冬凌草甲素通过调控LncRNA-H19抑制急性髓系白血病细胞增殖和促细胞凋亡研究

Research on oridonin inhibiting cell proliferation and promoting apoptosis of acute myeloid leukemia by regulating LncRNA-H19

目的探讨冬凌草甲素对急性髓系白血病HL-60细胞增殖、凋亡和自噬的影响并明确作用机制是否与调控LncRNA-H19表达相关。方法体外常规培养HL-60细胞,取处于对数生长期的细胞进行实验,观察不同浓度冬凌草甲素(5.0、10.0、15.0、20.0、25.0µmol/L)对HL-60细胞增殖、凋亡和LncRNA-H19 mRNA、相关蛋白表达的影响以及LncRNA-H19 siRNA对冬凌草甲素的干预作用。采用实时定量PCR法检测细胞中LncRNA-H19表达,CCK-8法检测细胞增殖,流式细胞术法检测细胞凋亡,Western Blot法检测Caspase-3、Bax、Bcl-2、LC3-Ⅱ、Beclin-1蛋白表达。结果与Control组比较,5.0~25.0µmol/L的冬凌草甲素呈浓度依赖性地抑制HL-60细胞增殖,诱导细胞凋亡,促进细胞中Caspase-3、Bax、LC3-Ⅱ、Beclin-1蛋白表达,抑制细胞中Bcl-2蛋白表达以及LncRNA-H19 mRNA表达。与si-NC组比较,si-LncRNA-H19组细胞增殖率明显降低,凋亡率明显增加,Caspase-3、Bax、LC3-Ⅱ、Beclin-1蛋白表达明显上调,Bcl-2蛋白表达明显下调,差异有统计学意义(P<0.05或P<0.01)。结论冬凌草甲素通过抑制LncRNA-H19的表达而阻止HL-60细胞增殖,促进细胞凋亡和自噬。

Objective To investigate the impacts of oridonin on proliferation,apoptosis and autophagy of HL-60 cells in acute myeloid leukemia and to determine whether the mechanism of action is related to the regulation of LncRNA-H19 expression.Methods HL-60 cells were routinely cultured in vitro,and cells in logarithmic growth phase were taken for experiments to observe the impacts of oridonin with different concentrations(5.0,10.0,15.0,20.0,25.0µmol/L)on HL-60 cell proliferation,apoptosis,LncRNA-H19 mRNA and related protein expression,and the intervention functions of LncRNA-H19 siRNA on oridonin.The expression of LncRNA-H19 in cells was detected by real-time quantitative PCR,cell proliferation was detected by CCK-8,apoptosis was detected by flow cytometry,and the expressions of Caspase-3,Bax,Bcl-2,LC3-Ⅱ,Beclin-1 protein were detected by Western Blot.Results Compared with the control group,oridonin of 5.0-25.0µmol/L inhibited HL-60 cell proliferation,induced cell apoptosis,promoted Caspase-3,Bax,LC3-Ⅱ,Beclin-1 protein expressions,inhibited Bcl-2 protein expression and LncRNA-H19 mRNA expression in cells in a concentration-dependent manner.Compared with the si-NC group,the cell proliferation rate in the si-LncRNA-H19 group significantly reduced,and apoptosis rate significantly increased.Caspase-3,Bax,LC3-Ⅱ,Beclin-1 protein expressions were significantly up-regulated,Bcl-2 protein expression was significantly down-regulated,and the differences were statistically significant(P<0.05 or P<0.01).Conclusion Oridonin inhibits proliferation of HL-60 cells and promotes apoptosis and autophagy by inhibiting the expression of LncRNA-H19.