芳香烃受体（AhR）在多发性骨髓瘤发生发展中的作用机制研究

The development and progression of multiple myeloma (MM) are closely related to the inflammatory microenvironment. Researches have demonstrated that Aryl hydrocarbon Receptor (AhR) can up-regulate the expression of inflammatory factors in tumor microenvironment and promote tumor growth and immune escape. The inflammatory factors also induce the expression of AhR in immune cells and tumor cells, but the detailed mechanism of AhR in the regulation of chronic inflammation and tumor remains unknown. In the preliminary research, we had found that the protein level of AhR in MM was highly expressed. Knockdown of AhR can significantly decrease the sensitivity of cells to inflammatory stimuli resulted in the down-regulation of inflammatory factors and inhibit the proliferation of MM cells. Based on the theoretical and preliminary experimental study, we hypothesize that AhR up-regulate chronic inflammation to promote the development and progression of MM by interacting with the signaling pathway of inflammation and tumor. To confirm the hypothesis, we plan to construct AhR-knockout MM cell line with CRISPR/Cas9 gene editing system to evaluate the expression of inflammatory factors and cell proliferation in AhR-knockout cell line compared with wild type. Co-IP and ChIP Assays will be performed to analyze the interaction between AhR and NF-κB or STAT3 and protein-DNA interaction. At the same time, the transcription regulation of NF-κB and STAT3 on AhR gene will be studied and the anti-MM effect of AhR small-molecule inhibitors will be examined both in vitro and vivo. This study is intended to clarify the mechanism of AhR in MM and provide a new effective therapeutic target.

多发性骨髓瘤(multiple myeloma, MM)的发生发展与炎症微环境密切相关。资料显示，芳香烃受体（AhR）能够上调肿瘤微环境中炎症因子的表达同时促进肿瘤的生长和免疫逃逸，但是关于AhR在“炎-癌”转化网络中具体的调控机制尚不清楚。在前期实验中我们发现，AhR在MM中表达水平较高，敲减AhR能够显著降低细胞对炎症刺激分子的敏感性，下调炎症因子的水平同时抑制MM细胞的增殖，因此我们推测 “AhR通过与‘炎-癌’信号通路相互作用上调非可控性炎症促进MM的发生发展”。为证实该假说，本课题拟利用CRISPR/Cas9构建AhR缺失的MM细胞，评价敲除后细胞增殖及炎症因子水平的改变，应用ChIP分析AhR与NF-κB和STAT3协同作用，同时研究NF-κB和STAT3对AhR蛋白表达的调控，最后验证AHR抑制剂的抗MM活性。本项目旨在阐明AhR在MM中的作用机制，为其有效治疗提供新的靶点。

炎症信号通路活化在多发性骨髓瘤的发生发展中起着重要功能，针对多发性骨髓瘤“炎-癌”信号转导网络的深入研究是发展新型抗炎和抗肿瘤药物的关键。芳香烃受体（AHR）在肿瘤的发生发展中起到复杂的调控作用，本项目的研究目标是利用CRISPR/Cas9进行基因敲除，在细胞和动物水平阐明AHR信号通路对多发性骨髓瘤生长的调控作用，并且揭示其分子机制。利用质粒和慢病毒转染系统将肿瘤细胞中AHR基因敲除后可以显著减缓该细胞的增殖，机制研究表明在多发性骨髓瘤中敲除AHR后可以显著降低炎症因子IL-6, IL-8和IL-1β的表达，其中IL-6在多发性骨髓瘤发生发展中起到至关重要的作用；建立小鼠动物模型，实验结果表明AHR基因敲除后可以显著抑制多发性骨髓瘤在小鼠体内的生长；在完成前期研究的基础上，我们还设计和制备了智能化载体递送敲除AHR的质粒，目的是通过基因敲除抑制多发性骨髓瘤在体内外的生长；针对IDO1-AHR信号通路在肿瘤发生发展中的作用，我们构建了靶向肿瘤组织的纳米载体递送IDO1抑制剂阻断IDO1-AHR信号通路，加强化疗药物的抗肿瘤活性，同时制备了具有IDO1响应型的纳米载体递送化疗药物降低药物的毒副作用。本研究揭示了AHR在多发性骨髓瘤发生发展中的作用机制，证明其可以通过上调炎症因子的表达促进肿瘤细胞增殖，同时炎症因子可以促进AHR表达；抑制IDO1-AHR信号通路可以显著抑制肿瘤生长，针对前面研究结果，后期进一步制备了智能化载体进行编辑基因的递送以期达到抗肿瘤目的；该研究为后期多发性骨髓瘤的防治与干预策略奠定了初步的理论依据和实验基础。