【作者】 刘可；

【导师】 段宣初；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 目的：探讨miR-34a能否通过HMGB1调控视网膜母细胞瘤细胞的凋亡及自噬,并增加DNA的损伤以提高化疗药物对视网膜母细胞瘤的治疗效果。方法：培养人视网膜母细胞瘤细胞Y79及Weri-RB1后分四部分完成本实验研究。1.miR-34a调节RB细胞HMGBl的表达：Y79及Weri-RB-1细胞转染miR-34a mimic或antagomir-34a后,qRT-PCR及Western-blotting分别检测HMGB1mRNA及蛋白表达水平改变,萤光素酶检测分析HMGB13’UTR报告基因活性改变。2.miR-34a通过调节HMGBl诱导凋亡并抑制自噬：Y79细胞转染miR-34a mimic、HMGB1shRNA或同时转染miR-34a和pUNO1-HMGB1cDNA72小时后Caspase3试剂盒及Annexin V/FITC试剂盒检测细胞凋亡及Caspase3活性,Western-blotting分析cleaved-cap3及cleaved PARP蛋白水平改变。Y79细胞转染miR-34a mimic或]HMGB1shRNA72小时后HBSS饥饿培养1小时,Western-blotting检测LC3、p62及HMGB1蛋白水平改变,免疫荧光观察并计数LC3点状聚集改变,透射电镜观察并计数自噬空泡数量改变。3.miR-34a通过抑制自噬增强RB细胞对化疗的敏感性：Y79细胞经长春新碱、依托泊苷或卡铂处理48小时后Western-blotting检测LC3及p62的蛋白水平。Y79细胞分别转染1miR-34a mimic、HMGB1shRNA、Beclin shRNA及Atg5shRNA72小时后长春新碱、依托泊苷或卡铂处理48小时,计数LC3点状聚集数并检测细胞生存能力。4.miR-34a通过抑制自噬增加RB细胞化疗后DNA的损伤：Y79细胞分别转染miR-34a mimic、HMGB1shRNA及Atg5shRNA后依托泊苷或卡铂处理48小时,Western-blotting检测γ-H2AX蛋白水平,Annexin V/FITC凋亡试剂盒检测凋亡水平改变。在Atg5基因干扰的细胞加入NAC再经依托泊苷或卡铂处理48小时,Western-blotting观察γ-H2AX蛋白及凋亡水平改变。结果：Y79细胞和Weri-RB1细胞在转染miR-34a mimic后HMGB1的mRNA水平、蛋白表达及3’UTR报告基因活性均下降,相反抑制miR-34a后HMGB1的mRNA水平、蛋白表达及3’UTR报告基因活性提高。转染mir-34a mimic或HMGB1shRNA后,Y79细胞的凋亡水平及Caspase3活性均明显提高,反映蛋白裂解水平的cleaved Cap3及cleaved PARP表达也增加。而通过转染pUNO1-HMGB1促使HMGB1过表达可以抑制由miR-34a mimic诱导的凋亡、Caspase3活性及cleaved PARP表达。HBSS饥饿培养Y79细胞后HMGB1的mRNA在1-2小时内增加,后逐渐下降至基线水平,而miR-34a一直处于增加状态。通过增加miR-34a或减少HMGB1能抑制由饥饿诱导的LC3II表达及LC3点状聚集,而使用溶酶体蛋白酶抑制剂E/P后可以恢复miR-34a mimic转染细胞内的LC3Ⅱ的表达水平。超微结构检查发现miR-34a mimic可以减少由饥饿产生的自噬空泡。Y79细胞在经过长春新碱、依托泊苷或卡铂治疗后,LC3Ⅱ表达水平增加而p62下降。通过观察LC3点状聚集可以发现miR-34a mimic及HMGB1shRNA抑制了由长春新碱、依托泊苷或卡铂诱导的自噬,但增加了化疗药物对RB的作用效果。通过抑制自噬重要的调节因子Beclin1和Atg5也可以达到增加化疗效果的作用。Y79细胞经依托泊苷或卡铂治疗后可以反映DNA损伤的γ-H2AX蛋白表达升高。miR-34a mimic、HMGB1shRNA及Atg5shRNA均可增加由化疗诱导的凋亡及γ-H2AX表达水平。而抗氧化剂NAC可以减少由依托泊苷或卡铂诱导的DNA损伤。结论：HMGB1是miR-34a在RB细胞内的作用目标之一。miR-34a可以减少]HMGB1的mRNA水平以促进RB细胞的凋亡并抑制自噬,通过增加细胞DNA的损伤来提高化疗药物对RB细胞的治疗效果,为临床治疗RB提供新的思路及方法。更多还原

【Abstract】 Purpose:To emphasize the mechanisms governing the regulation of HMGB1expression by microRNA, and their possible contribution to autophagy and drug resistance.Methods:HMGB1is a target of miR-34a in human retinoblastoma cells: After transfected by miR-34a mimic or antagomir-34a, pRT-PCR, Western-blotting and luciferase assays were perfomed on Y79and Weri-RB1cell lines.miR-34a induces apoptosis by modifying HMGB1expression: Y79cells were transfected with miR-34a mimic, HMGB1shRNA or pUNO1-HMGB1by72hours, apoptosis and Caspase3activities were detected, expression of cleaved-cap3and cleaved PARP was also evaluated by Western-blotting.Transfected by miR-34a mimic or HMGB1shRNA72for72hours, Y79cells were cultured in HBSS for1hour. Protein expression of LC3, p62and HMGB1were evaluated by Western-blotting, LC3puncta was analysed by immunofluorescence.Moreover, ultrastructural electron microscopy analysis was performed to detect autophagic vesicles.Inhibition of autophagy enhances chemotherapeutic