SOX7基因经Wnt通路对进展期MDS患者CD34/CD117骨髓细胞生物学功能的影响

Myelodysplastic syndrome (MDS) is a group of hematopoietic malignancies. Our previous study revealed that the canonical Wnt/β-catenin pathway was activated in the occurrence and development of MDS. Also, the methylation of a negative regulator of the Wnt signaling pathway, SOX7, represses its expression, and is correlated with the prognosis of MDS. Co-expression of CD34/CD117 had been found in part of MDS patients, stands for poor prognosis and associates with a higher risk of leukemic transformation.We will increase the expression of SOX7 in CD34+/CD117+ marrow cells extracted by flow cytometry through transduction with a lentivirus vector. We will test, in vitro, the effect of SOX7 on cell apoptosis, proliferation, colony formation and differentiation in CD34+/CD117+ marrow cells of RAEB-1 and RAEB-2. SOX7 is involved the development of MDS by inhibiting the combination of β-catenin and TCF/LEF, thus regulating the expression of downstream genes（cyclinD1、c-myc and survivin）of the Wnt signaling pathway. Therefore, elucidating the prognostic value of SOX7 may provide novel evidence for prognostic marker and therapeutic.

MDS为恶性血液系统疾病，课题组前期研究发现，经典Wnt通路异常激活参与MDS发生发展，该通路拮抗基因SOX7甲基化状态与MDS患者预后密切相关，同时发现甲基化修饰介导SOX7表达沉默。因部分MDS患者出现异常共表达CD34/CD117骨髓细胞，且该群细胞具有较高的转白风险和预示较差预后，我们将以此为标记分选RAEB-1和RAEB-2患者骨髓异常细胞，利用慢病毒转染增加其SOX7基因表达，以从体外功能学研究评价SOX7表达沉默对患者骨髓原始细胞的增殖、细胞周期及细胞凋亡的影响，并推测SOX7可影响患者原始细胞的分化。试验最终将证明SOX7在患者骨髓原始细胞中通过抑制β-catenin与TCF/LEF结合来调控经典Wnt信号通路下游基因（cyclinD1、c-myc及survivin）表达，而参与疾病的发生发展。阐明SOX7发挥预后价值的可能机制，为寻找MDS新的预后标记及治疗靶点提供依据。

Wnt信号通路为最为保守通路之一，参与胚胎发育、新陈代谢、血管再生、肿瘤生长等生理病理过程，被广大科研人员作为主要研究对象，对其研究不断被深入和拓展。β-catenin为Wnt通路核心蛋白，其在胞浆中富集，并向核内转移，引起Wnt下游蛋白表达。β-catenin活性由多种因素共同作用，拮抗和激活因素并存。前期研究表明上调SOX7通过拮抗Wnt信号通路，抑制肿瘤细胞生长。本课题通过实验证实SOX7可下调Wnt通路下游蛋白C-MYC、 Survivn、Cyclin D1，并抑制SKM-1细胞增值。我们通过免疫共沉淀发现，SOX7通过与β-catenin结合来抑制Wnt通路活性。但与β-catenin结合的蛋白众多，某些蛋白能拮抗SOX7对Wnt通路的抑制作用。BCL9作为癌基因在血液肿瘤和实体肿瘤中被广泛激活，促进肿瘤细胞增值、存活和转移，其机制为促进Wnt通路的激活。我们进一步通过免疫共沉淀和TOPFLASH/FOPLASH荧光报告载体技术，证明BCL9能与β-catenin结合并促进Wnt通路活性。为进一步阐述三者的关系，我们使用293T细胞株为研究对象，构建外源HA-SOX7、HA-BCL9和Flag-β-catenin质粒。转染细胞后发现，SOX7和BCL9可竞争结合β-catenin，并影响β-catenin转录活性，由此我们证实SOX7对Wnt通路的干扰作用，是由于直接竞争抑制BCL9对β-catenin的转录激活作用，这为高表达BCL9相关肿瘤的治疗提供新方向。