NS-398对HGF诱导的肝癌细胞株MMP-7,MMP-9,TIMP-1表达的影响

Effect of NS-398 on matrix metalloproteinase expression in hepatocyte growth factorinduced HepG2 cell line

目的:研究选择性COX-2抑制剂NS-398对肝癌细胞株MMP和TIMP表达的影响,探讨选择性COX-2抑制剂降低肿瘤细胞侵袭力的机制．方法:以不同浓度的NS-398(0,20,40,60, 80μmol/L)作用于HGF诱导的HepG2细胞,免疫组化法观察细胞中MMP-7,MMP-9,TIMP-1的蛋白质表达,ELISA法检测细胞外培养液中MMP-7,MMP-9,TIMP-1的含量,RT-PCR法分析3种蛋白酶的mRNA转录水平．结果:NS-398作用细胞48 h后,发现MMP-7, MMP-9,TIMP-1的蛋白质表达程度均下降, NS-398作用于HepG2细胞后,MMP-7在培养液中的含量和其在细胞内的mRNA相对水平分别为8．2±0．6,5．8±0．8,4．3±0．8,2．7±0．4, 1．7±0．4μg／L和0．58±0．06,0．42±0．03,0.37±0．01,0．36±0．01,0．33±0．01:MMP-9在培养液中未检出,其细胞内mRNA相对水平分别为0．32±0．02,0．23±0．02,0．21±0．01,0．17±0．01,0．13±0．01;TIMP-1在培养液中的含量和其在细胞内的mRNA相对水平分别为39．0±0．9,29．5±2．8,25．0±0．9,16．8±0．4,11．8±0．3μg/L和0．19±0．02,0．17±0．02,0．16±0．01, 0．13±0．01．0．结论:选择性COX-2抑制剂能够抑制MMP和TIMP基因转录,使TIMP/MMP比例失衡,这可能是其降低肿瘤细胞侵袭力的机制之一．

AIM： To investigate the effect of the selective cyclooxygenase-2 （COX-2） inhibitor NS-398 on the expression of matrix metalloproteinases （MMPs） and tissue inhibitor of metalloproteinases （TIMPs） in liver cancer cell line induced by hepatocyte growth factor （HGF）, and to explore the possible mechanism of NS-398 in depressing tumor cell invasiveness. METHODS： HGF-induced liver cancer cells HepG2 were treated with different concentrations of NS-398 （0, 20, 40, 60, 80μ mol/L）. The expression of MMP-7, MMP-9 and TIMP-1 inside the cells, the contents outside of the cells and the levels of mRNA were observed by immunohistochemistry staining, enzyme-linked immunosorbent assay （ELISA）, and reverse transcription-polymerase chain reaction （RT-PCR）, respectively. RESULTS： After HepG2 cells were treated with NS-398 for 48 h, the expression of MMP-7, MMP-9 and TIMP-1 protein in the cells were decreased; the content of MMP-7 in the culture fluid was 8.2 ± 0.6, 5.8 ± 0.8, 4.3 ± 0.8, 2.7± 0.4, and 1.7 ＋ 0.4μg/L, respectively, as 0, 20, 40, 60, and 80 μmol/L NS-398 was used; the relative level of MMP-7 mRNA in the cells was 0.58 ± 0.06, 0.42 ± 0.03, 0.37 ± 0.01, 0.36 ± 0.01, and 0.33 ±0.01, respectively; MMP-9 can not be detected in the culture fluid, but its relative mRNA level was 0.32 ± 0.02, 0.23 ± 0.02, 0.21 ± 0.01, 0.17 ± 0.01, and 0.13± 0.01, respectively, in the cells; the content of TIMP-1 in the culture fluid was 39.0 ±0.9, 29.5 ± 2.8, 25.0 ± 0.9, 16.8 ± 0.4, 11.8 ±0.3 μg/L and its relative mRNA level in the cells was 0.19 ± 0.02, 0.17 ± 0.02, 0.16 ± 0.01, 0.13 ± 0.01, and 0, respectively. CONCLUSION： The selective COX-2 inhibitor can inhibit the gene transcription of MMP and TIMP, and lead to the imbalance of TIMP and MMP percentage, which is one of the possible mechanisms in reducing tumor cell invasive ability.