1,25(OH)2D3协同顺铂通过RASAL2调控RAS信号转导通路抑制胃癌进展

Gastric cancer is the most common malignant tumor with high incidence and mortality in China. Advanced gastric cancer ranks mainly in clinical diagnosed patients. Cisplatin is widely used in the chemotherapy of advanced gastric cancer, while it is often combined used with other drugs because of its serious side effects. However, the clinical therapeutic effect is limited. It is indicated by our previous studies that 1,25(OH)2D3 played a synergistic role with cisplatin in promoting cell apoptosis and inhibiting cell proliferation in gastric cancer cells. With the help of microarray analysis, we found that RASAL2 was up-regulated in combined treatment cells by 1,25(OH)2D3 and cisplatin. In order to clarify the mechanism of 1,25(OH)2D3's synergistic anticaner effects, we designed this project. The project plans to silence or over-express RASAL2 gene and then explore the influences of combined treatment on cellular proliferation, apoptosis, migration and invasion of gastric cancer cells. Importantly, we will analyze the activity of RAS and RAS/RAF/MEK/ERK signal pathway after the combined treatment with 1,25(OH)2D3 and cisplatin. Gastric cancer nude mice xenograft model is to be established for studying the influence of 1,25(OH)2D3 and cisplatin on gastric cancer progress and to reveal the regulation of RAS signal pathway in this course. The project is predicted to further clarify the possible mechanism of 1,25(OH)2D3's synergistice anticancer role with cisplatin, which will be benefit for realize the anticancer effects of vitamin D.

胃癌是我国最常见的消化道恶性肿瘤，临床以进展期胃癌为主。顺铂为进展期胃癌的基本化疗药，因毒副作用大常与其他药物联用以降低毒副作用，但临床效果欠佳。前期研究发现，1,25(OH)2D3可协同顺铂抑制胃癌细胞增殖，促进胃癌细胞凋亡，此作用与RAS-ERK信号转导通路有关; 继而通过基因芯片筛选发现RASAL2基因在1,25(OH)2D3与顺铂联合作用胃癌细胞时呈现显著高表达。为明确1,25(OH)2D3的协同抑癌机制，本项目将通过沉默和过表达RASAL2基因，研究1,25(OH)2D3和（或）顺铂对胃癌细胞增殖与凋亡、迁移与侵袭的影响；分析RAS活性及RAS/RAF/MEK/ERK信号转导通路受到的调控；并建立人胃癌裸鼠移植瘤模型，在体研究1,25(OH)2D3协同顺铂对胃癌进展的抑制。本项目预期将进一步揭示1,25(OH)2D3协同顺铂抑制胃癌进展的机制，深入认识维生素D的抗癌功效。

本项目从细胞、分子水平和动物水平探讨活性维生素D 1,25(OH)2D3是否通过调节RASAL2信号通路发挥协同抑制胃癌进展的作用并探究其具体机制。本项目使用RASAL2 shRNA质粒和RASAL2过表达质粒转染胃癌BGC-823细胞株，以分别得到RASAL2基因沉默和过表达的胃癌细胞株，经筛选建立稳定转染的细胞株，用于细胞和动物实验。通过MTT实验、细胞周期试验、细胞凋亡实验发现，RASAL2过表达增强1,25(OH)2D3与顺铂联合作用下对胃癌细胞增殖与侵袭的作用，RASAL2敲低则显著降低1,25(OH)2D3的该协同抑癌作用。为进一步探讨RASAL2在活性维生素D协同抑制胃癌中的作用机制，通过RAS活性检测实验和免疫印迹实验发现，RASAL2过表达降低1,25(OH)2D3与顺铂联合作用下胃癌细胞RAS活性，通过RAS/MEK/ERK信号通路发挥作用。RASAL2过表达的胃癌细胞，在1,25(OH)2D3与顺铂共同作用下，p-ERK/ERK表达水平显著降低（P<0.05），p-MEK/MEK表达水平亦显著降低（P<0.05）。筛选获得稳定转染有RASAL2 shRNA或过表达RASAL2质粒的胃癌细胞，鉴定后皮下注射进行裸鼠成瘤实验，构建动物模型。从动物成瘤后给予1,25(OH)2D3与顺铂联合治疗或单独治疗，观察裸鼠体重变化及肿瘤大小变化。1,25(OH)2D3与顺铂联合治疗荷瘤小鼠，RASAL2过表达组的肿瘤体积显著低于其余各组的水平（P<0.05），较1,25(OH)2D3或顺铂单独作用荷瘤小鼠更显著抑制肿瘤生长的效果。实验结束后处死荷瘤小鼠，并对肿瘤剥离，对肿瘤组织进行免疫组化检测和免疫印迹检测，观察RASAL2表达、RAS、MEK、ERK蛋白表达水平。研究发现RASAL2在1,25(OH)2D3与顺铂联合治疗组中显著高于1,25(OH)2D3或顺铂单独治疗的水平（P<0.05）。在1,25(OH)2D3与顺铂联合治疗组中，pERK/ERK水平显著低于1,25(OH)2D3或顺铂单独治疗的水平（P<0.05），p-MEK/MEK的水平亦显著低于1,25(OH)2D3或顺铂单独治疗的水平（P<0.05）。本项目的研究进一步揭示了活性维生素D 1,25(OH)2D3协同增强顺铂对胃癌进展的抑制作用，将为维生素D用于胃癌的综合治疗提供实验依据。