反义RNA对胃癌细胞MT1-MMP基因表达和侵袭性的抑制作用

Inhibitory effect of antisense RNA on the gene expression of membrane-type 1 matrix metalloproteinase and invasiveness of human gastric carcinoma cells

目的:膜研究膜型-1基质金属蛋白酶(MT1- MMP)反义RNA对人胃癌细胞BGC823靶基因表达和侵袭特性的影响方法:利用基因重组技术构建人MT1-MMP反义RNA真核表达载体,转染人胃癌细胞BGC823,应用RT-PCR、MTT、明胶酶谱和体外侵袭实验等方法观察人胃癌细胞BGC823转染前后,MT1-MMP mRNA表达水平、细胞生长、明教酶A活性及细胞体外侵袭能力等指标的变化.结果:成功构建了MT1-MMP反义RNA真核表达载体pasMMP14,将其转染胃癌细胞BGC823后,与阴性对照组相比,实验组MT1- MMP mRNA表达水平降低,抑制率为36%.转染48 h,明教酶A的活化受到了明显抑制.转染72 h,细胞增殖明显受抑(t=2.358,P<0.01 vs空白组:t=2.727 P<0.01 vs阴性组).实验组的穿膜细胞数明显低于空白对照组和阴性对照组(t=5.744,P<0.01;t=5.695,P<0.01).结论:反义RNA对人胃癌细胞MT1-MMP基因表达和侵袭能力具有明显的抑制作用,MT1-MMP基因可作为胃癌抗侵袭治疗的分子靶点.

AIM： To investigate the influence on the gene expression of membrane-type 1 matrix metalloproteinase （MT1-MMP） and invasiveness of human gastric carcinoma cell line BGC823 by anfisense RNA. METHODS： The eukaryotic expression vector carrying MT1-MMP antisense RNA was constructed with recombinant technology and then transfected into human gastric cancer cell line BGC823. The changes of MT1-MMP mRNA expression, cell proliferation, activation of gelatinase A and cell invasion ability were examined by reverse transcription-polymerase chain reaction （RT-PCR）, MTT assay, zymography and Transwell invasion assay, respectively. RESULTS： The eukaryotic expression vector （named pasMMP14） containing MT1-MMP antisense RNA was successfully constructed and transfected into BGC823 cells. The expression of MT1-MMP mRNA was down-regulated with an inhibitory rate of 36%, in comparison with that in negative control group. At the 48th hour after transfection with pasMMP14, the activity of gelatinase A was dramatically inhibited. After 72 hours, cell proliferation was significantly decreased as compared with that in pcDNA3.0 group and normal control group （t = 2.358, P 〈 0.01; t = 2.727, P 〈 0.01）. Transwell invasion assay showed that the invasive property was greatly suppressed in pasMMP14-transfected group as compared with that in pcDNA3.0 group and normal control group （t = 5.744, P 〈 0.01; t = 5.695, P 〈 0.01）. CONCLUSION： Antisense RNA can evidently inhibit the gene expression of MT1-MMP and invasiveness of gastric cancer cells, suggesting that MT1-MMP gene may be a molecular target of anti-invasion therapy for gastric cancer.