目的探讨子痫前期孕妇血浆中超甲基化的Ras相关区域家族1A（ras association domain family 1A，RASSF1A）基因的水平变化及应用价值。方法选择晚期妊娠子痫前期患者60例为实验组，其中轻度和重度子痫前期各30例；正常晚期孕妇60名为对...目的探讨子痫前期孕妇血浆中超甲基化的Ras相关区域家族1A（ras association domain family 1A，RASSF1A）基因的水平变化及应用价值。方法选择晚期妊娠子痫前期患者60例为实验组，其中轻度和重度子痫前期各30例；正常晚期孕妇60名为对照组。提取血浆游离DNA，经甲基化敏感的限制性内切酶处理，实时定量检测酶切前、后RASSF1A基因的浓度，同时检测β肌动蛋白（β-actin）基因以确保酶的完全消化。结果子痫前期孕妇血浆中超甲基化的RASSF1A基因含量为正常妊娠组的3．31倍。轻、重度子痫前期患者浓度有差异，中位数分别为1659．00copies／mL及2036．50copies／mL（P〈0．05）。结论子痫前期孕妇血浆中的超甲基化RASSF1A基因含量明显升高，且浓度水平与子痫前期的严重程度相关。展开更多
目的:探讨微小RNA-9-5p(mi R-9-5p)调控肿瘤高甲基化基因1(hypermethylated in cancer 1,HIC1)对乳腺癌MDA-MB-231细胞对化疗药多柔比星敏感性的影响及其作用机制。方法:(1)使用慢病毒转染建立上调或下调mi R-9-5p细胞,加入不同浓度多...目的:探讨微小RNA-9-5p(mi R-9-5p)调控肿瘤高甲基化基因1(hypermethylated in cancer 1,HIC1)对乳腺癌MDA-MB-231细胞对化疗药多柔比星敏感性的影响及其作用机制。方法:(1)使用慢病毒转染建立上调或下调mi R-9-5p细胞,加入不同浓度多柔比星后,用CCK-8和流式细胞仪检测细胞活力和凋亡情况。(2)通过反转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)和蛋白质印迹试验检测上调或下调miR-9-5p时HIC1的表达情况。通过数据库资料、双荧光素酶报告基因系统验证miR-9-5p与HIC1的关系。(3)下调HIC1,观察加入多柔比星后细胞活力和凋亡情况。结果:(1)上调miR-9-5p可提高细胞活力、抑制凋亡。下调mi R-9-5p可降低细胞活力、促进凋亡。(2)miR-9-5p靶向HIC1,两者的表达呈负相关。(3)抑制HIC1的表达,可逆转miR-9-5p下调的作用。结论:miR-9-5p可通过下调HIC1降低乳腺癌细胞对多柔比星的敏感性。展开更多
目的:通过建立血浆游离DNA中肿瘤高甲基化基因1(hypermethylated in cancer 1, HIC-1)甲基化检测技术,评估其作为体液活检诊断方法在乳腺良恶性疾病鉴别中的价值。方法:收集35份乳腺疾病患者(其25例乳腺癌,10例乳腺良性疾病)及10份健康...目的:通过建立血浆游离DNA中肿瘤高甲基化基因1(hypermethylated in cancer 1, HIC-1)甲基化检测技术,评估其作为体液活检诊断方法在乳腺良恶性疾病鉴别中的价值。方法:收集35份乳腺疾病患者(其25例乳腺癌,10例乳腺良性疾病)及10份健康志愿者的血液样本,分离、提取血浆中的游离DNA。采用亚硫酸氢盐测序(bisulfite sequencing PCR,BSP)法,检测HIC-1基因启动子区域-636～-424 bp中16个CpG位点的甲基化水平。结果:在检测的16个CpG位点中,以-636～-617 bp中的4个位点甲基化最为明显。其中,乳腺癌组的这4个位点平均甲基化率为22.6%,而良性乳腺疾病组为8.5%,健康对照组为8.3%,3组间的差异有统计学意义(P<0.05)。进一步绘制受试者工作特征曲线(receiver operator characteristic curve, ROC曲线)进行分析,发现以这4个位点的平均甲基化率诊断乳腺癌时,其曲线下面积(area under the cure AUC)为0.794,证实其对乳腺癌具有一定的诊断价值。结论:检测血浆游离DNA中抑癌基因HIC-1启动子区甲基化水平,在乳腺癌诊断中有一定参考价值。展开更多
目的 :探讨抑癌基因肿瘤高甲基化基因1(hypermethylated in cancer 1,HIC-1)和致癌基因透明质酸介导的细胞游走受体(hyaluronan-mediated motility receptor,HMMR)在乳腺小叶增生、乳腺纤维腺瘤和乳腺癌组织中的表达情况,及其与乳腺癌...目的 :探讨抑癌基因肿瘤高甲基化基因1(hypermethylated in cancer 1,HIC-1)和致癌基因透明质酸介导的细胞游走受体(hyaluronan-mediated motility receptor,HMMR)在乳腺小叶增生、乳腺纤维腺瘤和乳腺癌组织中的表达情况,及其与乳腺癌临床病理参数间的关系。方法:应用免疫组织化学方法检测57例患者的乳腺手术切除标本(其中乳腺小叶增生17例,乳腺纤维腺瘤及乳腺癌各20例)中HIC-1与HMMR蛋白的表达情况,观察其染色结果以半定量分析,并结合临床病理学指标进行相关分析。结果:(1)乳腺小叶增生和乳腺纤维腺瘤组织中HIC-1蛋白表达呈中度阳性(平均计分均为5.65分);而乳腺癌组织中HIC-1表达减弱,呈弱阳性(平均计分为2.15分),二者间差异有统计学意义(P<0.001)。(2)乳腺小叶增生和纤维腺瘤组织中的HMMR均呈低表达(其中纤维腺瘤平均计分为0.3分,小叶增生平均积分为0),而乳腺癌组织中HMMR表达呈弱阳性(平均计分为2.3分),二者间差异有统计学意义(P<0.001)。(3)HIC-1及HMMR蛋白的表达与乳腺癌相关临床病理特征之间未见明显关联。结论:抑癌基因HIC-1蛋白在乳腺癌组织中呈明显低表达,而致癌基因HMMR在乳腺癌组织中呈较高表达,HIC-1及受HIC-1调控的下游基因HMMR可能与乳腺恶性肿瘤的发生、发展有关。展开更多
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 obtai...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.展开更多
