AIM:To investigate aberrant DNA methylation of CpG islands and subsequent low-or high-level DNA microsatellite instability(MSI)which is assumed to drive colon carcinogenesis. METHODS:DNA of healthy individuals,adenoma...AIM:To investigate aberrant DNA methylation of CpG islands and subsequent low-or high-level DNA microsatellite instability(MSI)which is assumed to drive colon carcinogenesis. METHODS:DNA of healthy individuals,adenoma(tu-bular or villous/tubulovillous)patients,and colorectal carcinoma patients who underwent colonoscopy was used for assessing the prevalence of aberrant DNA methylation of human DNA mismatch repair gene mutator L homologue 1(hMLH1),Cyclin-dependent kinase inhibitor 2A(CDKN2A/p16),and O-6-methylguanine DNA methyltransferase(MGMT),as well as their rela- tion to MSI. RESULTS:The frequency of promoter methylation for each locus increased in the sequence healthy tissue/adenoma/carcinoma.MGMT showed the highest frequency in each group.MGMT and CDKN2A/p16 presented a statistically significant increase in promoter methylation between the less and more tumorigenic forms of colorectal adenomas(tubular vs tubullovillous and villous adenomas).All patients with tubulovillous/villous adenomas,as well as all colorectal cancer patients,showed promoter methylation in at least one of the examined loci.These findings suggest a potentially crucial role for methylation in the polyp/adenoma to cancer progres- sion in colorectal carcinogenesis.MSI and methylation seem to be interdependent,as simultaneous hMLH1, CDKN2A/p16,and MGMT promoter methylation was present in 8/9 colorectal cancer patients showing the MSI phenotype. CONCLUSION:Methylation analysis of hMLH1,CD- KN2A/p16,and MGMT revealed specific methylation profiles for tubular adenomas,tubulovillous/villous adenomas,and colorectal cancers,supporting the use of these alterations in assessment of colorectal tumorigenesis.展开更多
BACKGROUND The integrinβ6 gene,which is expressed in epithelial cancer,plays a pivotal role in various aspects of cancer progression.The present research for integrinβ6 regulation mainly focuses on the post-transcri...BACKGROUND The integrinβ6 gene,which is expressed in epithelial cancer,plays a pivotal role in various aspects of cancer progression.The present research for integrinβ6 regulation mainly focuses on the post-transcription and translation related regulation mechanism and its role in tumorigenesis.The mechanisms of how the integrinβ6 gene is regulated transcriptionally,and the promoter and transcription factors responsible for basic transcription of integrinβ6 gene remain unknown.AIM To clone and characterize the integrinβ6 promoter.METHODS Software analysis was used to predict the region of integrinβ6 promoter.Luciferase reporter plasmids,which contained the integrinβ6 promoter,were constructed.Element deletion analysis was performed to identify the location of core promoter and binding sites for transcription factors.RESULTS The regulatory elements for the transcription of the integrinβ6 gene were located between-286 and-85 and contained binding sites for transcription factors such as STAT3 and Ets-1.CONCLUSION For the first time,we found the region ofβ6 core promoter and demonstrated the binding sites for transcription factors such as Ets-1 and STAT3,which are important for integrinβ6 promoter transcription activity.These findings are important for investigating the mechanism of integrinβ6 activation in cancer progression.展开更多
In order to analyze the Oslbglu4 phenotype, the inducible promoter of the transgenic rice which knock-down the Oslbglu4 expression was assessed. The result showed that 30 μM dexamethasone（DEX） had the stronger indu...In order to analyze the Oslbglu4 phenotype, the inducible promoter of the transgenic rice which knock-down the Oslbglu4 expression was assessed. The result showed that 30 μM dexamethasone（DEX） had the stronger induction effect than 10 μM DEX by β-Glucuronidase （GUS） staining, qRT-PCR further verified the Oslbglu4 gene deletion. The effect of DEX and its solvent absolute ethanol on seed development was measured, and no significant effect was observed. The conclusion is that final concentration of DEX at 30 μM is suitable for pOp6 promoter induction.展开更多
