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目的:研究Ras相关区域家族1A基因(ras association domain family 1A,RASSF1A)启动子区甲基化对结肠癌组织中该基因转录和表达的影响。方法:应用甲基化特异性PCR(Methylation-special PCR,MSP)、RT-PCR和Western blot方法检测30例结肠癌组织和癌旁组织中的RASSF1A基因启动子区甲基化状态、mRNA和蛋白表达水平。结果:①RASSF1A基因启动子区在结肠癌组织和正常组织中的甲基化频率分别为57%(17/30)和20%(6/30),甲基化频率在两组具有统计学差异(p<0.01),,结肠癌组织中RASSF1A基因启动子区甲基化频率显著高于癌旁正常组织(x2=8.531,p<0.01);②结肠癌组织中RASSF1A基因mRNA和蛋白表达均显著低于癌旁组织(癌组织和癌旁正常组织中mRNA相对表达量分别为0.2836±0.0493和0.5092±0.0433,P<0.001;以上组织中蛋白相对表达量分别为0.3124±0.0472和0.5320±0.0440,P<0.01);③在结肠癌组织中,甲基化组RASSF1A基因mRNA和蛋白表达明显低于非甲基化组(甲基化组和非甲基化组mRNA相对表达量分别为0.0686±0.0174和0.5511±0.0486,P<0.0001;以上组中蛋白相对表达量分别为0.1219±0.0326和0.5614±0.0380,P<0.0001)。结论:结肠癌组织中RASSF1A基因启动子区甲基化明显增高,与该基因蛋白表达减少显著相关,这可能是导致结肠癌中RASSF1A抑癌基因失活的主要原因。
AIM: To investigate the effect of methylation of ras association domain family 1A (RASSF1A) promoter on the transcription and expression of this gene in colon cancer tissues. Methods: Methylation-specific PCR (MSP), RT-PCR and Western blot were used to detect the methylation status of RASSF1A promoter in 30 cases of colorectal cancer tissues and adjacent tissues. The mRNA and protein The expression level. Results: ①The methylation frequency of RASSF1A promoter region was 57% (17/30) and 20% (6/30) in colon cancer tissues and normal tissues, respectively. The methylation frequency was statistically different between the two groups (P <0.01). The methylation frequency of RASSF1A promoter in colorectal cancer tissues was significantly higher than that in adjacent normal tissues (χ2 = 8.531, p <0.01). ② The mRNA and protein expressions of RASSF1A in colon cancer tissues were significantly lower The relative expression of mRNA in adjacent tissues (cancer tissues and adjacent normal tissues were 0.2836 ± 0.0493 and 0.5092 ± 0.0433 respectively, P <0.001; the relative expression of protein in the above tissues was 0.3124 ± 0.0472 and 0.5320 ± 0.0440 respectively, P < 0.01). (3) The expression of RASSF1A mRNA and protein in methylation group was significantly lower than that in non-methylation group (the relative expression levels of methylation group and non-methylation group were 0.0686 ± 0.0174 and 0.5511 ± 0.0486, P <0.0001; The relative expression of protein in the above groups were 0.1219 ± 0.0326 and 0.5614 ± 0.0380 respectively, P <0.0001). CONCLUSION: The methylation of RASSF1A promoter region in colon cancer tissues is significantly increased, which is significantly associated with the decreased expression of RASSF1A gene, which may be the main reason for the inactivation of RASSF1A tumor suppressor gene in colon cancer.