人原发性肺癌组织中Rab相互作用溶酶体蛋白基因甲基化检测及其临床意义

Detection of Rab interacting lysosomal protein gene methylation in human primary lung cancer and its clinical significance

目的分析Rab相互作用溶酶体蛋白(RILP)在肺癌患者癌组织及肺癌细胞系中的表达及其甲基化情况,探索RILP基因的生物学功能。方法收集重庆医科大学附属长寿人民医院88例肺癌患者的癌组织和癌旁组织标本,分别用免疫组化染色、荧光定量PCR检测RILP蛋白及mRNA表达水平。选取肺癌细胞系(H1299、A549、H358、H19936和H460)和正常肺上皮细胞系(BEAS-2B),用荧光定量PCR检测RILP mRNA表达水平。用甲基化特异性PCR检测肺癌组织及细胞中RILP的甲基化。分析肺癌患者一般临床资料与RILP甲基化的关系。用空载体或人源RILP表达载体转染A549细胞,用克隆形成实验检测细胞增殖活性。结果肺癌组织RILP高表达率为13.64%,低于癌旁组织的76.14%(P<0.05),癌旁组织RILP mRNA表达水平高于肺癌组织(P<0.05),正常肺上皮细胞系中RILP mRNA表达水平均高于肺癌细胞系(P<0.05)。与正常肺上皮细胞系相比,RILP基因在肺癌细胞系中出现了明显的甲基化。60.23%(53/88)的肺癌患者发生RILP基因甲基化。RILP甲基化与RILP非甲基化患者肿瘤分化程度比较,差异有统计学意义(P<0.05)。A549细胞过表达RILP后,其增殖活性明显受到抑制(P<0.05)。结论RILP可能作为抑癌基因在肺癌中发挥作用,并且受甲基化调控。

Objective To analyze the expression and methylation of Rab interacting lysosomal protein(RILP)in cancer tissues of lung cancer patients and lung cancer cell lines,and to explore the biological function of RILP gene.Methods The cancer tissue and paracancerous tissue samples of 88 patients with lung cancer in Changshou People's Hospital Affiliated to Chongqing Medical University were collected,and the expression levels of RILP protein and mRNA were detected by immunohistochemical staining and fluorescence quantitative PCR respectively.Lung cancer cell lines(H1299,A549,H358,H19936 and H460)and normal lung epithelial cell line(BEAS-2B)were selected,and the expression levels of RILP mRNA were detected by fluorescence quantitative PCR.The methylation of RILP in lung cancer tissues and cells were detected by methylation-specific PCR.The relationship between general clinical data and RILP gene methylation in patients with lung cancer was analyzed.A549 cells were transfected with empty vector or human RILP expression vector,and the cell proliferation activity was detected by clone formation assay.Results The high expression rate of RILP in cancer tissues was 13.64%,which was lower than 76.14%in paracancerous tissues(P<0.05).The expression level of RILP mRNA in paracancerous tissues was higher than that in lung cancer tissues(P<0.05).The expression level of RILP mRNA in normal lung epithelial cell line was higher than that in lung cancer cell lines(P<0.05).Compared with normal lung epithelial cell line,RILP gene was significantly methylated in lung cancer cell lines.Methylation of RILP gene occurred in 60.23%(53/88)lung cancer patients.The differences in tumor differentiation between RILP methylated patients and RILP non-methylated patients were statistically significant(P<0.05).After overexpression of RILP,the proliferation activity of A549 cells was significantly inhibited(P<0.05).Conclusion RILP may play a role as a tumor suppressor gene in lung cancer,and this gene is regulated by methylation.