IGFBP-3联合IL-24上调miR-218抑制mTORC2/AKT/FOXO通路活化对前列腺癌恶性进展及血管生成的作用机制研究

The growth, invasion and metastasis of cancer are all based on angiogenesis, which forms a highly combined ecological system with cancer cells. The mechanism of angiogenesis is not well elucidated to date, but it is supposed that cancer cell, endothelial cells, growth factor and extracellular matrix are all involved in this process.Some research confirm that miR-218 play a very important role of angiogensis,which is also regulated by miR-218 through miR-218/SLIT/ROBO signalling pathway. Prostate cancer presents the most complicated biological behavior among the male genitourinary system malignancies, and there is no ideal anti-angiogenesis therapy for clinical option now. In our previous project supported by national natural scientific foundation, we found that blocking AKT/mTOR signal pathway can inhibit angiogenesis of prostate cancer, and IGFBP-3 conjugated IL-24 can induce apoptosis of prostate cancer cells and suppress angiogenesis by inactivating AKT/mTOR signal pathway, at the same time, surprisingly,increase the miR-218 expression. However, AKT/mTOR signal pathway could be reactivated after Rictor, a key protein of mTORC2 complex, was recruited to regulate the phosphorylation of AKT/mTOR signal pathway. Bioinformatics has predicted and proven that Rictor is the target of tumor suppressor gene miR-218, whose expression is dramatically decreased in the progression of prostate cancer and elevated again after exposure to IGFBP-3 conjugated IL-24. The project is intende to use LNCaP/C4-2 cells and PCa specimens and orthotopic transplantation tumor animal model as the object material and use the experiment technique such as NimbleGen microarray，bioinformatics etc. On the basis of our previous research, this study will try to figure out the molecular mechanism of inhibiting prostate cancer angiogenesis by down regulating mTORC2/AKT/FOXO signal pathway using Rictor specific miR-218, and the possible relationship among abnormal miR-218 expression, prostate cancer progression and angiogenesis. Besides, we will build up in vivo and in vitro angiogenesis models to compare the differences between endogenous and exogenous miR-218, and discuss the possibility and feasibility of clinical application of miR-218, which will shed a new light and biomarker on prevention and treatment of prostate cancer.

miR-218是血管生成的重要调节因子,持续、异常上调的血管生成是前列腺癌（PCa）恶性进展的关键步骤。课题组前期证实IGFBP-3+IL-24抑制PCa AKT/mTOR通路活化,促进细胞凋亡并上调抑癌基因miR-218，但miR-218调控PCa恶性进展及血管生成机制不明。本项目拟利用模拟PCa临床进展的LNCaP/C4-2序列细胞、PCa病理标本和移植瘤裸鼠模型,结合高通量测序、甲基化芯片、ChIP-seq、体外血管形成实验等技术,求证IGFBP-3+IL-24通过影响表观遗传状态来调控miR-218表达的作用机制,探查miR-218异常表达与PCa恶性进展之间的关系以及作为PCa风险预测因子的价值，阐述miR-218靶向Rictor抑制mTORC2/AKT/FOXO通路活化而调控血管生成的调控网络并寻找其下游与血管生成相关的新靶点,探索应用miR-218诊治PCa的可能性和可行性。

研究证实miR-218可以抑制癌细胞增殖、迁移、侵袭和转移，是肿瘤抑制基因。然而miR-218对肿瘤新生血管形成的作用及其机制尚未见明确报道。本研究中我们对GEO数据库中前列腺癌microRNA表达高通量测序数据进行统计分析后发现miR-218的表达在前列腺癌组织中明显低于良性前列腺增生组织，且转移性前列腺癌组织中miR-218的表达量更低。在良性前列腺增生细胞BPH-1中miR-218表达水平显著高于前列腺癌细胞系LNCaP,C4-2及CWR22Rv1。构建miR-218过表达前列腺癌细胞，血管内皮细胞趋化实验结果显示在共培养体系和条件培养基条件下， LV3-miR-218细胞对血管内皮细胞HUVECs的趋化能力明显降低。在无血清条件下，LV3-NC细胞CM，而非LV3-miR-218细胞CM，可以促进HUVEC生长。体外内皮细胞形成血管样结构即tube-formation试验同样证实miR-218在体外对前列腺癌细胞的血管形成能力具有抑制作用。Q-PCR，Western blot及ELISA实验结果显示过表达miR-218可抑制前列腺癌细胞VEGFA的表达和分泌。通过TargetScan预测分析提示Rictor基因为miR-218潜在靶基因，双荧光素酶报告基因检测实验证实前列腺癌细胞中Rictor为miR-218的靶标基因。Q-PCR及Western blot结果显示miR-218可靶向抑制 RICTOR /AKT/mTOR/ HIF2α/VEGFA信号轴，从而抑制前列腺癌细胞血管形成能力。进一步利用CWR22Rv1-miR-218过表达细胞模型进行裸鼠皮下瘤实验，结果显示LV3-miR-218组肿瘤生长速度较慢，瘤体平均重量显著低于LV3-NC组，免疫组织化学染色结果提示LV3-miR-218组瘤体组织中新生血管marker CD31、细胞增殖marker PCNA、RICTOR及VEGFA的表达较LV3-NC组均明显降低。兔角膜血管形成实验结果显示LV3-NC细胞可在兔角膜中形成新生血管，而LV3-miR-218细胞则无明显血管形成。本研究揭示miR-218可靶向Rictor并负调控AKT/mTOR信号通路及其下游血管生成因子VEGFA，从而抑制前列腺癌肿瘤血管生成，提示miR-218可作为前列腺癌诊断及治疗新的策略切入点。