microRNA-223通过IKKα调控NF-κB信号通路导致慢性淋巴细胞白血病耐药的机制研究

Chronic lymphocytic leukemia (CLL) is still an incurable disease, drug resistance of tumor cells is the main reason results in patient death. CLL　had the abnormal expression of microRNA (miRNA) and continuous activation of NF-κB signaling pathway which contributed to the drug resistance.Our previous studies show that exprssion of miRNA-223 was significantly low in CLL, especially in the relapsed/refractory CLL patients and was an independent adverse prognostic factor. Simultaneously we also found IKKα ,which was an important activator of non-canonical NF-κB signaling pathway , was high expressed in CLL and was negatively correlated with the low expression of miRNA-223.Our preliminary functional study showed that IKKα might be the target of miRNA-223 in CLL.All these datas indicate that miRNA-223 might contribute to the drug resistance of CLL through activating NF-κB signaling pathway by targeting IKKα .So we design this project to identify ① low exprssion of miRNA-223 will lead to the drug resistance of CLL,② miRNA-223 target IKKα directly,③ miRNA-223 contributes to the drug resistance of CLL through activating NF-κB signaling pathway by targeting IKKα. We will identify the above outcomes in cell lines, NOD/SCID mouse model and the primary CLL cells. This project will elucidate the exact mechanism of miRNA-223 in CLL drug resistance, explain the possible reason of drug resistance in CLL, and provide a new therapeutic target for the treatment. So this project has great academic and applicative value.

慢性淋巴细胞白血病（CLL）目前仍是一种不可治愈的疾病，肿瘤细胞发生耐药是导致患者死亡的主要原因。microRNA(miRNA)表达紊乱和 NF-κB信号通路异常活化是CLL耐药的重要因素。我们前期研究显示miRNA-223在CLL中显著低表达，难治耐药患者中表达更低，为CLL独立不良预后因素；同时发现 NF-κB信号通路活化蛋白 IKKα在CLL中特别是难治耐药患者表达明显升高。初步功能研究显示miRNA-223可能通过靶向IKKα而异常活化NF-κB信号通路导致CLL细胞耐药。本课题拟在细胞系、动物体内及CLL患者原代细胞三个层面进一步验证miRNA-223 ①参与CLL耐药；②直接调控 IKKα；③通过调控IKKα异常活化NF-κB信号通路参与CLL耐药，从而明确miRNA-223在CLL耐药中的具体作用机制，阐明CLL潜在的耐药原因，为其治疗提供新靶点，具有重要的理论和实际意义。

慢性淋巴细胞白血病（CLL）是一种常发生于中老年人的淋巴细胞增殖性肿瘤。近年来，CLL发病率逐年增高，耐药是患者死亡的主要原因。我们前期研究发现 miRNA-223 在初治CLL中显著低表达，NF-κB 信号通路相关基因IKKα表达水平升高，在耐药患者中更为明显。 NF-κB 信号通路异常是B细胞淋巴瘤耐药的重要原因，因而我们推测miRNA-223 与CLL 耐药相关，且通过靶向 IKKα调控 NF-κB 信号通路来发挥其功能，本研究从细胞系、动物实验、CLL原代细胞三个方面验证该科学问题。细胞系水平：通过双荧光素酶报告实验验证miRNA-223是否靶向调控IKKα；上调或下调miRNA-223后通过Western Blot检测IKKα的表达，通过CCK8法、PI-FACS-细胞周期实验以及流式细胞术检测细胞周期、增殖及凋亡的变化；上调IKKα后，Western Blot检测NF-κB的表达。动物水平：应用上调或下调miRNA-223的细胞系皮下接种裸鼠成瘤后，通过qPCR检测IKKα相对表达量；CLL原代细胞水平：知情同意的前提下，收集CLL患者外周血标本，CD19磁珠分选CD19+ CLL细胞，通过qPCR检测miRNA-223、IKKα表达水平，统计分析初治和耐药CLL患者CD19+ 细胞miRNA-223 和 IKKα 表达水平的关系。双荧光素酶报告实验证实：miRNA-223靶向调控IKKα，IKKα是miRNA-223直接调控的靶基因。细胞系水平上，miRNA-223上调后，IKKα表达量下降，miRNA-223下调后，IKKα表达量上升；IKKα上调后，NF-κB表达下降。miRNA-223上调细胞系细胞较对照组增殖倍数显著减少，凋亡率显著增加，S期细胞比例减少，G1期细胞比例增高，G2/M期细胞比例减少。动物水平上，miRNA-223下调细胞系皮下接种裸鼠成瘤瘤体较对照组IKKα表达量显著增加。CLL原代细胞水平上，qPCR结果显示CD19+ CLL细胞中miRNA-223 与IKKα表达水平呈负相关，耐药CLL患者CD19+ 细胞较初治CLL患者miRNA-223表达更低 ，IKKα表达更高。综上结果显示：miRNA-223通过靶向调控IKKα、异常活化NF-κB信号通路，从而抑制有丝分裂及DNA合成、抑制细胞增殖、促进细胞凋亡导致CLL耐药。