INPP4B新互作蛋白Rac1协同肺癌肿瘤形成的机制研究

Lung cancer is the most common malignant tumor in the world, and it has becoming the first killer in China. INPP4B is a new tumor suppressor gene which mediated tumorigenesis through PI3K-Akt pathway and has been proved in breast and prostate cancer cells. In our previous work, we found that INPP4B can be transferred from the cytoplasm to the nucleus by oxidative stress, thus, we guess that there may be some potential protein substrates to interact with INPP4B. INPP4B may have some new tumorigenesis mechanisms which are independent of PI3K-Akt pathway. Our research is to discuss the new tumor suppressive mechanisms of INPP4B. We used immunoprecipitation and mass spectrometry analysis (LC-MS/MS) to determine the preferential binding proteins of INPP4B, and we choose Rac1 as the candidate protein. We intend to induce Rac1 expression by Tet-on system to determine the correlation between Rac1 and INPP4B entering into the nuclei and to determine the protein binding region of INPP4B by constructing different INPP4B mutants. We intend to application Crispr/Cas9 gene silencing technology to establish INPP4B and Rac1 knockdown cell lines. We detect γ-H2AX foci, Rad51 focus formation after DNA damage response, and cell sensitivity change (MTT). We use flow cytometry assay to detect the cell cycle change and apoptosis. We aim to determine the synergistic effect of INPP4B and Rac1 and provide new molecular mechanisms and individual drug targets for the development and treatment of lung cancer.

肺癌是世界范围内最常见的恶性肿瘤，已成为中国恶性肿瘤死亡的第一杀手。INPP4B是PI3K-Akt通路中的新肿瘤抑制基因，在乳腺癌、前列腺癌等多种肿瘤细胞中已被证实。我们发现氧化应激反应可促使INPP4B从细胞质迁移至细胞核中，猜测INPP4B具有潜在的蛋白质底物群，并不依赖于PI3K-Akt通路。我们应用免疫沉淀结合质谱分析，确定Rac1为INPP4B的优先候选结合蛋白。我们拟通过构建不同的INPP4B突变体，确定Rac1引发INPP4B入核的蛋白质结合区，并应用Crispr/Cas9基因沉默技术建立INPP4B和Rac1敲减细胞系，检测DNA损伤后细胞系中γ-H2AX聚焦点、Rad51聚焦点形成情况。利用流式细胞仪检测DNA损伤后各细胞系细胞周期和自身凋亡情况的改变。从而确定INPP4B与Rac1在DNA损伤、肿瘤形成中的协同作用。为肺癌的发生和治疗提供新型分子机制和个体化药物靶点。

肌醇聚磷酸4-磷酸酶B (INPP4B)是一种肿瘤抑制因子，我们在前期研究‘以人转录组为靶点的shRNA文库筛查肺上皮细胞的肿瘤抑制因子’中筛查得到。我们应用Crispr/Cas9基因编辑系统在A549细胞系中敲除INPP4B，经测序及Western blot实验验证INPP4B成功敲除。我们构建CTL A549、shINPP4B A549、Crispr-INPP4B A549细胞系，对以上细胞系用多聚甲醛固定并进行 X-gal染色，检测细胞 β-半乳糖苷酶阳性比例。我们发现当INPP4B缺失后，细胞老化率增高，而这种老化率增高在IR照射后显著增强。此外，我们还观察到IR照射会引发INPP4B从细胞质迁移到细胞核中，而INPP4B的缺失会导致A549细胞对IR照射的敏感性增强，细胞凋亡增加，DNA同源重组修复受损，以及DNA双链断裂。我们通过IP结合液质联用质谱分析发现INPP4B可结合Rad50，维持Rad50的稳定性，使DNA损伤修复能力增强。综上所述，我们揭示了肿瘤抑制因子INPP4B可通过促进Rad50介导的DNA双链断裂修复来抑制癌细胞老化。