结直肠癌新候选抑癌基因TRPM4的作用及分子机制研究

Colorectal cancer has high incidence and poor prognosis. The main reason is that the etiology and pathogenesis has not been fully elucidated. Our preliminary study found that TRPM4 gene, a member of the transient receptor potential melastatin family, was significantly down-regulated or silenced in colorectal cancer and its protein expression level was closely related to the prognosis of the patients. Restoring TRPM4 expression in colon cancer cells can dramatically increased calcium ion concentration in cytoplasm and significantly inhibited the tumor cell growth and enhanced the tumor cell apoptosis. TRPM4 are identified as calcium-activated nonselective cation channels channels. They are impermeable to calcium. Instead, they mediate transport of monovalent cations (such as Na+, K+) through regulating membrane depolarization and Ca2+ homeostasis, thereby strongly impact on cell physiology. This project will study the expression and the expression control mechanisms of TRPM4 gene, analyze the correlation of these with the clinicpathological features and explore by in vitro and in vivo experiments that whether TRPM4 gene plays an important biological role in carcinogenesis and progression of colorectal cancer and the molecular mechanisms especially the control of calcium ion signal pathway and cell death. This study will clarify the role of TRPM4 in the development of colorectal cancer and mechanisms and provide scientific basis for colorectal cancer diagnosis and theatment based on TRPM4.

结直肠癌发病率高，预后差，主要原因在于病因和发病机理仍未完全阐明。我们前期研究发现瞬时受体电位M通道家族成员TRPM4在结直肠癌组织中的表达较正常肠粘膜显著下降或沉默，并与结直肠癌患者的预后密切相关。TRPM4基因转染后能够明显增加肠癌细胞HT29中的胞浆钙离子浓度并显著抑制肿瘤的生长和促进肿瘤细胞凋亡。TRPM4是一种Ca2+激活的非选择性阳离子（如Na+、K+）通道, 其本身不能直接影响Ca2+通透性，但可通过改变膜电位值间接影响Ca2+内流，在细胞生理活动中起重要作用。本项目将检测TRPM4在结直肠癌组织中的表达及其调控机制，并通过体内外实验研究TRPM4在结直肠癌中所起的生物学作用及其分子机制，尤其是TRPM4是否通过调控钙离子信号通路影响细胞死亡方面发挥重要作用，阐明TRPM4在结直肠癌发生发展中的作用和机制，为基于TRPM4的结直肠癌诊断和治疗提供科学依据。

TRPM4是一种非选择性阳离子通道，在调节细胞内钙离子浓度方面起重要作用，已报道在多种肿瘤中异常表达并与肿瘤的迁移相关。本课题我们研究TRPM4在结直肠癌转移中的所起的作用，RT-PCR和MSP以及免疫组化分析TRPM4的表达及甲基化状态，TRPM4表达质粒转染、增殖和迁移检测，钙离子浓度和免疫荧光等检测相关的信号通路。我们的实验结果表明在大多数结直肠癌组织和肠癌细胞系中TRPM4表达降低甚至沉默，且在这些异常表达的肿瘤细胞及组织中检测到TRPM4基因启动子处的高度甲基化。TRPM4蛋白的表达和肿瘤的分期 （p<0.001）、淋巴结转移（p=0.008）、远处转移（p=0.025）和侵袭（p<0.001）显著相关。TRPM4阳性表达的患者术后生存率明显高于阴性表达者。肠癌细胞株转染TRPM4后，通过体内外实验发现TRPM4能显著抑制肿瘤细胞的增殖、迁移，并引起细胞骨架重构。流式细胞术和Western Blot结果显示TRPM4显著促进钙离子内流并激活钙蛋白酶（calpain），介导焦点粘附激酶FAK的蛋白水解并抑制其磷酸化，从而抑制下游PI3K/AKT/mTOR通路来抑制肿瘤细胞的迁移，TRPM4同时造成多种迁移相关蛋白的表达改变。这些抑制作用可被钙蛋白酶抑制剂解除，TRPM4有希望作为一种肿瘤标志物和潜在治疗靶点应用于结直肠癌的诊断和转移治疗。