CPNE3调控细胞自噬在膀胱癌侵袭转移中的作用及分子机制研究

The two main features of malignancy, invasion and metastasis, are the main cause of cancer recurrence and patient death in bladder cancer. The underlying mechanism remains to be elucidated. Autophagy is closely related to tumor invasion and metastasis.We previously observed significant elevated expression of CPNE3 gene in invasive bladder cancer tissues and bladder cancer cell lines with high potential of invasion. Further, the expression level of CPNE3 seems to be positively related to LC3 expression, a protein marker for invasion and autophagy in bladder cancer; and negatively related to P62. Therefore, we hypothesized that CPNE3 may play a regulatory role in invasion and metastasis of bladder cancer via autophagy, affecting the pathological progression of bladder cancer. In this study, we aim to establish CPNE3 overexpression/knockout cell line and animal model via lenti-viral and CRISPR-Cas9 approach; investigate the effect of altered CPNE3 expression on cellular functional phenotype and autophagy; and examine the impact of CPNE3 on autophagy and JNK signaling using autophagy and JNK inhibitors. We believe that our study will shed light on how autophagy and invasion are regulated by CPNE3 in a MAPK-JNK signaling dependent mechanism, providing experimental basis preliminary data and for the development of novel gene targeted therapy and drug discovery.

侵袭转移是影响膀胱肿瘤患者疗效和死亡的主要原因，其机制不明。有研究发现自噬与肿瘤侵袭转移密切相关。申请人前期工作表明CPNE3在侵袭性膀胱癌组织及高转移潜能癌细胞中异常高表达，其表达量与侵袭转移潜能及自噬标志蛋白LC3表达呈正相关，与P62表达负相关。故我们假设：CPNE3可能通过调控自噬而促进膀胱癌侵袭转移，以此调控膀胱癌的病理进程。本项目拟通过慢病毒转染及CRISPR/Cas9基因编辑技术构建CPNE3过表达/敲除的细胞和动物模型，探讨CPNE3表达改变对膀胱癌细胞功能表型及自噬形成的影响；结合自噬抑制剂和JNK抑制剂处理，进一步观察CPNE3对自噬和JNK信号通路的作用，从而阐明CPNE3通过MAPK/JNK信号通路调控细胞自噬，促进膀胱癌侵袭转移的分子机制，为寻找新的基因治疗靶点和药物开发提供理论基础和实验依据。

侵袭转移是影响膀胱肿瘤患者疗效和死亡的主要原因，其机制不明。有研究发现自噬与肿瘤侵袭转移密切相关。申请人前期工作表明CPNE3在侵袭性膀胱癌组织及高转移潜能癌细胞中异常高表达，其表达量与侵袭转移潜能及自噬标志蛋白LC3表达呈正相关，与P62表达负相关。故我们假设：CPNE3可能通过调控自噬而促进膀胱癌侵袭转移，以此调控膀胱癌的病理进程。本项目拟通过慢病毒转染及CRISPR/Cas9基因编辑技术构建CPNE3过表达/敲除的细胞和动物模型，探讨CPNE3表达改变对膀胱癌细胞功能表型及自噬形成的影响；结合自噬抑制剂和PI3K抑制剂处理，进一步观察CPNE3对自噬和PI3K信号通路的作用，从而阐明CPNE3通过PI3K/Akt/mTOR信号通路调控细胞自噬，促进膀胱癌发生发展的分子机制。本研究发现CPNE3在膀胱癌组织及癌细胞中异常高表达，其表达水平和膀胱癌患者预后密切相关；CPNE3基因被敲减后膀胱癌细胞的增殖能力明显受抑制，并促进其凋亡；其可能是通过激活PI3K/Akt/mTOR信号通路，促进LC3和Beclin-1并抑制P62表达，以此诱导自噬的发生，从而促进膀胱癌发生发展。本研究发现可为膀胱癌基因治疗靶点和药物开发提供理论基础和实验依据。