ITIH5基因启动子甲基化与胃癌生物学行为关系的研究

Relationship between aberrant methylation of ITIH5 promoter and biological behaviour of gastric cancer

目的ITIH5在多种肿瘤表达下调,且其表达下调与患者预后不良相关。我们既往研究发现ITIH5在胃癌发生发展过程中可能发挥重要的生物学作用。然而ITIH5在胃癌发生发展中的生物学意义尚不清楚。本文研究了ITIH5甲基化调控对胃癌生物学行为的影响。方法采用RT-PCR检测细胞ITIH5表达情况。用MS-qPCR分析胃癌组织中ITIH5基因启动子区甲基化水平。用BSP对胃癌细胞ITIH5启动子区进行甲基化测序。采用5-aza去甲基化以及过表达ITIH5处理细胞后,采用MTT法、transwell实验和划痕实验检测胃癌细胞增殖、侵袭和迁移能力。采用流式细胞仪检测细胞凋亡情况。结果胃癌细胞系中ITIH5的表达水平低于正常胃上皮细胞系。胃癌组织中ITIH5 mRNA表达与ITIH5启动子的甲基化水平呈负相关。胃癌细胞ITIH5的表达下调与其启动子区甲基化相关。5-aza去甲基化处理和过表达ITIH5,可以显著抑制胃癌细胞增殖,诱导细胞凋亡,并导致其侵袭和迁移能力减弱。结论胃癌中ITIH5基因的表达下调与启动子区甲基化修饰有关。ITIH5在胃癌的发生中可能起着抑癌基因的作用。

Objective ITIH5 is downregulated in a wide variety of human tumours,and survival data analysis indicates that low expression correlates with poor prognosis.In our previous study,we found that ITIH5 has an important role in the development and progression of gastric cancer.Yet,The clinical and biological implications of ITIH5 are poorly understood.Therefore,this study identified the value of aberrant DNA hypermethylation of ITIH5 and the impact of ITIH5 on the biological behavior of gastric cancer cells.Methods The real-time PCR technique was used to detect the expression of ITIH5.DNA methylation level of ITIH5 gene promoter region was analyzed in bisulfate-modified DNA with a quantitative methylation-specific PCR(MSP)assay.Methylation status of ITIH5 promoter was analyzed using bisulfite sequencing PCR(BSP).Cell lines were treated with ITIH5 overexpression vectors or 5-Aza.The cell proliferation assay was determined by the MTT method,migration and invasion were determined by transwell migration and matrigel invasion assays,apoptosis was measured by flow cytometry.Results We found that the expression level of ITIH5 in gastric cancer cell lines was significantly lower than that in human immortalized gastric epithelial cell(GES)line.The promoter methylation level of ITIH5 had a significant negative correlation with the expression level of ITIH5.In human gastric cancer,ITIH5 promoter was found hypermethylated and its expression inversely correlated with the methylation grade.In vitro,5-Aza and overexpression of ITIH5 inhibited gastric cancer cell proliferation,induced apoptosis,decreased invasion,and decreased migration.Conclusion ITIH5 was frequently methylated in human gastric cancer and its expression was regulated by promoter region methylation.ITIH5,which acts as an important tumor suppressor gene,played an important role in gastric cancer.