长链非编码RNA调节胶质瘤放化疗敏感性分子机制的研究

Radiotherapy and chemotherapy is the primary postoperative treatment for gliomas, and the sensitivities of radiotherapy and chemotherapy are the key to success. Glioma cell lines U251AR and U87AR resistant to radiotherapy and chemotherapy have been established, and their biological characteristics were already evaluated.Through long non-coding RNA (lncRNA) chip, the lncRNA expression profile of the radiotherapy and chemotherapy resistance cells and the parental cells has already been analyzed. Based on the preliminary work, we were about to perform the following experiments: 1) the differentially expressed lncRNA would be screened and verified by quantitative real time PCR (qRT-PCR); 2) effects of lncRNA on the proliferation, apoptosis and radiotherapy and chemotherapy sensitivities of glioma cells would be observed; 3) the target genes of lncRNA would be predicted through bioinformatics methods, the DNA, RNA and proteins combined with lncRNA would be detected by RNA pull down, RNA-RIP and ChIRP-seq methods, and co-expression networks would be established; 4) the expressions of lncRNAs and their target genes in the plasma and serum samples of patients proved in clinic to be enjoying sensitivity and resistance of radiotherapy and chemotherapy would be detected by qRT-PCR and immunohistochemistry, and their cell experiment results were verified. Our research will clear the molecular mechanisms of lncRNA affecting the sensitivities of radiotherapy and chemotherapy of gliomas, which can provide a new theoretical foundation for clarifying the regulation mechanisms of glioma cells resistant to chemotherapy and radiotherapy and find the new targets for glioma treatment.

放化疗是胶质瘤术后治疗主要手段，而放化疗敏感性是治疗成败关键。我们已构建并鉴定同期耐放化疗的胶质瘤细胞株U251AR和U87AR，并通过lncRNA芯片，比较了耐放化疗细胞和亲本细胞的lncRNA表达谱。本研究拟在此基础上：1）分析lncRNA芯片，筛选差异表达lncRNA并QRT-PCR验证；2）检测lncRNA对胶质瘤细胞增殖、凋亡及放化疗敏感性的影响；3）生物信息学方法预测lncRNA潜在靶基因，通过RNA pull down、RNA-RIP、ChIRP-seq等方法检测其靶基因的DNA、RNA、蛋白质，构建共表达分子网络；4）QRT-PCR和免疫组化检测已被临床验证的放化疗敏感/抵抗患者的血浆/血清样本中lncRNA及其靶基因的表达，验证细胞实验结果。本课题将明确lncRNA影响胶质瘤放化疗敏感性的分子机制，为胶质瘤细胞耐放化疗调控机制阐明提供新的理论基础，为胶质瘤治疗提供新靶点。

放化疗是胶质瘤术后治疗主要手段，而放化疗敏感性是治疗成败关键。本项目采用基因芯片分析了体外构建的耐放化疗胶质瘤细胞；筛选出差异表达于耐放化疗胶质瘤细胞中的ncRNAs与mRNAs；通过体内外研究验证胶质瘤耐放化疗差异分子靶点表达并构建共表达网络；进一步深入探讨差异表达分子靶点对胶质瘤耐放化疗的影响及其深层次调控机制。通过研究得到以下研究结果：一、成功筛选了胶质瘤耐放化疗细胞中差异表达的ncRNAs与miRNAs；二、初步明确了差异表达LncRNA RP11-838N2.4促进胶质瘤化疗增敏，并阐明LncRNA RP11-838N2.4通过抑制miR-10a表达介导胶质瘤化疗敏感性；三、证实了miR-203靶向SNAI2介导胶质瘤EMT进程在耐放化疗胶质瘤细胞化疗抵抗中发挥作用；四、发现了结缔组织生长因子CTGF表达与胶质瘤化疗抵抗密切相关，CTGF可通过介导TGF-β信号表达调控胶质瘤化疗抵抗。项目研究初步提示了胶质瘤放化疗抵抗是多因素共同参与的复杂分子调控网络；不同层面分子靶点均可影响胶质瘤放化疗敏感性；前期研究的相关分子靶点为胶质瘤放化疗抵抗的治疗提供了新靶点，进一步丰富与完善了胶质瘤放化疗抵抗分子调控机制。