细胞分裂周期蛋白CDC5L在AML中的作用及其分子机制研究

Acute myeloid leukemia is a kind of malignant tumor with uncontrolled cell proliferation. Blood cell cycle disorder is one of the main pathogenesis of AML. The role of cell division cycle protein 5 like (CDC5L) in cell cycle regulation and tumorigenesis and development has gradually become a hot research topic, but the role of AML proliferation is not clear. Our preliminary results showed that the expression of CDC5L in patients with AML was significantly higher than that in the normal population. Compared with normal peripheral blood cells, the expression level of CDC5L was significantly higher in AML cell lines. In vitro experiments showed that the proliferation of THP-1 cells decreased significantly after interfering with CDC5L. However, it is still necessary to study the mechanism of CDC5L regulating the abnormal proliferation of AML cells. In vitro, the molecular mechanism of CDC5L regulation of cyclin expression and AML cell proliferation was proposed. The effects of CDC5L on tumor growth, proliferation and angiogenesis in nude mice were studied in vivo. The molecular mechanism of AKT-FOXO3a/FOXM1 regulation of CDC5L expression was clarified. This paper provides an important basis for clarifying the molecular mechanism of CDC5L regulation of AML proliferation and finding new drug targets.

急性髓系白血病（Acute myeloid leukemia，AML）是一类以细胞增殖失控为主的恶性肿瘤。血细胞周期紊乱是AML主要发生机制之一。细胞分裂周期蛋白5L（CDC5L）在细胞周期调控和肿瘤发生、发展中的作用逐渐成为研究热点，而在AML增殖中的作用尚不清楚。我们前期实验结果发现CDC5L在AML患者中的表达明显高于正常人群。与正常人群外周血细胞相比，CDC5L的表达水平在AML细胞系中明显增高。体外实验发现干扰CDC5L后，THP-1细胞的增殖能力明显下降。但仍需研究CDC5L调节AML细胞异常增殖的作用机制。本课题拟在体外研究CDC5L调节周期蛋白表达及AML细胞增殖的分子机制；体内研究CDC5L对裸鼠体内肿瘤生长、增殖和血管生成的影响；并明确AKT-FOXO3a/FOXM1调节CDC5L表达的分子机理。为阐明CDC5L调节AML增殖的分子机制和寻找新药物靶点提供重要依据。

AML作为一种恶性肿瘤，它具有高度的异质性，以基因突变、无限增殖、分化和成熟障碍以及髓外浸润为特点。细胞周期紊乱是 AML 主要的发生机制之一。本课题首先通过Oncomine数据库对比发现，CDC5L在AML中的表达明显高于Normal。qPCR和Western Blot检测CDC5L在正常人群外周血和AML细胞系中表达。结果发现CDC5L的mRNA和蛋白水平在AML细胞系中表达明显增高。感染HL60细胞Ad-siCDC5L，用Western Blot检测CDC5L对于HL60细胞的增殖、侵袭和凋亡蛋白的影响。结果显示干扰CDC5L可明显抑制HL60细胞的增殖和侵袭相关蛋白表达，促进凋亡蛋白表达。感染Ad-CDC5L，用Western Blot检测过表达CDC5L对于HL60细胞的增殖和侵袭蛋白表达情况。结果显示过表达CDC5L可明显促进HL60细胞增殖和侵袭蛋白表达。HL60细胞分为两组做转录组学分析。结果显示KCNAB2经CDC5L过表达后，表达有显著上调。我们通过KM分析，免疫浸润等生信分析，结果显示与正常样本相比，KCNAB2在AML中高表达。KCNAB2高表达与不良预后相关。由此可见KCNAB2高表达可作为AML不良结局的潜在生物标志物。接下来，我们细胞水平验证KCNAB2和CDC5L对HL60细胞增殖能力的影响。结果显示，与对照组相比，Ad-CDC5L可明显促进HL60细胞增殖，而干扰KCNAB2则降低HL60增殖能力。与Ad-CDC5L组相比，CDC5L+si-KCNAB2-4组的增殖能力明显减弱，由此可见KCNAB2可以抑制CDC5L对于HL60细胞的增殖能力的影响。Bufalin具有抗肿瘤的效果。我们猜测Bufalin可以通过CDC5L调控AML的增殖。Bufalin刺激HL60细胞，发现CDC5L的表达明显降低，cleaved-caspase 3表达明显上升。由此推断，Bufalin可能通过CDC5L调控HL60的增殖能力。我们研究发现CDC5L可能通过KCNAB2影响AML的增殖，且Bufalin抑制CDC5L的表达。由此可见CDC5L在治疗AML进展中，可能作为一个有效的靶点。