重楼皂苷Ⅰ激活IGFBP1信号通路抑制肝癌细胞生长和增殖的作用机制

Polyphyllin Ⅰ inhibit cell growth and proliferation of hepatocellular carcinoma through activating IGFBP1 signaling pathway

目的:探讨中药单体重楼皂苷Ⅰ抑制肝癌细胞生长和增殖的分子作用机制。方法:采用MTT和EDU法检测肝癌Bel-7402细胞的生长和增殖情况;Western Blot法检测Bel-7402细胞中肿瘤相关特异蛋白1(Sp1)和胰岛素样生长因子结合蛋白1(IGFBP1)的表达水平;采用细胞瞬间转染技术将Sp1过表达质粒或IGFBP1 siRNA转染至细胞;双荧光素酶基因报告系统检测IGFBP1启动子活性。结果:MTT结果发现PPI处理肝癌Bel-7402细胞24、48、72 h后,显著抑制细胞的生长,且呈时间和剂量依赖关系;EDU实验结果显示PPI可显著抑制Bel-7402细胞的增殖(P<0.05);双荧光素酶检测结果表明PPI显著上调Bel-7402细胞中IGFBP1启动子活性(P<0.05);Western Blot结果证实,PPI显著下调Sp1蛋白表达水平(P<0.05),上调IGFBP1蛋白表达水平(P<0.05),过表达Sp1可阻断PPI对IGFBP1的上调作用(P<0.05),siRNA沉默IGFBP1有效逆转PPI对肝癌Bel-7402细胞的生长抑制作用(P<0.05)。结论:IGFBP1可能是PPI抑制肝癌Bel-7402细胞生长和增殖的关键靶蛋白,PPI通过调控Sp1/IGFBP1信号通路抑制肝癌Bel-7402细胞的生长和增殖。

Objective: To investigate the molecular mechanism of inhibiting the cell growth and proliferation of hepatocellular carcinoma(HCC) by Chinese herbal monomer Polyphyllin Ⅰ(PPI). Methods: MTT and EDU assay were performed to detect the cell growth and proliferation of HCC. Tumor-related proteins Sp1 and IGFBP1 in Bel-7402 cells were detected by Western Blot analysis. Sp1 overexpressed plasmid or IGFBP1 siRNA were transfected by using transient transfection technology.Dul-luciferase gene reporter system was used to measure the activity of IGFBP1 promoter. Results: MTT results showed that PPI significantly inhibited the growth of Bel-7402 cells in a time and dose dependent manner after treated with PPI for 24, 48 and 72 hours. EDU results revealed that PPI dramatically inhibited the proliferation of bel-7402 cells(P<0.05). Moreover, PPI raised the IGFBP1 promoter activity in Bel-7402 cells(P<0.05). Western Blot analysis suggested that PPI significantly down-regulated the protein expression of Sp1(P<0.05) and up-regulated the protein expression of IGFBP1(P<0.05). In addition, overexpression of Sp1 could block the up-regulation effect of PPI on IGFBP1 protein(P<0.05). Silencing of IGFBP1 effectively reversed PPI-induced cell growth inhibition of bel-7402 cells(P<0.05). Conclusion: IGFBP1 may be a key target for PPI inhibiting the growth and proliferation of Bel-7402 cells. PPI could inhibit the cell growth of HCC by regulating the Sp1/IGFBP1 signaling pathway.