DNA 甲基化抑制剂对肿瘤细胞中核糖核酸酶抑制因子表达下调的影响

摘要

背景与目的:人核糖核酸酶抑制因子 (ribonucleasE-inhibitor, RI) RI能有效抑制血管生成因子诱导的血管形成及某些可移植性实体瘤在动物体内的生长.然而, RI抗肿瘤的分子机制还未完全阐明.许多抑癌基因通过启动子区域异常的甲基化而使表达缺失,去甲基化抑制能使其表达恢复.为了进一步了解 RI的功能以及探讨 RI与肿瘤发生的关系,本实验拟研究甲基化抑制剂 5-aza-2′-deoxycytidinE-(5-Aza-CdR)对肿瘤细胞中 RI表达的影响.方法:用 5-Aza-CdR作用人乳腺癌细胞系 MCF-7、人胃癌细胞系 BGC-823、人的前列腺癌细胞系 DU-145和人结肠癌细胞系 HT-29.通过 RT PCR,蛋白免疫印迹法( Western blot) ,免疫荧光( immunofluorescence)和免疫细胞化学( immunocytochemistry)技术分析 RI基因的表达.结果:与对照组比较, 5-Aza-CdR能显著提高 RI基因在 MCF-7、 BGC-823和 DU 145细胞中的表达, RT PCR检测结果分别为 37.2%、 46.0%和 32.4%, Western blot检测结果分别为 26.4%、 20.9%和 24.4%( P< 0.01);但对 HT-29细胞没有明显的影响.结论: RI基因可能与胃癌、前列腺癌和乳腺癌的发生有关.%BACKGROUND ＆ OBJECTIVE: Human ribonuclease inhibitor (RI) could effectively block angiogenin induced angiogenesis, and inhibit growth of transplant solid tumors in animals. However, its exact molecular mechanism of antitumor has not been totally ascertained. Many tumor suppressor genes occur loss of expression by aberrant methylation in promoter region. Demethylation, treated with methylation inhibitor 5-aza-2 deoxycytidine (5-Aza-CdR),can restore the gene expression. To further explore functions of RI, and investigate relationship between RI and tumorigenesis, the study was designed to explorE-effects of 5 Aza CdR on expression of RI in cancer cell lines. METHODS: Human breast cancer cell line MCF-7, human gastric cancer cell linE-BGC-823, human prostatE-cancer cell line DU-145, and human colon cancer cell line HT-29 were treated with 5 Aza CdR. Expression of RI was analyzed by reverse transcriptase polymerase chain reaction (RT PCR), Western blot, immunofluorescence, and immunocytochemistry. RESULTS: Expression of RI significantly elevated by 5-Aza-CdR at both Mrna and protein level in MCF-7, BGC-823, and DU-145 cells (P< 0.01). Compared with control cells, Mrna levels of RI in MCF-7, BGC-823, and DU-145 cells treated with 5-Aza-CdR werE-increased by percentages of 37.2% , 46.0% , and 32.4% , respectively; protein cevels of RI were increased by percentages of 26.4% , 20.9% , and 24.4% , respectively; but no obvious changE-observed in HT 29 cells. CONCLUSION: RI genE-may Be-involved in tumorigenesis of gastric, prostate, and breast cancer.