[ERK/AP-1信号通路对PAR-2激动剂诱导的肝癌细胞增殖的影响]

[Effect of ERK/AP-1 signaling pathway on proliferation of hepatoma cells induced by PAR-2 agonists].

作者信息

Zheng Yan-min, Xie Li-qun, Li Xuan, Zhao Jun-yan, Chen Xiao-yi, Chen Li, Zhou Jing, Li Fei

机构信息

Department of Gastroenterology, Affiliated Hospital, Medical College of Chinese People's Armed Police Forces, Tianjin 300162, China.

出版信息

Zhonghua Yi Xue Za Zhi. 2009 Dec 1;89(44):3116-21.

PMID:20193273

Abstract

OBJECTIVE

To investigate the expression of protease activated receptor-2 (PAR-2) in human HepG2 hepatoma cells and elucidate the effects of trypsin and PAR-2 agonist peptide SLIGKV-NH(2) upon the proliferation of hepatoma cells and its intracellular signaling mechanism.

METHODS

PAR-2 protein and mRNA expression were detected by immunofluorescence and RT-PCR. The cells were treated with SLIGKV-NH(2), trypsin, reverse PAR-2 agonist peptide VKGILS-NH(2) or PD98059. The changes of cell cycle distribution were evaluated by flow cytometry. The proliferative potential of HepG2 cells was estimated by MTT. The changes of PAR-2, c-fos and PCNA mRNA expression were detected by RT-PCR. The changes of c-fos and PCNA protein expression were detected by Western blotting.

RESULTS

PAR-2 protein and mRNA were expressed in HepG2 cells. PAR-2 mRNA expression (PAR-2/beta-actin) were 0.70 +/- 0.04 and 0.99 +/- 0.05 respectively in cells treated with trypsin and SLIGKV-NH(2). They were both significantly higher than that in the control group (0.35 +/- 0.05, F = 135.534, P < 0.01). Percent G(0)/G(1) phase of HepG2 cells treated with trypsin or SLIGKV-NH(2) were significantly lower than those in the control group [(56.11 +/- 0.85)%, (57.85 +/- 0.46)% vs (79.12 +/- 0.67)%, both P < 0.01] Percent S phase, G(2)/M phase and proliferation index (PI) of HepG2 cells treated with trypsin or SLIGKV-NH(2) were significantly elevated (P < 0.01). The proliferation-enhancing effects and the up-regulation of mRNA and protein of c-fos and PCNA induced by trypsin or SLIGKV-NH(2) were significantly blocked by pretreatment with PD98059 (P < 0.01). There was no statistical significance in proliferation of HepG2 cells between the reverse PAR-2 agonist peptide VKGILS-NH(2) and control group (P > 0.05).

CONCLUSION

PAR-2 is expressed in HepG2 hepatoma cells. PAR-2 activation induced by trypsin or SLIGKV-NH(2) promotes the proliferation of HepG2 cells partially via the ERK/AP-1 pathway.

摘要

目的

研究蛋白酶激活受体-2（PAR-2）在人肝癌HepG2细胞中的表达，阐明胰蛋白酶和PAR-2激动剂肽SLIGKV-NH₂对肝癌细胞增殖的影响及其细胞内信号转导机制。

方法

采用免疫荧光和RT-PCR检测PAR-2蛋白和mRNA表达。细胞分别用SLIGKV-NH₂、胰蛋白酶、PAR-2反向激动剂肽VKGILS-NH₂或PD98059处理。通过流式细胞术评估细胞周期分布的变化。用MTT法评估HepG2细胞的增殖潜能。用RT-PCR检测PAR-2、c-fos和PCNA mRNA表达的变化。用蛋白质印迹法检测c-fos和PCNA蛋白表达的变化。

结果

PAR-2蛋白和mRNA在HepG2细胞中表达。胰蛋白酶和SLIGKV-NH₂处理的细胞中PAR-2 mRNA表达（PAR-2/β-肌动蛋白）分别为0.70±0.04和0.99±0.05。两者均显著高于对照组（0.35±0.05，F=135.534，P<0.01）。胰蛋白酶或SLIGKV-NH₂处理的HepG2细胞G₀/G₁期百分比显著低于对照组[（56.11±0.85）%，（57.85±0.46）%对（79.12±0.67）%，P均<0.01]。胰蛋白酶或SLIGKV-NH₂处理的HepG2细胞S期、G₂/M期百分比和增殖指数（PI）显著升高（P<0.01）。PD98059预处理可显著阻断胰蛋白酶或SLIGKV-NH₂诱导的增殖增强作用以及c-fos和PCNA mRNA及蛋白的上调（P<0.01）。PAR-2反向激动剂肽VKGILS-NH₂与对照组相比，HepG2细胞增殖无统计学意义（P>0.05）。