The P19CL6 mouse embryonic carcinoma cells efficiently differentiate into cardiac muscle cells in the presence of DMSO. A reporter plasmid for cardiac muscle differentiation was constructed by connecting the CMV enhan...The P19CL6 mouse embryonic carcinoma cells efficiently differentiate into cardiac muscle cells in the presence of DMSO. A reporter plasmid for cardiac muscle differentiation was constructed by connecting the CMV enhancer and a 250 bp MLC-2v promoter in front of the GFP gene to further evaluate the role of the CMV enhancer. This plasmid (pCBVenh/MLC-2vpro/EGFP) was stably introduced into P19CL6 cells, and the transfectant differentiated into cardiomyocytes with DMSO. Upon DMSO addition, GFP was immediately transcribed (within 2 days) and the amount of the transcript increased with cultivation. Concomitantly, GFP fluorescence was detected in the cells under a microscope. However, native MLC-2v was transcribed later on day 4. This expression time course is different from that of GFP. Clearly the CMV enhancer responded immediately to DMSO. Since GATA DNA-binding proteins play crucial roles in the initiation of cardiomyocyte differentiation, such a response could be ascribed to the presence of multiple GATA motifs in the enhancer sequence but not in the native MLC-2v promoter. Thus the CMV enhancer may be not only useful for gene therapy and monitoring cell differentiation but also the study of the role of GATA transcription factors expressed in P19CL6 cells.展开更多
Nanog protein is expressed in the interior cells of compacted morulae and maintained till epiblasts but downregulated by implantation stage. It is also expressed in embryonic stem cells, embryonic carcinoma cells and ...Nanog protein is expressed in the interior cells of compacted morulae and maintained till epiblasts but downregulated by implantation stage. It is also expressed in embryonic stem cells, embryonic carcinoma cells and embryonic germ cells but disappeared in differentiated ES cells. In this study, we have isolated, sequenced, and performed the first characterization of the Nanog promoter. The transcription start sites were mapped by primer extension analysis. Two promoter regions were found upstream the transcription start sites and the expression of major Nanog promoter/ reporter gene construct is abolished in differentiated F9 EC cells as compared to the undifferentiated counterpart. We also showed that a putative octamer motif (ATGCAAAA) is necessary for the major promoter activity. Gel shift and supershift assays showed that Oct-1, Oct-4 and Oct-6 protein selectively bind to the octamer motif.展开更多
O6-methylguanine DNA methyltransferase(MGMT), a DNA repair enzyme, has been reported in some congenital malformations, but it is less frequently reported in neural tube defects. This study investigated MGMT mRNA expre...O6-methylguanine DNA methyltransferase(MGMT), a DNA repair enzyme, has been reported in some congenital malformations, but it is less frequently reported in neural tube defects. This study investigated MGMT mRNA expression and methylation levels in the early embryo and in different embryonic stages, as well as the relationship between MGMT and neural tube defects. Spina bifida aperta was induced in rats by a single intragastric administration of all-trans retinoic acid on embryonic day(E) 10, whereas normal control rats received the same amount of olive oil on the same embryonic day. DNA damage was assessed by detecting γ-H2 A.X in spina bifida aperta rats. Real time-polymerase chain reaction was used to examine mRNA expression of MGMT in normal control and spina bifida aperta rats. In normal controls, the MGMT mRNA expression decreased with increasing embryonic days, and was remarkably reduced from E11 to E14, reaching a minimum at E18. In the spina bifida aperta