瞬时受体电位M8在前列腺癌骨转移中作用及其机制的研究

Prostate cancer will causes bone metastasis and skeletal-related events (SRE) in about 80% patients for which there is no satisfactory treatment. For lack of specifity, current pharmaceuticals may cause serious side-effects. So it is crucial to explore novel targeted therapy. As an important second massager, Ca2+ plays an important role in regulation of cell proliferation, differentiation, migration and invasion and has been a new target in cancer therapy. Transient receptor potential M8 (TRPM8) is an ion channel specifically expressed in prostate and play important role in regulation Ca2+ homostasis. Our previous studies demonstrated that TRPM8 regulated the proliferation and migration of prostate cancer cells both in vitro and in vivo, while latest study showed that TRPM8 and its natural agonist PSA played a crisis role in regulating bone metastasis of prostate cancer which maybe caused by the activation of PSA-TRPM8 pathway. Based on our previous results, we will, by using agonist and/or antagonist through coculture (in vitro) and athymic nude mice model (in vivo), explore the role of TRPM8 in the interaction of prostate cancer cells and bony microenvironment, namely, prostate cancer cells inducing differentiation and activation osteoblast and osteoclast, while osteoblast and osteoclast chemotaxis prostate cancer cells. Hence we may probe into the possibility of disturbing the so-called "vicious circle" between prostate cancer cells and bony microenvironment so as to inhibit the bone metastasis of prostate cancer by targeted therapy.

约80%前列腺癌患者最终会出现骨转移并死于骨相关事件。目前治疗骨转移药物大多因作用机制缺乏特异性可引起严重的副作用，探索新的靶向治疗方法已成当务之急。瞬时受体电位M8（TRPM8）是新近发现的前列腺特异性钙通道，申请人前期研究表明TRPM8通过调节细胞内Ca2+稳态而调控前列腺癌细胞增殖及迁移，可作为肿瘤治疗的新靶点。最新的研究表明TRPM8及其激动剂PSA参与前列腺癌骨转移调控，我们推测其与PSA激活TRPM8相关。本课题拟在前期基础上，采用基因调控及使用TRPM8通道激动剂和（或）阻断剂的方法，结合荷瘤裸鼠动物模型，揭示TRPM8对前列腺癌细胞与骨微环境相互作用的影响及TRPM8在成骨/破骨细胞定向趋化前列腺癌细胞中的作用。通过体内、体外实验相结合，探讨通过TRPM8这一靶点干预前列腺癌与骨微环境之间"恶性循环"，从而抑制前列腺癌骨转移的可能性，为前列腺癌的基因靶向治疗奠定理论基础。

约80%前列腺癌患者最终会出现骨转移并死于骨相关事件，目前治疗骨转移药物大多因作用机制缺乏特异性可引起严重的副作用。瞬时受体电位M8（TRPM8）是新近发现的前列腺特异性钙通道，申请人前期研究表明TRPM8 通过调节细胞内Ca2+稳态而调控前列腺癌细胞增殖及迁移，可作为肿瘤治疗的新靶点。研究内容包括：RT-PCR，Western blot检测trpm8在MC3T3-E1及Raw264.7细胞中的表达、电生理证实小鼠MC3T3-E1及Raw264.7细胞中Trpm8通道活性、 完成小鼠trpm8基因干扰慢病毒载体vshRNA构建及鉴定、trpm8在成骨细胞诱导前列腺癌细胞迁移中的作用研究、前列腺癌细胞中trpm8表达水平对MC-3T3-E1(成骨细胞)成熟及矿化的影响。结果：(1) 成功构建了LNCaP-shTRPM8、DU145-shTRPM8及PC3-TRPM8稳转细胞株，并通过Western blot检测LNCaP、LNCaP-shTRPM8、LNCaP-vector，以及PC3、PC3-vector、PC3-TRPM8中迁移通路相关蛋白的变化水平。(2) 外源性表达trpm8可增强PC-3细胞诱导MC-3T3-E1(成骨细胞)成熟及矿化。(3) 通过MC-3T3-E1(成骨细胞)分别与LNCaP、LNCaP-shTRPM8、 PC3及PC3-TRPM8共培养，结果发现成骨细胞对TRPM8表达水平高的LNCaP及PC3-TRPM8细胞，相对于LNCaP-shTRPM8 及PC3，具有更强的趋化作用。科学意义：前列腺癌骨转移是前列腺癌-骨微环境之间相互作用的结果，寻找在前列腺癌细胞及骨微环境中特异性表达并发挥重要作用的靶点是干预前列腺癌-微环境间“恶性循环”，抑制骨转移的理想治疗方法。trpm8表达于前列腺癌细胞及成骨、破骨细胞，促进前列腺癌细胞增殖和迁移、增强成骨和破骨细胞趋化前列腺癌细胞，并促进成骨细胞分化成熟，因而是靶向干预前列腺癌骨转移的理想靶点。本研究揭示了TRPM8通道在前列腺癌骨转移中的作用，为靶向干预前列腺癌骨转移提供了理论基础。