靶向Notch-1的miRNA在浸润性膀胱癌中的功能及分子机制研究

Our previous research revealed that the expression level of Notch-1 was significantly decreased in invasive bladder cancer tissues compared with the normal bladder transitional epithelium. The effects and underlying mechanism of Notch-1 on bladder cancer remain unknown. In current study, we interrogated MicroRNAs (miRNAs) arrays of two human invasive bladder cancer cell lines T24 and 5637 and human uroepithelial immortalized cell line SV-HUC-1.We identified 36 significantly up-regulated miRNAs in the invasive bladder cancer.. MiRNAs inhibit translation or induce mRNA degradation in general by binding to the 3' untranslational region (3'UTR) of target mRNAs. We analyzed potential miRNAs targeting Notch-1 through picTar, miRanda and Targetscan. Among the 36 elevated miRNAs, we found 5 of them matched with Notch-1 3'UTR. Overexpression of miR-34a could decrease the expression of Notch1 and its downstream HES-1. . Further, we try to investigate the underlying functions of miRNAs targeting Notch-1 in invasive bladder cancer. Real-time PCR, western-Blot and immunofluorescence were used to analyze their expression. Lentivirus system will be employed to elevate or knockdown the expression of mir-34a and its effects on tumorigenesis and metastasis will be assessed both in vitro and in nude mice. Apoptosis will be examined by TUNEL assay and Annexin V-FITC kit（annxin V staining）. Wound-healing and matrigel invasion assay will be used to determine migration and invasion potentials of bladder cancer cells. Furthermore, the correlations of miR-34a expression with clinicopathological characteristics, prognosis as well as Notch-1 expression level will be evaluated. We would investigate the potential of serum and urine miRNAs as biomarkers for invasive bladder cancer.. Our findings will present functional and mechanistic insight into the critical role of miRNAs targeting Notch-1 in the progression and metastasis of invasive bladder cancer. It lays the groundwork for exploring the importance of Notch signaling in invasive bladder cancer. The Notch pathway could be a worthwhile target for further bladder cancer therapy. Serum and urine miRNAs may have strong potential as novel biomarkers for invasive bladder cancer.

前期研究发现，与正常膀胱粘膜相比浸润性膀胱癌中Notch-1表达显著下降，miRNA基因芯片分析显示浸润性膀胱癌细胞T24和5637与人膀胱上皮永生化细胞株SV-HUC-1相比有5种靶向Notch-1的miRNA显著升高，其中过表达miR-34a后Notch-1及下游HES-1蛋白表达显著下调。本课题拟进一步通过miRNA基因芯片筛选，生物信息学分析，Real-time PCR, pMIR-REPORT荧光素酶报告系统，miRNA功能缺失及过表达，膀胱癌细胞皮下成瘤裸鼠动物模型等手段，研究浸润性膀胱癌中靶向Notch-1的miRNA的功能及分子机制；系统分析这些miRNA的差异表达与膀胱癌的临床分期、病理分级、患者预后等的关系,评价血清及尿液中miRNA作为标志物的价值。从miRNA入手阐明浸润性膀胱癌中Notch-1异常表达的具体机制可为膀胱癌的治疗提供新的靶点和理论依据。

Notch信号通路调节细胞增殖，分化等重要生物学过程，其表达和功能的异常与多种肿瘤的发生及进展密切相关，但在泌尿系统肿瘤中的生物学功能及调节机制并不明确。在浸润性膀胱癌中，通过miRNA芯片联合Notch信号通路芯片分析，发现Notch1及其配体DLL1、JAG2在膀胱癌细胞中表达显著下降。实时定量PCR及免疫组化证实Notch1及DLL1在膀胱癌组织中表达低于癌旁组织。在肾透明细胞癌中，和癌旁组织相比，Notch1表达显著升高。通过siRNA干扰下调Notch1的表达或者上调Notch1的表达证实Notch1显著促进细胞增殖，细胞周期、迁移及侵袭能力。Notch1可通过靶基因Hes1抑制PTEN的表达，进而激活AKT信号通路。在肾癌中Notch1是重要的癌基因，有望成为肿瘤治疗的新靶点。