TPX2通过PI3K/Akt信号通路调控肝癌细胞增殖及凋亡  

TPX2 regulates proliferation and apoptosis of hepatocarcinoma cells through PI3K/Akt signaling pathway

作　　者：黄丹红 吴建兵[2] 简捷[3] 张岩 刘利珍 HUANG Danhong;WU Jianbing;JIAN Jie;ZHANG Yan;LIU Lizhen(Department of Laboratory,Jiading District Central Hospital Afliated Shanghai University of Medicine&Health Sciences,Shanghai 201800,China;Department of Oncology,Second Affiliated Hospital of Nanchang University,Jiangxi Provincial Key Laboratory of Molecular Medicine,Nanchang 330006,Jiangxi Province,China;Department of Gastroenterology,Tird Affiliated Hospital of Nanchang University,Nanchang 330008,Jiangxi Province,China;Department of Oncology,Jiading District Central Hospital Affiliated Shanghai University of Medicine&Health Sciences,Shanghai 201800,China)

机构地区：[1]上海健康医学院附属嘉定区中心医院检验科,上海201800 [2]南昌大学第二附属医院肿瘤科,江西省分子医学重点实验室,江西南昌330006 [3]南昌大学第三附属医院消化内科,江西南昌330008 [4]上海健康医学院附属嘉定区中心医院肿瘤科,上海201800

出　　处：《肿瘤》2019年第11期897-907,共11页Tumor

摘　　要：目的:探讨爪蟾驱动蛋白样蛋白2靶蛋白(targeting protein for Xenopus kinesin-like protein 2,TPX2)在肝癌细胞中表达及对肝癌细胞增殖和凋亡的调控作用,以及可能的作用机制。方法:应用实时荧光定量PCR法及蛋白质印迹法分别检测正常肝细胞L-02及肝癌Huh7、Hep3B、HepG2和MHCC97-H细胞中TPX2 mRNA及蛋白表达。将靶向TPX 2的重组质粒pMagic4.1-shRNA-TPX2分别转染肝癌Huh7和Hep3B细胞后,应用MTT法检测肝癌细胞的增殖能力,FCM法检测肝癌细胞周期及细胞凋亡率。应用实时荧光定量PCR法检测靶向沉默TPX 2后,肝癌细胞中TPX2、磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)、蛋白激酶B(protein kinase B,PKB,又称Akt)、p21、Bcl-2、c-Myc和Cyclin D1 mRNA表达,及蛋白质印迹法检测TPX2、PI3K、磷酸化-Akt(phosphorylation-Akt,p-Akt)、Akt、p21和Bcl-2蛋白表达。结果:肝癌Huh7、Hep3B、HepG2和MHCC97-H细胞中TPX2 mRNA及蛋白表达水平均明显高于正常肝细胞L-02(P值均<0.01)。重组质粒pMagic4.1-shRNA-TPX2成功转染后,肝癌Huh7和Hep3B细胞中TPX2mRNA及蛋白表达水平均明显下调(P值均<0.01)。靶向沉默TPX 2后,Huh7和Hep3B细胞增殖能力均明显下降(P值均<0.01),细胞周期被阻滞于G0/G1期(P值均<0.01),细胞凋亡率明显增加(P值均<0.01);Huh7和Hep3B细胞中PI3K、Bcl-2、c-Myc和Cyclin D1 mRNA表达水平明显下调(P值均<0.05),PI3K、p-Akt和Bcl-2蛋白表达水平明显下调(P值均<0.05),p21 mRNA及蛋白表达水平均明显上调(P值均<0.01)。结论:TPX2通过PI3K/Akt信号通路调控肝癌细胞增殖及凋亡,有望成为肝癌治疗的潜在靶点。Objective: To investigate the expression of TPX2(targeting protein for Xenopus kinesin-like protein 2) in hepatocarcinoma cells and its regulating effects on the proliferation and apoptosis of hepatocellular carcinoma cells, as well as its possible mechanism.Methods: The expressions of TPX2 mRNA and protein in normal liver L-02 cells and hepatocarcinoma Huh7, Hep3 B, HepG2 and MHCC97-H cells were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. After the recombinant plasmid pMagic4.1-shRNA-TPX2 targeting TPX 2 gene was transfected into hepatocarcinoma Huh7 and Hep3 B cells, the proliferation of hepatocarcinoma cells was detected by MTT assay, the cell cycle and apoptosis rate of hepatocarcinoma cells were detected by FCM.The expressions of TPX2, phosphatidylinositol 3-kinase(PI3 K), protein kinase B(PKB, Akt),Bcl-2, c-Myc and Cyclin D1 mRNAs in hepatocarcinoma cells after silencing TPX 2 gene were detected by real-time fluorescent quantitative PCR, and the expressions of TPX2, PI3 K,phosphorylation-Akt(p-Akt), Akt, p21 and Bcl-2 proteins were detected by Western blotting.Results: The expression levels of TPX2 mRNA and protein in hepatocarcinoma Huh7, Hep3 B,HepG2 and MHCC97-H cells were significantly higher than those in normal hepatocytes L-02(all P < 0.01). After the recombinant plasmid pMagic4.1-shRNA-TPX2 was successfully transfected into Huh7 and Hep3 B cells, the expression levels of TPX2 mRNA and protein were significantly down-regulated(both P < 0.01). After silencing TPX 2 gene, the proliferation ability of Huh7 or Hep3 B cells was significantly decreased(both P < 0.01), the cell cycle was arrested at G0/G1 phase(both P < 0.01), and the apoptotic rate was obviously increased(both P < 0.01). The expression levels of PI3 K, Bcl-2, c-Myc and Cyclin D1 mRNAs in Huh7 and Hep3 B cells after silencing TPX 2 gene were down-regulated(all P < 0.05), the expression levels of PI3 K, p-Akt and Bcl-2 protein were down-regulated(all P < 0.05), whereas the expression levels of p21 m

关 键 词：肝肿瘤 细胞增殖 细胞凋亡 PI3K/AKT信号通路 TPX2 

分 类 号：R735.7[医药卫生—肿瘤]