efficacy in retinoblastoma cells:After treated by VCR, ETO and CBP, expression of LC3and p62was detected by Western-blotting. Cells tranfected with miR-34a mimic,MGB1shRNA,Beclin shRNA and Atg5shRNA were treated by VCR, ETO and CBP for48hours, then LC3puncta and cell viability were monitored.Inhibition of autophagy enhances DNA damage in chemotherapy:After transfected by miR-34a mimic,HMGB1shRNA or Atg5shRNA, Y79cells were treated with ETO and CBP for48hours,y-H2AX expression and apoptosis activity were evaluated.Results:The mRNA levels of HMGB1decreased following miR-34a mimic treatment, whereas antagomir-34a increased HMGB1expression. miR-34a mimic inhibited HMGB1luciferase activities, whereas antagomir-34a increased HMGB1luciferase activities in Y79cells.Consistently, the protein levels of HMGB1decreased following miR-30mimic treatment, whereas antagomir-34a increased HMGB1protein expression in Y79cellsSuppression of HMGB1expression promotes apoptosis by flow cytometric analysis of the percentage of the cells that are Annexin V-positive.Knockdown of HMGB1or miR-34a mimic increased activity of caspase3, cleaved-caspase3, and cleaved-PARP.Furthermore, overexpression of HMGB1by transfection with pUNO1-HMGB1inhibited miR-34a mimic-induced apoptosis, caspase3activity, and cleaved-PARP.Starvation increased mRNA expression of HMGB1at one to two hours, and returned to the baseline levels at six hours when miR-34a expression was increased in the retinoblastoma cell. miR-34a mimic or knockdown of HMGB1inhibited starvation-induced LC3-II expression and LC3puncta number.In contrast, treatment with lysosomal protease inhibitorsE/Prestored LC3-II expression in miR-34a-transfection cells.Moreover, ultrastructural electron microscopy analysis revealed that miR-34a mimic decreased the number of autophagic vesicles after starvation.Vincristine (VCR), etoposide (ETO), and carboplatin (CBP)increased LC3-II expression and decreased p62expression in retinoblastoma cells. miR-34a mimic and knockdown of HMGB1significantly decreased VCR, ETO, and CBP-induced autophagy by monitoring LC3puncta, but enhanced chemotherapeutic efficacy in retinoblastoma cells.Inhibition of autophagy by knockdown of beclin1and ATG5also increased chemotherapeutic efficacy in retinoblastoma cells.Exposure of Y79cells to ETO and CBP resulted in the phosphorylation of histone H2A at Ser139(y-H2AX), a marker for DNA damage. miR-34a mimic, knockdown of HMGB1, and knockdown of ATG5increased chemotherapy drug-induced γ-H2AX expression.miR-34a mimic, knockdown of HMGB1, and knockdown of ATG5increased chemotherapy-induced apoptosis. Moreover, the antioxidant NAC attenuated ETO-and CBP-induced DNA damage and apoptosis in ATG5knockdown cells.Conclusions:miR-34a targets HMGB1mRNA, leading to translational repressionof HMGB1. miR-34a is a potent inhibitor of autophagy, which promotes chemotherapeutic agent-induced DNA damage and apoptosis.更多还原