膀胱癌组织MAGE-A3表达临床意义研究

Expression of MAGE-A3 in bladder cancer and its clinical significance

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及不同膀胱癌细胞系中黑色素瘤抗原-A3(melanoma-associated antigen-A3,MAGE-A3)的表达情况,探讨MAGE-A3表达水平与膀胱癌分期、分级及无进展生存期等的关系,分析MAGE-A3的表达水平对不同膀胱癌细胞系增殖的影响。方法选取2013-06-01-2016-05-31武汉市第一医院泌尿外科膀胱癌患者120例。培养膀胱癌细胞系5367、HT1376及EJ-28。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、癌旁组织、正常膀胱组织及膀胱癌细胞系中MAGE-A3mRNA和蛋白表达水平;免疫组化检测MAGE-A3蛋白阳性率;采用小干扰RNA(small interfering RNA,siRNA)敲减膀胱癌细胞系中MAGE-A3的表达水平,细胞集落形成实验检测MAGE-A3对细胞活力的影响。结果膀胱癌组织中MAGE-A3mRNA的表达水平为3.31±1.28,高于癌旁组织的1.38±0.57和正常组织的0.94±0.36,F=22.85,P<0.05;膀胱癌组织中MAGE-A3蛋白的表达水平为0.91±0.32,显著高于癌旁组织的0.22±0.07和正常组织的0.16±0.11,F=30.48,P<0.05。120例膀胱癌患者中,56例膀胱癌组织MAGE-A3蛋白表达阳性,阳性率为46.7%;MAGE-A3mRNA在5367和HT1376细胞系表达水平较高,在EJ-28细胞未检测到表达。在不同病理分期(χ2=13.718,P=0.008)、病理分级(χ2=11.345,P=0.01)和无进展生存期(χ2=4.262,P=0.039)之间,MAGE-A3表达量差异均有统计学意义;随病理分期、分级的升高和无进展生存期缩短而升高;敲减MAGE-A3后,5367和HT1376细胞增殖能力明显减弱,EJ-28增殖能力无明显变化。结论 MAGE-A3在膀胱癌组织中高表达,且与膀胱癌分期、分级、无进展生存期及肿瘤细胞增殖活力密切相关,有望成为膀胱癌免疫治疗的靶点。

OBJECTIVE Bladder cancer is one of the most common malignancies, which is endangering human health seriously. This study aims to investigate the expressions of melanoma associated antigen-A3 （MAGE-A3） in bladder cancer tissues and different bladder cancer cell lines,explore the relationship between MAGE-A3 expressions and bladder cancer staging,grade and progression-free survival,and analyze the effect of MAGE-A3 expression on the proliferation of different bladder cancer cell lines. METHODS Totally 120 cases of bladder cancer were collected from June 1,2013 to May 31,2016 in Urology of the First Hospital in Wuhan City according to the inclusion and exclusion criteria, and the bladder cancer cell lines 5367, HT1376 and EJ-28 were cultured. Real-time fluorescence quantitative PCR（RT-qPCR） and western blot were used to detect the expressions of MAGE-A3 mRNA and protein in bladder cancer tissues,adjacent tis sues,normal bladder tissues and bladder cancer cell lines. Immunohistochemistry was used to detect the positive rate of MAGE-A3 protein. The expression levels of MAGE-A3 in bladder cancer cell line were knockdown by small interfering RNA（siRNA）, and the cell colony formation assay was used to detect the effect of MAGE-A3 on cell viability. RESULTS The expressions of MAGE-A3 mRNA and protein in bladder cancer tissues （3.31±1.28;0.91±0.32） were significantly higher than that in adjacent tissues （1. 38±0. 57;0. 22±0. 07） and normal tissues （0. 94±0. 36;0. 16±0.11） （F=22.85,P〈0.05;F=30.48,P〈0.05）. In 120 cases of bladder cancer patients,56 cases of bladder cancer tis- sue MAGE-A3 protein expression was positive,the positive rate was 46.7%. MAGE-A3 mRNA expression was higher in 5367 and HT1376 cell lines,and no expression was detected in EJ-28 cells. The differences of MAGE-A3 expression be- tween different pathological stages, pathological grade and progressiomfree survival were statistically significant （X^2= 13. 718,P=0. 008;X^22 = 11. 345 ,P=0.01 ,%2 =4. 262,P=0. 039） ,which increased with the increase of pathological stage, grading and the decrease of progression free survival. The proliferation of 5367 and HT1376 cells was significantly de creased after MAGE-A3 knockdown （t=8. 753,P=0. 000 9;t=3. 725,P=0. 020 4） ,and the proliferation of EJ 28 had no significant change （t=0. 465 6, P= 0. 665 7）. CONCLUSIONS MAGE A3 is highly expressed in bladder cancer tissues and is closely related to the stage, grade, progression-free survival and tumor cell proliferation of bladder cancer, which is expected to be the target of bladder cancer immunotherapy.