CSN6/Snail1通路调控上皮间质转化及其在前列腺癌进展中的作用

CSN6 can stabilized MDM2, overexpression of MDM2 can promote the tumor metastasis. But the relationship and the underlying mechanism between CSN6 and tumor metastasis is unclear. The present study show that CSN6 induce EMT in PCa DU145 cells, and up regulates the expression of MDM2. Another report show that regulation of the degradation of target protein is the valide way to execute the biological function of E3 ligase MDM2. Foxo3a is a target proteion of MDM2. Repress the expression of Foxo3a can induce EMT in cancer cells. Sanil can promote EMT through directly repress the transcription of E-cadherin. In this study, we report, there is downregulated expression of Foxo3a and upregulated expression of Snail in CSN6-overexpressed cells. By JASPAR database, Foxo3a binding sites in the Snail1 promoter were detected. Therefore, We will identify CSN6 functions as a regulator of EMT in PCa cells. CSN6 can induce EMT via stablizing MDM2, down-regulating the expression of Foxo3a, the inhibation of the transcription of Snail1 was decreased, up-regulating the snail1, then repress the expression of E-cadherin, at last promote EMT and cell invasion in PCa cells. The hypothesis will also be investigated in animal experiment and human prostate cancer tissues. If it is verified, our study may provide a new target for prostate.

CSN6可增强MDM2稳定性，过表达MDM2可促进肿瘤转移，但CSN6与肿瘤转移关系及分子机制未见报道；本研究显示，CSN6可诱导前列腺癌细胞(PCa)发生EMT，同时上调MDM2。研究表明，MDM2为泛素E3连接酶，通过调控靶蛋白降解发挥其功能；Foxo3a是MDM2底物蛋白，抑制Foxo3a可诱导癌细胞发生EMT；Snail1能抑制E-cadherin转录并启动EMT。本研究显示，PCa细胞中过表达CSN6，Foxo3a表达下调，Snail1表达升高； 通过JASPAR数据库，发现Snail1启动子区存在Foxo3a结合位点。因此我们推测CSN6是PCa细胞EMT发生的重要调节分子，通过稳定MDM2，下调Foxo3a表达，进而降低其对Snail1的转录抑制，Snail1表达升高，下调E-cadherin，诱导EMT，最终促进细胞侵袭和转移。上述机制如证实，将为PCa治疗提供新靶点。

本研究采用前列腺癌细胞为研究对象，研究COP9 信号小体第六亚基（CSN6）对前列腺癌细胞EMT发生的诱导作用及其对前列腺癌细胞侵袭能力的影响；探讨CSN6对前列腺癌细胞生长功能的影响及其分子机制；最后阐述CSN6在紫杉醇治疗前列腺癌中的作用进行初步探索。本研究首先利用RT-PCR及western blotting技术检测CSN6在前列腺癌细胞中的表达， 然后建立稳定下调及过表达CSN6的前列腺癌细胞系，利用倒置显微镜观察CSN6对细胞形态学的影响、Western blotting检测CSN6对前列腺癌细胞中E-Cadherin、Vimentin、MDM2、Foxo3a及Snail1表达的影响，划痕实验、Matrigel实验检测细胞的迁移和侵袭能力。利用MTT实验、克隆形成实验测定CSN6对前列腺癌细胞增殖活力的影响；流式细胞仪检测细胞周期分布情况；利用western blotting检测CSN6对细胞周期相关蛋白和凋亡相关蛋白表达的影响；透射电镜实验、JC-1实验和流式细胞凋亡实验检测，CSN6对前列腺癌细胞凋亡情况的影响。利用MTT实验、透射电镜实验、JC-1实验和流式细胞凋亡实验检测靶向抑制CSN6在紫杉醇治疗前列腺癌中的作用。以上实验结果表明，CSN6在前列腺细胞中广泛表达，且在转移程度高的细胞中高表达，提示CSN6可能在人前列腺癌细胞的生物学行为中发挥重要作用。CSN6可诱导前列腺癌细胞发生EMT，从而促进癌细胞侵袭能力，影响MDM2稳定性及表达水平，促进Foxo3a的降解，下调其表达水平，从而减弱对snail1基因的转录抑制，提高snail1的表达，进而下调E-cadherin表达，诱导EMT发生，促进前列腺癌细胞的侵袭和转移；CSN6可调控前列腺癌细胞生长，影响细胞周期G1/S期进程，并通过调控Cyclin E1、Cyclin D1、p27、survivin和p-Bcl-2的表达而实现的；抑制CSN6可协同紫杉醇促进前列腺癌细胞凋亡。