Wu Ming-Fu, Liao Guo-Ning, Jia Ping, Xi Ling, Lu Yun-Ping, Ma Ding
Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430 030, P.R. China.
Ai Zheng. 2004 Nov;23(11):1263-6.
BACKGROUND & OBJECTIVE: Membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP-14) is a newly discovered enzyme, which plays a key role in tumor metastasis. This study was to observe inhibitory effect of MT1-MMP antisense nucleotide on proliferation and invasive potential of human highly metastatic ovarian carcinoma cell line SW626.
Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify MT1-MMP cDNA fragments with 2 different restriction sites at its 5c-end. RT-PCR products were cloned into plasmid pcDNA3.1 in antisense direction. The recombinant pMMP14as was transfected into SW626 cells. Changes of cell proliferation, MT1-MMP protein expression, activities of MMP-2 and MMP-9, and cell invasion ability were detected by MTT assay, Western blot, optimized gelatin zymography, and matrigel in vitro invasion assay, respectively.
Antisense MT1-MMP eukaryotic expression vector pMMP14as was constructed successfully. After 48-h transfection with pMMP14as, proliferation of pMMP14as-transfected SW626 cells was significantly lower than that of control cells. Compared with control cells, the expression of endogenous MT1-MMP protein in pMMP14as-transfected cells was decreased with a inhibition rate of 65.8%. The activation of proMMP-2 was remarkably inhibited, and the mean invasive cell percentage was (63.3+/-5.8)% in pMMP14as-transfected cells, which was far less than (97.6+/-7.5)% in control cells (P< 0.05).
Both cell proliferation and invasive potential of SW626 cells were inhibited effectively by antisense MT1-MMP, suggesting that MT1-MMP may be a proper molecular target of anti-invasion therapy for human ovarian cancer.
膜型1基质金属蛋白酶（MT1-MMP/MMP-14）是一种新发现的酶，在肿瘤转移中起关键作用。本研究旨在观察MT1-MMP反义核苷酸对人高转移卵巢癌细胞系SW626增殖和侵袭能力的抑制作用。
采用逆转录聚合酶链反应（RT-PCR）扩增MT1-MMP cDNA片段，其5'端带有2个不同的限制性酶切位点。将RT-PCR产物反向克隆到质粒pcDNA3.1中。将重组质粒pMMP14as转染至SW626细胞。分别采用MTT法、蛋白质免疫印迹法、改良明胶酶谱法和基质胶体外侵袭实验检测细胞增殖、MT1-MMP蛋白表达、MMP-2和MMP-9活性以及细胞侵袭能力的变化。
成功构建了反义MT1-MMP真核表达载体pMMP14as。pMMP14as转染SW626细胞48小时后，转染pMMP14as的SW626细胞增殖明显低于对照细胞。与对照细胞相比，pMMP14as转染细胞内源性MT1-MMP蛋白表达降低，抑制率为65.8%。前MMP-2的激活受到显著抑制，pMMP14as转染细胞的平均侵袭细胞百分比为（63.3±5.8）%，远低于对照细胞的（97.6±7.5）%（P<0.05）。
反义MT1-MMP可有效抑制SW626细胞的增殖和侵袭能力，提示MT1-MMP可能是人类卵巢癌抗侵袭治疗的合适分子靶点。
