Crim1通过瘤细胞FAK-ERK1/2-VEGF信号和内皮VEGFR2磷酸化双重调节宫颈鳞癌微血管生成的研究

Angiogenesis is the basis fro the growth, invasion and metastasis of solid tumor. The tumor cells and endothelial cells can modulate tumor angiogenesis through a paracrine pathway and an autocrine pathway, respectively. However, few key molecules have been identified to be able to regulate both of the two pathways to modulate the angiogenesis of tumor. Crim1 can activate FAK which can activate ERK to up-regulate the expression of VEGF, and FAK-mediated VEGF regulation is the major mechanism through which tumor cells induce angiogenesis. Moreover, Crim1 expressed in endothelial cells enhances the level of phosphorylated VEGFR2 under the autocrine VEGF signaling, and thus promote the tube formation of endothelial cells. Therefore, Crim1 may promote the angiogenesis-inducing capacity of tumor cells by activating the FAK/ERK/VEGF signaling, and may also promote the vessel formation ability of endothelial cells through elevating the level of phosphorylated VEGFR2. With gene transfection, RNAi, endothelial tube formation assay and establishment of animal models and other research methods, the dual regulatory roles of Crim1 on the angiogenesis of cervical squamous carcinoma and the related molecular mechanisms will be deeply explored to demonstrate that Crim1 is the critical modulating molecule that can regulate the paracrine pathway of tumor cells and autocrine pathway of endothelial cells to promote the angiogenesis of cervical squamous cell carcinoma.

微血管生成是实体肿瘤生长和转移的基础。瘤细胞和内皮细胞分别通过旁分泌和自分泌途径调节肿瘤微血管生成，但目前较少鉴定出能同时调节此两方面途径的关键性分子。Crim1能激活FAK，瘤细胞FAK通过激活ERK1/2上调VEGF表达是其诱生血管的重要机制，提示Crim1可能通过FAK-ERK1/2-VEGF信号促进瘤细胞旁分泌性血管诱生能力。此外，内皮细胞表达的Crim1能提高内皮自分泌性VEGF作用下VEGFR2磷酸化水平，从而促进内皮血管形成。故Crim1可能通过活化FAK-ERK1/2-VEGF信号促进肿瘤诱生血管，且能通过增强VEGFR2磷酸化而调节内皮血管形成。本课题通过基因转染、RNAi、内皮管腔形成实验和动物模型构建等方法，深入研究Crim1对宫颈鳞癌微血管生成的双重调节作用及分子机制，证实Crim1是同时调节瘤细胞旁分泌和内皮细胞自分泌途径而双重促进宫颈鳞癌微血管生成的关键性分子。

CRIM1 (Cysteine-rich motor neuron1）可通过影响VEGFA的活性调节器官发育过程中血管的生成，提示它可能是一个血管生成调节因子。本课题探讨CRIM1 对宫颈鳞癌微血管生成的调节作用及分子机制。. 本课题首先观察临床宫颈鳞癌中CRIM1表达情况及其与肿瘤微血管密度的关系；进而观察不同水平CRIM1对癌细胞血管诱生和内皮细胞血管生成能力的影响及机制；最后观察CRIM1对活体肿瘤的生长、新生血管含量(CD105表达)的影响。. 实验结果显示，子宫颈鳞癌组织中CRIM1表达水平显著高于正常宫颈上皮，且其表达与肿瘤微血管密度正相关。CRIM1基因转染后癌细胞上清液刺激下，内皮细胞增殖、迁移和管腔形成能力分别高于正常癌细胞上清液刺激下的内皮细胞。CRIM1基因转染后癌细胞和细胞上清液中VEGF水平分别高于正常癌细胞，且此癌细胞中p-ERK水平显著高于未转染组，利用U0126抑制ERK活化后再行CRIM1基因转染，癌细胞中VEF表达水平显著低于正常转染组。转染组癌细胞中p-FAK水平明显高于未转染组，利用Y15抑制FAK活化后再行基因转染，此癌细胞中p-ERK和VEGF水平也分别低于正常转染组。外源性感染CRIM1蛋白对癌细胞VEGF表示无影响。转染了CRIM1基因的内皮迁移能力和管腔形成能力显著高于未转染的内皮细胞，且转染后的内皮细胞中p-VEGFR2的表达水平也相应的高于未转染组。高表达CRIM1的癌细胞活体肿瘤生长速度、瘤体重量及新生微血管含量皆高于对照组，且瘤体组织中VEGF、p-FAK和p-ERK的表达水平也分别明显高于对照组。. 本课题首次揭示CRIM1高表达于临床子宫颈鳞癌，能通过活化FAK/ERK/VEGF信号通路增强肿瘤旁分泌性的血管诱生能力，并同时通过提高内皮VEGFR2的磷酸化水平从而增强内内皮细胞自分泌性的血管生成能力。