Rce1在膀胱癌的表达及其对T24细胞凋亡的影响

Expression of Rce1 in bladder cancer and its influence on the apoptosis of T24 cells

目的:探讨Rce 1(Ras and a-factor converting enzyme 1)在膀胱癌组织及对应癌旁组织中的表达差异及其意义;探究慢病毒介导sh RNA沉默Rce1基因对人膀胱癌细胞株T24细胞凋亡的影响。方法:采用q RT-PCR和Western blot技术检测Rce1在12例膀胱癌组织及对应癌旁组织中m RNA和蛋白表达情况,采用免疫组化技术检测76例膀胱癌组织及对应癌旁组织中Rce1表达情况;结合临床病理资料,分析Rce1表达与临床病理特征的关系。设计针对Rce1基因的sh RNA慢病毒干扰载体感染T24细胞,经嘌呤霉素筛选后建立稳定感染细胞系,实时荧光定量PCR、Western blot分别检测感染后各组细胞Rce1 m RNA和蛋白表达水平;应用流式细胞仪检测T24细胞凋亡改变情况。结果:q RT-PCR和Western blot显示在12对癌及癌旁组织中Rce1差异性表达,差异有统计学意义(P=0.00);78例癌组织中Rce1阳性表达率为70.5%(55/78),癌旁组织中阳性率为19.0%(4/21),组间差异有统计学意义(P=0.000);统计学分析表明,Rce1表达量与肿瘤组织学分级、T分期和肿瘤是否远处转移存在相关关系(P<0.05);Kaplan-Meier生存分析显示,Rce1阳性表达与病人术后生存时间呈负相关关系(P=0.016)。与空白对照组和阴性对照组相比,感染sh RNA慢病毒干扰载体细胞组Rce1 m RNA和蛋白表达水平明显下调(P=0.000),细胞凋亡明显增加。结论:Rce1在膀胱癌组织中表达高于癌旁组织,Rce1在膀胱癌的发生发展中可能发挥重要作用;慢病毒介导sh RNA沉默Rce1基因能有效下调Rce1在T24细胞中表达,并诱导细胞凋亡;Rce1有望成为膀胱癌临床诊治和预后的一个新指标。

Objective:To investigate the expression and clinical significance of Ras and a-factor converting enzyme 1(Rce1)in tissues of bladder urothelial carcinoma(UHC)and pericarcinoma(PC)and to explore the effect of lentivirus-mediated sh RNA silencing Rce1 gene on apoptosis and cell cycle of human bladder cancer T24 cells. Methods:Immunohistochemical staining for Rce1 expression was performed in bladder urothelial carcinoma(UCB) and adjacent pericarcinoma tissues;the methods of q RT-PCR and Western blot were utilized to investigate m RNA/protein expression of Rce1 in UCB. Rce1(+)and Rce1(-)patients were compared in terms of p TNM grade and differentiation grade. Lentiviral interference vectors of sh RNA-Rce1 were constructed successfully and were transfected into T24 cells,then the T24 cells stably knockdown Rce1 gene expression were screened by puromycin. Expression of Rce1 m RNA and protein in T24 cells was detected by q RT-PCR and Western blot,respectively. Apoptosis of T24 cells was examined by flow cytometry. Results:The expressions of Rce1 were highly up-regulated at both m RNA and protein level in carcinoma tissues when compared with those of adjacent ones through q RT-PCR and Western blot analysis. By IHC,positive expression of Rce1 was examined in55/76(70.5%)of UCBs and in 4/21(19.0%)of paired pericarcinoma tissues. Positive expression of Rce1 was correlated with poorer differentiation,higher T classification and tumor metastasis(P<0.05). In Kaplan-Meier survival analysis,significant association between positive expression of Rce1 and shortened patients' survival was found(P<0.001). Compared with those in control group and negative control group,expressions of Rce1 m RNA and protein were significantly reduced after Rce1-sh RNA lentiviral interference vectors being infected(P=0.000)and apoptosis was increased significantly(P=0.000). Conclusion:The expression of Rce1 is significantly higher in UCBs when compared with that of pericarcinoma tissues;positive expression of Rce1 also correlated with prognosis of patients with UCB. Lentivirus-mediated sh RNA silencing Rce1 gene can down regulate the expression of Rce1 gene,promote apoptosis in T24 cells. Rce1 may serve as a biomarker of diagnosis and prognosis in patients with UCB.