假基因UBDP1竞争性结合microRNA调控胶质瘤恶性增殖侵袭的机制研究

Gliomas are the most common primary brain tumors and characterized by rapid proliferation and invasive growth. Despite current aggressive treatment, almost all of gliomas can recur which makes patients face a poor clinical outcome. Therefore, it is of very high priority to explore the vital genetic changes and core pathways involved in glioma progression, which will pave new ways to developing targeted therapies. Previously, we have found a pseudogene UBDP1 dramatically upregulated in glioma based on lncRNA microarray and its expression positively related to the parent gene UBD in glioma tissues and cells. Moreover, both of UBDP1 and UBD could significantly promote glioma cell proliferation. Bioinformatic analysis was then performed and showed that there existed common miRNA (such as miRNA-4747) binding sites within UBD and UBDP1 sequences. Exogenous miRNA-4747 could obviously suppress the expression of UBD and UBDP1 in glioma cells. Accordingly, we speculate that the pseudogene UBDP1 can compete with UBD for miRNA binding which will result in increased expression of oncogenic UBD in glioma. Here, this project intends to elucidate the UBDP1-miRNA-UBD regulatory network involved in glioma progression and explore core pathways underlying the downstream of UBDP1 with multiple methods and materials including CRISPR-Cas9, next-generation sequencing, glioma stem cell, and patient-derived xenograft. We hope to take deep insights into the oncogenic mechanisms driven by pseudogenes in glioma and identify novel targets for drug development, both of which will help promote the prognosis of glioma patients in the future.

脑胶质瘤是最常见原发中枢神经系统肿瘤，恶性程度高，极易复发，患者死亡率高，现有治疗方式仍未有突破性进展。深入研究胶质瘤基因调控网络变化，筛选关键信号通路，对靶向药物开发、患者生存期延长等具有重要意义。申请人前期用长链非编码RNA芯片筛选出胶质瘤中显著上调的假基因UBDP1，并在样本和细胞系中验证了其与UBD表达相关，两者均具有促进胶质瘤恶性增殖的能力；信息分析发现UBDP1和UBD有共同miRNA结合区域，如miRNA-4747可同时调控两者表达。基于此我们提出“假基因UBDP1通过竞争性结合miRNA促进癌基因UBD表达，进而促使胶质瘤恶性进展”的科学假设。本项目拟通过基因敲除、转录组测序、胶质瘤干细胞、PDX模型等技术，探索UBDP1-miRNA-UBD网络共同调控胶质瘤恶性表型机制及下游信号通路，明确靶向假基因调控网络治疗胶质瘤临床转化潜能，为提高胶质瘤患者预后提供分子理论依据。

研究目的：本研究旨在深入探索假基因影响胶质母细胞瘤恶性进展的关键分子机制，为胶质母细胞瘤的靶向治疗提供新的可能。.研究方法：首先，通过对前期工作中已完成的胶质母细胞瘤和正常脑组织基因表达谱芯片测序数据（GEO dataset: GSE51146）进行进一步分析，最终筛选出差异表达的假基因UBDP1；通过胶质母细胞瘤样本和正常脑组织样本的QPCR实验验证UBDP1及其真基因UBD在胶质母细胞瘤中的异常表达，并进行Kaplan-Meier单因素生存分析以及Cox多因素生存分析。接下来，我们通过FISH+蛋白免疫荧光实验来探索UBDP1和UBD在胶质母细胞瘤细胞中的分布；通过CCK-8细胞增殖实验和transwell细胞迁移、侵袭实验来探索UBDP1和UBD对胶质母细胞瘤细胞功能的调控作用；通过荷瘤裸鼠的生存分析和裸鼠脑内肿瘤样本的免疫组化实验来探索UBDP1和UBD在体内对胶质母细胞瘤恶性进展的调控作用。最后，我们通过生信分析及western blot实验筛选出UBDP1和UBD相同结合位点的miR-6072；通过荧光素酶报告基因实验来探索UBDP1、UBD与miR-6072在细胞中的结合情况。.研究结果：30例胶质母细胞瘤样本和15例正常脑组织样本的QPCR实验结果显示，UBDP1和UBD在胶质母细胞瘤中的表达较正常脑组织均显著升高； Kaplan-Meier生存分析以及Cox生存分析结果显示，UBDP1、UBD在胶质母细胞瘤中的表达均是胶质母细胞瘤患者预后的独立影响因素；FISH+蛋白免疫荧光实验结果显示，UBDP1和UBD主要分布于胶质母细胞瘤细胞的胞浆中；细胞功能实验结果显示，UBDP1和UBD均促进胶质母细胞瘤的细胞增殖、迁移和侵袭；脑内荷瘤裸鼠的生存分析及肿瘤免疫组化结果显示，UBDP1、UBD过表达的脑内荷瘤裸鼠的生存时间显著低于对照组，UBDP1、UBD过表达的肿瘤样本中的ki-67比例显著高于对照组。western blot实验结果显示，miR-6072抑制UBD的表达；荧光素酶报告基因实验的结果显示，UBDP1、UBD均能和miR-6072在细胞中结合。.研究结论：假基因UBDP1通过竞争性结合miR-6072促进UBD的表达，从而促进胶质母细胞瘤的增殖、迁移和侵袭。