AIM： To identify the relationship between DNA hyper- methylation and histone modification at a hyperme- thylated, silenced tumor suppressor gene promoter in human gastric cancer cell lines and to elucidate whether al...AIM： To identify the relationship between DNA hyper- methylation and histone modification at a hyperme- thylated, silenced tumor suppressor gene promoter in human gastric cancer cell lines and to elucidate whether alteration of DNA methylation could affect histone modification. METHODS： We used chromatin immunoprecipitation （CHIP） assay to assess the status of histone acetylation and methylation in promoter regions of the p16 and rnutL homolog 1 （MLH1） genes in 2 gastric cancer cell lines, SGC-7901 and MGC-803. We used methylation- specific PCR （MSP） to evaluate the effect of 5-Aza-2＇- deoxycytidine （5-Aza-dC）, trichostatin A （TSA） or their combination treatment on DNA methylation status. We used RT-PCR to determine whether alterations of histone modification status after 5-Aza-dC and TSA treatment are reflected in gene expression. RESULTS： For thep16 and MLH1 genes in two cell lines, silenced loci associated with DNA hypermethylation were characterized by histone H3-K9 hypoacetylation and hypermethylation and histone H3-K4 hypomethylation. Treatment with TSA resulted in moderately increased histone H3-K9 acetylation at the silenced loci with no effect on histone H3-K9 methylation and minimal effects on gene expression. In contrast, treatment with 5-Aza- dC rapidly reduced histone H3-K9 methylation at the silenced loci and resulted in reactivation of the two genes. Combined treatment with 5-Aza-dC and TSA was synergistic in reactivating gene expression at the loci showing DNA hypermethylation. Similarly, histone H3-K4 methylation was not affected alter TSA treatment, andincreased moderately at the silenced loci after 5-Aza-dC treatment. CONCLUSION： Hypermethylation of DNA in promoter CpG islands is related to transcriptional silencing of tumor suppressor genes. Histone H3-K9 methylation in different regions of the promoters studied correlates with DNA methylation status of each gene in gastric cancer cells. However, histone H3-K9 acetylation and H3-K4 methylation inversely correlate 展开更多
AIM：To evaluate the methylation status of CDH1, FHIT, MTAP and PLAGL1 promoters and the association of these findings with clinico-pathological characteristics.METHODS： Methylation-specific PCR （MSP） assay was per...AIM：To evaluate the methylation status of CDH1, FHIT, MTAP and PLAGL1 promoters and the association of these findings with clinico-pathological characteristics.METHODS： Methylation-specific PCR （MSP） assay