LIMK1：罗格列酮抑制人胃癌细胞增殖、迁移及侵袭的作用靶点

Rosiglitazone (ROS) is a PPARγ agonist, the potential for development of anticancer drugs, our preliminary work confirmed, ROS can obvious inhibit proliferation and induced apoptosis in human gastric MGC803 cells and can reverse the multidrug resistance of SGC7901 /VCR cell in vitro and in vivo, ROS can inhibit the migration and invasion of human gastric cancer cell.LIMK1 play an important role in invasiveness of tumor cells, so far, there is not research reports about LIMK1 play a role in ROS inhibit the proliferation, migration and invasion of human gastric cancer cell. The subject on the basis of the preliminary studies to verify the LIMK1 expression of MGC803 cells treat with ROS by recombinant cell lines, siRNA, RT-PCR, real-time PCR, Western blot analysis, confocal laser cell adhesion, cell persistent, cell invasion assay, tissue microarray technology, Further demonstrate that the ROS downregulates the Rho1-Rock1-LIMK1-cofilin signaling pathway, suppressing human gastric cancer cell proliferation, migration and invasion, and to demonstrate that the molecular mechanisms and confirm LIMK1 is the role of the key targets.

罗格列酮（Rosiglitazone, ROS）是一种PPARγ激动剂，具有开发潜力的抗肿瘤药物，我们前期工作证实，ROS能体内外明显抑制人胃癌MGC803细胞增殖，诱导其凋亡，能逆转SGC7901/VCR细胞多药耐药，抑制人胃癌细胞迁移和侵蚀能力；LIMK1对肿瘤细胞的侵袭性起重要作用，迄今，尚未见LIMK1对ROS抑制人胃癌细胞增殖、迁移与侵袭作用的研究。本课题在前期研究的基础上，验证ROS处理MGC803细胞的LIMK1表达，通过重组细胞系、siRNA、RT-PCR、Real-time PCR、Western blot、激光共聚焦、细胞粘附、细胞迁延、细胞侵袭实验、组织芯片等技术，进一步研究ROS通过下调LIMK1抑制人胃癌细胞增殖、迁移与侵袭，影响Rho1-Rock1-LIMK1-cofilin途径，阐明其分子机制，证实LIMK1是作用的关键靶点。

本研究通过构建含有高表达LIMK1的EX-C0763-M98-5真核表达载体并用其转染MGC803细胞。用RT-PCR及Western blot验证ROS处理前后细胞中LIMK1 mRNA及蛋白的表达。检测ROS处理前后，过表达LIMK1对MGC803细胞周期、细胞增殖、迁移及侵袭能力的作用。并通过应用慢病毒介导的RNA干扰技术下调LIMK1后，观察沉默LIMK1对罗格列酮（Rosiglitazone，ROS）抑制人胃癌MGC803细胞增殖、迁移与侵袭的影响及对人胃癌MGC803细胞裸鼠皮下移植瘤生长的抑制作用。最后通过qRT-PCR检测ROS对人胃癌MGC803细胞Rac1、Rock、Pak1、LIMK1和Cofilin-1 mRNA表达水平的影响；Western blot检测ROS对人胃癌MGC803细胞Rac1、Rock、Pak1、LIMK1、Cofilin-1和P-cofilin-1蛋白表达水平的影响，探讨ROS抑制人胃癌细胞增殖、迁移与侵袭的作用的可能靶分子。研究得出，LIMK1高表达可促进MGC803 细胞增殖、迁移及侵——ROS能抑制MGC803细胞增殖、迁移及侵袭，LIMK1可能是ROS抑制人胃癌MGC803细胞增殖、迁移及侵袭作用。慢病毒介导的 RNAi可有效沉默人胃癌 NGC803细胞中 LIMK1的表达.沉默LIMK1 可抑制人胃癌MGC803细胞增殖、迁移与侵袭，沉默LIMK1 增强罗格列酮抑制人胃癌MGC803细胞增殖、迁移与侵袭，并能抑制人胃癌MGC803细胞裸鼠皮下移植瘤生长。ROS抑制胃癌细胞的作用，可通过下调Rac1，抑制Rock、Pak1表达，减少LIMK1磷酸化cofilin-1，从而降低cofilin-1活性，抑制人胃癌细胞增殖、迁移与侵袭，从而证实LIMK1可能是ROS抑制人胃癌细胞增殖、迁移与侵袭的作用靶分子。