表达的影响。方法 应用甲基化特异性聚合酶链反应和实时定量逆转录-聚合酶链反应技术,检测33例肝细胞癌及相应的癌旁组织和20例正常对照肝组织中WIF-1基因启动子甲基化状态和mRNA表达水平,分析上述组织WIF-1基因甲基化及mRN表达与临床资料的关系并分析甲基化对mRNA表达的影响。结果 WIF-1基因在癌组织中甲基化率明显高于癌旁组织和正常组织（Х^2=4．89,P〈0．05）,而癌旁组织和正常组织中WIF-1基因甲基化率相比无明显差异;癌组织中WIF-1基因甲基化与乙肝表面抗原、肝硬化有关;WIF-1基因mRNA表达量在三种组织中比较无明显差异。结论 WIF-1基因启动子甲基化与HCC的发生相关;WIF-1基因启动子甲基化与其mRNA表达的关系有待进一步研究。