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目的探讨磷脂酰肌醇蛋白多糖-3(GPC3)对Hippo信号通路的调控机制及其对人肝癌Huh7细胞生物学行为的影响。方法分别构建4种靶向GPC3和YAP1基因的sh RNA序列,然后转染入肝癌Huh7细胞,并筛选稳定表达的细胞株。采用荧光定量PCR法和蛋白印迹法检测转染后的Huh7细胞中GPC3和YAP1的表达,以筛选有效沉默GPC3和YAP1的sh RNA。观察沉默GPC3和YAP1以及人重组YAP1(rh YAP1)对肝癌细胞增殖、侵袭和凋亡的影响。GPC3 sh RNA转染实验分为6组:未转染组、空载体组、GPC3-714-sh RNA组、GPC3-647-sh RNA组、GPC3-1718-sh RNA组、GPC3-2134-sh RNA组。YAP1 sh RNA转染实验分为6组:未转染组、空载体组、YAP1-906-sh RNA组、YAP1-1363-sh RNA组、YAP1-1666-sh RNA组、YAP1-2895-sh RNA组。GPC3对YAP1的调控实验分为5组:未转染组、空载体组、GPC3-1718-sh RNA组、GPC3-1718-sh RNA+rh YAP1组、YAP1-1666-sh RNA组。结果 1成功构建了可有效沉默GPC3和YAP1表达的GPC3-1718-sh RNA和YAP1-1666-sh RNA质粒。2 GPC3sh RNA各转染组细胞中GPC3 m RNA和蛋白表达均明显低于未转染组(P<0.05)和空载体组(P<0.05),而GPC3-1718-sh RNA组又明显低于其他转染组(P<0.05)。YAP1 sh RNA各转染组细胞中YAP1 m RNA和蛋白表达均明显低于未转染组(P<0.05)和空载体组(P<0.05),而YAP1-1666-sh RNA组又明显低于其他转染组(P<0.05)。3 GPC3-1718-sh RNA组和YAP1-1666-sh RNA组细胞中YAP1 m RNA和蛋白表达均明显低于未转染组(P<0.05)和空载体组(P<0.05);而GPC3-1718-sh RNA+rh YAP1组细胞中YAP1 m RNA和蛋白表达明显高于GPC3-1718-sh RNA组(P<0.05)和YAP1-1666-sh RNA组(P<0.05)。4与未转染组和空载体组比较,GPC3-1718-sh RNA组和YAP1-1666-sh RNA组的细胞增殖和侵袭能力明显降低(P<0.05),细胞凋亡明显增加(P<0.05);而GPC3-1718-sh RNA+rh YAP1组细胞增殖、侵袭和凋亡均得到明显改善(P<0.05)。结论 GPC3很有可能通过对Hippo信号通路YAP1的调控,实现其对人肝癌细胞Huh7生物学行为的影响。
Objective To investigate the regulatory mechanism of Glypican-3 (GPC3) on Hippo signaling pathway and its effect on the biological behavior of human hepatoma Huh7 cells. Methods Four shRNA sequences targeting GPC3 and YAP1 were constructed and transfected into Huh7 cells. The stable cell lines were screened. The expression of GPC3 and YAP1 in transfected Huh7 cells was detected by fluorescence quantitative PCR and Western blotting to screen sh RNAs that effectively silence GPC3 and YAP1. To observe the effects of silencing GPC3 and YAP1 and human recombinant YAP1 on the proliferation, invasion and apoptosis of hepatocellular carcinoma cells. GPC3 shRNA transfection experiments were divided into 6 groups: untransfected group, empty vector group, GPC3-714-sh RNA group, GPC3-647-sh RNA group, GPC3-1718-sh RNA group, GPC3-2134-sh RNA group. The YAP1 shRNA transfection experiments were divided into 6 groups: untransfected group, empty vector group, YAP1-906-sh RNA group, YAP1-1363-sh RNA group, YAP1-1666-sh RNA group, YAP1-2895-sh RNA group. GPC3 YAP1 regulation experiments were divided into five groups: untransfected group, empty vector group, GPC3-1718-sh RNA group, GPC3-1718-sh RNA + rh YAP1 group, YAP1-1666-sh RNA group. Results 1 GPC3-1718-sh RNA and YAP1-1666-sh RNA plasmids were successfully constructed which can effectively silence the expression of GPC3 and YAP1. The expression of GPC3m RNA and protein in GPC3shRNA transfected group was significantly lower than that in the untransfected group (P <0.05) and empty vector group (P <0.05), and GPC3-1718-sh RNA group was significantly lower than Other transfection group (P <0.05). The YAP1 mRNA and protein expression levels in YAP1 sh RNA transfected group were significantly lower than those in untransfected group (P <0.05) and empty vector group (P <0.05), while YAP1-1666-sh RNA group was significantly lower than Other transfection group (P <0.05). The mRNA and protein expressions of YAP1 in GPC3-1718-sh RNA group and YAP1-1666-sh RNA group were significantly lower than those in untransfected group (P <0.05) and empty vector group (P <0.05) The expression of YAP1 m RNA and protein in 1718-sh RNA + rh YAP1 group was significantly higher than those in GPC3-1718-sh RNA group and YAP1-1666-sh RNA group (P <0.05). Compared with untransfected group and empty vector group, GPC3-1718-sh RNA group and YAP1-1666-sh RNA group showed a significant decrease in cell proliferation and invasion (P <0.05) and a significant increase in apoptosis (P <0.05 ), While the proliferation, invasion and apoptosis of GPC3-1718-sh RNA + rh YAP1 group were significantly improved (P <0.05). Conclusion GPC3 is likely to exert its effect on the biological behavior of human hepatocellular carcinoma Huh7 by regulating the Hippo signaling pathway YAP1.