白细胞介素-18抑制肝癌细胞增殖、促进细胞凋亡的机制研究

Mechanism of IL-18 inhibiting proliferation and promoting apoptosis of hepatoma cells

目的探讨白细胞介素-18(IL-18)通过调控Wnt/β-连环素(β-catenin)信号通路影响肝癌细胞增殖及凋亡的作用机制。方法培养肝癌细胞株HepG2、Bel-7402与正常肝细胞株L02,实时荧光定量逆转录聚合酶链反应(qRT-PCR)与蛋白质印迹法(Western blotting)分别检测细胞IL-18 mRNA及蛋白表达。预实验中筛选出HepG2细胞为实验细胞,分别转染pcDNA、pcDNAIL-18,Western blotting验证转染效率。四甲基偶氮唑盐微量酶反应比色法(MTT法)与膜联蛋白V(Annexin V)-异硫氰酸荧光素(FITC)/碘化丙啶(PI)染色实验分别检测HepG2细胞增殖及凋亡情况。qRT-PCR与Western blotting分别检测HepG2细胞中B淋巴细胞瘤-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)、β-catenin、细胞周期蛋白D1(Cyclin D1)表达水平。HepG2细胞中转染IL-18过表达质粒后再加入Wnt/β-catenin通路激活剂(SKL2001),并检测其对HepG2细胞增殖及凋亡的影响。结果与L02细胞相比,肝癌HepG2、Bel-7402细胞中IL-18 mRNA及蛋白表达均显著降低[(1.02±0.12)比(0.39±0.09)(/0.45±0.12);(0.41±0.14)比(0.11±0.13)(/0.19±0.14)](P<0.05);IL-18过表达后HepG2细胞吸光度[24 h(0.31±0.10)比(0.27±0.13);48 h(0.63±0.12)比(0.39±0.14);72 h(0.98±0.18)比(0.54±0.15)]、Bcl-2及β-catenin、Cyclin D1蛋白表达水平均明显低于pcDNA组,而细胞凋亡率[(6.93±0.22)%比(20.54±3.97)%]及Bax蛋白表达水平均明显升高;SKL2001可激活Wnt/β-catenin信号通路进而逆转IL-18对肝癌细胞增殖的抑制作用及其对细胞凋亡的促进作用。结论IL-18可下调肝癌细胞β-catenin表达而抑制Wnt/β-catenin信号通路进而减弱肝癌细胞增殖能力并提高细胞凋亡水平。

Objective To investigate the mechanism of interleukin-18(IL-18)affecting the proliferation and apoptosis of hepatoma cells by regulating Wnt/β-catenin signaling pathway.Methods Hepatoma cell lines HepG2,Bel-7402 and normal liver cell line L02 were cultured,and IL-18 mRNA and protein expression were detected by qRT-PCR and Western blotting,respectively.In the prelimi⁃nary experiment,HepG2 cells were selected as experimental cells,transfected with pcDNA and pcDNA-IL-18,respectively,and the transfection efficiency was verified by Western blotting.MTT and Annexin V-FITC/PI staining experiments were used to detect the pro⁃liferation and apoptosis of HepG2 cells.The expression levels of Bax,Bcl-2,β-catenin and Cyclin D1 in HepG2 cells were detected by qRT-PCR and Western blotting,respectively.HepG2 cells were transfected with IL-18 overexpression plasmid and then Wnt/β-catenin pathway activator(SKL2001)was added to detect the proliferation and apoptosis of HepG2 cells.Results Compared with L02 cells,IL-18 mRNA and protein expression in HepG2 and Bel-7402 cells were significantly decreased[(1.02±0.12)vs.(0.39±0.09)/(0.45±0.12);(0.41±0.14)vs.(0.11±0.13)/(0.19±0.14)](P<0.05).After IL-18 overexpression,the OD value of HepG2 cells[24 h(0.31±0.10)vs.(0.27±0.13);48 h(0.63±0.12)vs.(0.39±0.14);72 h(0.98±0.18)vs.(0.54±0.15)]and the expression levels of Bcl-2,β-catenin and Cyclin D1 were significantly lower than those of pcDNA group,while the apoptosis rate[(6.93±0.22)%vs.(20.54±3.97)%]and Bax protein ex⁃pression level were significantly increased.SKL2001 could activate Wnt/β-catenin signaling pathway to reverse the inhibitory effect of IL-18 on hepatoma cell proliferation and its promotion of apoptosis.Conclusion IL-18 can down-regulate the expression ofβ-catenin in hepatocellular carcinoma cells and inhibit Wnt/β-catenin signaling pathway,thereby attenuating the proliferation of hepatoma cells and increasing the level of apoptosis.