多肽探针研究Nav1.7调节前列腺癌PC3细胞转移的分子机制

Metastasis is the primary factors contributing to deaths in patients with cancer,and is the research focus in medical research in the present study. Voltage-gated sodium channel subtype Nav1.7 has been found to be highly expressed in the prostate canncer cell line PC3 and to modulate the metastasis of this cell line, but the mechanism underlying this process is not clarified so far. The research of sodium channel complex and phosphorylation has become a hot theme in the sodium channel related field, and some studies indicated that Nav1.7 complex and phosphorylation play important rols in its regulation of metastasis. Our previous work showed that two spider peptide toxins, JZTX-I and HWTX-IV, could inhibit the inactivation and activation of Nav1.7 and then promoted and supressed the metastasis of PC3 cells, respectively, which would provide us an novel probes for the investigation of PC3 metastasis regulated by Nav1.7. A combination of several proteomic techniques, including BN/SDS-PAGE， phosphorylation identification, will be used to identify Nav1.7-interacted proteins and phosphorylated sites in the PC3 cells treated with JZTX-I and HWTX-IV. Moreover, 2-3 proteins and phosphorylation sites will be further studied. This study might help to elucidate the mechanism of Nav1.7 regulated PC3 metastasis, to identify potential protein markers and novel mechanisms of metastasis of cancer cells, and to establish a novel stratedy for invastigating the regulation of ion channels on physiological and pathological processes.

肿瘤转移是导致肿瘤患者死亡的首要因素，是当前国际医学研究重点。国际同行研究表明钠通道亚型Nav1.7在高转移前列腺癌PC3细胞上特异性高表达，并促进其转移活性，但分子机制尚不清楚。蛋白质相互作用复合物和磷酸化修饰是钠通道功能执行和调控的必要途径，阐明PC3细胞转移过程Nav1.7复合物和磷酸化修饰变化，是揭示钠通道在生理/病理过程的功能所必须的。本课题基于前期研究发现蜘蛛多肽毒素JZTX-I和HWTX-IV分别增强和减弱PC3转移活性，创新性提出利用JZTX-I与HWTX-IV作为分子探针，通过蛋白质组学技术包括BN/SDS-PAGE膜蛋白复合物分离、磷酸化修饰质谱鉴定，比较JZTX-I导致的转移增强组和HWTX-IV导致的转移减弱组Nav1.7互作蛋白和磷酸化修饰位点变化，为深入解释Nav1.7调节肿瘤转移的途径、发现新药物靶标提供依据，为探讨肿瘤相关离子通道在病理过程作用提供新思路新方法

肿瘤转移是导致肿瘤患者死亡的首要因素并受到众多因素的影响及调控。Nav1.7在高转移性前列腺癌细胞(PC3/Mat-LyLu)上特异性高表达并促进其转移活性,阐明前列腺癌细胞转移过程中Nav1.7互作蛋白和磷酸化修饰能够为Nav1.7调节其细胞转移的分子机制提供理论基础。本研究中从蜘蛛粗毒中分离并鉴定了JZTX-I和HNTX-III，JZTX-I抑制Nav1.7的失活导致Mat-LyLu细胞转移活性增强；而HNTX-III抑制Nav1.7的激活导致Mat-LyLu细胞转移活性减弱。细胞迁移与侵袭能力检测表明，5μM的JZTX-I处理细胞24h，迁移能力与侵袭能力分别增强50.2%及19.0%；5μM的HNTX-III处理细胞24h，迁移能力及侵袭能力分别减弱44.0%及60.1%。以JZTX-I和HNTX-III为分子探针，2DE结合质谱分析增强和减弱Mat-LyLu细胞转移过程中质膜蛋白质的差异表达，得到108个差异表达蛋白质，包括与细胞粘附相关的 Annexin、Muskelin等，与肿瘤相关的Cofilin-1、Moesin、Fascin等。选取与肿瘤转移相关的差异表达蛋白Fascin、Muskelin、Annexin A2、Cofilin-1进行Western Blot检测表明与2DE-MS结果一致。功能分析表明这些蛋白参与调节细胞骨架及其相关蛋白，导致细胞转移能力改变，其中Rho GTPase在信号通路起着中心调控作用，当抑制Rac或Rho的活性均导致细胞转移活性的减弱及相应的蛋白差异表达。JZTX-I和HNTX-III处理细胞，结合TiO2富集与iTRAQ标记，鉴定比较了差异磷酸化蛋白质，包括1919条非冗余磷酸化肽段，3519个磷酸化位点，657个非冗余磷酸化蛋白质。这些蛋白主要参与细胞生理进程和代谢，行使各种分子结合及转录共激活功能。通过对磷酸化肽段进行基于序列的motif富集分析，发现13种丝氨酸motif、4种苏氨酸motif及1种酪氨酸motif序列。分析overlap磷酸化蛋白质的定量信息，得到120个差异磷酸化蛋白质，通过蛋白互作网络及所涉及的相关信号通路分析表明，这些信号通路均与肿瘤的生长、增殖、存活、运动、侵袭及转移相关。我们的研究结果为深入解释Nav1.7调节肿瘤转移的途径提供了实验依据。