下调RhoC表达对多发性骨髓瘤血管生成的影响及机制研究

The role of RhoC (Ras homolog gene family, member C) in cancer invasion and metastasis has been investigated intensively. More recently, multiple studies have also shown that RhoC may actively participate in the process of neovascularization and it is essential for angiogenesis induced by vascular endothelial growth factor. Our preliminary studies show that RhoC is highly expressed in neoplastic plasma cells as well as endothelial cells and thus, it may critically be involved in the angiogenic processes in multiple myeloma (MM). In order to determine if regulating the level of RhoC expression in endothelial cells will affect, in vitro, proliferation, pseudopodia formation, migration and tube formation of endothelial cells, and in vivo, the tumor growth and/or angiogenesis of grafted MM in mice, we intend to modify the RhoC expression in tumors cells and endothelial cells and then to evaluate the biological and functional properties of endothelial cells and the growth rate of grafted tumor in mice. We are also going to explore the possible mechanism involved in the regulation processes. We plan to isolate endothelial cells from MM to perform primary culture and further characterization. With the cultured human umbilical vein endothelial cells (HUVEC) as control, we are going to determine if modifying the expression of RhoC in endothelial cells will affect the proliferation, cell cycle, and ability of invasion of cultured myeloma cell line (RPMI8266), as well as to explore the possible mechanism of the regulation processes. Our studies will lay a solid foundation for the development of novel cancer therapeutic approaches targeting tumor endothelial cells.

RhoC与肿瘤浸润转移关系的研究已有较多文献报道，近年研究表明其还参与血管新生的全过程。我们前期研究发现RhoC在多发性骨髓瘤（MM）细胞中高表达，同时也表达于MM血管内皮细胞中，且与MM血管生成密切相关。那么调控RhoC表达，体外是否对血管内皮细胞增殖、伪足形成、迁移和成管能力产生影响？体内是否影响MM移植瘤生长和瘤体血管生成？其可能机制是什么？本项目拟通过调节血管内皮细胞中RhoC表达，体外观察对血管内皮细胞增殖、伪足形成、迁移和成管能力的影响；下调RhoC表达后体内对移植瘤生长、瘤细胞增殖和血管生成的影响，并探讨其潜在机制；分离、鉴定和原代培养MM血管内皮细胞，以传代培养的HUVEC为对照，观察调节血管内皮细胞RhoC表达后，对MM RPMI 8226细胞增殖、细胞周期及侵袭能力的影响，并探讨其可能机制。为开展以肿瘤内皮细胞为靶点的肿瘤治疗奠定理论基础。

为探讨RhoC表达与多发性骨髓瘤血管生成的关系及其可能机制。我们在测RhoC在MM组织、RPMI8226细胞及HUVECs中表达的基础上，构建RhoC shRNA质粒和慢病毒载体，转染骨髓瘤血管内皮细胞（MVECs）和HUVECs；采用WB和RT-PCR测细胞中RhoC表达情况；鬼笔环肽进行骨架染色，荧光共聚焦显微镜观察细胞骨架；扫描电镜观察内皮细胞伪足形态和数量；划痕实验测细胞迁移能力；基质胶上观察细胞成环能力；微载体观察细胞出芽数目；WB和RT-PCR测ROCK和MAPK表达情况；转染MVECs及HUVECs，收集内皮细胞培养上清液培养RPMI8226细胞；WB和RT-PCR测RPMI8226-S组RhoC表达；CCK-8法测细胞增殖；小室测细胞侵袭能力；流式细胞术测细胞周期；WB测PI3K、CDK、CyclinD1、MMP2、9蛋白表达。建裸鼠移植瘤模型，WB和RT-PCR测移植瘤RhoC表达含量；计数基质胶内VEGF诱导新生血管数；免疫细胞化学测移植瘤组织中Ki-67、CD31表达。结果显示：转染MVECs和HUVECs内RhoC表达均降低，RhoC S组较NC组细胞骨架规则、丝状伪足数目明显减少；RhoC S组较NC组细胞迁移能力明显降低，二维成管数目以及出芽数目和能力降低；RhoC S组较NC组细胞ROCK和MAPK表达明显降低；条件培养后RPMI8226细胞中RhoC表达降低；RPMI8226-S组较NC组细胞增殖、侵袭能力降低，处于S期的细胞比例降低；RPMI8226-S组较NC组PI3K、CDK、CyclinD1、MMP2及MMP9等蛋白表达含量均出现降低；RPMI8226-S组较NC组基质胶内VEGF诱导的新生血管数下降，移植瘤中RhoC蛋白和mRNA表达含量降低，Ki-67、CD31表达降低。结果表明：下调MVECs中RhoC表达，可影响MVECs的骨架和伪足形成，抑制血管内皮细胞血管网格管状结构形成和出芽能力；其机制可能与RhoC/ROCK、RhoC/MAPK通路有关；下调 MVECs中RhoC表达可下调RPMI8226内RhoC表达，影响RPMI8226的细胞周期，增殖和侵袭能力；其机制可能与PI3K/Akt、CDK/CyclinD1、MMP2/MMP9通路相关。体内实验，下调RPMI8226的RhoC可抑制MM细胞增殖，降低移植瘤血管新生