microRNAs调控IL-9介导的免疫耐受机制在慢性淋巴细胞白血病中的研究

Background: B-cell chronic lymphocytic leukemia (CLL) is a disease caused by a clonal expansion of small, mature B lymphocytes. It is associated with a profound immune defect and immune reconstitution. MicroRNA (miR), such as miR-155 and miR-21, are emerging as important gene expression regulators often involved in a variety of pathogenesis such as cancers and autoimmunity. Our unpublished data have found that there was high levels of IL-9 in sera of CLL patients. Signal transducers and activators of transcription (STAT) proteins are the principle signaling proteins for many cytokines and growth factors, thereby play a critical role in regulating immune cell homeostasis, differentiation and cellular functions.Former studies have demonstrated that STAT3 and STAT6 contributed to the secretion and function of IL-9. ..Objective: In this study, we aim to explore the effects of microRNAs on IL-9 mediated immunosuppression of CLL, establish the interaction network of miR-155, miR-21, STAT3, STAT6 and IL-9 in immune tolerance of CLL. ..Methods: Expression of miR-155, miR-21, STAT3, STAT6 and IL-9 both in CLL cell lines and primary CLL cells are detected by both qRT-PCR and Western-Blot. The proportion of IL-9 secreting cells (Th2, Th9, Th17 and Treg) in CLL is measured by FACS. To explore the effects of miR-155 and miR-21 on CLL cells and IL-9 secreting cells, they are isolated and transfected with miR-155 and miR-21, respectively. The promoter and inhibitor of STAT3 and STAT6 are used to validate the roles of STAT3 and STAT6 in the production and function of IL-9. The effects of microRNA in CLL development are further studied by animal model with CLL...Expected results: (1) To elucidate the expression of miR-155, miR-21, STAT3, STAT6 and IL-9 in CLL, analyze their relationship with clinical characteristics, explore their potential for clinical application as diagnosis, therapy response and prognosis biomarkers. (2) To demonstrate the effects of miR-155 and miR-21 on IL-9 secreting cells and involved mechanisms, explain the importance of STAT3 and STAT6 in this process,and establish miR-155 and miR-21 related interaction network in IL-9 production and function. (3) To discuss the importance and possibility of miR-155, miR-21, STAT3, STAT6 and IL-9 in CLL diagnosis, treatment and prognosis and provide novel insight for the immune tolerance and possible therapeutic strategy of CLL.

慢性淋巴细胞白血病(CLL)患者体内存在T、B细胞构成比例、免疫耐受及免疫调节功能紊乱，但其机制仍不甚清。已证实miR-155及miR-21参与CLL免疫调控机制。我们的前期实验发现IL-9在CLL患者中高表达，STAT3与STAT6能够调节IL-9的产生及活性，前期生物信息学分析证实miR-155、miR-21与STAT3、STAT6及IL-9之间存在密切联系。因此我们设想miR-155、miR-21可能通过STAT3、STAT6的介导来实现对IL-9表达的调控，进而在CLL免疫耐受中发挥作用。本课题拟通过探讨CLL中miR-155、miR-21与STAT3、STAT6对于IL-9分泌和功能发挥的影响，揭示CLL细胞中免疫紊乱及免疫耐受的确切机制，通过动物实验进一步验证miRNAs对CLL的免疫调控，阐述IL-9可能作为逆转CLL免疫耐受靶标的可能性，为CLL治疗提供理论依据。

慢性淋巴细胞白血病(CLL)是单克隆性小淋巴细胞恶性增殖性疾病，患者体内存在T、B细胞构成比例、免疫耐受及免疫调节功能紊乱，但其机制仍不甚清。已证实miR-155及miR-21参与CLL免疫调控机制。我们的前期实验发现IL-9在CLL患者中高表达，STAT3与STAT6能够调节IL-9的产生及活性，前期生物信息学分析证实miR-155、miR-21与STAT3、STAT6及IL-9之间存在密切联系。在这项研究中，我们旨在探索microRNAs在IL-9介导的CLL免疫抑制中发挥的作用，建立CLL免疫耐受中miR-155、miR-21与STAT3、STAT6及IL-9之间的相互作用网络。.主要研究内容包括检测CLL标本及细胞中miR-155、miR-21、STAT3、STAT6、IL-9等的表达；阐明STAT3、STAT6对miR-155、miR-21和IL-9的相互作用和影响，证实在CLL中miR-155、miR-21对相关免疫细胞产生IL-9及相关因子的调控是通过STAT3、STAT6信号通路来实现的；并结合上述研究结果开展动物实验，进一步在小鼠模型中进行体内验证。.研究结果显示CLL患者中miR-155、miR-21及IL-9表达水平明显升高，且CLL患者及细胞株中存在STAT3、STAT6异常活化。上调miR-155及miR-21经IL-9预处理的细胞株IL-9分泌增加，STAT3抑制剂可消除这种作用。上调miR-155及miR-21可也调节经IL-9处理CLL细胞株增殖及凋亡，而STAT3抑制剂可以阻断miR-155及miR-21上调对细胞增殖和凋亡的作用。STAT3抑制剂可以抑制预处理CLL细胞IL-9的分泌，且IL-9通过介导STAT3、STAT6的磷酸化调控CLL细胞的增殖及凋亡。淋巴瘤动物模型中存在IL-9的异常表达。.本项研究首次将miR-155、miR-21、STAT3、STAT6、IL-9及免疫细胞相关功能基因、蛋白在CLL中进行整合；通过开展免疫细胞、细胞因子及基因组学综合分析研究，结合肿瘤免疫及基因组学探索CLL细胞的免疫耐受机制，为阐述CLL的发生发展机制提供依据。建立了上述指标之间相互作用网络，揭示了其作为CLL治疗靶标的可行性，阐述临床开发及应用潜能，为CLL的免疫治疗提供新的治疗靶点，为最终达到治愈CLL目标奠定基础。