was performed in 13 nonneoplastic gastric adenocarcinorna, 30 intestinal-type gastric adenocarcinorna and 35 diffuse-type gastric adenocarcinorna samples from individuals in Northern Brazil. Statistical analyses were performed using the chi-square or Fisher＇s exact test to assess associations between rnethylation status and clinico-pathological characteristics.RESULTS： Hypermethylation frequencies of CDH1, FHIT, MTAPand PLAGL1 promoter were 98.7%, 53.9%, 23.1% and 29.5%, respectively. Hyperrnethylation of three or four genes revealed a significant association with diffuse-type gastric cancer compared with nonneoplastic cancer. A higher hyperrnethylation frequency was significantly associated with H pylori infection in gastric cancers, especially with diffuse-type. Cancer samples without lymph node metastasis showed a higher FHIT hypermethylation frequency. MTAP hypermethylation was associated with H pylori in gastric cancer samples, as well as with diffuse-type compared with intestinal-type. In diffuse-type, MTAP hypermethylation was associated with female gender.CONCLUSION： Our findings show differential gene methylation in tumoral tissue, which allows us to conclude that hypermethylation is associated with gastric carcinogenesis. MTAP promoter hypermethylation can be characterized as a marker of diffuse-type gastric cancer, especially in women and may help in diagnosis, prognosis and therapies. The H pylori infectious agent was present in 44.9% of the samples. This infection may be correlated with the carcinogenic process through the gene promoter hypermethylation, especially the MTAP promoter in diffuse-type. A higher H pylori infection in diffuse-type may be due to greater genetic predisposition.展开更多
AIM:To investigate the relation between RECK methylation and clinicopathological characteristics of gastric cancer patients and evaluate the role of RECK methylation in peritoneal metastasis of gastric cancer.METHODS:...AIM:To investigate the relation between RECK methylation and clinicopathological characteristics of gastric cancer patients and evaluate the role of RECK methylation in peritoneal metastasis of gastric cancer.METHODS:Methylation of RECK gene in 40 paired samples of gastric cancer and its corresponding adjacent normal mucosa,lymph nodes and peritoneal irrigation fluid was detected by methylation-specific polymerase chain reaction.RESULTS:Aberrant methylation of RECK gene was detected in 27.5%(11/40)of the adjacent normal mucosa samples,in 47.5%(19/40)of gastric cancer samples,in 57.1%(12/21)of the lymph node samples,and in 35%(14/40)of peritoneal irrigation fluid samples,respectively,with a significant difference between the