MicroRNA调控自噬对胶质母细胞瘤放疗敏感性的作用和机制研究

Autophagy is considered to be one of the important mechanisms of resistance to radiotherapy. Our previous studies have demonstrated inhibition of autophagy can enhance anti-tumor treatment in glioblastoma. miRNA microarray analysis revealed that there are levels of different miRNA between radioresistant glioblastoma cells and the parental cell from which they were derived, suggesting that miRNA may be the key factor that autophagy affect radiosensitivity. In this study, quantitative real time PCR was used to evaluate the expression the different miRNA. Using integrated bioinformatic alogorithms to predict the potential target gene associated with autophagy of the miRNAs. We transfected glioblastoma cells with miRNA mimics or inhibitor, overexpression target gene or siRNA plasmid and evaluated their effect on cell radiosensitivity and autophagy in vitro and in vivo (subcutaneous and in situ tumor xenograft). Finally, we investigate the correlation among the candidate miRNA, target gene and autophagy activity in human glioblastoma tissues, and its relationship with radiotherapy effect, clinical staging and prognosis. This study is important to elucidate the molecular mechanism of autophagy affect radiosensitivity in glioblastoma and to provide the novel target of cancer treatment, which is meanfingful in clinical.

自噬近年来被认为是恶性肿瘤放疗抗拒的重要机制之一。我们前期研究证实抑制自噬的发生能增强胶质母细胞瘤的抗肿瘤治疗效果，通过miRNA芯片分析胶质母细胞瘤放疗抗拒细胞和亲本细胞，发现4个可能与自噬相关的候选miRNA表达存在差异，提示miRNA可能是自噬影响胶质母细胞瘤放疗抗拒的关键因子。本研究拟通过实时定量PCR鉴定miRNA表达差异，运用生物信息软件分析这些候选miRNA与自噬通路相关的靶基因并验证，利用miRNA 模拟/抑制、过表达/沉默靶基因的方法观察胶质母细胞瘤放疗敏感性和自噬变化情况，并通过皮下移植瘤和原位瘤动物模型进一步确认。最后，分析临床标本的候选miRNA与相应的靶基因、自噬相关基因表达的相关性，以及与放疗疗效、临床分期和预后的关系。本项目能阐明自噬影响胶质母细胞瘤放疗敏感性的具体分子机制，为肿瘤治疗抗拒提供新的治疗靶点，具有重要的临床意义。

自噬被认为是恶性肿瘤放疗抗拒的重要机制之一，抑制自噬可逆转放疗抗性，利用小分子miRNA抑制自噬是确切可行的手段。我们前期研究证实抑制自噬的发生能增强胶质母细胞瘤的抗肿瘤治疗效果。本研究中，通过miRNA芯片分析胶质母细胞瘤放疗抗拒细胞和亲本细胞，发现多个可能与自噬相关的候选miRNA表达存在差异，并通过实时定量PCR验证了miRNA表达差异，运用生物信息软件分析miR-181a和miR-124-3p与自噬通路相关的靶基因密切相关。荧光酶活性检测结果证实ATG-5是miR-181a的靶基因，而MST-4是miR-124-3p的靶基因。相对于对照miRNA，胶质母细胞瘤放疗抗拒细胞U87MG-R分别转染miR-181a或miR-124-3p后，ATG-5或MST-4蛋白表达水平都明显降低，经照射的U87MG-R细胞自噬发生明显减少，细胞增殖活性明显下降，细胞凋亡发生明显增多。皮下移植瘤动物实验显示：miR-181a或miR-124-3p与放疗联合治疗U87MG-R移植瘤取得更好抑制肿瘤生长的效果，提高了移植瘤的放疗敏感性。miR-181a通过下调自噬通路关键分子ATG-5的表达，抑制细胞自噬；而miR-124-3p通过下调MST4（STK26)表达，抑制自噬通路的ATG4B的磷酸化，影响细胞自噬的发生，最终逆转胶质瘤细胞的放疗抗性。通过临床样本检测发现miR-124-3p的表达与胶质瘤预后呈正相关。本研究从细胞自噬作为着手点，发现miR-181a/ATG5和miR-124-3p/MST4/ATG4B两条通路通过抑制细胞自噬，从而影响胶质瘤细胞的放疗敏感性。研究结果将为胶质瘤的治疗提供新的靶点，为提高胶质瘤放疗敏感性提供了新的研究思路，具有重要的临床意义。