miR-802负调控CYLD促进膀胱尿路上皮癌病理发生与发展

Effect of miR-802 on pathogenesis and progression of bladder urothelial carcinoma by inhibition of CYLD

目的:本研究主要是miR-802促进上皮-间质转化(EMT)的发展抑制CYLD激活NF-κB信号通路,促进膀胱尿路上皮癌发生与发展。方法:qRT-PCR检测miR-802在膀胱尿路上皮癌肿瘤组织和癌旁正常组织中的表达,膀胱尿路上皮癌细胞系(T24、5637、J82、BIU-87、SW870、UM-CM-3)与非肿瘤正常尿路上皮细胞系SVHUC-1的表达及与临床特征因素的相关性;预测靶基因CYLD,用荧光素酶报告基因法及Western blot检测与miR-802的调控关系;Western blot检测3种EMT相关蛋白表达水平(E-cadherin、N-cadherin及Vimentin)与miR-802的相关性;qRT-PCR检测127例膀胱尿路上皮癌肿瘤组织和癌旁正常组织及膀胱尿路上皮癌细胞系T24、5637、J82、BIU-87、SW870、UM-CM-3与非肿瘤正常尿路上皮细胞系SV-HUC-1中的CYLD mRNA表达;Western blot检测膀胱尿路上皮癌肿瘤组织中CYLD蛋白表达水平;用CCK8法、流式细胞法、Tranwell迁移实验检测了膀胱尿路上皮癌细胞系T24的增殖、迁移及周期S期的聚集;采用Western blot检测CYLD及TNF-α对NF-κB信号通路及相关分子的调控作用。结果:miR-802在膀胱尿路上皮癌患者及膀胱尿路上皮癌各株细胞系中表达均呈现升高趋势,而同时与危险因素分级、TNM分期、淋巴结转移也呈现正相关;miR-802mimics转染组较miR-NC对照组CYLD蛋白的表达量明显降低;miR-802过表达导致N-cadherin与Vimentin的蛋白表达显著升高,而E-cadherin的表达显著降低;CYLD可以作用于膀胱尿路上皮癌,对其有抑制效果;miR-802通过促进EMT的发展,而促进膀胱尿路上皮癌细胞的增殖、迁移及周期S期的聚集,但该效果被CYLD回补;CYLD抑制NF-κB信号通路活性。结论:miR-802促进EMT的发展抑制CYLD激活NF-κB信号通路,促进膀胱尿路上皮癌发生与发展,本研究miR-802有可能成为诊断和预测膀胱尿路上皮癌的潜在生物标志物。

Objective:In this study,miR-802 promoted the development of epithelial-mesenchymal transition(EMT),inhibited the CYLD in activating NF-κB signaling pathway,and promoted the occurrence and development of bladder urothelial carcinoma.Method:QRT-PCR was used to detect the expression of miR-802 in tumor tissues of bladder urothelial carcinoma and paracancerous normal tissues,and the expression of bladder urothelial carcinoma cell lines(T24,5637,J82,BIU-87,SW870,UM-CM-3)and non-tumor normal urothelial cell line SVHUC-1 and its correlation with clinical features.The target gene CYLD was predicted,and the regulatory relationship between miR-802 and the target gene CYLD was detected by using the luciferase reporter gene method and Western blot.Western blot was used to detect the correlation between the expression levels of three EMT-related proteins(E-cadherin,N-cadherin and Vimentin)and miR-802.The mRNA expression of CYLD in 127 patients with bladder urothelial carcinoma and paracancerous normal tissue and bladder urothelial carcinoma cell lines T24,5637,J82,BIU-87,SW870,UM-CM-3 and non-tumor normal urothelial cell line SV-HUC-1 was detected by qRT-PCR.The expression level of CYLD protein in bladder urothelial carcinoma was detected by Western blot.CCK8,flow cytometry and transwell migration assay were used to detect the proliferation,migration and aggregation of T24 in urinary tract epithelial carcinoma cell line.Western blot was used to detect the regulatory effects of CYLD and TNF-infection on NF-negative B signaling pathways and related molecules.Result:The expression of miR-802 was increased in all cell lines of bladder urothelial carcinoma and bladder urothelial carcinoma,and was positively correlated with risk factor grade,TNM stage and lymph node metastasis.The expression level of CYLD protein in the miR-802 mimics transfection group was significantly lower than that of the miR-NC control group.Overexpression of miR-802 led to a significant increase in protein expression of N-cadherin and Vimentin,while E-cadherin expression decreased.CYLD can inhibit bladder urothelial carcinoma.By promoting the development of EMT,miR-802 promoted the proliferation,migration and aggregation of bladder urothelial cancer cells in the S phase,but the effect was reversed by CYLD.CYLD inhibits the activity of NF-treated B signaling pathway.Conclusion:MiR-802 promotes the development of EMT,inhibits the CYLD in activating NF-κB signaling pathway,and promotes the occurrence and development of bladder urothelial carcinoma.In this study,miR-802 may become apotential biomarker for the diagnosis and prediction of bladder urothelial carcinoma.