温度敏感TRP离子通道蛋白参与调控人类黑素瘤发生发展的机制研究

Temperature-sensitive TRP ion channels, or thermoTRPs, are an important group of membrane proteins that sense temperature changes and many physical and chemical stimuli. They have been confirmed to participate in temperature sensation and regulation as well as additional crucial physiological processes. Importantly, thermoTRPs play an important role in cancer development. Indeed, transformation of normal cells to tumorigenic cells is tightly associated with the expression of ion channels, which is dynamically regulated by complex physiological and pathological factors. Previously we have demonstrated that co-expressing of TRPV1 and TRPV3 subunits yielded heteromeric channels that serve as novel cellular sensors for heat and chemical stimuli. Based on these findings, in the proposed study we plan to examine the expression pattern and functional properties of thermoTRP channels in human melanocyte and malignant melanoma cells. We will further investigate the correlation between thermoTRPs and melanoma as well as the physiological and pathological roles that may participate in melanoma progression, such as cell cycle, migration, invasion and cell death. In this process, we aim to develop pharmacological assessment methods targeting thermoTRPs in melanoma cells. We anticipate that the study will provide novel knowledge of thermoTRPs as valuable diagnostic and/or prognostic marker, as well as promising targets for pharmaceutical intervention of melanoma cancers.

离子通道蛋白同细胞增殖及癌症的发生和演进关系密切，部分温度敏感TRP离子通道蛋白在人类黑素瘤中表达异常，但由其异常表达引起通道功能紊乱在参与调控黑素瘤发生和发展中的作用机制尚不清楚。申请人前期研究证实温度敏感TRPV离子通道蛋白亚基可以异源组装形成杂合通道(J.Gen.Physiol.2007,129:191封面文章)，纠正了以前的错误认识，阐述了杂合通道在温度及化学激活过程中的功能特性(J.Biol.Chem.2012, 287:7279)。在此基础上，本项目拟通过蛋白表达结合电生理膜片钳技术检测由异常表达在黑素瘤中的温度敏感TRP离子通道所致通道功能增强或减弱的特性；选择通道激活剂及拮抗剂刺激细胞，观察细胞中钙离子变化，阐明离子通道的激活和失活在黑素瘤发生发展中的关键作用机制；并利用通道靶点对多种物理、化学干预的高度敏感性，寻找黑素瘤靶向凋亡诱导剂，为选择定向干预的新药开发提供依据。

离子通道蛋白同细胞增殖及癌症的发生和演进关系密切，部分温度敏感TRP 离子通道蛋白在人类黑素瘤中表达异常，但由其异常表达引起通道功能紊乱在参与调控黑素瘤发生和发展中的作用机制尚不清楚。本项目通过对温度敏感TRP离子通道蛋白的表达结合钙离子成像技术检测由异常表达在黑素瘤中的温度敏感TRP 离子通道所致通道功能增强或减弱的特性；选择通道激活剂及拮抗剂刺激细胞，观察细胞中钙离子变化，研究离子通道的激活和失活在黑素瘤发生发展中的关键作用机制；并利用通道靶点对多种物理、化学干预的高度敏感性，寻找黑素瘤靶向凋亡诱导剂，为选择定向干预的新药开发提供依据。在研究中，我们筛查了人类黑素细胞和恶性黑色素瘤细胞中thermoTRP通道蛋白的表达模式和功能特性。我们也进一步检查了thermoTRPs在黑色素瘤发生发展中的作用，如细胞周期，迁移，入侵和细胞死亡的生理和病理之间的相关性。在此过程中，应用蛋白质印迹和数字PCR来分别研究蛋白质和RNA水平的表达谱。结果表明，TRPM8，TRPV2和TRPV4均在黑素瘤细胞和黑素细胞中有表达。然而，与黑素细胞相比，黑素瘤细胞中TRPV4蛋白和mRNA水平增加。此外，通过MTT测试使用TRPV4的激活剂GSK1016790A，可以导致黑色素瘤A375细胞的细胞活力的下降，而通过流式细胞术测试细胞周期揭示GSK不影响A375黑素瘤细胞的细胞周期。此外，钙成像表明TRPV4激活通过GSK诱导黑色素瘤细胞的凋亡。总体而言，我们的研究发现TRPV4在黑色素瘤中上调并与黑素细胞的致瘤性相关。激活TRPV4可以抑制A375黑素瘤细胞的增殖。因此，TRPV4可以作为有价值的诊断和/或预后标记，以及用于黑素瘤治疗的药物干预的有希望的靶标。