摘要
目的：干扰TP53诱导的糖酵解及凋亡调节蛋白（TIGAR）表达可以增加人脑胶质瘤细胞的放射敏感性，但其机制尚不明了。硫氧还原蛋白-1（TRX1）是一个通过核转运参与电离辐射后DNA损伤修复过程的重要的还原蛋白。由于TRX1依赖NADPH保持还原状态从而发挥功能，而在氧化应激条件下，增加TIGAR表达有助于产生更多的NADPH从而帮助细胞对抗氧化。本课题试图通过改变TIGAR的表达水平，研究TIGAR对TRX1核转运的影响及其在调节胶质瘤细胞放射敏感性中的作用，从而阐述TIGAR调节胶质瘤细胞放射敏感性的机制；通过比较TIGAR在胶质瘤细胞与正常胶质细胞中功能的差异，探讨TIGAR作为胶质瘤放疗增敏靶点的可能性；最后，通过建立人脑胶质瘤裸鼠原位种植模型，验证TIGAR调节细胞放射敏感性的机制。达到阐明干扰TIGAR表达增加人脑胶质瘤放射敏感性机制的目的。
方法：采用Western blot技术检测电离辐射对细胞TIGAR表达水平及p53蛋白水平的影响；通过实时荧光定量PCR技术检测电离辐射后细胞TIGAR、TP53转录水平的改变；通过转染TIGAR siRNA干扰TIGAR表达，构建pcDNA3.1-TIGAR瞬时过表达TIGAR；采用流式细胞技术、NADPH检测试剂盒检测电离辐射前后细胞的氧化还原水平；通过转染野生型TRX1过表达质粒（pcDNA3.1-WT-TRX1）及突变型TRX1过表达质粒（pcDNA3.1-MT-TRX1）、G418筛选得到野生型及突变型TRX1稳定过表达细胞株；采用克隆形成试验检测电离辐射后细胞的克隆存活率；通过免疫荧光技术评价电离辐射后不同时间点（0.5-12h）胶质瘤细胞DNA损伤的修复水平；通过Western blot技术检测干扰TIGAR表达对细胞TRX1核转运的影响；采用氧化还原Western blot技术检测电离辐射前后TRX1蛋白的氧化还原水平。最后通过原位种植胶质瘤细胞建立人脑胶质瘤裸鼠原位种植模型；通过原位注射TIGAR shRNA慢病毒实现基因治疗，并采用核磁共振成像（MRI）技术评价基因治疗联合放疗的治疗效果。
结果：（1）电离辐射后，p53野生型人脑胶质瘤A172细胞内TIGAR表达水平在照后1h开始增加，照后8h回落至基础水平；p53蛋白水平于照后0.5h开始增加，先于TIGAR表达增加。而在受照的p53突变型（M237I）T98G细胞中，TIGAR与p53蛋白水平均无显著增加。克隆形成试验显示，干扰TIGAR表达显著降低受照A172、T98G细胞克隆存活率。（2）干扰TIGAR表达显著增加受照胶质瘤细胞内的活性氧（ROS）水平并造成受照细胞内NADPH水平耗竭，在单纯照射组细胞内NADPH的含量仅较照射前下降了40%，而TIGAR干扰联合照射组细胞内NADPH的含量较照射前下降了约75%。相似的，干扰TIGAR表达也引起了受照胶质瘤细胞内GSH/GSSG比例的进一步下降，而TIGAR过表达则可显著增加受照胶质瘤细胞内的NADPH含量与GSH/GSSG的比例。Western blot结果显示，电离辐射后细胞内TRX1可发生核转运，核转运的峰值时间为照后2h。而干扰TIGAR表达可显著抑制受照胶质瘤A172、T98G细胞内的TRX1核转运。（3）干扰TIGAR表达无法抑制野生型TRX1过表达A172细胞发生电离辐射诱导的TRX1核转运。氧化还原Western blot结果表明A172细胞受照后，胞浆内TRX1于照后0.5h内被氧化，于照后8h回复到还原状态，而干扰TIGAR表达可显著延缓细胞浆内TRX1的还原进程，结果显示胞浆内TRX1于照后8h仍处于氧化状态。而在野生型TRX1过表达细胞内，TIGAR干扰无法延缓TRX1的还原进程，电离辐射后细胞核内TRX1的氧化还原进程与细胞浆内TRX1类似，也可被干扰TIGAR表达延缓。克隆形成试验的结果显示，野生型TRX1过表达可显著抑制TIGAR干扰引起的放射增敏效应，而突变型TRX1过表达联合TIGAR干扰无法在单纯干扰TIGAR表达的基础上进一步减少受照胶质瘤细胞的克隆存活率。更为关键的是，增加TIGAR表达对TRX1突变型细胞的放射敏感性无显著影响，表明TRX1核转运在TIGAR调节胶质瘤细胞放射敏感性中发挥了重要作用。（4）免疫荧光实验的结果显示，受照A172γ-H2AX的点状荧光在照后4h内消失，而在TIGAR干扰组A172细胞内γ-H2AX点状荧光直到照后12h依然存在，表明细胞内DNA损伤修复进程被显著延迟。与Western blot的结果相似，免疫荧光实验的结果显示受照A172细胞内TRX1核转运的峰值时间为照后2h，而TIGAR干扰组细胞内TRX1核转运则完全消失。在野生型TRX1过表达细胞中，干扰TIGAR表达导致的DNA损伤修复延迟与TRX1核转运抑制均得到了显著恢复。（5）与胶质瘤细胞不同，改变TIGAR表达无法改变正常脑胶质细胞的放射敏感性。并且尽管电离辐射后，星形胶质细胞内可以观察到TRX1核转运现象，但干扰TIGAR表达无法抑制电离辐射引起的星形胶质细胞TRX1核转运，也无法延缓受照星形胶质细胞内TRX1的还原进程。氧化还原Western blot的结果还显示，受照星形胶质细胞内TRX1的氧化程度显著低于相同照射条件下脑胶质瘤细胞内TRX1的氧化还原程度。（6）通过TIGARshRNA慢病毒基因治疗，证实了干扰TIGAR表达可增加荷瘤小鼠颅内胶质瘤的放疗敏感性。
