微纤维相关蛋白5调控上皮-间质转化相关基因促进膀胱癌细胞迁移和侵袭

目的 探讨微纤维相关蛋白5（MFAP5）在膀胱癌中的表达水平及其对膀胱癌细胞的生物学作用。方法 收集上海交通大学附属第一人民医院确诊的膀胱癌患者手术切除标本，通过免疫组织化学染色检测MFAP5的表达情况。分别从膀胱肿瘤组织和正常膀胱组织分离纯化得到肿瘤相关成纤维细胞（CAF）和正常成纤维细胞（NF），通过qRT-PCR检测CAF、NF及人膀胱癌细胞系（T24、5637、UMUC3、J82）中MFAP5 mRNA的相对表达量。通过美国癌症基因组图谱（TCGA）数据库及基因表达汇编（GEO）数据库GSE13507数据集中的膀胱癌表达谱数据，分析MFAP5表达与膀胱癌进展和恶性程度的相关性。用重组人MFAP5蛋白处理膀胱癌细胞系T24、5637，检测其对细胞迁移和侵袭能力及迁移相关蛋白表达的影响。结果 MFAP5在膀胱癌组织中主要表达于肿瘤间质，其表达水平与膀胱癌T分期和肿瘤分级有关（P均<0.05），并且CAF中MFAP5 mRNA相对表达量高于NF、T24、5637、UMUC3、J82细胞（P均<0.01）。TCGA和GSE13507数据库分析结果均显示，高表达MFAP5的膀胱癌患者总生存期短于低表达患者（P均<0.05）。基因集差异分析结果显示MFAP5表达水平随肿瘤恶性程度的升高而升高（P<0.01），并与上皮-间质转化相关基因钙黏蛋白2、Twist1、基质金属蛋白酶9（MMP9）、E盒结合锌指蛋白1（ZEB1）密切相关（P均<0.01）。重组人MFAP5蛋白刺激能促进T24、5637细胞中包括N-钙黏蛋白、MMP9、ZEB1、Twist在内的上皮-间质转化相关蛋白表达（P<0.05，P<0.01），同时增强细胞的迁移和侵袭能力（P均<0.01）。结论 MFAP5可能是预测膀胱癌发生侵袭进展的潜在分子标志物。

Microfibril-associated protein 5 regulates epithelial-mesenchymal transition-related genes to promote migration and invasion of bladder cancer cells

Objective To investigate the expression of microfibril-associated protein 5 (MFAP5) in bladder cancer tissues and its biological effects on bladder cancer cells. Methods The surgical resection specimens of bladder cancer patients, who were diagnosed in Shanghai General Hospital of Shanghai Jiao Tong University, were collected, and the expression level of MFAP5 was detected using immunohistochemistry. The cancer-associated fibroblasts (CAFs) and normal fibroblasts (NFs) were isolated and purified from bladder tumor tissues and normal bladder tissues, respectively. The mRNA expression levels of MFAP5 in CAFs, NFs, and four kinds of human bladder tumor cell lines (T24, 5637, UMUC3 and J82) were detected using quantitative real-time polymerase chain reaction. The correlation of MFAP5 expression with the progression and malignancy of bladder cancer was analyzed using the bladder cancer expression profile data derived from the Cancer Genome Atlas (TCGA) database and the GSE13507 database of Gene Expression Omnibus (GEO). The T24 and 5637 cells were treated with recombinant human MFAP5 protein to detect the effect on cell migration and invasion, as well as the expression of migration-associated proteins. Results MFAP5 was mainly expressed in the stroma of bladder cancer tissues, and its expression level was related to T stage and tumor grade of bladder cancer (both P<0.05). The mRNA expression level of MFAP5 in CAFs was significantly higher than those in NFs, and T24, 5637, UMUC3 and J82 cells (all P<0.01). The results of TCGA and GSE13507 database both showed that the overall survival of bladder cancer patients with high expression of MFAP5 was significantly shorter than that of patients with low expression of MFAP5 (both P<0.05). Gene set variation analysis showed that the expression level of MFAP5 increased with the degree of tumor malignancy (P<0.01), and its expression was closely related to expression of epithelial-mesenchymal transition-related genes such as cadherin 2, Twist1, matrix metalloproteinase 9 (MMP9), and zinc-finger E-box-binding protein 1 (ZEB1) (all P<0.01). In T24 and 5637 cells, the stimulation of recombinant human MFAP5 protein could up-regulate the expression of epithelial-mesenchymal transition-related proteins, including N-cadherin, MMP9, ZEB1 and Twist, and enhance the migration and invasion abilities (all P<0.01). Conclusion MFAP5 may be a potential molecular marker for predicitng the invasion and progression of bladder cancer.