慢病毒介导miR-34a表达对视网膜母细胞瘤自噬的影响及机制研究

Effect of lentivirus mediated miR-34a expression on autophagy in retinoblastoma and its mechanism

目的探讨慢病毒介导miR-34a表达对视网膜母细胞瘤自噬的影响并对其机制进行研究。方法取对数生长期的Y79细胞,将转染miR-34a的慢病毒载体、转染高迁移率族蛋白1(high mobility group protein 1,HMGB1)载体、共转染miR-34a+HMGB1的慢病毒载体及培养液接种至Y79细胞构建转染miR-34a细胞系。按照接种载体分组,将Y79细胞株分为4组,分别为转染miR-34a组、转染HMGB1组、共转染miR-34a+HMGB1组及阴性对照组。用qRT-PCR检测miR-34a的表达,采用Capase3检测试剂盒检测Caspase3活性。采用流式细胞仪及单丹磺酰尸胺(monodansyl cadaverin,MDC)试剂盒在488 nm荧光下检测MDC荧光率,利用透射电子显微镜观察自噬超微结构。结果 miR-34a转染结果显示,转染miR-34a组miR-34a的相对表达量(2. 04±0. 46)显著高于阴性对照组(1. 03±0. 21)(P <0. 05),共转染miR-34a+HMGB1组的HMGB1的相对表达量(0. 42±0. 08)显著低于转染HMGB1组(1. 08±0. 16),差异有统计学意义(P <0. 05)。Caspase3活性测试显示,转染miR-34a组Caspase3活性(10. 75±2. 87)明显高于阴性对照组(3. 48±0. 74),转染HMGB1组Caspase3活性(2. 46±0. 94)低于共转染miR-34a+HMGB1组(6. 21±1. 74),差异有统计学意义(P <0. 05)。流式细胞仪检测结果显示,转染miR-34a组MDC阳性率(56. 94%)明显高于阴性对照组(2. 15%),共转染miR-34a+HMGB1组的MDC阳性率(43. 11%)明显高于转染HMGB1组(10. 32%)(P <0. 05)。透射电子显微镜观察自噬超微结构显示,阴性对照组无明显改变,共转染miR-34a+HMGB1组可见Y79细胞双层膜结构,转染miR-34a组可见自噬溶酶体结构。结论 miR-34a促进视网膜母细胞瘤的自噬凋亡,其机制可能为抑制HMGB1的表达。

Objective To investigate the effect of lentivirus mediated miR-34a expression on autophagy in retinoblastoma and study its mechanism.M ethods The logarithmic growth stage of Y79 cells were cultured and transfected with miR-34a vector,transfected with HMGB1 vector,transfected with miR-34a + HMGB1 vector to construct transfected miR-34a cell line.According to the inoculation vector group,Y79 cell lines were divided into 4 groups,which were miR-34a transfected group,HMGB1 transfected group,miR-34a + HMGB1 co-transfected group and negative control group.The expression of miR-34a was detected by qRT-PCR.Caspase 3 activity was measured using Capase 3 Assay Kits.The MDC fluorescence rate was measured by flow cytometry and monodansyl cadaverine( MDC) kit at 488 nm fluorescence.The autophagy ultrastructure was observed by transmission electron microscopy.Results Transfection results were showed that the relative expression of miR-34a in miR-34a transfected group( 2.04 ± 0.46) was significantly higher than that in negative control group( 1.03 ±0.21)( P < 0.05).The relative expression of HMGB1 in miR-34a + HMGB1 cotransfected group( 0.42 ± 0.08) was significantly lower than that in HMGB1 transfected group( 1.08 ± 0.16),The difference was statistically significant( P < 0.05).The Caspase3 activity test was showed that the activity of Caspase3 in the miR-34a transfected group was significantly higher than that of the negative control group,and the Caspase3 activity in the HMGB1 transfected group was lower than that of the miR-34a+ HMGB1 co-transfected group,and the difference was statistically significant( P < 0.05).The flow cytometry showed that the positive rate of MDC in miR-34a transfected group( 56.94 %) was significantly higher than that in the negative control group( 2.15 %),and the positive rate of MDC in the miR-34a + HMGB1 co-transfected group( 43.11 %) was significantly higher than that of the HMGB1 transfected group( 10.32 %)( P < 0.05).The ultrastructure of autophagy was observed by transmission electron microscopy,there was no obvious change in the negative control group,and the double membrane structure was found in the miR-34a + HMGB1 co-transfected group,as well as the structure of autophagosome lysosome was found in the miR-34a transfected group.Conclusion miR-34a promotes the autophagy and apoptosis of retinoblastoma,and its mechanism may be related to inhibit the expression of HMGB1.