沉默YAP通过Wnt/β-catenin信号通路影响胃癌BGC823细胞的凋亡

Effect of Yes-associated Protein Knockdown on Apoptosis of Gastric Cancer BGC823 Cells Through Wnt/β-catenin Signaling Pathway

目的本研究探讨Yes相关蛋白(YAP)对胃癌BGC823细胞凋亡的影响。方法 BGC823细胞中转染YAP小干扰RNA(YAP siRNA1、YAP siRNA2),用qRT-PCR和蛋白质印迹法分别测定细胞中YAP表达,筛选干扰效果较好的YAP siRNA1做后续实验。MTT测定细胞增殖,平板克隆实验测定克隆形成能力,流式细胞术测定凋亡情况,分光光度法检测Caspase-3、Caspase-9活性,蛋白质印迹法检测β-catenin、c-myc、CyclinD1蛋白水平。用Wnt/β-catenin的激活剂处理下调YAP后的BGC823细胞,按照上述方法测定细胞增殖凋亡和克隆形成能力。结果 YAP siRNA1、YAP siRNA2下调BGC823细胞中YAP的转录和表达,YAP siRNA1对YAP表达的干扰效率较好。敲低YAP后的细胞增殖能力、克隆形成能力降低,细胞凋亡率由(6.32±0.58)%升高至(32.71±2.64)%,Caspase-3、Caspase-9活性升高,β-catenin、c-myc、cyclinD1蛋白表达减少,与正常培养的BGC823细胞相比,差异有统计学意义(P<0.05)。Wnt/β-catenin的激活剂处理可以部分逆转敲低YAP对BGC823细胞的凋亡、增殖、克隆形成能力和细胞中Caspase-3、Caspase-9活性的影响。结论 YAP敲低诱导胃癌细胞凋亡,且作用机制与抑制Wnt/β-catenin通路激活有关。

Objective To explore the effect of Yes-associated protein(YAP)on the apoptosis of gastric cancer BGC823 cells and related mechanism.Methods YAP small interference RNA(YAP siRNA1,YAP siRNA2)were transfected into BGC823 cells.qRT-PCR and Western blot were used to determine the expression of YAP,respectively.YAP siRNA1,which had better interference effect,was selected for the follow-up experiment.MTT,plate clone formation assay and flow cytometry were applied to determine cells proliferation,cloning ability and apoptosis,respectively.The activities of Caspase-3 and Caspase-9 were detected by spectrophotometry.Western blot was applied to detectβ-catenin,c-myc and CyclinD1 protein levels.BGC823 cells of down-regulated YAP were treated with Wnt/β-catenin activator.The cell proliferation,apoptosis and clonogenic ability were determined.Results YAP siRNA1 and YAP siRNA2 downregulated the transcription and expression of YAP in BGC823 cells.The interference efficiency of YAP siRNA1 was better.After knocking down YAP,the abilities of cell proliferation and clone formation were decreased;the apoptosis rate was increased from(6.32±0.58)%to(32.71±2.64)%;the activities of Caspase-3 and Caspase-9 were increased;the expression ofβ-catenin,c-myc and cyclinD1 protein were decreased,with statistically significant difference compared with normal BGC823 cells(P<0.05).The activator treatment of Wnt/β-catenin could partly reverse the effect of YAP knockdown on the apoptosis,proliferation,cloning formation of BGC823 cells and the activities of Caspase-3 and Caspase-9.Conclusion YAP knockdown induces the apoptosis of gastric cancer cells,and the mechanism is related to the inhibition of Wnt/β-catenin pathway activation.