model, γ-H2 A.X protein expression was increased, and mRNA expression of MGMT was markedly decreased on E14, E16, and E18. Bisulfite sequencing polymerase chain reaction for MGMT promoter methylation demonstrated that almost all CpG sites in the MGMT promoter remained unmethylated in both spina bifida aperta rats and normal controls, and there was no significant difference in methylation level between the two groups on either E14 or E18. Our results show that DNA damage occurs in spina bifida aperta rats. The mRNA expression of MGMT is downregulated, and this downregulation is independent of promoter DNA methylation.展开更多
Recently, redox factor-1 (Ref-1) has received considerable attention as an enzyme for stimulating tumor cell growth. We hypothesized that Ref-1 is upregulated during the early stage of tumorigenesis. Utilizing both tu...Recently, redox factor-1 (Ref-1) has received considerable attention as an enzyme for stimulating tumor cell growth. We hypothesized that Ref-1 is upregulated during the early stage of tumorigenesis. Utilizing both tumor promotion sensitive P+ and promotion resistant P? mouse skin epidermal JB6 cells, we found that Ref-1 expression was only induced in tumor promotion sensitive P+ cells following TPA treatment. Consistent with that, Ref-1 knockdown suppressed skin cell transformation. Interestingly, Ref-1 knockdown suppressed nuclear translocation of NF-kappaB subunit p65, and inhibited production of proinflammatory cytokines. These results suggest Ref-1 may promote early tumorigenesis and serve as a target for chemoprevention.展开更多
目的:探讨中国人群中IL-6基因启动子中单核苷酸多态性与HBV感染的遗传易感性关联.方法:提取160例HBV感染者及212例健康献血者外周血基因组DNA,用PCR-RFLP方法检测两组人群中G-174C、G-572C和G-597A三个多态性位点的基因型.性别、吸烟、...目的:探讨中国人群中IL-6基因启动子中单核苷酸多态性与HBV感染的遗传易感性关联.方法:提取160例HBV感染者及212例健康献血者外周血基因组DNA,用PCR-RFLP方法检测两组人群中G-174C、G-572C和G-597A三个多态性位点的基因型.性别、吸烟、饮酒以及Hardy-Weinberg等采用Chi-square Test检测,多态性与HBV感染者风险关联及亚组关联的统计学分析采用非条件Logistic回归并同时校正混杂因素.结果:PCR-RFLP检测结果显示:IL-6基因启动子中G-174C和G-597A两个位点在中国人群中不存在多态性,而G-572C处的多态性在人群中普遍存在,其多态性位点等位基因频率分布符合Hardy-Weinberg平衡定律.统计分析表明该位点的多态性在两组人群中有明显的差异(G/C vs G/G,OR=2.65,P<0.05;C/C vs G/G,OR=3.31,P<0.05);亚组分析中表明<30岁的年龄组中差异显著(G/G vs G/C或C/C,OR=16.92,P<0.05).结论:在中国汉族人群中,IL-6基因启动子中-572处的多态性对于乙型肝炎的发生有着显著的易患关系,同时年龄对于这种易感关联有着协同作用.展开更多
基金Supported by A 2-year grant of the Greek Ministry of Health and Welfare,No.111K/56
文摘AIM:To investigate aberrant DNA methylation of CpG islands and subsequent low-or high-level DNA microsatellite instability(MSI)which is assumed to drive colon carcinogenesis. METHODS:DNA of healthy individuals,adenoma(tu-bular or villous/tubulovillous)patients,and colorectal carcinoma patients who underwent colonoscopy was used for assessing the prevalence of aberrant DNA methylation of human DNA mismatch repair gene mutator L homologue 1(hMLH1),Cyclin-dependent kinase inhibitor 2A(CDKN2A/p16),and O-6-methylguanine DNA methyltransferase(MGMT),as well as their rela- tion to MSI. RESULTS:The frequency of promoter methylation for each locus increased in the sequence healthy tissue/adenoma/carcinoma.MGMT showed the highest frequency in each group.MGMT and CDKN2A/p16 presented a statistically significant increase in promoter methylation between the less and more tumorigenic forms of colorectal adenomas(tubular vs tubullovillous and villous adenomas).All patients with tubulovillous/villous adenomas,as well as all colorectal cancer patients,showed promoter methylation in at least one of the examined loci.These findings suggest a potentially crucial role for methylation in the polyp/adenoma to cancer progres- sion in colorectal carcinogenesis.MSI and methylation seem to be interdependent,as simultaneous hMLH1, CDKN2A/p16,and MGMT promoter methylation was present in 8/9 colorectal cancer patients showing the MSI phenotype. CONCLUSION:Methylation analysis of hMLH1,CD- KN2A/p16,and MGMT revealed specific methylation profiles for tubular adenomas,tubulovillous/villous adenomas,and colorectal cancers,supporting the use of these alterations in assessment of colorectal tumorigenesis.
