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
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摘要
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.
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.
引文
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[2]BARRETT A,HERMANN G J.A caenorhabditis elegans homologue of LYST functions in endosome and lysosome-related organelle biogenesis[J].Traffic,2016,17(5):515-535.
[3]LI J,HU S B,WANG L Y,ZHANG X,ZHOU X,YANG B,et al.Autophagy-dependent generation of Axin2+cancer stem-like cells promotes hepatocarcinogenesis in liver cirrhosis[J].Oncogene,2017,36(48):6725-6737.
[4]CARCHMAN E H,MATKOWSKYJ K A,MESKE L,LAMBERT PF.Dysregulation of autophagy contributes to anal carcinogenesis[J].PLoS One,2016,11(10):e0164273.
[5]GAO Z H,JIN P N,CHEN X X,LI H Y,WANG Y,CHEN Z,et al.Significance of dynamic changes in high mobility group box-1 and epithelial neutrophil activating peptide-78 in cerebrospinal fluid and serum of children with purulent meningitis[J].J Appl Clin Pediatr,2016,31(12):949-950.禚志红,靳培娜,陈晓昕,李海英,王越,陈铮,等.细菌性脑膜炎患儿脑脊液及血清高迁移率族蛋白-1、中性粒细胞活化肽-78动态变化的意义[J].中华实用儿科临床杂志,2016,31(12):949-950.
[6]ALBULESCU R,NEAGU M,AIBULESCU L,TANASE C.Tissular and soluble miRNAs for diagnostic and therapy improvement in digestive tract cancers[J].Expert Rev Mol Diagn,2011,11(1):101-120.
[7]ZHANG J.Effect of oxaliplatin on human retinoblastoma cell proliferation and apoptosis[J].J Xinxiang Med Univ,2015,32(11):989-991.张坚.奥沙利铂对人视网膜母细胞瘤细胞增殖及凋亡的影响[J].新乡医学院学报,2015,32(11):989-991.
[8]HAJI MOHD YASIN N A,GRAY A R,BEVIN T H,KELLY L E,MOITENO A C.Choroidal melanoma treated with stereotactic fractionated radiotherapy and prophylactic intravitreal bevacizumab:The Dunedin Hospital experience[J].J Med Imaging Radiat Oncol,2016,60(6):756-763.
[9]ZAKARIA O M,DAOUD M Y,FARRAG S H,MULHIM A S.Efficacy of different protocols in treatment of nephroblastoma:A revisit[J].Gulf J Oncolog,2016,1(21):55-60.
[10]JI Q S,YU Q,SUN S Q,KE G J,WEN Y C.Expression of miR-34a/SIRT1 in human lens epithelial cells during H2O2-induced oxidative stress[J].Rec Adv Ophthalmol,2017,37(8):728-731.季青山,俞茜,孙思勤,柯根杰,温跃春.miR-34a/SIRT1在H2O2诱导人晶状体上皮细胞氧化应激中的表达[J].眼科新进展,2017,37(8):728-731.
[11]LOU G,LIU Y,WU S,XUE J,YANG F,FU H,et al.The p53/miR-34a/SIRT1 positive feedback loop in quercetin-induced apoptosis[J].Cell Physiol Biochem,2015,35(6):2192-2202.
[12]CHENG Y,WANG D,WANG B,LI H,XIONG J,XU S,et al.HMGB1 translocation and release mediate cigarette smoke-induced pulmonary inflammation in mice through a TLR4/My D88-dependent signaling pathway[J].Mol Biol Cell,2016,28(1):201-209.
[13]LIAO H,XIAO Y,HU Y,XIAO Y,YIN Z,LIU L.Suppression of cellular proliferation and invasion by HMGB1 knockdown in bladder urothelial carcinoma cells[J].Oncol Res,2014,22(5-6):235-245.
[14]WANG S S,CHEN Y H,CHEN N,WANG L J,CHEN D X,WENG H L,et al.Hydrogen sulfide promotes autophagy of hepatocellular carcinoma cells through the PI3K/Akt/m TOR signaling pathway[J].Cell Death Dis,2017,8(3):e2688.
[15]YANG R,OUYANG Y,LI W,WANG P,DENG H,SONG B.Autophagy plays a protective role in tumor necrosis factor-α-induced apoptosis of bone marrow-derived mesenchymal stem cells[J].Stem Cells Dev,2016,25(10):788-797.
[16]DU H,CHE J,SHI M,ZHU L,HANG J B,CHEN Z,et al.Beclin1 expression is associated with the occurrence and development of esophageal squamous cell carcinoma[J].Oncol Lett,2017,14(6):6823-6828.
[17]ADAMS B D,WALI V B,CHENG C J,INUKAI S,BOOTH C J,AGAWAL S,et al.miR-34a silences c-SRC to attenuate tumor growth in triple-negative breast cancer[J].Cancer Res,2016,76(4):927-939.
[18]GAUR S,WEN Y,SONG J H,PARIKH N U,MANGALA L S,BLESSING A M,et al.Chitosan nanoparticle-mediated delivery of miRNA-34a decreases prostate tumor growth in the bone and its expression induces non-canonical autophagy[J].Oncotarget,2015,6(30):29161-29177.
[19]WEN X R,TANG M,QI D S,HUANG X J,LIU H Z,ZHANG F,et al..Butylphthalide suppresses neuronal cells apoptosis and inhibits JNK-Caspase3 signaling pathway after braini schemia/reperfusion in rats[J].Cell Mol Neurobiol,2016,36(7):1087-1095.
[20]WU H M,SHAO L J,JIANG Z F,LIU R Y.Gemcitabine-induced autophagy protects human lung cancer cells from apoptotic death[J].Lung,2016,194(6):959-966.
[21]XUE E,ZHANG Y,SONG B,XIAO J,SHI Z.Effect of autophagy induced by dexamethasone on senescence in chondrocytes[J].Mol Med Rep,2016,14(4):3037-3044.