结论：本课题的研究首次提出在人脑胶质瘤细胞内干扰TIGAR表达显著抑制电离辐射引起的TRX1核转运，此现象很可能与干扰TIGAR表达的放射增敏机制有关。干扰TIGAR表达抑制TRX1核转运的原因可能与破坏细胞内氧化还原稳态、导致受照细胞内TRX1过氧化及还原进程延缓有关。干扰TIGAR表达无法抑制野生型TRX1过表达胶质瘤细胞内TRX1发生核转运。干扰TIGAR表达对正常脑胶质细胞的放射敏感性无显著影响，并且干扰TIGAR表达可能增加胶质瘤的放疗敏感性。
Objective: TP53-induced glycolysis and apoptosis regulator (TIGAR) knockdownis proven to radiosensitize glioma cells, but the mechanisms are not fully understood.Thioredoxin-1(TRX1) is a redox-sensitive oxidoreductase, which plays critical roles inDNA damage signal transduction via nuclear translocation in irradiated cells. Becausethe TRX1-dependent DNAdamage signaling pathway relies on NADPH to maintain thereduced state of TRX1, and TIGAR functions to increase NADPH generation underoxidative stress, in this study, the role of TRX1in TIGAR abrogation-inducedradiosensitization was investigated. Primary astrocytes were used in order to elaboratethe functions of TIGAR in irradiated normal human glial cells. Finally, human xenograftorthotopic models were used to prove whether TIGAR interference could radiosensitizeglioma in vivo.
Methods: Protein levels of TIGAR and p53in glioma cells being irradiated weredetermined with Western blot analysis. Real-time PCR revealed the transcriptionalactivity of TIGAR and TP53at different points in glioma cells suffered by ionizingradiation. TIGAR siRNA and pcDNA3.1-TIGAR were synthesized and transfected intoglioma cells to make TIGAR abrogated or over-expressed. Forty-eight hourspost-transfection, glioma cells were irradiated and the reactive oxygen species (ROS)was determined by flow cytometry. Glioma cells over-expressing wild-type (WT) ormutant-type (MT) TRX1were estabolished by transfecting pcDNA3.1-WT-TRX1orpcDNA3.1-MT-TRX1and by G418selection. To evaluate the radiosensitivity of gliomacells suffer by TIGAR interference, clonogenic assay was performed to examine the cellsurvival fractions at14days post-IR, and immunofluoresence assay was carried out toquantify γ-H2AX foci in0.5-12h post-IR. To elaborate the mechanism of TIGARknockdown-induced radiosensitization of glioma cells, IR-induced TRX1nucleartranslocation was determined by Western blot. Redox Western blot indicated the redox state of cytoplasmic and nuclear TRX1in irradiated glioma cells. Finally, by usingTIGAR shRNA lentivirus-based gene therapy, TIGAR abrogation-inducedradiosensitization of glioma in vivo was illustrated by MRI.
