蛋白酶激活受体2激动剂对食管癌EC109细胞侵袭转移的促进作用

Protease-activated receptor-2 agonists promote cell invasion and metastasis in human esophageal cancer cell line EC109

目的:探讨人食管癌细胞株EC109蛋白酶激活受体-2(PAR-2)的表达,以及内源性PAR-2激动剂胰蛋白酶和PAR-2激动肽SLIGKV对该细胞侵袭转移的影响及其可能的分子机制.方法:采用RT-PCR法和免疫细胞化学染色法检测EC109细胞中PAR-2mRNA及蛋白的表达情况;胰蛋白酶和SLIGKV对细胞施加干预后,MTT法检测细胞增殖情况,Transwell小室法检测细胞侵袭和迁移能力的变化,RT-PCR法检测PAR-2、MMP-2/MMP-9mRNA表达的变化,明胶酶谱法检测MMP-2、MMP-9明胶酶活性的变化.结果:食管癌细胞EC109在胰蛋白酶和SLIGKV刺激后,PAR-2mRNA表达较对照组显著上调(0.781±0.045、0.653±0.029vs0.491±0.032,均P<0.01);胰蛋白酶在1-10nmol/L、SLIGKV在1-50μmol/L时可促进细胞增殖(P<0.01或0.05),呈剂量依赖性;Transwell小室实验中胰蛋白酶组及SLIGKV组的细胞侵袭和迁移能力显著高于对照组及反肽组(72.5±9.2、59.4±8.7vs31.6±6.6、36.2±9.8,均P<0.01);胰蛋白酶及PAR-2激动肽作用后细胞MMP-9mRNA的表达量明显高于对照组及反激动肽组(0.719±0.034、0.466±0.042vs0.341±0.032、0.370±0.021,均P<0.01),细胞水解明胶的能力也明显高于对照组及反激动肽组(75.6±6.1、60.4±4.6vs44.9±4.2、39.3±5.2,均P<0.01).而对MMP-2的mRNA表达和水解明胶的能力无明显影响(P>0.05).结论:食管癌EC109细胞表达PAR-2受体,且内源性PAR-2激动剂胰蛋白酶和PAR-2激动肽SLIGKV可能通过激活PAR-2受体上调MMP-9表达,进而促进食管癌细胞侵袭转移.

AIM：To determine the expression of protease activated receptor-2 （PAR-2） in human esophageal cell line EC109,and to evaluate the effects of PAR-2 activation on cell invasion and migration. METHODS：The expression of PAR-2 protein and mRNA in EC109 cells was determined byimmunocytochemistry and reverse transcription-polymerase chain reaction （RT-PCR）,respectively. Methyl thiazol tetrazolium （MTT） assay,cell invasion and migration assay,semi-quantitative PCR and zymographic analysis were performed to examine whether endogenous PAR-2 activator trypsin and PAR-2 activating-peptide SLIGKV could alter cell proliferation,invasion,migration,and matrix metalloproteinase （MMP） production. RESULTS：Both PAR-2 mRNA and protein were expressed in EC109 cells. PAR-2 mRNA was up-regulated in cells treated with trypsin or PAR-2-activating peptide SLIGKV （0.781 ± 0.045 and 0.653 ± 0.029 vs 0.491 ± 0.032,both P 0.01）,but not in those treated with control peptide VKG-ILS （P 0.05）. Trypsin and SLIGKV promoted the proliferation of EC109 cells in a dose-and time-dependent manner. No significant difference was noted in cell proliferation between untreated cells and cells treated with control peptide VKGILS. Treatment with trypsin or SLIGKV significantly increased the number of EC109 cells that passed through the Millicell inserts in the migration assay （72.5 ± 9.2 vs 31.6 ± 6.6,and 59.4 ± 8.7 vs 36.2 ± 9.8,both P 0.01）. Compared with untreated cells and cell treated with con-trol peptide,trypsin and SLIGKV significantly increased the mRNA expression （0.719 ± 0.034 vs 0.341 ± 0.032,and 0.466 ± 0.042 vs 0.370 ± 0.021,both P 0.01） and gelatinolytic activity （75.6 ± 6.1 vs 44.9 ± 4.2,and 60.4 ± 4.6 vs 39.3 ± 5.2,both P 0.01） of MMP-9.CONCLUSION：PAR-2 is expressed in EC109 cells. PAR-2 activation may be able to promote the invasion and metastasis of human esopha-geal carcimoma cells by stimulating MMP-9 pro-duction.