STAT/AKT/NFκB串话调控前列腺癌细胞化疗耐药通路的机制研究

Chemoresistance is one of the important causes leading to metastasis of CRPC (castration-resistant prostate cancer).It is becoming a tendency to target at some signaling pathways closely related to its underlying mechanisms. However, the specific targets and regulation mechanisms between pathways are still unclear. Our previous work shows that the cross talk between Stat3/AKT plays a critical role in angiogenesis in CRPC; the cross talk between Stat3/NF-κB affects cell apoptosis and growth; the expression of STAT3 varies in our established chemoresistannt cell lines. According to these results, we assume that the cross talk among Stat3/AKT/ NF-ΚB three signaling pathways could regulate the chemoresistance in CRPC and JAK may plays the key role in the whole pathways. Based on our established chemoresistannt cell lines, we propose to adopt small molecule JAK inhibitor and siRNA to prove that JAK is the key target in Stat3/AKT/ NF-ΚB cross talk by both traditional methods such as Western Blot，EMSA and new technology means such as LCM(Laser-Capture Microdissection) and Nano Pro. Also, this project aims to explore the related negative regulatory pathway and elucidate the mechanisms of chemoresistance to lay the foundation and provide a new way for the combination of target and Chemotherapy of CRPC.

化疗耐药是导致CRPC（去势抵抗性前列腺癌）转移的重要原因之一，针对相关作用通路进行干预成为趋势，但具体靶点及通路间的调控机制仍不清楚。我们前期工作发现：Stat3/AKT串话对CRPC血管形成具有相互作用；Stat3/NFκB串话具调节细胞凋亡与增殖的作用；自建化疗耐药细胞株STAT3表达有变化。据此提出假设：Stat3/AKT/NFκB三条细胞信号传导通路串话可能对CRPC化疗耐药具调控作用，而JAK是整个传导环路中的关键。我们拟在自建化疗耐药细胞株基础上，采用小分子JAK抑制剂阻断及siRNA干扰的方式，通过Western Blot、EMSA、LCM等传统方法及Nano Pro等新技术手段，证明JAK系Stat3/AKT/NFκB串话影响CRPC化疗耐药的关键靶点。本研究对阐明CRPC化疗耐药机制，揭示CRPC靶向治疗规律具有重要意义，将为CRPC化疗联合靶向药物治疗提供新思路。

化疗耐药是导致包括去势抵抗性前列腺癌等恶性肿瘤转移的重要原因之一。我们前期工作发现：Stat3/AKT串话对CRPC血管形成具有相互作用；Stat3/NFκB串话具调节细胞凋亡与增殖的作用；自建化疗耐药细胞株STAT3表达有变化。我们认为Stat3/AKT/NFκB三条细胞信号传导通路串话可能对CRPC化疗耐药具调控作用，而JAK是整个传导环路中的关键。.通过Western blotting检测STAT3, p-STAT3, E-cadherin, N-cadherin, survivin, and Bcl-xL的蛋白表达水平. 用共聚焦及RT-PCR检测E-cadherin和N-cadherin表达情况.我们发现与常氧环境下相比，PC-3在低氧环境下对多西他赛更加耐受，并且伴有p-STAT3 和 N-cadherin 上调, 而 E-cadherin 下调. 通过用S3I-201抑制STAT3表达，阻断了低氧诱导的EMT。低氧环境下，STAT3的上调促进几种生存因子的表达（survivin和Bcl-xL）.并且STAT3抑制剂阻断了低氧诱导的化疗耐药。因而在低氧环境下可能是通过上调STAT3促进化疗耐受及EMT. 而抑制STAT3表达可能是逆转低氧条件下化疗耐受及EMT的靶点。.通过Western Blot技术检测JAK2抑制剂和IL-6处理后的食管鳞癌细胞中STAT3、磷酸化STAT3、NF-κB p65、磷酸化NF-κB p65 和COX-2蛋白表达。用荧光定量RT-PCR法检测STAT3和NF-κB siRNA转染食管鳞癌细胞后STAT3、NF-κB及COX-2的mRNA水平。我们发现STAT3在三种食管鳞癌细胞系中不同程度地激活，阻断JAK/STAT3信号通路可以抑制食管鳞癌的细胞生长、血管生成及调节细胞周期。同时，阻断JAK/STAT3信号通路可以通过下调NF-κB p65的磷酸化、COX-2蛋白水平及IL-6的含量抑制食管鳞癌的肿瘤相关性炎症。另外，IL-6可以激活STAT3及NF-κB通路从而促进食管鳞癌的肿瘤相关性炎症及肿瘤生长。我们还发现COX-2是STAT3及NF-κB共同的下游基因。我们认为JAK/STAT3信号通路可以通过IL-6以及与NF-κB p65及COX-2的交互作用连接肿瘤相关性炎症与肿瘤生长，它有望成为恶性肿瘤预防及治疗的靶点。