Results:(1) In wild-type p53-expressing A172cells TIGAR expression wasincreased1h post-IR and decreased to basal level8h post-IR. Meanwhile, p53expression was increased within0.5h post-IR, which was earlier than TIGARupregulation. However, in mutant-type p53-expressing (M237I) T98G cells, thereseemed to be no IR-induced increase in p53and TIGAR expression. Clonogenic assayrevealed that the survival fractions of both A172and T98G cells treated with TIGARsiRNAs were significantly lower than that of parental cells.(2) ROS generation wassignificantly increased by TIGAR abrogation in irradiated glioma cells. Meanwhile,TIGAR interference reduced by approximately75%cellular NADPH in irradiatedglioma cells, compared with a reduction of less than40%in cells irradiated alone.Similarly, the ratio of GSH/GSSG was significantly further decreased by TIGARinterference in cells exposed to IR. In addition, both NADPH level and the ratio ofGSH/GSSG rebounded by TIGAR overexpression. Western blot analysis proved thatTRX1was imported into nuclei in irradiated cells. The peak time of TRX1nucleartranslocation was approximately2h post-IR. However, IR-induced nucleartranslocation of TRX1was strikingly hindered by TIGAR interference.(3) Interestingly,TIGAR silencing-induced blockage of TRX1nuclear translocation could be abolishedby WT-TRX1overexpression. Redox Western blot indicated that in irradiated A172cells, the levels of cytoplasmic oxidized TRX1were raised within0.5h post-IR andreturned to their original reducing state within8h post-IR. TIGAR knockdown coulddelay the reduction process of oxidized cytoplasmic TRX1. Cytoplasmic TRX1remained oxidized at8h post-IR by TIGAR interference. However, WT-TRX1overexpression preserved the reducing state of TRX1in cells treated with TIGARsiRNA. The alteration in redox state of nuclear TRX1in response to IR showed thesame pattern as that of cytoplasmic TRX1. Clonogenic survival assay determined thatTIGAR interference-induced radiosensitization of glioma cells was notably diminishedby WT-TRX1overexpression. Data also revealed that the combination of MT-TRX1overexpression and TIGAR interference could not further radiosensitize glioma cellsany more. Importantly, TIGAR overexpression also could not rescue MT-TRX1overexpression-induced radiosensitization, indicating that TRX1nuclear translocation might play a major role in TIGAR-regulated radiosensitivity of glioma cells.(4) Byusing immunofluoresence assay, it was shown that γ-H2AX foci disappeared within4hpost-IR in control A172cells. However, the γ-H2AX foci remained even at12h post-IRin TIGAR low-expressing cells, indicating the delayed DNA damage repair (DDR)process. The nuclear translocation of TRX1was observed with a peak time of2hpost-IR in cells overexpressing scramble siRNA. However, in TIGAR-silenced A172cells, there seemed to be no TRX1transported into the nuclei. InWT-TRX1-overexpressing A172cells, both TIGAR-silencing-induced delay of theDDR process and blockage of TRX1nuclear translocation were abolished.(5)Clonogenic survival assay showed that there was no difference in radiosensitivityamong primary astrocytes over-expressing scramble siRNA, pcDNA3.1, TIGAR siRNA,and pcDNA3.1-TIGAR. Although IR-induced TRX1nuclear translocation was observedin primary astrocytes, Western blot assay showed that neither IR-induced TRX1nucleartranslocation nor the reduction process of oxidized TRX1was hindered by TIGARknockdown in primary astrocytes. Data also showed that in primary astrocytes theoxidative levels of both cytoplasmic and nuclear TRX1induced by IR wereconsiderably lower than those in glioma cells.(6) By using TIGAR shRNAlentivirus-based gene therapy, it was proven TIGAR interference could radiosensitizeglioma in vivo.
Conclusion: The present study demonstrated that TIGAR knockdown significantlynot only inhibits IR-induced nuclear translocation of TRX1in glioma cells butconsiderably defers the reduction process of oxidized TRX1in irradiated glioma cells.Both the blockage of TRX1nuclear translocation and radiosensitization induced byTIGAR interfering are abolished by wild-type TRX1overexpression. TIGARabrogation considerably radiosensitizes glioma cells but has no effect on radiosensitivityof primary astrocytes. Finally, TIGAR interference might radiosensitize glioma in vivo.
引文
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