基于增强子驱动的lnc-TNT经顺式作用正反馈调控介导胶质瘤耐药的机制研究

替莫唑胺化疗耐药是影响脑胶质瘤患者预后的重要因素。申请人前期利用lncRNA芯片、染色质免疫共沉淀-高通量测序等技术发现：耐药胶质瘤中高表达的新型长链非编码RNA lnc-TNT结合ASH1L，潜在顺式调控ERC1基因，参与NF-κB通路激活。在此基础上，申请人拟利用细胞生物学实验验证lnc-TNT介导胶质瘤耐药；染色质共沉淀-高通量测序及染色体构象俘获等技术探究增强子元件对lnc-TNT表达水平的影响；纯化RNA染色质分离实验等明确lnc-TNT结合ASH1L经组蛋白修饰调控替莫唑胺耐药相关基因；进一步解析lnc-TNT经顺式作用调控ERC1，激活ERC1/NF-κB通路，正反馈调节lnc-TNT表达，影响胶质瘤细胞自我更新、DNA损伤修复和凋亡抵抗，介导胶质瘤化疗耐药。本研究可揭示新型增强子驱动的lnc-TNT通过顺式作用及正反馈调控介导胶质瘤替莫唑胺耐药的分子机制。

Temozolomide (TMZ) resistance mainly contributes to the unfavorable prognosis of patients with glioma. Previously, we used lncRNA array, RNAi, ChIP-seq, ChIRP-MS and RIP to find that TMZ resistant glioma cells expressed higher level of a novel lncRNA lnc-TNT compared with the parental glioma cells and that the novel enhancer element of lnc-TNT gene was marked with H3K27ac, an enhancer-specific modification. Lnc-TNT could potentially bind to ASH1L, a H3K4 methyltransferase, mediate the tri-methylation in the promoter regions of ERC1 gene and activate the NF-κB signaling pathway in glioma cells. Based on these results, we aim to explore the molecular mechanism of lnc-TNT regulated by a novel enhancer element contributing to TMZ resistance via ASH1L-mediated histone modification in glioma. We will verify the lnc-TNT-mediated TMZ resistance in glioma cells, and investigate its mechanism regulating TMZ resistance with DNA-pulldown, co-IP, ChIP-PCR, RNA microarray, 3C and so on. We will study the role of enhancer-specific modifications H3K27ac regulating lnc-TNT expression, clarify the gene expression profiling regulated by the lnc-TNT/ASH1L axis and explore the process of lnc-TNT cis-regulating ERC1 expression, activating ERC1/NF-κB signaling axis and mediating the self-renewal, DNA damage repair and apoptosis resistance in glioma. This study is expected to elucidate a new theoretical evidence for the mechanism of TMZ resistance, excavate the therapeutic targets against TMZ resistance and provide a new approach to the treatment of glioma.