Ikaros蛋白通过调控岩藻糖基转移酶Fut4转录影响儿童急性淋巴细胞白血病患者预后的分子机制探究

Ikaros, as an early lymphoid-specific transcription factor, had been proved to be associated with poor prognosis of acute lymphoblastic leukemia (ALL), but the mechanism still unidentified. On the basis of previous and our own work, we hypothesize that Ikaros affects the prognosis of pediatric acute lymphoblastic leukemia, by transcriptional control of fucosyltransferase Fut4. Ikaros can recruit HDAC1 repressor complex to Fut4 promoter and inhibit its trascritption. Once Ikaros’s function was destroyed, caused by gene alternative splicing, repressor complex disassembled and activator complex formed in Fut4 promoter instead, which lead to the over-expression of Fut4. Higher level of Fut4 can promote cell proliferation, inhibit cell apoptosis and increase drug resistance. . Alterations in cell surface glycosylation are associated with tumor prognosis and recurrence, and Lewis antigens are functionally important terminal glycan epitopes, which synthesized by fucosyltransferase (Futs). In order to figure out why Ikaros mutation can lead to poor prognosis, Anti-tumor effects of Ikaros were evaluated by animal models and cultured cell lines. Ikaros isoforms in ALL patients were identified and the rate between Ikaros functional and disfunctional types was calculated. Fut4 expression level was quantified by Q-PCR. Data showed that Fut4 and its catalyzed product LeX increased with the rise of Ikaros disfunctional rate. Results from transfected cell lines also agreed with that. pCDNA3.1-Ik6 (Ikaros disfunctional type) and pCDNA3.1-Ik1 (Ikaros functional type) recombinant plasmids were developed and transfected into leukemia cell line, the level of Fut4 and LeX in Ik6 cell line was higher than Ik1. Since Fut4 over-expression can stimulate cell proliferation and lead to drug resistance in solid cancer cell line, such as breast caner and hepatocarcinoma, we predicted that the effect of Ikaros mutation on prognosis of ALL patients was related with Fut4 dysregulation. . In order to clarify the regulatory mechanism of Ikaros on Fut4 gene transcription, the experiments were planned to conduct as follows: Dual luciferase reporter assay was used to identify the regulation of Ikaros on Fut4 promoter; EMSA and Chip were used to certify the Ikaros binding site on Fut4 promoter; CO-IP and Chip were carried out to show Ikaros’s role in recruiting HDAC1 transcriptional repressor complex to Fut4 promoter; Fut4 over-expression and interference were implemented to study Fut4’s effect on the biological activity of leukemia cell line, eg, cell proliferation, apoptosis, cell cycle and drug sensibility. To explore the functional role of Ikaros in the prognosis of ALL will help to design individualized treatment plan and improve the prognosis of patients.

在总结前人与自身工作基础上，我们假设：Ikaros蛋白通过抑制Fut4基因转录发挥其抗肿瘤效应，Ikaros基因突变削弱自身蛋白调控功能，解除对Fut4基因的抑制作用，导致Fut4蛋白过表达，引起一系列细胞生物学行为的改变—细胞生长加快、凋亡受抑、药敏性降低，是导致患者预后不良的原因之一。小鼠模型和细胞实验证实Ikaros蛋白的抗肿瘤效应。临床样本和培养细胞检测结果显示Ikaros蛋白功能降低伴随着Fut4蛋白及肿瘤细胞表面多糖（LeX寡糖，Fut4催化产物）表达水平的增加。构建pEGFP-N3-Fut4和Fut4-shRNA干扰腺病毒，分别转染细胞，以阐明Fut4表达水平对细胞的影响和意义。为进一步探讨分子机制，奠定良好的理论与实验基础，分子生物学和表观遗传学实验技术被用来研究Ikaros基因突变影响Fut4转录的调控机制。明确Ikaros基因突变影响预后的机制将为靶向治疗提供理论依据。

本项目紧紧抓住骨髓微环境耐药之关键环节，沿着“Ikaros突变→FUT4转录抑制解除，细胞岩藻糖基化与LeX生成增加，糖蛋白整合素α5β1的岩藻糖基化水平增加→白血病细胞与微环境基质fibronectin黏附增强，FAK通路过度活化→白血病细胞侵袭能力增强” 这条主线，首先以临床样本为研究对象，测定转录因子Ikaros的表达谱，确认FUT4 表达水平与Ikaros无功能亚型之间的正相关性；构建Ikaros功能亚型（IK1）重组腺病毒表达载体并转染Jurkat得转基因细胞，检测细胞FUT4及LeX的表达量，萤光素酶实验、体外/体内结合实验和结合动力学实验证明Ikaros能够直接结合在FUT4 启动子上，发挥转录抑制功能；然后构建Ikaros无功能亚型（IK6）转基因细胞，重复检测细胞FUT4及LeX的表达量，证明IK6能够解除对FUT4的抑制；构建FUT4过表达和敲减转基因细胞，证实FUT4能够岩藻糖基化整合素α5β1，并增强其介导的白血病细胞与基质的交互作用；接着建立白血病细胞-骨髓基质体外共培养模型，检测整合素-FAK信号通路，及白血病细胞侵袭情况；最后用IK6转基因细胞，重复上述实验。.本项目首次发现、证实肿瘤细胞表面的LeX糖链能够增强骨髓微环境中白血病细胞与基质的交互作用，激活肿瘤生存信号转导通路，导致了骨髓耐药性的产生。本研究阐明了Ikaros突变引起不良预后的分子机制，加深了对这种新发现的高危白血病亚型的认识，为发现新的药物作用靶点奠定良好的理论与实验基础。.本课题全面完成研究计划，发表学术论文9篇（标注资助），并培养硕士生3名。