低氧诱导假基因PDIA3P1/miR-124-3p相关通路促进胶质母细胞瘤间充质表型转化的机制研究

As a key characteristic of Glioblastoma （GBM） microenvironment, hypoxia promotes the mesenchymal transition that is the main cause for the bad prognosis of GBM. However, the precise mechnism remain unclear. Our pilot experiment showed that hypoxia upregulated the expression of pseudogene PDIA3P1, which enhanced the invasion and migration ability of GBM cells and was related with the mesenchymal transition. The pseudogenes are one kind of long-chain non-coding RNAs and played an important regulatory role in tumorigenesis and progression by downstream miRNAs. Our research suggested that the miR-124-3p and its downstream target gene RELA might be involved in the mesenchymal transition effects of PDIA3P1 by competitive endogenous RNA (ceRNA) regulatory pathways. This study will firstly explore the expressing level and role of PDIA3P1 in clinical glioma tissues and GBM cell lines. Then with the help of gene expression profile and bioinformatics prediction, we will screen and verify the key targets involved in the GBM mesenchymal transition promoted by the upregulated PDIA3P1 in vitro experiments; at the end the new mechanism that hypoxia promote GBM mesenchymal transition through the “PDIA3P1/miR-124-3p/RELA” related pathway will be validated by further in vitro and in vivo experiments. Hopefully, our study will provide new theoretical basis for the malignant progression and therapy of GBM.

低氧作为胶质母细胞瘤（GBM）微环境重要特征之一，可促进GBM间充质表型转化，是导致GBM预后差的主要原因，但具体机制尚不明确。我们预实验发现，低氧诱导假基因PDIA3P1表达上调，增强细胞侵袭迁移能力，与GBM间充质表型转化相关。而假基因作为长链非编码RNA主要依赖下游miRNA在肿瘤发生发展中发挥重要调控作用。进一步研究提示miR-124-3p与其下游靶基因RELA等组成的竞争性内源RNA（ceRNA）调控通路可能参与了PDIA3P1的上述作用。本课题拟先利用临床胶质瘤标本和GBM细胞系明确PDIA3P1表达水平及作用；然后将表达谱芯片与生物信息学预测相结合，利用体内、外实验全面筛选并验证PDIA3P1促间充质表型转化的关键靶点。由此揭示低氧通过PDIA3P1/miR-124-3p/RELA相关通路促进GBM间充质表型转化的全新机制，以期为GBM恶性进展研究和治疗提供新的理论依据。

低氧作为胶质母细胞瘤（GBM）微环境重要特征之一，可促进GBM间充质表型（MES）转化，是导致GBM预后差的主要原因，但具体机制尚不明确。胶质瘤干细胞（GSCs）是被认为是肿瘤起始细胞，在GBM恶性生物表型的进展和演化中发挥关键性作用。 GBM具有显著异质性的特征，其含有前神经元（PN）和MES两种表型的GSCs，GSCs PN向MES表型的转化（PMT）是介导GBM治疗抵抗的重要原因之一，因此，探究介导GSCs PMT的潜在分子机制能够为GBM提供新的治疗策略。.主要研究内容包括一下两部分：.1、低氧诱导PDIA3P1上调，促进GBM MES表型转化的机制研究。.长链非编码RNA（LncRNAs）是影响胶质瘤发生和发展的关键因素。我们发现：PDIA3P1的表达与胶质瘤患者的肿瘤分级和预后密切相关。功能富集分析发现PDIA3P1与MES表型转化相关。在GBMs中过表达PDIA3P1显著增强了胶质瘤的侵袭迁移能，敲减PDIA3P1后则发挥相反的效果。进一步研究发现PDIA3P1通过海绵吸附结合miR-124-3p，调节RELA的表达，进而激活下游NF-κB通路促进GBM的MES表型转化。此外，我们还研究发现低氧通过HIF1促进PDIA3P1的转录表达。结论：PDIA3P1是通过PDIA3P1-miR-124-3p-RELA轴，将低氧微环境和胶质瘤MES转变相连接的关键因素。.2、PDIA3P1促进GSCs PMT表型转化进而促进GBM化疗抵抗的分子机制研究.替莫唑胺（TMZ）化疗耐药性是导致GBM术后复发的一个重要原因。我们研究发现：PDIA3P1在TMZ耐受型GBM细胞系中表达上调；过表达PDIA3P1能够促进GBMs对TMZ的治疗耐受性。我们发现PDIA3P1通过竞争性结合C/EBPβ抑制其泛素化降解，进而维持C/EBPβ的稳定性，促进GSCs PMT。此外我们发现PDIA3P1的表达会随着TMZ治疗出现上调，其中p38α-MAPK信号通路在PDIA3P1的应激性上调中发挥关键作用。TMZ与p38α靶向药物（NEF）联用发挥协同抗肿瘤效应。结论：PDIA3P1通过稳定C/EBPβ促进PMT，降低GBM细胞对TMZ治疗的敏感性。NEF可抑制PDIA3P1的表达上调，NEF与TMZ联合使用可提供更好的抗肿瘤效果。