基金Supported by National Sciences Foundation of Shandong Province,No. ZR2014HM101
文摘BACKGROUND The integrinβ6 gene,which is expressed in epithelial cancer,plays a pivotal role in various aspects of cancer progression.The present research for integrinβ6 regulation mainly focuses on the post-transcription and translation related regulation mechanism and its role in tumorigenesis.The mechanisms of how the integrinβ6 gene is regulated transcriptionally,and the promoter and transcription factors responsible for basic transcription of integrinβ6 gene remain unknown.AIM To clone and characterize the integrinβ6 promoter.METHODS Software analysis was used to predict the region of integrinβ6 promoter.Luciferase reporter plasmids,which contained the integrinβ6 promoter,were constructed.Element deletion analysis was performed to identify the location of core promoter and binding sites for transcription factors.RESULTS The regulatory elements for the transcription of the integrinβ6 gene were located between-286 and-85 and contained binding sites for transcription factors such as STAT3 and Ets-1.CONCLUSION For the first time,we found the region ofβ6 core promoter and demonstrated the binding sites for transcription factors such as Ets-1 and STAT3,which are important for integrinβ6 promoter transcription activity.These findings are important for investigating the mechanism of integrinβ6 activation in cancer progression.
基金Supported by Guizhou International Cooperation Project on Science and Technology[No.QiankehewaiG(2013)7040]The 20th Project of The Joint Committee on Scientific and Technical Cooperation between The Government of the Kingdom of Thailand and The Government of the People’s Republic of China(No.20-606J)China.Suranaree University of Technology grant number SUT3-304-54-12-29,Thailand
文摘In order to analyze the Oslbglu4 phenotype, the inducible promoter of the transgenic rice which knock-down the Oslbglu4 expression was assessed. The result showed that 30 μM dexamethasone（DEX） had the stronger induction effect than 10 μM DEX by β-Glucuronidase （GUS） staining, qRT-PCR further verified the Oslbglu4 gene deletion. The effect of DEX and its solvent absolute ethanol on seed development was measured, and no significant effect was observed. The conclusion is that final concentration of DEX at 30 μM is suitable for pOp6 promoter induction.
文摘The P19CL6 mouse embryonic carcinoma cells efficiently differentiate into cardiac muscle cells in the presence of DMSO. A reporter plasmid for cardiac muscle differentiation was constructed by connecting the CMV enhancer and a 250 bp MLC-2v promoter in front of the GFP gene to further evaluate the role of the CMV enhancer. This plasmid (pCBVenh/MLC-2vpro/EGFP) was stably introduced into P19CL6 cells, and the transfectant differentiated into cardiomyocytes with DMSO. Upon DMSO addition, GFP was immediately transcribed (within 2 days) and the amount of the transcript increased with cultivation. Concomitantly, GFP fluorescence was detected in the cells under a microscope. However, native MLC-2v was transcribed later on day 4. This expression time course is different from that of GFP. Clearly the CMV enhancer responded immediately to DMSO. Since GATA DNA-binding proteins play crucial roles in the initiation of cardiomyocyte differentiation, such a response could be ascribed to the presence of multiple GATA motifs in the enhancer sequence but not in the native MLC-2v promoter. Thus the CMV enhancer may be not only useful for gene therapy and monitoring cell differentiation but also the study of the role of GATA transcription factors expressed in P19CL6 cells.
文摘Nanog protein is expressed in the interior cells of compacted morulae and maintained till epiblasts but downregulated by implantation stage. It is also expressed in embryonic stem cells, embryonic carcinoma cells and embryonic germ cells but disappeared in differentiated ES cells. In this study, we have isolated, sequenced, and performed the first characterization of the Nanog promoter. The transcription start sites were mapped by primer extension analysis. Two promoter regions were found upstream the transcription start sites and the expression of major Nanog promoter/ reporter gene construct is abolished in differentiated F9 EC cells as compared to the undifferentiated counterpart. We also showed that a putative octamer motif (ATGCAAAA) is necessary for the major promoter activity. Gel shift and supershift assays showed that Oct-1, Oct-4 and Oct-6 protein selectively bind to the octamer motif.
