AIM: To evaluate the genetic and epigenetic inactivation mechanism of the RASSF1A tumor suppressor gene at 3p21.3 in extrahepatic cholangiocarcinoma. METHODS: RT-PCR was used to investigate the transcriptional express...AIM: To evaluate the genetic and epigenetic inactivation mechanism of the RASSF1A tumor suppressor gene at 3p21.3 in extrahepatic cholangiocarcinoma. METHODS: RT-PCR was used to investigate the transcriptional expressing and re-expression of RASSFIA. RASSFIA mutation was analyzed with SSCP and selective sequencing. PCR was performed to detect the loss of heterozygosity (LOH) at the region of chromosome 3p21.3. Genomic DNA were modificated bisulfite and the frequency of methylation of CpG islands in RASSFIA promoter were evaluated by methylation specific PCR (MS-PCR). RESULTS: In all 48 samples and one cell lines of extrahepatic cholangiocarcinoma, the RASSFIA mutation is rare (6.12%, 3/49), 33 samples (68.75%) and QBC-939 cell lines (X2= 14.270, P= 0.001<0.01) showed RASSFIA express inactivation with LOH at microsatellite loci D3S4604. Among these 33 samples and QBC-939, 28 of 33 (84.85%) tumor samples and 1 cell lines were methylated for majority of 16 CpGs, the average frequency is 73.42%. CONCLUSION: The data we present suggest that RASSFIA which we have been searching for at 3p21.3 may be one of the key tumor suppressor gene and play an important role in the pathogenesis of extrahepatic cholangiocarcinoma, and the promoter methylation and allelic loss are the major mechanism for inactivation of RASSFIA.展开更多
目的探讨肺癌患者痰标本中FH IT、P16、MGMT、RASSF1A和APC等抑癌基因启动子异常甲基化及其联合检测在肺癌筛查及早期诊断中的价值。方法采用甲基化特异性PCR(methylation specific PCR,MSP)法,检测47例肺癌组织及对应的痰标本FHIT、P16...目的探讨肺癌患者痰标本中FH IT、P16、MGMT、RASSF1A和APC等抑癌基因启动子异常甲基化及其联合检测在肺癌筛查及早期诊断中的价值。方法采用甲基化特异性PCR(methylation specific PCR,MSP)法,检测47例肺癌组织及对应的痰标本FHIT、P16,MGMT、RASSF1A和APC基因启动子区甲基化状态。24例肺良性疾病患者痰标本作为对照。结果 47例肺癌组织标本中FHIT、P16、MGMT、RASSF1A、APC基因启动子区甲基化检出率分别为40.4%(19/47)、53.2%(25/47)、36.2%(17/47)、21.3%(10/47)和38.3%(18/47);对照的痰标本中五者甲基化检出率分别为38.3%(18/47)、48.9%(23/47)、36.2%(17/47)、17.0%(8/47)和29.8%(14/47),两组甲基化检出率存在着一致性[P>0.05;κ(0.8～1.0)]。24例肺良性病变痰标本中未检测到任何异常甲基化,与肺癌组比较差异有统计学意义(P<0.05)。五项指标联合检测可明显提高肺癌检测的灵敏度(80.9%)和特异度(100.0%)。FHIT和P16基因痰标本甲基化检出率与患者吸烟指数有相关性(P<0.05)。结论痰标本中多个肺癌相关基因甲基化联合检测有望成为肺癌筛查、早期诊断简便有效的指标。展开更多
基金Supported by the National High Technology Research and Development Program of China (863 Program), No. 2002AA214061
文摘AIM: To evaluate the genetic and epigenetic inactivation mechanism of the RASSF1A tumor suppressor gene at 3p21.3 in extrahepatic cholangiocarcinoma. METHODS: RT-PCR was used to investigate the transcriptional expressing and re-expression of RASSFIA. RASSFIA mutation was analyzed with SSCP and selective sequencing. PCR was performed to detect the loss of heterozygosity (LOH) at the region of chromosome 3p21.3. Genomic DNA were modificated bisulfite and the frequency of methylation of CpG islands in RASSFIA promoter were evaluated by methylation specific PCR (MS-PCR). RESULTS: In all 48 samples and one cell lines of extrahepatic cholangiocarcinoma, the RASSFIA mutation is rare (6.12%, 3/49), 33 samples (68.75%) and QBC-939 cell lines (X2= 14.270, P= 0.001<0.01) showed RASSFIA express inactivation with LOH at microsatellite loci D3S4604. Among these 33 samples and QBC-939, 28 of 33 (84.85%) tumor samples and 1 cell lines were methylated for majority of 16 CpGs, the average frequency is 73.42%. CONCLUSION: The data we present suggest that RASSFIA which we have been searching for at 3p21.3 may be one of the key tumor suppressor gene and play an important role in the pathogenesis of extrahepatic cholangiocarcinoma, and the promoter methylation and allelic loss are the major mechanism for inactivation of RASSFIA.
文摘目的探讨肺癌患者痰标本中FH IT、P16、MGMT、RASSF1A和APC等抑癌基因启动子异常甲基化及其联合检测在肺癌筛查及早期诊断中的价值。方法采用甲基化特异性PCR(methylation specific PCR,MSP)法,检测47例肺癌组织及对应的痰标本FHIT、P16,MGMT、RASSF1A和APC基因启动子区甲基化状态。24例肺良性疾病患者痰标本作为对照。结果 47例肺癌组织标本中FHIT、P16、MGMT、RASSF1A、APC基因启动子区甲基化检出率分别为40.4%(19/47)、53.2%(25/47)、36.2%(17/47)、21.3%(10/47)和38.3%(18/47);对照的痰标本中五者甲基化检出率分别为38.3%(18/47)、48.9%(23/47)、36.2%(17/47)、17.0%(8/47)和29.8%(14/47),两组甲基化检出率存在着一致性[P>0.05;κ(0.8～1.0)]。24例肺良性病变痰标本中未检测到任何异常甲基化,与肺癌组比较差异有统计学意义(P<0.05)。五项指标联合检测可明显提高肺癌检测的灵敏度(80.9%)和特异度(100.0%)。FHIT和P16基因痰标本甲基化检出率与患者吸烟指数有相关性(P<0.05)。结论痰标本中多个肺癌相关基因甲基化联合检测有望成为肺癌筛查、早期诊断简便有效的指标。
