微小RNA-30a作为膀胱尿路上皮癌的预后因素，通过拮抗Notch1抑制细胞恶性肿瘤。
MicroRNA-30a as a prognostic factor in urothelial carcinoma of bladder inhibits cellular malignancy by antagonising Notch1.
作者信息
Zhang Chao, Ma Xin, Du Jun, Yao Zhiyong, Shi Taoping, Ai Qing, Chen Xusheng, Zhang Zhenting, Zhang Xu, Yao Xin
机构信息
Department of Genitourinary Oncology, National Clinical Research Center of Cancer, Key Laboratory of Cancer Prevention and Therapy Tianjin, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China.
Department of Urology, China PLA General Hospital, Beijing, China.
出版信息
BJU Int. 2016 Oct;118(4):578-89. doi: 10.1111/bju.13407. Epub 2016 Feb 21.
OBJECTIVE
To explore the relation between microRNA-30a (miR-30a) and Notch1, and to evaluate the potential prognostic role of miR-30a in invasive urothelial carcinoma of the bladder (UCB).
PATIENTS AND METHODS
In all, 50 invasive UCB tissue specimens, along with the adjacent bladder tissue specimens were obtained, and the clinical parameters of the 50 patients were analysed. Bioinformatics analysis was performed and miR-30a was selected as a potential miRNA targeting Notch1, with a luciferase assay performed to verify the binding site between miR-30a and Notch1. Quantitative real-time reverse transcriptase-polymerase chain reaction was used to assess the RNA expressions of miR-30a and Notch1, while Western Blotting and immunohistochemical staining were used to assess the protein expression of Notch1. Finally, cell proliferation, cell cycle, cell migration and invasion assays were used to evaluate the cellular effects of miR-30a and Notch1 on the UCB cell lines T24 and 5637.
RESULTS
MiR-30a was downregulated in tumour tissues when compared with adjacent bladder tissues (P < 0.001), negatively correlating with Notch1 messenger RNA (R(2) 0.106, P = 0.021) in invasive UCB, and miR-30a expression further decreased in patients with shorter overall survival and disease-free survival (P = 0.039 and P = 0.037, respectively). The luciferase assay showed that miR-30a inhibited the Notch1 3'-untranslated region reporter activities in the T24 and 5637 cell lines (both P < 0.001). Both miR-30a and small interfering RNA Notch1 negatively regulated cell proliferation (P = 0.002 and P = 0.035 in T24; P = 0.029 and P = 0.037 in 5637 cell lines), activated cell cycle arrest (both P < 0.001 in T24; both P < 0.001 in 5637 cell lines), and prevented cellular migration (both P < 0.001 in T24; P = 0.003 and P < 0.001 in 5637 cell lines) and invasion (P = 0.009 and P = 0.006 in T24; P = 0.006 and P = 0.002 in 5637 cell lines) abilities. Ectopic Notch1 without the 3'untranslated region partially rescued the above-mentioned cellular effects of over-expressed miR-30a on T24 and 5637 cells.
CONCLUSIONS
MiR-30a lessens cellular malignancy by antagonising oncogene Notch1 and plays an effective prognostic role in invasive UCB.
目的
探讨微小RNA - 30a（miR - 30a）与Notch1之间的关系，并评估miR - 30a在膀胱浸润性尿路上皮癌（UCB）中的潜在预后作用。
患者与方法
共获取50例膀胱浸润性UCB组织标本及相邻膀胱组织标本，并分析50例患者的临床参数。进行生物信息学分析，选择miR - 30a作为靶向Notch1的潜在微小RNA，并进行荧光素酶测定以验证miR - 30a与Notch1之间的结合位点。采用定量实时逆转录 - 聚合酶链反应评估miR - 30a和Notch1的RNA表达，同时采用蛋白质印迹法和免疫组织化学染色评估Notch1的蛋白质表达。最后，通过细胞增殖、细胞周期、细胞迁移和侵袭实验评估miR - 30a和Notch1对UCB细胞系T24和5637的细胞效应。
结果
与相邻膀胱组织相比，肿瘤组织中miR - 30a表达下调（P < 0.001），在膀胱浸润性UCB中与Notch1信使RNA呈负相关（R² = 0.106，P = 0.021），且在总生存期和无病生存期较短的患者中miR - 30a表达进一步降低（分别为P = 0.039和P = 0.037）。荧光素酶测定显示，miR - 30a抑制T24和5637细胞系中Notch1 3'非翻译区报告基因活性（均为P < 0.001）。miR - 30a和小干扰RNA Notch1均对细胞增殖有负向调节作用（T24细胞系中P = 0.002和P = 0.035；5637细胞系中P = 0.029和P = 0.037），激活细胞周期阻滞（T24细胞系中均为P < 0.001；5637细胞系中均为P < 0.001），并抑制细胞迁移（T24细胞系中均为P < 0.001；5637细胞系中P = 0.003和P < 0.001）及侵袭能力（T24细胞系中P = 0.009和P = 0.006；5637细胞系中P = 0.006和P = 0.002）。不含3'非翻译区的异位Notch1部分挽救了过表达miR - 30a对T24和5637细胞上述的细胞效应。
结论
miR - 30a通过拮抗癌基因Notch1减轻细胞恶性程度，并在膀胱浸润性UCB中发挥有效的预后作用。
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