基金supported by the National Natural Science Foundation of China,No.81671469,81171072(to ZWY)the National Basic Research Program of China(973 Program),No.2013CB945402(to ZWY)the Program for Liaoning Innovative Research Team in University of China,No.LT2013016(to ZWY)
文摘O6-methylguanine DNA methyltransferase(MGMT), a DNA repair enzyme, has been reported in some congenital malformations, but it is less frequently reported in neural tube defects. This study investigated MGMT mRNA expression and methylation levels in the early embryo and in different embryonic stages, as well as the relationship between MGMT and neural tube defects. Spina bifida aperta was induced in rats by a single intragastric administration of all-trans retinoic acid on embryonic day(E) 10, whereas normal control rats received the same amount of olive oil on the same embryonic day. DNA damage was assessed by detecting γ-H2 A.X in spina bifida aperta rats. Real time-polymerase chain reaction was used to examine mRNA expression of MGMT in normal control and spina bifida aperta rats. In normal controls, the MGMT mRNA expression decreased with increasing embryonic days, and was remarkably reduced from E11 to E14, reaching a minimum at E18. In the spina bifida aperta model, γ-H2 A.X protein expression was increased, and mRNA expression of MGMT was markedly decreased on E14, E16, and E18. Bisulfite sequencing polymerase chain reaction for MGMT promoter methylation demonstrated that almost all CpG sites in the MGMT promoter remained unmethylated in both spina bifida aperta rats and normal controls, and there was no significant difference in methylation level between the two groups on either E14 or E18. Our results show that DNA damage occurs in spina bifida aperta rats. The mRNA expression of MGMT is downregulated, and this downregulation is independent of promoter DNA methylation.
文摘Recently, redox factor-1 (Ref-1) has received considerable attention as an enzyme for stimulating tumor cell growth. We hypothesized that Ref-1 is upregulated during the early stage of tumorigenesis. Utilizing both tumor promotion sensitive P+ and promotion resistant P? mouse skin epidermal JB6 cells, we found that Ref-1 expression was only induced in tumor promotion sensitive P+ cells following TPA treatment. Consistent with that, Ref-1 knockdown suppressed skin cell transformation. Interestingly, Ref-1 knockdown suppressed nuclear translocation of NF-kappaB subunit p65, and inhibited production of proinflammatory cytokines. These results suggest Ref-1 may promote early tumorigenesis and serve as a target for chemoprevention.
文摘目的:探讨中国人群中IL-6基因启动子中单核苷酸多态性与HBV感染的遗传易感性关联.方法:提取160例HBV感染者及212例健康献血者外周血基因组DNA,用PCR-RFLP方法检测两组人群中G-174C、G-572C和G-597A三个多态性位点的基因型.性别、吸烟、饮酒以及Hardy-Weinberg等采用Chi-square Test检测,多态性与HBV感染者风险关联及亚组关联的统计学分析采用非条件Logistic回归并同时校正混杂因素.结果:PCR-RFLP检测结果显示:IL-6基因启动子中G-174C和G-597A两个位点在中国人群中不存在多态性,而G-572C处的多态性在人群中普遍存在,其多态性位点等位基因频率分布符合Hardy-Weinberg平衡定律.统计分析表明该位点的多态性在两组人群中有明显的差异(G/C vs G/G,OR=2.65,P<0.05;C/C vs G/G,OR=3.31,P<0.05);亚组分析中表明<30岁的年龄组中差异显著(G/G vs G/C或C/C,OR=16.92,P<0.05).结论:在中国汉族人群中,IL-6基因启动子中-572处的多态性对于乙型肝炎的发生有着显著的易患关系,同时年龄对于这种易感关联有着协同作用.
