肿瘤微环境中ROS介导IgG表达对膀胱癌细胞增殖和侵袭能力的影响

摘要 目的：探讨肿瘤微环境（TME）中活性氧（ROS）介导免疫球蛋白G（IgG）表达对膀胱癌EJ细胞增殖、迁移和侵袭能力的影响。方法：临床收集的18例膀胱癌患者样本，通过Western blot法检测膀胱癌和癌旁正常组织样本中IgG表达量。利用免疫荧光染色（IF）技术分别对膀胱癌组织和癌旁正常组织中ROS和IgG分子进行共定位和相对定量分析。将活性氧清除剂N-乙酰基-L-半胱氨酸（NAC）加入膀胱癌细胞EJ中，实验分为3组：空白组（EJ细胞）、阴性对照组（EJ+PBS）、实验组（EJ+PBS+NAC），10 mM NAC药物处理48小时后，运用DHE-ROS荧光探针技术和Western blot实验检测药物NAC对ROS和IgG相对表达水平的影响；通过克隆集落形成实验、划痕实验、Transwell实验检测去除ROS后对膀胱癌细胞增殖、迁移和侵袭的影响。结果：人体膀胱癌组织中ROS和IgG分子表达水平显著高于癌旁正常组织（P<0.001）；荧光显微镜显示膀胱肿瘤组织中正常膀胱尿路上皮细胞组织被肿瘤细胞严重破坏，结构紊乱不规则，IgG和ROS表达水平均升高，而癌旁组织膀胱尿路上皮组织的结构均匀规则；NAC药物处理EJ细胞后，与空白组和阴性对照组相比ROS和IgG表达显著降低，同时实验组细胞的增殖、迁移和侵袭能力明显下降（P<0.01）。结论：ROS和IgG在临床膀胱癌组织和体外膀胱癌细胞株EJ中均显著高表达，在肿瘤微环境中ROS通过调控IgG表达，从而促进膀胱癌细胞的增殖、迁移、侵袭。ROS和IgG可能成为膀胱癌早期诊断和生物治疗的临床新靶点。

Effects of ROS-mediated IgG Expression in Tumor Microenvironment on Cell Proliferation and Invasion of Bladder Cancer Cells

ABSTRACT Objective: To investigate the effect of reactive oxygen species(ROS)-mediated immunoglobulin G (IgG) expression on the proliferation, migration and invasion of bladder cancer cell line EJ in the tumor microenvironment(TME). Methods: The IgG expression in bladder cancer and adjacent normal tissue samples was detected by Western blot from 18 clinical patients with bladder cancer. Then, the co-localization and relative quantification analysis of ROS and IgG molecules were measured with immunofluorescence staining technology(IF) in bladder cancer tissues and adjacent normal tissues, respectively. The bladder cancer cells EJ were treated with the drug N-acetyl-L-cysteine (NAC), a reactive oxygen species scavenger, for 48 hours with 10mM NAC, and then the experiment was divided into three groups: the blank group (EJ cells), the negative control group (EJ+PBS), the experimental group (EJ+PBS+NAC). The DHE-ROS fluorescent probe technology and Western blot was used to detect the effect of drug NAC on the relative expression levels of ROS and IgG. The effects of removing ROS on the proliferation, migration and invasion of bladder cancer cell line EJ were detected by clonal colony formation assay, wound healing assay, and Transwell assay. Results: The expression levels of ROS and IgG in human bladder cancer tissues were significantly higher than those in para-carcinoma tissues(P<0.001). Immunofluorescence stain of bladder cancer tissues showed that the normal bladder urothelial cells in tumor tissue was severely damaged by tumor cells, with disordered and irregular structure, and the expression levels of IgG and ROS were increased, while the structure of bladder urothelial cells was uniform and regular in para-carcinoma tissue. After the drug NAC treatment of bladder cancer EJ cells, compared with the blank group and the negative control group, the expressions levels of ROS and IgG were significantly decreased, and the proliferation, migration and invasion abilities of bladder cancer EJ cells in the experimental group were significantly declined(P<0.01). Conclusion: ROS and IgG were significantly highly expressed in clinical bladder cancer tissues and vitro EJ cells. In the tumor microenvironment, ROS via regulating IgG expression promotes the proliferation, migration and invasion of bladder cancer cells. ROS and IgG may be new clinical targets for the early diagnosis and biological therapy of bladder cancer.