miR-144靶向APP调控AML1/ETO+白血病细胞髓外浸润的实验研究

Extramedullary infiltration is closely related with leukemia relapse and refractory. We analyzed clinical data of AML1/ETO + leukemia patients and found that patients with high expression of APP got significantly higher incidence of extramedullary infiltration than those who with low expression. In kasumi-1 cell line we found that cell migration was significantly decreased after interference of APP expression. Analyzing the difference of expression profiles between before and after interference, also we used KEGG, at last we focused on p-ERK/c-Myc/MMP-2 pathway.We used software to predict APP gene is the target gene of miR-144. And, we found that there was a negative correlation between APP and miR-144 in different leukemia cells. Therefore, we hypothesized that miR-144 target APP to regulate AML1/ETO + leukemia cells extramedullary infiltration by p-ERK/c-Myc/MMP-2 pathway. This study is designed to identify: 1) the relationship of miR-144 with extramedullary leukemia metastasis and survival, and confirmed miR-144 targets APP; 2) APP through p-ERK/c-Myc/MMP-2 pathway to regulate cell migration and regualte bone marrow mesenchymal cell barrier. This research will further clarify the mechanism of the development of extramedullary leukemia and help us to identify whether miR-144 and APP can be used as markers of extramedullary leukemia or not, it will be very important..

髓外浸润与白血病的复发和难治密切相关。我们分析AML1/ETO+白血病患者资料发现APP高表达者髓外浸润发生率显著升高。前期在kasumi-1细胞证实siRNA干扰APP表达后细胞迁移力降低，对比干扰前后表达谱变化，KEGG分析聚焦于p-ERK/c-Myc/MMP-2通路。同时软件预测APP是miR-144的靶基因，且在不同白血病细胞发现APP与miR-144呈负相关。因此提出假说：miR-144靶向APP通过p-ERK/c-Myc/MMP-2调控AML1/ETO+白血病细胞髓外浸润。本项目拟验证：1)miR-144与白血病髓外转移及生存的相关性，并证实其靶向调节APP；2)APP通过p-ERK/c-Myc/MMP-2调节白血病细胞迁移及对间充质细胞骨髓屏障的调节。此研究将进一步阐明髓外白血病的发生发展机制，明确miR-144和APP是否可作为监测髓外白血病的分子指标，具有重要的临床意义。

白血病是一种累及造血干/祖细胞的恶性克隆性疾病，白血病细胞以异常增生及浸润为主要特征，在我国发病率为3-4/10万，在肿瘤中居第七位，死亡率极高。近年来，随着免疫学、细胞遗传学、分子生物学等先进诊断技术的应用及化疗方案的不断改进和新型化疗药物的推出，使得白血病的治疗取得巨大进步；然而，初诊时伴有髓外浸润的患者化疗效果欠佳且易复发，髓外白血病亦成为造血干细胞移植后复发的根源。因此，白血病细胞髓外浸润成为了临床上亟待解决的难点。 . 急性髓系白血病（AML）伴t(8;21)（q22；q22）形成AML1/ETO 融合基因，被国内外学者认为是预后良好的指标，属于低危组白血病，但我们在临床工作中发现其临床转归差别很大，尤其是APP基因高表达者，髓外白血病发生率显著升高，中位生存时间短，复发率高。本研究通过扩大AML1 / ETO +临床病例，分析证实APP基因表达水平与髓外浸润存在正相关，进而影响患者长期生存率。体外细胞模型中，利用microRNA技术干扰APP的表达后，kasumi-1细胞迁移运动能力显著下降，证实APP参与白血病细胞的髓外浸润过程。为求进一步探索其机制，我们利用western-Blot、PCR等技术发现APP调控p-ERK、c-Myc、MMP-2信号通路分子的表达，相反地，MMP-9、CXCR4不受APP调控。利用ERK、c-Myc抑制剂进一步证实APP通过p-ERK/c-Myc/MMP-2信号通路调控白血病细胞髓外浸润的过程。将miR-144质粒转入kasumi-1细胞中，应用荧光素酶报告实验发现，miR-144负性调节APP的表达，进而证实miR-144靶向APP调控p-ERK/c-Myc/MMP-2信号通路，调控白血病细胞髓外浸润的过程。. 目前尚无法对髓外白血病进行预测和全面监测。因此，从分子水平探寻白血病髓外浸润的机制，找出关键靶向分子，对髓外白血病的早期诊断和防治意义重大。本课题研究结果为AML1/ETO+白血病的分层诊断和精准治疗提供理论依据。