MALAT1/miR-181d海绵调控NF-κB信号通路参与胶质瘤治疗抵抗的机制研究

Mesenchymal transformation is an important mechanism for therapy resistance and recurrence of gliomas. Our previous studies have found that miR-181d can alter the transcriptional activity of the NFκB pathway, thus changes the mesenchymal phenotype of giomas and relieves the radiation and chemotherapy resistance. Further large sample sequencing analysis and preliminary experiments showed that lncRNA-MALAT1 and miR-181d showed potential negative regulation relationship; MALAT1 expression was associated with the activated state of NFκB signaling, and was further up-regulated in the gliomas with therapy resistance. Based on our recent findings, we hypothesize that MALAT1/miR-181d sponge mediate therapy resistance by regulating NFκB signaling in gliomas. To test the hypothesis, we intend to carry out the following studies: techniques such as co-IP, luciferase reporter assay will be conducted to analyze MALAT1 as a molecular sponge of miR-181d in regulating the NFκB pathways at post-transcription regulation level, as well as the epigenetic regulation of NFκB signaling via PRC2 compounds; techniques such as ChIP, EMSA assays will be performed to verify the mechanism of the positive feedback network of MALAT1 activation mediated by NFκB transcription. Finally, primary cultured glioma cells and animal models will be utilized to verify the effect of radiation and chemotherapy resistance mediated by the regulatory network. This study is expected to interpret the mechanism of therapy resistance in gliomas from the perspective of mesenchymal transition regulated by non-coding RNAs.

间质表型转化是胶质瘤治疗抵抗和复发的重要机制。前期研究发现，miR-181d能够通过下调NFκB通路的转录活性，改变胶质瘤间质性表型并改善放化疗抵抗。进一步大样本测序分析及预实验发现，lncRNA-MALAT1与miR-181d呈潜在负性调控关系，MALAT1表达与NFκB通路的激活状态相关，其表达在治疗抵抗的胶质瘤中进一步上调。我们提出假说：MALAT1/miR-181d海绵调控NFκB信号参与胶质瘤治疗抵抗。拟运用荧光素酶报告、co-IP等试验分析MALAT1作为分子海绵吸附miR-181d发挥对NFκB通路的转录后调控，以及通过PRC2复合物对NFκB信号的表观遗传调控；运用凝胶迁移、ChIP等技术验证NFκB转录激活MALAT1形成正反馈调控；最后用原代培养细胞及动物模型验证上述调控网络在胶质瘤放化疗抵抗中的作用。本研究有望从非编码RNA调控间质转化的角度诠释胶质瘤治疗抵抗机制。

胶质瘤是脑和脊髓最常见的原发性恶性肿瘤，RNA表观遗传学调控在其恶性进展及化疗抵抗中发挥关键作用。该课题研究进行中，LncRNA MALAT1参与的NFκB激活促进胶质瘤间质表型转化的机制逐渐被揭示，然而其上游的调控机制不明。结合最新研究进展，我们基于胶质瘤RNA-seq大数据挖掘发现，RNA甲基化修饰关键调控分子的表达水平与NF-κB激活以及胶质瘤恶性进展及放化疗敏感性密切相关。我们向国家自然基金委员会报备后，将研究重点调整为RNA m6A修饰参与的NFκB上游调控的机制。. 基于团队构建的胶质瘤基因组数据库（CGGA）及TCGA数据库的生物信息分析，我们首先从胶质瘤基因表达谱角度系统分析了13种主要m6A相关调控蛋白的表达及潜在功能，发现m6A修饰与胶质瘤的恶性进展、化疗敏感性及NF-κB激活密切相关。进一步体外及体内模型中研究中发现，m6A甲基化酶METTL3能够促进IDH野生型胶质瘤恶性进展及化疗抵抗，深入的机制研究揭示了METTL3在HuR辅助下通过m6A修饰增加MALAT1稳定性，进而激活NF-κB参与胶质瘤恶性进展的新机制。.RNA甲基化酶的功能需要RNA识别蛋白的参与。我们进一步研究发现m6A关键识别蛋白YTHDF2可通过识别NF-κB通路抑制因子UBXN1的mRNA，抑制其表达从而激活下游的NF-κB通路；METTL3介导的UBXN1 mRNA 3’端m6A修饰可调控YTHDF2对UBXN1 mRNA的识别，并导致UBXN1 mRNA的降解。该研究发现揭示了YTHDF2通过识别METTL3介导的m6A修饰促进UXBN1 mRNA降解，从而激活NF-κB促进胶质瘤恶性进展的分子机制。. 该课题系列研究成果阐述了胶质瘤中RNA甲基化调控与肿瘤恶性进展及治疗抵抗的内在关系，并从RNA m6A修饰水平阐明了NF-κB信号激活的上游调控机制，为从RNA表观遗传角度深入理解胶质瘤的恶性进展、开发新疗法提供了重要理论依据。.