miR-26a对胶质母细胞瘤细胞放疗敏感性的影响及其机制研究

发布时间：2018-09-11 16:18

【摘要】：目的：研究miR-26a对胶质母细胞瘤（glioblastoma multiforme，GBM）细胞放疗敏感性的影响，并探索其相关分子学机制，深入了解miR-26a在GBM放疗敏感性方面的潜在意义，为GBM的个体化治疗提供更多的分子学基础。方法：利用Real-time PCR检测U251、A172、T98g三种放疗敏感性不同的GBM细胞系中miR-26a的表达水平；构建miR-26a过表达和敲低的GBM细胞系稳定株，进行克隆形成实验；利用流式细胞术测定miR-26a过表达后的GBM细胞周期变化；通过BrdU细胞增殖实验观察miR-26a对GBM细胞增殖的影响；流式细胞术测定放疗之后miR-26a过表达的GBM细胞的凋亡情况；单细胞免疫荧光实验（γH2AX fociassay）以及彗星实验判定miR-26a对GBM细胞DNA修复的影响；通过miRanda预测miR-26a的可能靶点，并结合双荧光素酶报告实验进一步验证； Western Blot检测在过表达和敲低miR-26a之后，ATM以及下游因子p-CHK2和p53的变化；构建原位胶质瘤动物模型，，利用MRI磁共振成像观察放疗前后GBM肿瘤的体积变化，并进行生存分析。结果：U251、A172、T98g三种GBM细胞系中，miR-26a的表达水平与细胞放疗敏感性相一致；相对于空载体对照组而言，miR-26a敲低的U87和U251细胞接受放疗后具有较多的克隆数(P＜0.05)，而miR-26a过表达后则克隆数减少(P＜0.05)；过表达miR-26a之后， U87细胞的S期缩短(P＜0.05)，细胞增殖能力增强(P＜0.05)，且接受放疗之后的细胞凋亡比例较增高（P＜0.001）；接受放疗之后，相对于空载体对照组，miR-26a敲低的U87细胞中γH2AX foci的数量较少（P＜0.05），彗星尾的长度较短（P＜0.05），而在miR-26a过表达的U87细胞中则相反；miR-26a可靶向作用于ATM，并进一步诱使其下游因子p-CHK2和p53的表达水平降低；动物实验显示miR-26a过表达的原位胶质瘤小鼠在接受放疗后肿瘤体积较小（P＜0.05），生存周期更长（P＜0.05）。结论：miR-26a可靶向作用于ATM，进而影响同源重组修复通路，降低GBM细胞的DNA修复能力，并最终增强了GBM细胞的放疗敏感性。
[Abstract]:Objective: to study the effect of miR-26a on the radiosensitivity of glioblastoma (glioblastoma multiforme,GBM) cells and explore its molecular mechanism, to understand the potential significance of miR-26a in GBM radiosensitivity, and to provide more molecular basis for the individualized treatment of GBM. Methods: Real-time PCR was used to detect the expression of miR-26a in three GBM cell lines with different radiosensitivity to radiotherapy, and to construct a stable cell line of GBM with miR-26a overexpression and knockdown. The changes of GBM cell cycle after overexpression of miR-26a were measured by flow cytometry, the effect of miR-26a on the proliferation of GBM cells was observed by BrdU cell proliferation assay, the apoptosis of GBM cells with miR-26a overexpression after radiotherapy was measured by flow cytometry. Single cell immunofluorescence assay (纬 H2AX fociassay) and comet assay were used to determine the effect of miR-26a on DNA repair in GBM cells. The possible targets of miR-26a were predicted by miRanda and further verified by double luciferase report. Western Blot was used to detect the changes of miR-26a and its downstream factors, p-CHK2 and p53 after overexpression and knockdown. An in situ glioma model was established, and the volume changes of GBM tumors before and after radiotherapy were observed by MRI magnetic resonance imaging, and survival analysis was performed. Results the expression level of miR-26a in the three GBM cell lines was consistent with the radiosensitivity of the cells. Compared with the control group of empty vector, U87 and U251 cells with low knockout of miR-26a had more clones after radiotherapy (P < 0. 05), but the number of clones decreased after overexpression of miR-26a (P < 0. 05). After overexpression of miR-26a, the S phase of U87 cells was shortened (P < 0. 05), the proliferation of U87 cells was increased (P < 0. 05), and the percentage of apoptosis was increased after radiotherapy (P < 0.001). Compared with the empty vector control group, the number of 纬 H2AX foci in U87 cells was lower than that in the control group (P < 0. 05), and the length of comet tail was shorter (P < 0. 05), whereas in U87 cells with overexpression of miR-26a, the number of 纬 H2AX foci was lower (P < 0. 05). MiR-26a could target ATM, and further induce the lower expression of p-CHK2 and p53, and the animal experiment showed that the tumor volume of miR-26a overexpression was smaller and the survival period was longer (P < 0. 05). ConclusionTwo miR-26a can target ATM, and then affect the homologous recombination repair pathway, reduce the DNA repair ability of GBM cells, and enhance the radiosensitivity of GBM cells.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.4

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