金属硫蛋白-1G对肝癌细胞增殖的影响

Proteomics analysis of the effects of metallothionein-1G on the proliferation of hepatocellular carcinoma cells

目的探讨金属硫蛋白-1G(MT1G)在肝细胞癌(HCC)恶化过程中发挥的调节作用及调节机制。方法利用无标记定量蛋白质组学技术比较MT1G过表达与参照细胞株之间蛋白质组差异;利用细胞计数试剂盒(CCK-8)、胸腺嘧啶核苷类似物染色试剂盒(EDU法)观察细胞表型变化;利用蛋白质印迹法(Western blot)检测相关信号通路关键因子表达量变化。样本间的比较采用t检验进行统计分析。结果首先成功构建出MT1G过表达细胞株。经质谱(MS)鉴定结果表明与参照细胞比共有182种蛋白在MT1G过表达细胞中出现表达量差异有统计学意义(阈值≥5.0倍或≤0.2倍,且P<0.05)。GO分析显示这些差异表达蛋白(DEPs)可能参与细胞周期调控和磷脂酰肌醇3激酶-蛋白激酶B(PI3K-Akt)信号通路。定量聚合酶链反应(PCR)验证表明与参照细胞比,MT1G在MT1G过表达细胞中上调表达,差异均有统计学意义(294.9±33.3)倍(t=8.837,P<0.01);吡多醛激酶(PDXK)上调表达(34.02±2.59)倍(t=13.148,P<0.01);UBX结构域蛋白7(UBXN)上调表达(19.08±0.47)倍(t=40.652,P<0.01);跨膜蛋白9家族成员4(TM9S4)上调(8.91±0.43)倍(t=20.485,P<0.01),这些结果与MS鉴定结果趋势一致,差异均有统计学意义。CCK-8和EDU染色结果揭示MT1G过表达可以显著抑制HCC细胞的增殖,而这种抑制作用是通过抑制Akt和雷帕霉素靶蛋白(mTOR)的磷酸化实现的。结论MT1G通过调节PI3K-Akt信号通路活性抑制肝细胞癌细胞的增殖。

Objective To investigate the biological function of metallothionein-1G(MT1G)and its underlying mechanisms.Methods Label-free quantitative proteomic approach was applied to analyze the proteomic differences between HepG2 cells with MT1G overexpression and control cells.Cell counting kit-8(CCK-8)assat and 5-ethynyl-2’-deoxyuridine(EDU)staining were applied to examine the effects of MT1G on the proliferation of hepatocellular carcinoma(HCC)cells.Western blotting was employed to detect the expression of key regulators.t-test was used for comparison between two groups.Results A total of 182 differential expression proteins(DEPs)were identified by mass spectrometry(MS)in MT1G overexpression group(Threshold:DEP>5.0 folds or<0.2 folds,P<0.05).GO analysis suggested that these DEPs might be involved in cell cycle and phosphoinositide-3-kinase(PI3K)-protein kinase(Akt)signaling pathway.Afterwards,real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)assay showed that the expression of MT1G,pyridoxal kinase(PDXK),Ubx domain protein 7(UBXN)and transmembrane protein 9 family member 4(TM9S4)was up-regulated by(294.9±33.3)folds(t=8.837,P<0.01),34.02±2.59 folds(t=13.148,P<0.01),(19.08±0.47)folds(t=40.652,P<0.01)and(8.91±0.43)folds(t=20.485,P<0.01)respectively as compared with that of control cells,which was consistent with the results obtained by MS.The results from CCK-8 assay and EDU staining revealed that MT1G significantly inhibited the proliferation of HCC cells.Western blotting indicated that MT1G could inhibit the phosphorylation of Akt and mammalian target of rapamycin(mTOR).Conclusion MT1G inhibits the proliferation of HCC cells via PI3K-Akt signaling pathway.