siRNA沉默Livin基因对胶质瘤TJ905干细胞多重耐药蛋白1和3表达的影响

摘要

目的 探讨siRNA沉默Livin基因后,替莫唑胺干预胶质瘤TJ905干细胞对多重耐药蛋白(MRP1、MRP3)基因表达的影响,揭示Livin基因与化疗耐药之间的关系.方法 分离培养胶质瘤TJ905干细胞,RNA干扰技术构建靶向Livin-siRNA,通过慢病毒转染胶质瘤TJ905干细胞建立实验组,另设空病毒转染建立对照组,用不同浓度(0、25、50、100、200、400μmol/L)的替莫唑胺干预胶质瘤TJ905干细胞72h,细胞增殖实验检测细胞增殖变化趋势,流式细胞技术检测细胞周期变化,实时荧光定量PCR检测Livin、MRP1、MRP3基因表达情况.结果 成功构建靶向Livin-siRNA模型,实验组TJ905干细胞增殖受到明显抑制,存活率显著低于对照组(P<0.05);使用不同浓度的替莫唑胺干预TJ905干细胞,对照组和实验组的细胞周期均停滞于G0/G1期,且实验组更加明显(P<0.05);与对照组相比较,实验组TJ905干细胞Livin基因表达较对照组下降(P<0.05);MRP1基因表达量下降(F=195.975,P<0.05)以及MRP3基因表达量下降(F=719.521,P<0.05).结论 TJ905干细胞MRP1、 MRP3高表达可能与Livin基因密切相关,靶向siRNA沉默Livin基因可有效降低MRP1、MRP3基因的表达从而逆转胶质瘤TJ905干细胞对化疗药物的耐药性,提示Livin基因可能在化疗耐药机制中发挥重要作用.%Objective To explore the effects of small interference RNA(siRNA) targeting Livin on genes of MRP1 and MRP3 in TJ905 stem cells,and reveal the relationship between Livin and chemotherapy resistance.Methods CD133+ stem-like cells were isolated by immunomagnetic beads from TJ905 cells.RNA interference technology was used to construct the siRNA targeting Livin (Livin-siRNA),and the experimental group was established by lentivirus transfected glioma TJ905 stem cells.The control group was established by empty virus transfection.Cell counting kit-8 assay,flow cytometry, realtime fluoresence quantitative PCR technology were used to detect and analyze the TJ905 stem cell proliferation and cell cycle and investigate the mRNA expression levels of Livin,MRP1 and MRP3.Results Targeting Livin-siRNA model was successfully constructed.The survival rate of TJ905 stem cells was significantly lower than that of the control group (P<0.05).The cell cycle of the control group and the experimental group were stopped by using different concentrations of temozolomide for glioma stem cells,and the experimented group was more obvious (P<0.05).Compared with the control group,the expression of Livin gene in TJ905 stem cells was significantly lower than that in the control group (P<0.05),and the expression of multiple drug resistant MRP1 gene (F=195.975,P<0.05) and the expression of MRP3 gene was significantly decreased (F=719.521,P<0.05).Conclusion Livin gene may be closely related to the high expression of MRP1 and MRP3 genes.Livin-siRNA can effectively reduce the expression of MRP1 and MRP3 to reverse the drug resistance of glioma TJ905 stem cells to chemotherapeutic drugs.It is suggested that Livin may play an important role in the mechanism of chemotherapy resistance.