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目的:观察人胃癌、癌旁组织与胃癌AGS细胞中人音猬因子相互作用蛋白(human hedgehog interacting protein,HHIP)基因启动子区域CpG岛的甲基化水平,探索其与胃癌发生的关系。方法:RT-PCR检测30例人胃癌组织、癌旁组织及AGS细胞中HHIP mRNA的表达,免疫组织化学方法和甲基化特异性PCR(methylation specific PCR,MSP)分别检测胃癌组织和癌旁组织的HHIP表达和HHIP基因启动子区域甲基化状态。AGS细胞予甲基化转移酶抑制剂5-氮杂-2’-脱氧胞苷(5-Aza-2’-deoxycitydine,5-Aza-dc)处理前后,RT-PCR、MSP和硫化测序PCR(bisulfite sequencing PCR,BSP)分别检测AGS细胞中HHIP mRNA表达、启动子区域甲基化水平变化、CpG岛甲基化位点数量的变化;分析HHIP基因启动子区CpG岛甲基化水平变化与HHIP mRNA表达水平变化之间的相关性。结果:胃癌组织中的HHIP mRNA(0.82±0.38 vs 1.60±0.26,P=0.000)和蛋白(0.51±0.03 vs 0.83±0.27,P<0.05)的表达均低于癌旁组织,并且与年龄、性别、TNM分期、分化程度、淋巴结转移均无显著相关性(均P>0.05)。癌旁组织中HHIP基因启动子区甲基化水平显著低于胃癌和AGS细胞[(17.7±3.59)%vs(62.9±6.14)%、(99.7±0.67)%,均P<0.05]。AGS细胞在5-Aza-dc干预后HHIP mRNA表达明显增高(4.68±0.22 vs 0.21±0.12,P<0.01),HHIP基因启动子区甲基化水平明显下降[(10.1±0.21)%vs(90.2±0.67)%,P<0.01],CpG岛甲基化位点明显减少,并且HHIP基因启动子区甲基化水平与mRNA表达呈负相关(r=-0.693,P=0.00)。结论:HHIP基因启动子区CpG岛的高甲基化水平可能通过抑制HHIP基因表达参与胃癌的发生。
OBJECTIVE: To investigate the methylation level of CpG island in promoter region of human hedgehog interacting protein (HHIP) gene in human gastric cancer, adjacent non-cancerous tissues and gastric cancer AGS cells, and to explore its relationship with gastric carcinogenesis. Methods: HHIP mRNA expression was detected by RT-PCR in 30 human gastric cancer tissues, adjacent non-cancerous tissues and AGS cells. Immunohistochemistry and methylation-specific PCR (MSP) were used to detect the expression of HHIP mRNA in gastric cancer tissues and adjacent tissues HHIP expression and HHIP gene promoter region methylation status. RT-PCR, MSP and sulf-PCR were performed before and after AGS cells were treated with 5-Aza-2’-deoxycitydine (5-Aza-dc) bisulfite sequencing PCR and BSP were used to detect the changes of HHIP mRNA expression, promoter methylation and CpG island methylation in AGS cells respectively. The changes of methylation level of CpG island in promoter region of HHIP gene and HHIP Correlation between mRNA expression changes. Results: The expressions of HHIP mRNA (0.82 ± 0.38 vs 1.60 ± 0.26, P = 0.000) and protein (0.51 ± 0.03 vs 0.83 ± 0.27, P <0.05) in gastric cancer tissues were lower than those in paracancerous tissues, , TNM stage, degree of differentiation and lymph node metastasis (all P> 0.05). The methylation level of HHIP gene promoter in paracancer tissues was significantly lower than that in gastric cancer and AGS cells [(17.7 ± 3.59)% vs (62.9 ± 6.14)%, (99.7 ± 0.67)%, respectively, P <0.05]. The expression of HHIP mRNA in AGS cells was significantly increased after intervention with 5-Aza-dc (4.68 ± 0.22 vs 0.21 ± 0.12, P <0.01) and the methylation level of HHIP gene promoter was significantly decreased (10.1 ± 0.21)% vs (90.2 ± 0.67)%, P <0.01]. The methylation of CpG islands was significantly reduced, and the methylation of HHIP promoter was negatively correlated with mRNA expression (r = -0.693, P = 0.00). CONCLUSION: Hypermethylation of CpG island in HHIP promoter may be involved in the carcinogenesis of gastric cancer by inhibiting the expression of HHIP gene.