adjacent normal mucosa and lymph node samples(P=0.023).Presence of RECK methylation in the primary tumor samples was significantly correlated with tumor invasion(P=0.023).The accuracy of RECK methylation in peritoneal lavage fluid samples for the diagnosis of peritoneal metastasis of gastric cancer was 72.5%(26/40),with a sensitivity of 66.7%(6/9) and a specificity of 74.2%(23/31).CONCLUSION:Aberrant methylation of RECK gene may provide useful information for the early diagnosis and treatment of peritoneal metastasis of gastric cancer.展开更多
文摘目的探讨子痫前期孕妇血浆中超甲基化的Ras相关区域家族1A（ras association domain family 1A，RASSF1A）基因的水平变化及应用价值。方法选择晚期妊娠子痫前期患者60例为实验组，其中轻度和重度子痫前期各30例；正常晚期孕妇60名为对照组。提取血浆游离DNA，经甲基化敏感的限制性内切酶处理，实时定量检测酶切前、后RASSF1A基因的浓度，同时检测β肌动蛋白（β-actin）基因以确保酶的完全消化。结果子痫前期孕妇血浆中超甲基化的RASSF1A基因含量为正常妊娠组的3．31倍。轻、重度子痫前期患者浓度有差异，中位数分别为1659．00copies／mL及2036．50copies／mL（P〈0．05）。结论子痫前期孕妇血浆中的超甲基化RASSF1A基因含量明显升高，且浓度水平与子痫前期的严重程度相关。
文摘目的:探讨微小RNA-9-5p(mi R-9-5p)调控肿瘤高甲基化基因1(hypermethylated in cancer 1,HIC1)对乳腺癌MDA-MB-231细胞对化疗药多柔比星敏感性的影响及其作用机制。方法:(1)使用慢病毒转染建立上调或下调mi R-9-5p细胞,加入不同浓度多柔比星后,用CCK-8和流式细胞仪检测细胞活力和凋亡情况。(2)通过反转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)和蛋白质印迹试验检测上调或下调miR-9-5p时HIC1的表达情况。通过数据库资料、双荧光素酶报告基因系统验证miR-9-5p与HIC1的关系。(3)下调HIC1,观察加入多柔比星后细胞活力和凋亡情况。结果:(1)上调miR-9-5p可提高细胞活力、抑制凋亡。下调mi R-9-5p可降低细胞活力、促进凋亡。(2)miR-9-5p靶向HIC1,两者的表达呈负相关。(3)抑制HIC1的表达,可逆转miR-9-5p下调的作用。结论:miR-9-5p可通过下调HIC1降低乳腺癌细胞对多柔比星的敏感性。
文摘目的:通过建立血浆游离DNA中肿瘤高甲基化基因1(hypermethylated in cancer 1, HIC-1)甲基化检测技术,评估其作为体液活检诊断方法在乳腺良恶性疾病鉴别中的价值。方法:收集35份乳腺疾病患者(其25例乳腺癌,10例乳腺良性疾病)及10份健康志愿者的血液样本,分离、提取血浆中的游离DNA。采用亚硫酸氢盐测序(bisulfite sequencing PCR,BSP)法,检测HIC-1基因启动子区域-636～-424 bp中16个CpG位点的甲基化水平。结果:在检测的16个CpG位点中,以-636～-617 bp中的4个位点甲基化最为明显。其中,乳腺癌组的这4个位点平均甲基化率为22.6%,而良性乳腺疾病组为8.5%,健康对照组为8.3%,3组间的差异有统计学意义(P<0.05)。进一步绘制受试者工作特征曲线(receiver operator characteristic curve, ROC曲线)进行分析,发现以这4个位点的平均甲基化率诊断乳腺癌时,其曲线下面积(area under the cure AUC)为0.794,证实其对乳腺癌具有一定的诊断价值。结论:检测血浆游离DNA中抑癌基因HIC-1启动子区甲基化水平,在乳腺癌诊断中有一定参考价值。
文摘目的 :探讨抑癌基因肿瘤高甲基化基因1(hypermethylated in cancer 1,HIC-1)和致癌基因透明质酸介导的细胞游走受体(hyaluronan-mediated motility receptor,HMMR)在乳腺小叶增生、乳腺纤维腺瘤和乳腺癌组织中的表达情况,及其与乳腺癌临床病理参数间的关系。方法:应用免疫组织化学方法检测57例患者的乳腺手术切除标本(其中乳腺小叶增生17例,乳腺纤维腺瘤及乳腺癌各20例)中HIC-1与HMMR蛋白的表达情况,观察其染色结果以半定量分析,并结合临床病理学指标进行相关分析。结果:(1)乳腺小叶增生和乳腺纤维腺瘤组织中HIC-1蛋白表达呈中度阳性(平均计分均为5.65分);而乳腺癌组织中HIC-1表达减弱,呈弱阳性(平均计分为2.15分),二者间差异有统计学意义(P<0.001)。(2)乳腺小叶增生和纤维腺瘤组织中的HMMR均呈低表达(其中纤维腺瘤平均计分为0.3分,小叶增生平均积分为0),而乳腺癌组织中HMMR表达呈弱阳性(平均计分为2.3分),二者间差异有统计学意义(P<0.001)。(3)HIC-1及HMMR蛋白的表达与乳腺癌相关临床病理特征之间未见明显关联。结论:抑癌基因HIC-1蛋白在乳腺癌组织中呈明显低表达,而致癌基因HMMR在乳腺癌组织中呈较高表达,HIC-1及受HIC-1调控的下游基因HMMR可能与乳腺恶性肿瘤的发生、发展有关。
基金A grant offered by Mashhad University of Medical Sciences, No. 84129
文摘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.
