茶黄素通过调节Wnt/β-catenin和Hedgehog信号通路对HepG2细胞增殖和凋亡的影响

Effects of theaflavins on HepG2 cell proliferation and apoptosis by regulating Wnt/β-catenin and Hedgehog signaling pathway

目的 探讨茶黄素对肝癌HepG2细胞增殖、凋亡及Wnt/β-catenin和Hedgehog信号通路的影响。方法 肝癌HepG2细胞用0、2.5、5、10、20、40、80、160、320μmol/L的茶黄素分别培养24、48、72 h,采用CCK-8法检测HepG2细胞活性。根据HepG2细胞活性实验结果将后续实验分为空白对照组,茶黄素低剂量(20μmol/L)组、中剂量(40μmol/L)组和高剂量(80μmol/L)组,细胞培养24 h。克隆形成实验检测HepG2细胞增殖;流式细胞术检测HepG2细胞凋亡;Western blot检测HepG2细胞中Ki67、PCNA、cleaved caspase-3、cleaved caspase-9、GLi1、SMO、PTch1、β-catenin、c-myc和Cyclin D1的蛋白表达量;实时荧光定量PCR(qRT-PCR)检测HepG2细胞中GLi1、SMO、PTch1、β-catenin、c-myc和Cyclin D1 mRNA相对表达量。结果 与空白对照组相比,茶黄素组HepG2细胞增殖显著降低(P<0.05)、凋亡率显著增加(P<0.05),GLi1、SMO、PTch1、β-catenin、c-myc和Cyclin D1 mRNA相对表达量均显著下调(P<0.05),Ki67、PCNA、GLi1、SMO、PTch1、β-catenin、c-myc和Cyclin D1的蛋白表达量显著下调(P<0.05),而cleaved caspase-3和cleaved caspase-9的蛋白表达量显著上调(P <0.05)。结论 茶黄素可通过调控Wnt/β-catenin和Hedgehog信号通路抑制HepG2细胞的增殖并促进其凋亡。

Objective To investigate the effects of theaflavins on the proliferation,apoptosis and Wnt/β-catenin and Hedgehog signaling pathway in hepatocellular carcinoma HepG2 cells.Methods HepG2 cells were treated with different concentrations of theaflavins(0,2.5,5,10,20,40,80,160 and 320μmol/L)and cultured for 24,48 or 72 hours.The viability of HepG2 cells was detected by CCK-8 assay.According to the results of HepG2 cell viability experiment,the subsequent experiments were divided into blank control group,low dose of theaflavins(20μmol/L)group,middle dose of theaflavins(40μmol/L)group and high dose of theaflavins(80μmol/L)group,and HepG2 cells were cultured for 24 hours.Cell proliferation was detected by colony formation assay and cell apoptosis was detected by flow cytometry.The levels of Ki67,PCNA,cleaved caspase-3,cleaved caspase-9,GLi1,SMO,PTch1,β-catenin,c-myc and cyclin D1 protein were detected by western blot.The levels of GLi1,SMO,PTch1,β-catenin,c-myc and cyclin D1 mRNA expression were detected by qRT-PCR.Results Compared with the control group,the HepG2 cell activity of theaflavins group was decreased,the cell apoptosis rate was increased,and the levels of GLi1,SMO,PTch1,β-catenin,c-myc and cyclin D1 mRNA expression were significantly downregulated(P<0.05).The levels of Ki67,PCNA,GLi1,SMO,PTch1,β-catenin,c-myc and cyclin D1 protein were downregulated,while the levels of cleaved caspase-3 and cleaved caspase-9 protein were significantly upregulated in theaflavins group(P<0.05).Conclusion Theaflavins could inhibit HepG2 cell proliferation and promote cell apoptosis by regulating the Wnt/β-catenin and Hedgehog pathway.