ROBO1基因突变通过CDC42/PAK1调控MDS细胞恶性生物学特征的分子机制

Axon guidance molecules ROBO is evolutionarily highly conserved transmembrane protein, ROBO and its ligand SLIT play important regulatory role in the process of axon guidance, angiogenesis, tumor invasion and metastasis. However, its role in the pathogenesis of MDS has not been reported yet. The mechanisms of MDS progressed from low-risk to high-risk remain unclear. Our previous studied identified ROBO1 as novel progression-related somatic mutations using whole-exome sequencing and multivariate analysis shows that ROBO1 mutations are independent factors for predicting poor survival. Preliminary functional experiments showed that ROBO1 was a tumor suppressor gene. Gene expression profile showed that PAK1 gene expression was significantly increased in patients with low ROBO1 expression. PAK1 can promote cell proliferation and inhibit cell apoptosis through MYC, which is mainly regulated by CDC42. Therefore, it is speculated that the ROBO1 mutations activate PAK1 to regulate the malignant biological characteristics of MDS clonal cells through activating CDC42. Based on the preliminary works, in this study, lentivirus mediated gene interference / over expression, constructing ROBO1 mutated hematopoietic cell and transplantation mice model, mRNA-sequencing, FCM, PCR, WB and IP will be used to further explore the molecular mechanism of ROBO1 mutations in the role of MDS disease progression.

ROBO是进化上高度保守的跨膜蛋白，ROBO及其配体SLIT对神经轴突导向、血管生成、肿瘤浸润和转移等具有重要的调节作用，但其在MDS发病中的作用尚无报道。MDS从低危进展到高危的机制未明，我们前期对从低危进展到高危MDS患者的全外显子测序，首次在进展期MDS发现ROBO1基因突变，且提示疾病预后不良。初步细胞功能实验提示ROBO1为抑癌基因，基因表达谱芯片显示：ROBO1低表达MDS患者PAK1表达升高，而PAK1能通过上调癌基因MYC表达进而促进细胞增殖、抑制凋亡，PAK1主要受CDC42调控。因此，推测ROBO1突变可能通过CDC42激活PAK1上调MYC表达而调控MDS细胞恶性生物学特征。本项目将采用慢病毒介导基因干扰/过表达、构建Robo1突变小鼠造血细胞移植模型、mRNA-sequencing、FCM、PCR、WB和IP等实验，探讨ROBO1突变促进MDS疾病进展的分子机制。

ROBO是进化上高度保守的跨膜蛋白，ROBO及其配体SLIT对神经轴突导向、血管生成、肿瘤浸润和转移等具有重要的调节作用。MDS从低危进展到高危的机制未明，我们前期对从低危进展到高危MDS患者的全外显子测序，首次在进展期MDS发现ROBO1基因突变，且提示疾病预后不良。初步细胞功能实验提示ROBO1为抑癌基因，基因表达谱芯片显示：ROBO1低表达MDS患者PAK1表达升高，而PAK1能通过上调癌基因MYC表达进而促进细胞增殖、抑制凋亡，PAK1主要受CDC42调控。因此，我们推测ROBO1突变可能通过CDC42激活PAK1上调MYC表达而调控MDS细胞恶性生物学特征。本项目通过慢病毒载体RNA干扰方法导入ROBO1突变体，在体外观察细胞凋亡、增殖、集落形成情况。我们发现ROBO1突变细胞凋亡抵抗，增殖过度，集落形成明显增加。进一步机制研究证实ROBO1突变细胞CDC42、PAK1、MYC表达明显升高，而P21、P15表达下降。通过成功构建小鼠Robo1-/-小鼠模型，证实Robo1-/-小鼠骨髓增生明显高于对照，血小板升高，骨髓检查巨核细胞增多及纤维组织增生。分析Robo1-/-小鼠骨髓细胞造血干/祖细胞显示Robo-/-小鼠LSK细胞比例较高，其中以CMP、MEP细胞比例升高明显。Robo-/-小鼠体内机制研究进一步证实ROBO1功能性缺失使其招募下游srGAP1功能异常，CDC42-GTP在细胞内聚集，并进而活化PAK1，上调下游癌基因MYC表达，从而导致MDS细胞异常增殖、凋亡抵抗并进而导致疾病进展。