目的:本研究旨在深入探究增殖相关蛋白2G4(proliferation-associated protein 2G4,PA2G4)通过PI3K/AKT/p53信号通路对人胆囊癌细胞GBC-SD增殖、迁移能力及凋亡过程的影响机制。方法:利用慢病毒转染技术构建PA2G4表达下调的胆囊癌细胞系...目的:本研究旨在深入探究增殖相关蛋白2G4(proliferation-associated protein 2G4,PA2G4)通过PI3K/AKT/p53信号通路对人胆囊癌细胞GBC-SD增殖、迁移能力及凋亡过程的影响机制。方法:利用慢病毒转染技术构建PA2G4表达下调的胆囊癌细胞系。采用RT-qPCR、Western blot对转染效率进行验证。运用CCK-8法、集落形成实验评估PA2G4对于GBC-SD细胞的增殖活性。用Transwell实验、划痕愈合实验评估PA2G4对于GBC-SD细胞的迁移能力影响。通过Western blot技术检测PA2G4对PI3K/AKT/p53及其相关磷酸化蛋白的表达水平,并检测凋亡相关蛋白Caspase-3、Bcl-2及Bax的表达水平。结果:GBC-SD细胞经PA2G4敲低后,其PA2G4的mRNA及蛋白表达水平均显著下降。敲低PA2G4显著抑制了GBC-SD细胞的增殖活性和迁移能力,同时促进了细胞凋亡。此外,PA2G4敲低还伴随p-PI3K、pAKT表达水平的下调,以及p-p53表达水平的上调。结论:本研究表明,PA2G4通过下调PI3K/AKT/p53抑制胆囊癌细胞的增殖与迁移,并诱导其凋亡。展开更多
The proteasome is a major protein-degrading enzyme, which catalyzes degradation of oxidized and aged proteins, signal transduction factors and cleaves peptides for antigen presentation. Proteasome exists in the equili...The proteasome is a major protein-degrading enzyme, which catalyzes degradation of oxidized and aged proteins, signal transduction factors and cleaves peptides for antigen presentation. Proteasome exists in the equilibrium of 26S and 20S particles. Proteasome function is altered by ethanol metabolism, depending on oxidative stress levels： low oxidative stress induces proteasome activity, while high oxidative stress reduces it. The proposed mechanisms for modulation of proteasome activity are related to oxidative modification of proteasomal proteins with primary and secondary products derived from ethanol oxidation. Decreased proteolysis by the proteasome results in the accumulation of insoluble protein aggregates, which cannot be degraded by proteasome and which further inhibit proteasome function. Mallory bodies, a common signature of alcoholic liver diseases, are formed by liver cells, when proteasome is unable to remove cytokeratins. Proteasome inhibition by ethanol also promotes the accumulation of pro-apoptotic factors in mitochondria of ethanol-metabolizing liver cells that are normally degraded by proteasome. In addition, decreased proteasome function also induces accumulation of the negative regulators of cytokine signaling （I-~B and SOCS）, thereby blocking cytokine signal transduction. Finally, ethanol-elicited blockade of interferon type 2 and 2 signaling and decreased proteasome function impairs generation of peptides for MHC class Ⅰ-restricted antigen presentation.展开更多
文摘目的:本研究旨在深入探究增殖相关蛋白2G4(proliferation-associated protein 2G4,PA2G4)通过PI3K/AKT/p53信号通路对人胆囊癌细胞GBC-SD增殖、迁移能力及凋亡过程的影响机制。方法:利用慢病毒转染技术构建PA2G4表达下调的胆囊癌细胞系。采用RT-qPCR、Western blot对转染效率进行验证。运用CCK-8法、集落形成实验评估PA2G4对于GBC-SD细胞的增殖活性。用Transwell实验、划痕愈合实验评估PA2G4对于GBC-SD细胞的迁移能力影响。通过Western blot技术检测PA2G4对PI3K/AKT/p53及其相关磷酸化蛋白的表达水平,并检测凋亡相关蛋白Caspase-3、Bcl-2及Bax的表达水平。结果:GBC-SD细胞经PA2G4敲低后,其PA2G4的mRNA及蛋白表达水平均显著下降。敲低PA2G4显著抑制了GBC-SD细胞的增殖活性和迁移能力,同时促进了细胞凋亡。此外,PA2G4敲低还伴随p-PI3K、pAKT表达水平的下调,以及p-p53表达水平的上调。结论:本研究表明,PA2G4通过下调PI3K/AKT/p53抑制胆囊癌细胞的增殖与迁移,并诱导其凋亡。
基金Supported by the National Institute on Alcohol Abuse and Alcoholism, grant number 5R21 AA015379-02
文摘The proteasome is a major protein-degrading enzyme, which catalyzes degradation of oxidized and aged proteins, signal transduction factors and cleaves peptides for antigen presentation. Proteasome exists in the equilibrium of 26S and 20S particles. Proteasome function is altered by ethanol metabolism, depending on oxidative stress levels： low oxidative stress induces proteasome activity, while high oxidative stress reduces it. The proposed mechanisms for modulation of proteasome activity are related to oxidative modification of proteasomal proteins with primary and secondary products derived from ethanol oxidation. Decreased proteolysis by the proteasome results in the accumulation of insoluble protein aggregates, which cannot be degraded by proteasome and which further inhibit proteasome function. Mallory bodies, a common signature of alcoholic liver diseases, are formed by liver cells, when proteasome is unable to remove cytokeratins. Proteasome inhibition by ethanol also promotes the accumulation of pro-apoptotic factors in mitochondria of ethanol-metabolizing liver cells that are normally degraded by proteasome. In addition, decreased proteasome function also induces accumulation of the negative regulators of cytokine signaling （I-~B and SOCS）, thereby blocking cytokine signal transduction. Finally, ethanol-elicited blockade of interferon type 2 and 2 signaling and decreased proteasome function impairs generation of peptides for MHC class Ⅰ-restricted antigen presentation.
