IGF-1R-Stat3信号通路通过激活中期因子表达促进肝癌细胞活力、迁移和侵袭

IGF-1R-Stat3 signaling pathway enhances viability,migration and invasion of hepatocelluar carcinoma cells by activating midkine expression

目的:探讨胰岛素样生长因子1受体(IGF-1R)-信号转导及转录激活蛋白3(Stat3)信号通路和中期因子(midkine)在肝细胞癌中的作用以及IGF-1R-Stat3信号通路与midkine表达之间的关系。方法:(1)采用RTqPCR检测人肝癌细胞株Huh7和Hep3B、人正常肝细胞株HL-7702、人肝细胞癌组织(n=32)、配对的癌旁组织(n=32)及正常成人肝组织(n=13)中IGF-1R、Stat3和midkine的mRNA表达水平,并应用Pearson相关分析评估肝细胞癌组织中三者表达水平之间的关系。(2)以Huh7细胞作为空白对照,在瞬时上调IGF-1R表达、稳定敲减IGF-1R表达及稳定敲减IGF-1R表达+瞬时上调Stat3表达的Huh7细胞中,采用RT-qPCR及Western blot检测Stat3和midkine mRNA表达水平及Stat3、磷酸化Stat3和midkine蛋白水平;以Huh7细胞作为空白对照,在瞬时上调或瞬时敲减Stat3表达的Huh7细胞中,采用RT-qPCR及Western blot分别检测midkine mRNA和蛋白表达水平。(3)采用MTT法和Transwell实验检测IGF-1R-Stat3信号通路和midkine表达变化对Huh7细胞活力、迁移和侵袭的影响。结果:分别与正常肝细胞和癌旁组织/正常肝组织相比,肝癌细胞株和肝细胞癌组织中IGF-1R、Stat3和midkine的mRNA表达水平显著升高(P<0.01);肝细胞癌组织中Stat3和midkine的mRNA表达水平均与IGF-1R的mRNA表达水平呈正相关(r=0.716和r=0.681,P<0.01),midkine mRNA表达水平与Stat3 mRNA表达水平也呈正相关(r=0.657,P<0.01)。在Huh7细胞中,IGF-1R通过上调Stat3表达并增加其磷酸化水平而促进midkine表达(P<0.01);而且IGF-1R通过激活Stat3、上调midkine表达而增强细胞活力、迁移能力和侵袭能力(P<0.01)。结论:IGF-1R-Stat3信号通路通过激活midkine表达增强肝癌细胞活力,促进细胞迁移和侵袭。

AIM:To investigate the role of insulin-like growth factor-1 receptor(IGF-1 R)-signal transducer and activator of transcription 3(Stat3)signaling pathway and midkine in hepatocelluar carcinoma(HCC)and the association between IGF-1 R-Stat3 signaling pathway and midkine expression.METHODS:The mRNA expression levels of IGF-1 R,Stat3 and midkine in HCC cell lines Huh7 and Hep3 B,normal liver cell line HL-7702,human HCC tissues(n=32),matched adjacent nontumorous tissues(n=32)and normal adult liver tissues(n=13)were detected by RT-qPCR,and the relationship among the mRNA expression of IGF-1 R,Stat3 and midkine in the HCC tissues was evaluated by Pearson correlation analysis.Using Huh7 cells as blank control,the Huh7 cells with transient over-expression of IGF-1 R,with stable knock-down of IGF-1 R,and with stable knock-down of IGF-1 R plus transient over-expression of Stat3 were constructed,and then the mRNA expression of Stat3 and midkine,and the protein levels of Stat3,phosphorylated Stat3 and midkine in these cells were determined by RT-qPCR and Western blot.Moreover,using Huh7 cells as blank control,the mRNA and protein levels of midkine in the Huh7 cells with transient knock-down or over-expression of Stat3 were also detected by RTqPCR and Western blot.The effects of IGF-1 R-Stat3 signaling pathway and midkine expression changes on HCC cell viability,migration and invasion were studied by MTT assay and Transwell chamber assays.RESULTS:The over-expression of IGF-1 R,Stat3 and midkine was found in human HCC cell lines and tissues(P<0.01).The mRNA levels of Stat3 and midkine were positively correlated with IGF-1 R mRNA level(r=0.716 and r=0.681,P<0.01),and midkine mRNA level was also positively correlated with Stat3 mRNA level in the HCC tissues(r=0.657,P<0.01).IGF-1 R promoted midkine expression by up-regulating Stat3 expression and increasing Stat3 protein phosphorylation in Huh7 cells(P<0.01).IGF-1 R enhanced Huh7 cell viability,migration and invasion by up-regulating Stat3 expression and activity,and subsequently activating midkine expression(P<0.01).CONCLUSION:IGF-1 R-Stat3 signaling pathway enhances tumor cell viability,migration and invasion by activating midkine expression in HCC.