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    下调Yes相关蛋白对胃癌细胞克隆形成能力和能量代谢的影响

    Effect of the down-regulation of Yes-associated protein on the cloning ability and energy metabolism of gastric cancer cells

      摘要
    • 摘要:
      目的研究下调Yes相关蛋白（Yes-associated protein，YAP）对胃癌细胞克隆形成能力及能量代谢的影响。
      方法胃癌细胞BGC823转染YAP小干扰RNA（YAP siRNA）和小干扰RNA阴性对照（siRNA-NC），同时以不做转染的细胞为对照（Con），qRT-PCR测定细胞中YAP mRNA水平，Western blotting测定细胞中YAP蛋白水平，噻唑蓝（MTT）测定细胞增殖活性，平板克隆实验测定细胞克隆形成能力，试剂盒测定细胞中三磷酸腺苷（ATP）水平及培养液上清中乳酸含量，Western blotting测定能量代谢相关蛋白己糖激酶2（HK2）、丙酮酸激酶M2亚型（PKM2）和活化的含半胱氨酸的天冬氨酸蛋白水解酶3（Cleaved Caspase-3）的水平。
      结果YAP siRNA细胞中YAP mRNA和蛋白水平均明显低于Con（P < 0.05），siRNA-NC细胞中YAP mRNA和蛋白水平与Con差异均无统计学意义（P>0.05）。YAP siRNA细胞增殖活性、克隆形成率明显低于Con（P < 0.05），细胞凋亡率明显高于Con（P < 0.05），细胞中ATP水平低于Con（P < 0.05），上清中乳酸含量低于Con（P < 0.05），细胞中HK2、PKM2蛋白水平也低于Con（P < 0.05），而Cleaved Caspase-3水平高于Con（P < 0.05）。
      结论下调YAP可以降低胃癌细胞克隆形成能力和增殖活性，诱导Caspase-3介导的胃癌细胞凋亡，干扰糖酵解关键酶HK2、PKM2的表达，降低细胞ATP水平，抑制胃癌细胞糖酵解。

       

      Abstract:
      ObjectiveTo investigate the effects of the down-regulation of Yes-associated protein (YAP) on the cloning ability and energy metabolism of gastric cancer cells.
      MethodsThe YAP small interfering RNA (YAP siRNA) and negative small interfering RNA (siRNA-NC) were transfected into the gastric cancer cell line BGC823, respectively, and the cells without transfection was set as the control (con).The levels of mRNA and protein level of YAP in cells were detected using the qRT-PCR and western blotting, respectively.The cell proliferation activity and cloning ability were determined using the MTT and plate cloning assay, respectively.The level of ATP in cells and content of lactic acid in supernatant of culture medium were determined by kit.The levels of related energy metabolism proteins HK2, PKM2 and apoptotic protein cleaved caspase-3 were determined using western blotting.
      ResultsThe levels of YAP mRNA and protein in YAP siRNA cells were significantly lower than those in Con cells (P < 0.05), and the differences of the levels of YAP mRNA and protein between siRNA-NC and Con cells were not statistically significant (P>0.05).The cell proliferation and colony formation rate in YAP siRNA cells were significantly lower than those in Con cells (P < 0.05), and the apoptosis rate in YAP siRNA cells was significantly higher than that in Con cells (P < 0.05).The level of ATP in cells, content of lactic acid in supernatant and levels of HK2 and PKM2 protein in YAP siRNA cells were lower than those in Con cells (P < 0.05), and the level of cleaved caspase-3 in YAP siRNA cells was higher than that in Con cells (P < 0.05).
      ConclusionsThe down regulation of YAP can reduce the abilities of clone formation and proliferation, induce the caspase-3 mediated apoptosis, interfer the expression of key enzymes HK2 and PKM2, decrease the level of ATP and inhibit the glycolysis in gastric cancer cells.

       

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