NF-κB/miR-130b-301b cluster/USP13信号轴介导PTEN蛋白降解促进膀胱癌进展的机制研究

NF-κB活化大量促癌因子诱导肿瘤发生，PTEN抑制PI3K/Akt通路并下调NF-κB活性。申请者发现：NF-κB与miR-130b-301b 基因簇（cluster）启动子结合促进其转录活化；miR-130b-301b cluster抑制PTEN野生型膀胱癌细胞系PTEN蛋白表达；信息学及前期实验提示去泛素化酶USP13可能作为中间因子介导miR-130b-301b cluster对PTEN蛋白的翻译后调节。因此NF-κB是否通过上调miR-130b-301b cluster抑制USP13进而诱导PTEN蛋白降解有待进一步阐明。本项目拟在PTEN野生/缺失及野生/突变型细胞模型及动物模型中，通过免疫沉淀、EMSA、原位杂交等研究方法，力图证明NF-κB/miR-130b-301b cluster/USP13信号轴诱导PTEN蛋白降解，反馈性活化NF-κB并促进膀胱癌恶性进展的假说。

Constitutive activation of NF-κB signaling stimulated aberrant cell proliferation and resistance to apoptosis thus promotes cancer initiation and progression. As a tumor suppressor, PTEN suppresses PI3K/Akt signal and nucleus translocation of NF-κB. We have discovered that: NF-κB upregulates the expression of miR-130b-301b cluster through binding to the gene promoter region; miR-130b-301b cluster upregulation leads to a suppressed PTEN protein expression in PTEN wild-type bladder cancer (BC) cell lines; Bioinformation analysis suggests that USP13 is the downstream target of both miR-130b and miR-301b, furthermore, as deubiquitinating enzyme, USP13 plays an important role in maintaining the stability of PTEN protein. Therefore, whether miR-130b-301b cluster induces the ubiquitylation degradation of PTEN protein through suppressing USP13 expression still calls for further investigation. In this project, we aim to investigate the regulating function of NF-κB/miR-130b-301b cluster/USP13 signaling in inducing PTEN protein degradation and promoting BC progression in a PTEN positive/deletion or PTEN positive/mutation BC cell model using molecular biological methods such as chromatin immunoprecipitation, electrophoretic mobility shift assay and detection of ubiquitylated protein level, mice xenograft tumor model is also concluded to verify our hypothesis. This study investigates the underlying mechanism of how NF-κB avoids the negative feedback and contributes to the progression of BC through inducing PTEN protein degradation. The finding of our project will help to better understand the role of NF-κB in the progression of BC, especially during early stage with normal expression of PTEN gene.