靶向lncRNA-XIST/miR-92a轴诱导AML细胞坏死性凋亡的作用及分子机制研究

The relapse and drug resistance of acute myeloid leukemia (AML) are closely related to apoptotic resistance. Necroptosis is a novel form of regulated cell death different from apoptosis. Recent studies indicate that induction of necroptosis is an important strategy for eliminating apoptosis-resistant cancer cells. However, the understanding of molecular mechanisms and induction methods of necroptosis is still limited. Our previous studies have shown that lncRNA XIST can inhibit proliferation and induce necroptosis in AML cells, which may be related to the interaction of XIST and miR-92a. In this research, we aim to elucidate the expression pattern and clinical significance of XIST in AML, to determine the interaction between XIST and miR-92a，and to explore the downstream molecules of XIST/miR-92a axis. Furthermore, we will evaluate the anti-leukemia effect of targeting XIST/miR-92a axis in AML xenograft mouse model. Taken together, completion of our study will demonstrate novel mechanisms of necroptosis based on XIST/miR-92a axis, and may offer new ideas for treating AML, especially the patients with relapse or drug resistance.

急性髓细胞性白血病（AML）的复发、耐药现象与肿瘤细胞凋亡耐受密切相关。坏死性凋亡是不同于凋亡的细胞主动死亡方式，被认为是清除耐凋亡肿瘤细胞的新途径，但目前对其分子调控机制和诱导途径的了解十分有限。我们的前期研究发现lncRNA XIST能抑制AML细胞增殖并诱导坏死性凋亡，且可能通过与miR-92a互作形成XIST/miR-92a轴来调控这一过程。本课题拟在前期工作基础上进一步阐明XIST在AML中的表达特征和临床意义；明确XIST与miR-92a的相互作用，找寻下游靶分子，阐明坏死性凋亡信号调控网络；同时，还将建立AML小鼠荷瘤模型验证靶向XIST/miR-92a轴在体内的抗肿瘤效应。本课题的顺利实施将深入揭示坏死性凋亡的分子机制，为AML，特别是复发、耐药患者的靶向治疗提供全新思路，具有重要的理论价值和应用潜力。

长链非编码RNA（lncRNA）和微小RNA（microRNA）在肿瘤细胞表观遗传学调控中发挥重要作用。XIST是我们前期筛选到的与血液肿瘤相关的lncRNA，初步研究提示XIST具有抑制急性髓系白血病（AML）细胞生长增殖并诱导坏死性凋亡的功能，且可能与miR-92a-3p结合形成XIST/miR-92a-3p信号轴共同调控AML 细胞坏死性凋亡过程，迄今尚无相关报道。本研究主要围绕XIST/miR-92a-3p轴调控急性髓细胞白血病细胞坏死性凋亡的作用及分子机制展开研究。我们基于CRISPR/Cas9-SAM系统构建XIST基因过表达慢病毒载体，同时构建miR-92a-3p-inhibition慢病毒载体，并将上述慢病毒分别转染AML细胞株进一步研究相关机制。通过CCK8、RT-qPCR、Western blot、流式细胞术、透射电镜、双荧光素酶报告基因、RNA pull-down以及小鼠体内实验等技术，初步证实lncRNA XIST是AML细胞潜在的抑癌基因，并可通过XIST/ miR-92a-3p/CYLD信号轴调控坏死性凋亡的发生；XIST低表达与AML不良预后相关，人为上调XIST表达水平可对AML细胞发挥杀伤效应。本研究结果具有较强的理论价值和应用前景，可为今后以靶向XIST为基础的AML治疗策略提供全新的思路。