Analysis of chemokine/chemokine system involoved in the growth of human hepatocellular carcinoma

TaqMan PCR检测了人HCC细胞系、HuH7、PLC/PRF/5和HepG2细胞中CCR6 mRNA的高水平表达。流式细胞术分析中，CCR6在HuH7、PLC/PRF/5和HepG2细胞表面表达增强，与TaqMan PCR mRNA表达结果相似。CCR6的配体（趋化因子）CCL20的mRNA表达在HuH7、PLC/PRF/5和HepG2细胞中被证实，它们都与表达CCR6的细胞系相同。CCL20可分泌到HuH7、PLC/PRF/5、HepG2细胞的培养上清液中，但在其他细胞系的培养上清液中检测不到。MTT实验显示，在HuH7细胞培养基中添加CCL20显著（约170%，p<0.01）刺激了表达CCR6的HuH7细胞的增殖。Western blotting分析表明，HuH7细胞中CCL20/CCR6的下游信号转导途径是由p44/42 MAP激酶介导的，而不是由p38 MAP激酶或SPAK/JNK介导的。趋化因子CCL20对其表面表达高水平受体CCR6的肝癌细胞系没有显着的趋化活性。目前的研究表明，部分人肝癌细胞通过趋化因子-趋化因子受体系统介导的自分泌或旁分泌机制刺激自身生长。

High level of CCR6 mRNA expression was quantified in human HCC cell lines, HuH7, PLC/PRF/5, and HepG2 cells as examined by TaqMan PCR. In flow cytometric analysis, also, CCR6 expression was enhanced on the surface of three HCC cell lines, HuH7, PLC/PRF/5, and HepG2 cells, similar to the results of mRNA expression by TaqMan PCR. The mRNA expression of CCL20, the ligand (chemokine) for CCR6, was demonstrated in HuH7, PLC/PRF/5, and HepG2 cells, all are identical to the cell lines expressing CCR6. CCL20 was secreted into the culture supernatant of HuH7, PLC/PRF/5, and HepG2 cells, but it was undetectable in the culture supernatant of other cell lines. MTT assay revealed that addition of CCL20 to the culture medium of HuH7 cells significantly (by approximately 170%, p<0.01) stimulated the proliferation of these cells expressing CCR6. Western blotting analysis clarified that the downstream signal transduction pathway of CCL20/CCR6 in HuH7 cells was mediated by p44/42 MAP kinase, but not by p38 MAP kinase or SPAK/JNK. Chemokines CCL20 did not show significant chemotactic activity towards hepatoma cell lines expressing high levels of their receptors, CCR6 on their surface. The present study indicates that some of the human hepatoma cells stimulate their own growth via the autocrine or paracrine mechanism mediated by chemokine-chemokine receptor system.