用带有质粒pGIH(35S-intron-GUS/ Hptr)的根癌农杆菌EHA101转化玉米愈伤组织,获得潮霉素抗性植株,再生性好的苏玉1 图7 Southern blot分析HpaII消化的质粒pGIH 和gusA基因沉默的愈伤组织的总DNA Fig.7 Southern blot analysis of plasmi...用带有质粒pGIH(35S-intron-GUS/ Hptr)的根癌农杆菌EHA101转化玉米愈伤组织,获得潮霉素抗性植株,再生性好的苏玉1 图7 Southern blot分析HpaII消化的质粒pGIH 和gusA基因沉默的愈伤组织的总DNA Fig.7 Southern blot analysis of plasmid pGIH(P) and plant genomic DNA of gusA gene si- lence callus(T)digested with HpaII号转化率可以达到8.1%。对转化植株进行GUS染色分析及PCR和Southern杂交检测证明外源基因已经整合,并能够稳定表达。反向PCR分析的三个转化植株,T-DNA插入片段均为单拷贝。部分失去分化能力的抗性愈伤组织,Southern blot分析发现其基因组中有gusA基因插入,但X-gluc染色呈阴性,经HpaII酶切分析发现整合的gusA基因发生了高度甲基化。展开更多
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...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.展开更多
To determine the possible role of the epigenetic mechanisms in carcinogenesis of the hepatocellular carcinoma, we methylation-profiled the promoter CpG islands of twenty four genes both in HCC tumors and the neighbori...To determine the possible role of the epigenetic mechanisms in carcinogenesis of the hepatocellular carcinoma, we methylation-profiled the promoter CpG islands of twenty four genes both in HCC tumors and the neighboring non-cancerous tissues of twenty eight patients using the methylation-specific PCR (MSP) method in conjunction with the DNA sequencing. In comparison with the normal liver tissues from the healthy donors, it was found that while remained unmethylated the ABL, CAV, EPO, GATA3, LKB1, NEP, NFL, NIS and p27^(KIP1) genes, varying extents of the HCC specific hypermethylation were found associated with the ABO, AR, CSPG2, cyclin al, DBCCR1, GALR2, IRF7, MGMT, MT1A, MYOD1, OCT6, p57^(KIP2), p73, WT1 genes, and demethylation with the MAGEA1 gene, respectively. Judged by whether the hypermethylated occurred in HCC more frequently than in their neighboring normal tissues, the hypermethylation status of the AR, DBCCR1, IRF7, OCT6, and p73 genes was considered as the event specific to the late stage, while that the rest that lacked such a distinguished contrast, as the event specific to the early stage of HCC carcinogenesis. Among all the clinical pathological parameters tested for the association with, the hypermethylation of the cyclin al gene was more prevalent in the non-cirrhosis group (P=0.021) while the hypermethylated p16^(INK4a) gene was more common in the cirrhosis group (P=0.017). The concordant methylation behaviors of nineteen genes, including the four previously studied and their association with cirrhosis has been evaluated by the best subgroup selection method. The data presented in this report would enable us to shape our understanding of the mechanisms for the HCC specific loss of the epigenetic stability of the genome, as well as the strategy of developing the novel robust methylation based diagnostic and prognostic tools.展开更多
AIM: To investigate the rrelationship between methylation of Syk (spleen tyrosine kinase) gene in promoter region and oncogenesis, metastasis of gastric carcinoma. The relation between silencing of the Syk gene and me...AIM: To investigate the rrelationship between methylation of Syk (spleen tyrosine kinase) gene in promoter region and oncogenesis, metastasis of gastric carcinoma. The relation between silencing of the Syk gene and methylation of Syk promoter region was also studied. METHODS: By using methylation-specific PCR (MSP) technique, the methylation of Syk promoter region in specimens from 61 gastric cancer patients (tumor tissues and adjacent normal tissues) was detected. Meanwhile, RTPCR was used to analyse syk expression exclusively. RESULTS: The expression of the Syk gene was detected in all normal gastric tissues. Syk expression in gastric carcinoma was lower in 14 out of 61 gastric cancer samples than in adjacent normal tissues (X^2=72.3, P<0.05). No methylation of Syk promoter was found in adjacent normal tissues, hypermethylation of Syk gene in promoter was detected 21 cases in 61 gastric carcinoma patients. The rate of methylation of Syk promoter in gastric carcinoma was higher than that in adjacent normal tissues (X^2=25.1, P<0.05). In 31 patients with lymph node metastasis, 17 were found with Syk promoter methylation. A significant difference was noted between two groups (X^2=11.4,P<0.05). CONCLUSION: Hypermethylation leads to silencing of the Syk gene in human gastric carcinoma. Methylation of Syk promoter is correlated to oncogenesis and metastasis of gastric carcinoma. Syk is considered to be a potential tumor suppressor and anti-metastasis gene in human gastric cancer.展开更多
文摘用带有质粒pGIH(35S-intron-GUS/ Hptr)的根癌农杆菌EHA101转化玉米愈伤组织,获得潮霉素抗性植株,再生性好的苏玉1 图7 Southern blot分析HpaII消化的质粒pGIH 和gusA基因沉默的愈伤组织的总DNA Fig.7 Southern blot analysis of plasmid pGIH(P) and plant genomic DNA of gusA gene si- lence callus(T)digested with HpaII号转化率可以达到8.1%。对转化植株进行GUS染色分析及PCR和Southern杂交检测证明外源基因已经整合,并能够稳定表达。反向PCR分析的三个转化植株,T-DNA插入片段均为单拷贝。部分失去分化能力的抗性愈伤组织,Southern blot分析发现其基因组中有gusA基因插入,但X-gluc染色呈阴性,经HpaII酶切分析发现整合的gusA基因发生了高度甲基化。
文摘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.
