膀胱癌组织MAGE-A3表达变化机制研究

Expression and mechanisms of MAGE-A3 in bladder carcinoma

目的膀胱癌是最常见的恶性肿瘤之一,严重危害人类健康。本研究检测膀胱癌组织中黑色素瘤抗原3(melanoma-associated antigen 3,MAGE-A3)的表达变化并探讨其内在机制,为膀胱癌的防治提供理论基础。方法收集2013-06-01-2016-05-31武汉科技大学附属武昌医院泌尿外科膀胱癌患者120例,取术中切取的膀胱癌组织和正常膀胱组织(距离肿瘤边缘>3cm)。实时荧光定量PCR(Real-time fluorescence quantitative PCR,RT-qPCR)和蛋白质印迹法检测膀胱癌组织和正常膀胱组织中MAGE-A3,DNA甲基转移酶1(DNA methyltransferase 1,DNMT1)及常染色体组蛋白赖氨酸甲基转移酶2(euchromatic histone-lysine N-methyltransferase 2,EHMT2)mRNA和蛋白表达变化;巢式降落式甲基化特异性PCR(nested methylated specific PCR,nMS-PCR)检测膀胱癌组织和正常膀胱组织中MAGE-A3启动子区DNA甲基化水平变化;染色质免疫共沉淀(chromatin immunoprecipitation,CHIP)检测膀胱癌组织和正常膀胱组织中MAGE-A3启动子区组蛋白H3K9二甲基化(H3K9me2)水平。结果膀胱癌组织中MAGE-A3mRNA相对表达量为5.41±0.85,蛋白相对表达量为0.76±0.08;正常膀胱组织中MAGE-A3mRNA相对表达量为0.92±0.15,蛋白相对表达量为0.20±0.07,两组比较差异有统计学意义,t值分别为15.56和15.60,均P=0.001。膀胱癌组织中DNMT1mRNA相对表达量为0.37±0.10,蛋白相对表达量为0.36±0.10;EHMT2mRNA相对表达量为0.81±0.09,蛋白相对表达量为0.27±0.08。正常膀胱组织中DNMT1mRNA相对表达量为1.02±0.25,蛋白相对表达量为0.91±0.15,RNA相对表达量为4.32±0.82,蛋白的相对表达量为1.09±0.17,两组比较差异有统计学意义,t值分别为5.95,9.16,11.58,11.08,均P<0.001。膀胱癌组织中MAGE-A3启动子区DNA甲基化水平为0.27±0.07,H3K9me2水平为0.25±0.13;正常膀胱组织MAGE-A3启动子区DNA甲基化水平为0.73±0.08,H3K9me2水平为0.61±0.24,两组比较差异有统计学意义,t值分别为7.189和3.860,均P<0.001。结论膀胱癌组织中MAGE-A3启动子区DNA甲基化及H3K9me2水平降低使MAGE-A3表达量升高,此机制可能参与膀胱癌的发生发展。

OBJECTIVE Bladder cancer is one of the most common malignancies which seriously endanger human health.This study was to detect the expression of MAGE-A3 in bladder cancer and explore its mechanisms,which provided a theoretical basis for the prevention and treatment of bladder cancer.METHODS According to the inclusion and exclusion criteria,120 cases of urinary surgical bladder cancer from Department of Urology,Wuchang Hospital,Wuhan University of Science and Technology from June 1,2013 to May 31,2016 were collected.Real-time fluorescence quantitative PCR(RT-qPCR)and Western Blot were used to detect the expression of MAGE-A3(Melanoma-associated antigen 3),DNMT1(DNA Methyltransferase 1)and EHMT2(Euchromatic histone-lysine N-methyltransferase 2)mRNA and protein in bladder cancer tissues and normal bladder tissues.Nested methylated specific PCR(nMS-PCR)was used to detect the changes of DNA methylation level in MAGE-A3 promoter region in bladder cancer tissues and normal bladder tissues.Chromatin immunoprecipitation(CHIP)was used to detect the levels of H3K9dimethylation(H3K9me2)in the MAGEA3 promoter region of bladder cancer tissues and normal bladder tissues.RESULTS The expression of MAGE-A3mRNAs(5.41±0.85)and protein(0.76±0.08)in bladder cancer tissue was higher than mRNA(0.92±0.15)and protein(0.20±0.07)in normal bladder tissues,while the mRNA and protein expression levels of DNMT1(0.37±0.10,0.36±0.10)and EHMT2(0.81±0.10,0.27±0.07)were lower than those of normal bladder tissues(1.02±0.25,0.91±0.15;4.32±0.82,1.10±0.17),P<0.001.The level of DNA methylation(0.27±0.07)and H3K9me2(0.25±0.13)in MAGE-A3 promoter region was lower than that in normal bladder tissues(0.73±0.08,0.61±0.21),P<0.001.CONCLUSION These results suggest that the expression levels of DNMT1 and EHMT2are reduced in bladder cancer,and the methylation of MAGE-A3 promoter region and H3K9me2 level are reduced,all these lead to upregulaion of MAGE-A3.