GJIC介导Rac1蛋白失活抑制白血病干细胞干性的作用及机制研究

Leukemia stem cells (LSCs) play central role in disease relapse and chemotherapy resistance. Loss of gap junctional intercellular communication (GJIC) among bone marrow stromal cells (BMSC) is correlated with leukemia relapse and chemotherapy resistance. Our previous studies have found up-regulation of GJIC in leukemia BMSC can lead to improvement of calcium influx, the increased intracellular calcium can be transmitted into leukemia cells, and led to proliferation repression of leukemia cells. The current proposal provided that declined calcium influx induced by GJIC is upstream signals regulating stemness of LSCs. Rac1 is recognized as critical factor in maintenance of stemness of tumor stem cell. Several studies have demonstrated that its activation can be regulated by calcium influx. Our pre-experiments showed that calcium influx of LSCs could inhibit activation of Rac1, but the detailed underlying mechanism is poorly known. We aim to demonstrated that up-regulation of GJIC can inhibit stemness of LSCs by improvement of calcium influx in vivo and vitro. Meanwhile, using Rac knock-down and up-regulation lentiviral and gene array analysis, we will investigate the important role of rac1 in biological effects mediated by up-regulation of GJIC in LSCs. Our studies will help the field to understand the molecular mechanism of stemness of LSCs induced by GJIC dysregulation, and highlight the significance of novel and potential targets for further therapeutic approaches in cancer.

白血病干细胞(LSCs)是白血病复发耐药的根源。骨髓基质细胞间隙连接细胞间通讯（GJIC）功能缺失与LSCs增殖失控关系密切。前期研究发现，上调白血病骨髓基质GJIC引起基质细胞内钙离子浓度（[Ca2+]i）显著增加并传递至共培养的白血病细胞，抑制细胞增殖，提示GJIC介导的胞内[Ca2+]i变化可能是影响LSCs干性的上游因素。Rac1蛋白是维持肿瘤干细胞干性的关键蛋白，其活性受胞内[Ca2+]i负向调控。预实验发现，LSCs内[Ca2+]i 增加可阻抑Rac1蛋白激活，但该效应对LSCs干性的影响及机制不明。本课题拟通过体内、外实验证实上调GJIC，增加胞内[Ca2+]i可抑制LSCs干性；进一步通过Rac1基因沉默/过表达、基因芯片结合生物信息学、表型拯救等实验证实Rac1是GJIC调控LSCs干性的关键调节蛋白并阐明其分子通路；探讨上调骨髓基质GJIC抑制LSCs干性的分子机制。

白血病干细胞（leukemia stem cells，LSCs）在白血病的发生、发展、化疗耐药和复发中起着关键作用，但对其克隆性增殖、分化、归巢及体内成瘤能力等干性维持的分子调节机制尚不明确。有必要深入研究LSCs的生物学调控机制，为探索针对LSCs的靶向治疗提供新的思路。我们的实验发现上调GJIC功能，胞内[Ca2+]i增加对LSCs的增殖、凋亡和分化都能有效抑制，说明CX43的上调能够上调GJIC功能，并通过胞内[Ca2+]i影响LSCs的生理活性。进一步实验证实，LSCs的成瘤性与Rac1的表达明显相关，Rac1过表达后，LSCs能更快的在免疫缺陷鼠中成瘤，与对照组相比有明显区别。此外，Rac1的表达也对CX43的表达有一定影响，说明Rac1在LSCs干性维持中可能通过CX43起作用。Rac1作用的下游靶基因及信号通路尚不明确，还需要进一步后续实验探索这一通路在LSCs干性维持中的作用。