尿液修饰核苷作为膀胱肿瘤标志物的筛选鉴定及分子机制研究

Modified nucleoside exists in RNA, and produced by a variety of specific modified enzymes act on the poly- nucleoside molecules, It is susceptible to external factors less and t excreted with the urine.Research shows that urine modified nucleosides as tumor markers to diagnose a variety of tumors is of great significance. Our initial work shows that 1-methyl adenosine (MlA) and 1-methylinosine (1-MeI) joint test in the early diagnosis of bladder cancer have high sensitivity (92.45%) and specificity (87.50%), However urine modified nucleosides as bladder tumor markers and its mechanism in bladder cancer is not clear. Research shows that urine modified nucleosides generation is correlate to tumor cells tRNA methylation enzymes. Then we put forward the hypothesis: bladder cancer is related to methylation . This topic intends to study :1) Screen for urine modified nucleosides tumor markers as early diagnosis of bladder cancer;2) Testing the differences of tRNA modified nucleoside,methylation enzyme' (miA58 methylation shift enzyme, S-adenosine base methionine enzyme) level and activity between bladder cancer organizations and normal cancer adjacent tissues;3)How siRNA interference silence the gene of methylase influence the modified nucleoside level. The research will help further opened the molecular mechanism of urine modified nucleoside in bladder cancer patients, and provides the basis for clinical diagnosis of bladder cancer and predict its early risk of recurrence.

修饰核苷存在于RNA中，由多种特定修饰酶作用于多聚核苷分子形成，其受外界因素影响少，在尿中定量排放。研究表明尿液修饰核苷对多种肿瘤早期诊断具有重大意义。我们前期工作表明1-甲基腺苷(MlA)和1-甲基次黄苷（1-MeI）联合检测对膀胱癌早期诊断具有极高的灵敏度和特异性，然而尿液修饰核苷作为膀胱癌特异性肿瘤标记物及其在膀胱癌发生机制中所起作用尚不清楚。研究表明尿液修饰核苷的产生与肿瘤细胞中tRNA甲基化酶有关。由此提出假说：甲基化修饰与膀胱癌的发生相关。本课题拟研究：1）筛选膀胱癌早期诊断的尿液修饰核苷肿瘤标志物；2）检测膀胱癌组织与正常癌旁组织中tRNA修饰核苷、甲基化酶（m1A58甲基化转移酶、S-腺苷基蛋氨酸酶）的水平及活性的差异；3）siRNA干扰沉默甲基化酶基因对修饰核苷水平的影响。该研究将有助于揭开膀胱癌患者尿液修饰核苷产生的分子机制，为早期诊断膀胱癌和预测其复发风险提供依据

研究表明尿液修饰核苷做为非常有潜力的肿瘤标记物对多种肿瘤的早期诊断、病情监测及预后判断具有重大意义。我们前期的工作表明：1-甲基腺苷（m1A）和1-甲基次黄苷（1-meI）联合检测对膀胱癌早期诊断具有极高的灵敏度和特异性。m1A58甲基转移酶（hTrm6p/hTrm61p）由两个亚基组成的异源四聚体，在人体内催化tRNA58位的腺苷（A）成为1-甲基腺苷（m1A）。该研究为证实hTrm6p/hTrm61p在膀胱癌发病中所起到的作用，测定了膀胱癌组织及癌旁正常组织中hTrm61 mRNA和蛋白的表达量，结果发现膀胱癌组织中hTrm61亚基表达明显高于癌旁正常组织，且与尿液中m1A的量呈正相关关系。在膀胱癌细胞株EJ、T24、5637及人胚胎肾脏细胞株HEK-293中筛选出了hTrm61 mRNA高表达的细胞株5637，用siRNA干扰技术在膀胱癌5637细胞株中敲除hTrm61，结果发现膀胱癌5637细胞株的增殖及凋亡均受到了明显影响，提示hTrm61可能参与并促进了膀胱癌的发病过程。该研究为进一步探讨m1A58甲基转移酶的表达调控及其生物学功能奠定了基础。