Zheng Yan-min, Xie Li-qun, Li Xuan, Zhao Jun-yan, Chen Xiao-yi, Chen Li, Zhou Jing, Li Fei
Department of Gastroenterology, Affiliated Hospital, Medical College of Chinese People's Armed Police Forces, Tianjin 300162, China.
Zhonghua Yi Xue Za Zhi. 2009 Dec 1;89(44):3116-21.
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.
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.
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).
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.
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。
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。
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）。
PAR-2在肝癌HepG2细胞中表达。胰蛋白酶或SLIGKV-NH₂诱导的PAR-2激活部分通过ERK/AP-1途径促进HepG2细胞增殖。
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