GPX2在肝内胆管癌中的表达及对其进展的影响

Expression of GPX2 in intrahepatic cholangiocarcinoma and its effect on progression

目的探究谷胱甘肽过氧化物酶2(GPX2)在肝内胆管癌(ICC)发生发展中的作用。方法利用Omicshare网站分析GPX2在ICC中的表达水平,通过实时定量反转录聚合酶链式反应(RT-qPCR)、Western blot及免疫组化染色验证其在ICC中的表达水平。构建稳定敲低和过表达GPX2的HuCCT1细胞系并进行体外实验。通过平板克隆实验、细胞计数试剂盒8(CCK-8)法、细胞划痕实验、Transwell实验、流式细胞术等探究GPX2对ICC细胞增殖、迁移、凋亡和上皮间充质转化(EMT)的影响。最后构建小鼠胆管癌模型,检测GPX2在小鼠胆管癌中的表达水平。结果Omicshare网站分析结果显示GPX2在ICC中普遍上调(P<0.001)。Western blot、RT-qPCR和免疫组化实验显示,与癌旁组织相比,GPX2在ICC中表达升高(P<0.0001)。平板克隆、CCK-8实验结果显示GPX2敲低后,细胞克隆形成率下降(P<0.01),增殖能力降低(P<0.001);GPX2过表达后,细胞克隆形成率升高(P<0.01),增殖能力增强(P<0.01)。细胞划痕、Transwell和Western blot实验结果显示GPX2敲低后,划痕愈合速度减慢、迁移能力降低、E-cadherin升高(P<0.01),N-cadherin(P<0.01)、Vimentin(P<0.05)降低;GPX2过表达后,划痕愈合速度增快、迁移能力增强、E-cadherin降低(P<0.05),N-cadherin(P<0.01)、Vimentin(P<0.001)升高;凋亡流式细胞术和Western blot实验结果显示GPX2敲低后,细胞凋亡率增多、Bcl-2降低(P<0.001),BAX升高(P<0.01);GPX2过表达后,细胞凋亡率降低(P<0.01),Bcl-2升高(P<0.0001),BAX降低(P<0.001)。最后在小鼠胆管癌模型中观察到GPX2升高。结论GPX2在人和小鼠的胆管癌组织中高表达,并可能增强胆管癌细胞的增殖和迁移能力,促进肿瘤细胞EMT,抑制肿瘤细胞凋亡。

Objective To investigate the role of Glutathione peroxidase 2(GPX2)in the occurrence and progression of intrahepatic cholangiocarcinoma(ICC).Methods The Omicshare website was used to analyze GPX2 expression levels in ICC.The expression levels in ICC were validated using quantitative real-time reverse transcription PCR(RT-qPCR),Western blot,and immunohistochemistry.Stable GPX2 knockdown and overexpression HuCCT1 cell lines were constructed.The effects of GPX2 on ICC cell proliferation,migration,apoptosis,and epithelial-mesenchymal transition(EMT)were investigated using colony formation assays,cell counting kit-8(CCK-8)assays,wound healing assays,Transwell assays,and flow cytometry.A mouse model of cholangiocarcinoma was constructed to assess GPX2 expression in mouse cholangiocarcinoma tissues.Results Based on the analysis results from the Omicshare website,GPX2 was generally upregulated in intrahepatic cholangiocarcinoma(ICC)(P<0.001).Western blot(P<0.0001),RT-qPCR(P<0.001),and immunohistochemistry experiments showed that,compared to adjacent non-cancerous tissues,the expression of GPX2 was significantly elevated in ICC.When GPX2 was knocked down,the colony formation rate of cells decreased significantly(P<0.01),and the proliferation capacity was reduced(P<0.001).Conversely,overexpression of GPX2 led to a significant increase in colony formation rate(P<0.01)and enhanced proliferation capacity(P<0.01).Results from wound healing and Transwell assays demonstrated that GPX2 knockdown slowed down cell wound healing(P<0.01)and reduced migration ability(P<0.01).Additionally,GPX2 knockdown resulted in an increase in E-cadherin(P<0.01)and a decrease in N-cadherin(P<0.01)and Vimentin(P<0.05).On the other hand,overexpression of GPX2 accelerated wound healing(P<0.05)and enhanced migration ability(P<0.05),while E-cadherin expression decreased(P<0.05)and N-cadherin(P<0.01)and Vimentin(P<0.001)expression increased.Flow cytometry for apoptosis and Western blot experiments indicated that GPX2 knockdown increased the apoptosis rate(P<0.001),decreased the expression of Bcl-2(P<0.001),and increased the expression of BAX(P<0.01).But overexpression of GPX2 reduced the apoptosis rate(P<0.01),increased Bcl-2 expression(P<0.0001),and decreased BAX expression(P<0.001).Finally,elevated levels of GPX2 were observed in a mouse model of cholangiocarcinoma.Conclusion GPX2 is highly expressed in human and mouse cholangiocarcinoma tissues,and it may enhance cholangiocarcinoma cell proliferation and migration,promote tumor cell EMT,and inhibit tumor cell apoptosis.