基金supported by the National High Technology Research and Development Program of China(863 Program)(2001AA217011,2002AA2Z3352)the Major State Basic Research Development Program of China (973 Program)(G1998051004)the Science Foundation of Shanghai Municipal Government(02DJ14056)to JingDe ZHU.
文摘To determine the possible role of the epigenetic mechanisms in carcinogenesis of the hepatocellular carcinoma, we methylation-profiled the promoter CpG islands of twenty four genes both in HCC tumors and the neighboring non-cancerous tissues of twenty eight patients using the methylation-specific PCR (MSP) method in conjunction with the DNA sequencing. In comparison with the normal liver tissues from the healthy donors, it was found that while remained unmethylated the ABL, CAV, EPO, GATA3, LKB1, NEP, NFL, NIS and p27^(KIP1) genes, varying extents of the HCC specific hypermethylation were found associated with the ABO, AR, CSPG2, cyclin al, DBCCR1, GALR2, IRF7, MGMT, MT1A, MYOD1, OCT6, p57^(KIP2), p73, WT1 genes, and demethylation with the MAGEA1 gene, respectively. Judged by whether the hypermethylated occurred in HCC more frequently than in their neighboring normal tissues, the hypermethylation status of the AR, DBCCR1, IRF7, OCT6, and p73 genes was considered as the event specific to the late stage, while that the rest that lacked such a distinguished contrast, as the event specific to the early stage of HCC carcinogenesis. Among all the clinical pathological parameters tested for the association with, the hypermethylation of the cyclin al gene was more prevalent in the non-cirrhosis group (P=0.021) while the hypermethylated p16^(INK4a) gene was more common in the cirrhosis group (P=0.017). The concordant methylation behaviors of nineteen genes, including the four previously studied and their association with cirrhosis has been evaluated by the best subgroup selection method. The data presented in this report would enable us to shape our understanding of the mechanisms for the HCC specific loss of the epigenetic stability of the genome, as well as the strategy of developing the novel robust methylation based diagnostic and prognostic tools.
基金Supported by the Science Fundof Department of Science and Technology of Jiangsu Province.03KJD320138
文摘AIM: To investigate the rrelationship between methylation of Syk (spleen tyrosine kinase) gene in promoter region and oncogenesis, metastasis of gastric carcinoma. The relation between silencing of the Syk gene and methylation of Syk promoter region was also studied. METHODS: By using methylation-specific PCR (MSP) technique, the methylation of Syk promoter region in specimens from 61 gastric cancer patients (tumor tissues and adjacent normal tissues) was detected. Meanwhile, RTPCR was used to analyse syk expression exclusively. RESULTS: The expression of the Syk gene was detected in all normal gastric tissues. Syk expression in gastric carcinoma was lower in 14 out of 61 gastric cancer samples than in adjacent normal tissues (X^2=72.3, P<0.05). No methylation of Syk promoter was found in adjacent normal tissues, hypermethylation of Syk gene in promoter was detected 21 cases in 61 gastric carcinoma patients. The rate of methylation of Syk promoter in gastric carcinoma was higher than that in adjacent normal tissues (X^2=25.1, P<0.05). In 31 patients with lymph node metastasis, 17 were found with Syk promoter methylation. A significant difference was noted between two groups (X^2=11.4,P<0.05). CONCLUSION: Hypermethylation leads to silencing of the Syk gene in human gastric carcinoma. Methylation of Syk promoter is correlated to oncogenesis and metastasis of gastric carcinoma. Syk is considered to be a potential tumor suppressor and anti-metastasis gene in human gastric cancer.
