PI3K/AKT/NF-κB轴调控胃癌细胞HGC-27增殖

PI3K/AKT/NF-κB Axis Regulates Gastric Cancer Cells Proliferation

目的:构建磷酸化AKT1(Ser473)位点突变真核表达载体,并检测PI3K/AKT/NF-κB信号通路轴对胃癌细胞增殖的影响。方法:以带有pcDNA3.0-Flag标签的AKT1质粒为模板,扩增出AKT1(Ser473A)(丝氨酸突变成丙氨酸)位点突变编码序列,将其插入pcDNA3.0-Flag载体中,双酶切和测序验证后瞬时转染人胚肾293T细胞,Western印迹检测其表达情况;将突变质粒与空载体分别转染胃癌细胞HGC-27,通过Western印迹检测其下游基因核转录因子κB(NF-κB)在蛋白水平的变化;通过CCK-8法检测对细胞生长曲线的影响。结果:双酶切和测序结果表明,pcDNA3.0-Flag-AKT1(Ser473A)真核表达质粒构建成功;转染293T细胞后获得表达;转染胃癌细胞HGC-27后,Western印迹验证去磷酸化AKT1(Ser473A)可下调NF-κB的蛋白水平(P<0.01);细胞生长曲线结果显示,转染pcDNA3.0-Flag-AKT1(Ser473A)较空载体细胞生长慢(P<0.01)。结论:PI3K/AKT/NF-κB信号通路轴在胃癌发生发展过程中发挥重要作用。

Objective：To construct AKT（Ser473） site mutations eukaryotic expression vectors and detect its function in gastric cancer cells.Methods：AKT（Ser473） was amplified from pcDNA3.0-Flag-AKT1 by recombinant PCR and was inserted into pcDNA3.0-Flag vector.The recombinant plasmids were identified by enzyme digestion and sequencing,and were transfected into HEK293T cells and detected by Western blot.Western blot was used to detect the effect of AKT（Ser473） on NF-κB protein expression.CCK-8 assay was performed to investigate the effect of the site mutation on gastric cancer of gastric cancer cell proliferation.Results：The AKT1 site mutation eukaryotic expression vector was successfully cloned and expressed.AKTl（Ser473A） could inhibit NF-κB expression and proliferation of gastric cancer cells.Conclusion：PI3K/AKT/NF-κB axis plays a key role in gastric cancer development and progression.