基金National Natural Science Foundation of China,No.30271477,No.30572162
文摘AIM： To identify the relationship between DNA hyper- methylation and histone modification at a hyperme- thylated, silenced tumor suppressor gene promoter in human gastric cancer cell lines and to elucidate whether alteration of DNA methylation could affect histone modification. METHODS： We used chromatin immunoprecipitation （CHIP） assay to assess the status of histone acetylation and methylation in promoter regions of the p16 and rnutL homolog 1 （MLH1） genes in 2 gastric cancer cell lines, SGC-7901 and MGC-803. We used methylation- specific PCR （MSP） to evaluate the effect of 5-Aza-2＇- deoxycytidine （5-Aza-dC）, trichostatin A （TSA） or their combination treatment on DNA methylation status. We used RT-PCR to determine whether alterations of histone modification status after 5-Aza-dC and TSA treatment are reflected in gene expression. RESULTS： For thep16 and MLH1 genes in two cell lines, silenced loci associated with DNA hypermethylation were characterized by histone H3-K9 hypoacetylation and hypermethylation and histone H3-K4 hypomethylation. Treatment with TSA resulted in moderately increased histone H3-K9 acetylation at the silenced loci with no effect on histone H3-K9 methylation and minimal effects on gene expression. In contrast, treatment with 5-Aza- dC rapidly reduced histone H3-K9 methylation at the silenced loci and resulted in reactivation of the two genes. Combined treatment with 5-Aza-dC and TSA was synergistic in reactivating gene expression at the loci showing DNA hypermethylation. Similarly, histone H3-K4 methylation was not affected alter TSA treatment, andincreased moderately at the silenced loci after 5-Aza-dC treatment. CONCLUSION： Hypermethylation of DNA in promoter CpG islands is related to transcriptional silencing of tumor suppressor genes. Histone H3-K9 methylation in different regions of the promoters studied correlates with DNA methylation status of each gene in gastric cancer cells. However, histone H3-K9 acetylation and H3-K4 methylation inversely correlate
基金Supported by Fundao de Amparo à Pesquisa do Estado de So Paulo, Coordenao de Aperfeioamento de Pessoal de Nível Superior and Conselho Nacional de Desenvolvimento Científico e Tecnológico
文摘AIM：To evaluate the methylation status of CDH1, FHIT, MTAP and PLAGL1 promoters and the association of these findings with clinico-pathological characteristics.METHODS： Methylation-specific PCR （MSP） assay was performed in 13 nonneoplastic gastric adenocarcinorna, 30 intestinal-type gastric adenocarcinorna and 35 diffuse-type gastric adenocarcinorna samples from individuals in Northern Brazil. Statistical analyses were performed using the chi-square or Fisher＇s exact test to assess associations between rnethylation status and clinico-pathological characteristics.RESULTS： Hypermethylation frequencies of CDH1, FHIT, MTAPand PLAGL1 promoter were 98.7%, 53.9%, 23.1% and 29.5%, respectively. Hyperrnethylation of three or four genes revealed a significant association with diffuse-type gastric cancer compared with nonneoplastic cancer. A higher hyperrnethylation frequency was significantly associated with H pylori infection in gastric cancers, especially with diffuse-type. Cancer samples without lymph node metastasis showed a higher FHIT hypermethylation frequency. MTAP hypermethylation was associated with H pylori in gastric cancer samples, as well as with diffuse-type compared with intestinal-type. In diffuse-type, MTAP hypermethylation was associated with female gender.CONCLUSION： Our findings show differential gene methylation in tumoral tissue, which allows us to conclude that hypermethylation is associated with gastric carcinogenesis. MTAP promoter hypermethylation can be characterized as a marker of diffuse-type gastric cancer, especially in women and may help in diagnosis, prognosis and therapies. The H pylori infectious agent was present in 44.9% of the samples. This infection may be correlated with the carcinogenic process through the gene promoter hypermethylation, especially the MTAP promoter in diffuse-type. A higher H pylori infection in diffuse-type may be due to greater genetic predisposition.
基金Supported by National Natural Science Foundation of China,No.30572162the Foundation of Education Bureau of Liaoning Province,China,No.2008S240
文摘AIM:To investigate the relation between RECK methylation and clinicopathological characteristics of gastric cancer patients and evaluate the role of RECK methylation in peritoneal metastasis of gastric cancer.METHODS:Methylation of RECK gene in 40 paired samples of gastric cancer and its corresponding adjacent normal mucosa,lymph nodes and peritoneal irrigation fluid was detected by methylation-specific polymerase chain reaction.RESULTS:Aberrant methylation of RECK gene was detected in 27.5%(11/40)of the adjacent normal mucosa samples,in 47.5%(19/40)of gastric cancer samples,in 57.1%(12/21)of the lymph node samples,and in 35%(14/40)of peritoneal irrigation fluid samples,respectively,with a significant difference between the adjacent normal mucosa and lymph node samples(P=0.023).Presence of RECK methylation in the primary tumor samples was significantly correlated with tumor invasion(P=0.023).The accuracy of RECK methylation in peritoneal lavage fluid samples for the diagnosis of peritoneal metastasis of gastric cancer was 72.5%(26/40),with a sensitivity of 66.7%(6/9) and a specificity of 74.2%(23/31).CONCLUSION:Aberrant methylation of RECK gene may provide useful information for the early diagnosis and treatment of peritoneal metastasis of gastric cancer.
