Jiang Xiaoling, Dutton Charyl M, Qi Wen-Ning, Block Joel A, Garamszegi Nandor, Scully Sean P
Department of Orthopedic Research, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.
J Cell Physiol. 2005 Mar;202(3):723-30. doi: 10.1002/jcp.20162.
Matrix metalloproteinases (MMPs) play a crucial role in tumor cell invasion and metastasis. Expression of MMP-1 has been reported as a prognostic predictor of recurrence in human chondrosarcoma, and studies using human chondrosarcoma cell lines indicate that MMP-1 expression levels correlate with in vitro invasiveness. These observations suggest that MMP-1 activity has a central role in cell egress from the primary tumor at an early step in the metastatic cascade. In this study, siRNA was used to investigate whether knock down of the MMP-1 gene could be used to inhibit invasiveness in a human chondrosarcoma cell line. The inhibitory effect of siRNA on endogenous MMP-1 gene expression and protein synthesis was demonstrated via RT-PCR, Northern blotting, Western blotting, collagenase activity assay, and an in vitro cell migration assay. The siRNA inhibited MMP-1 expression specifically, since it did not affect the expression of endogenous glyceraldehyde phosphate dehydrogenase (GAPDH) nor other collagenases. Most importantly, the siRNA mediated reduction in MMP-1 expression correlated with a decreased ability of chondrosarcoma cells to invade a Type I collagen matrix. The reduction of invasive behavior demonstrated by human chondrosarcoma cells transfected with MMP-1 siRNA and the specificity of this inhibition supports the hypothesis that this metalloproteinase molecule is involved in initiation of chondrosarcoma metastasis.
基质金属蛋白酶（MMPs）在肿瘤细胞侵袭和转移中起关键作用。已有报道称MMP - 1的表达可作为人类软骨肉瘤复发的预后预测指标，并且利用人类软骨肉瘤细胞系进行的研究表明，MMP - 1的表达水平与体外侵袭性相关。这些观察结果表明，MMP - 1活性在转移级联反应的早期阶段，对于肿瘤细胞从原发肿瘤中逸出起着核心作用。在本研究中，使用小干扰RNA（siRNA）来研究敲低MMP - 1基因是否可用于抑制人类软骨肉瘤细胞系的侵袭性。通过逆转录聚合酶链反应（RT - PCR）、Northern印迹法、蛋白质印迹法、胶原酶活性测定以及体外细胞迁移试验，证实了siRNA对内源性MMP - 1基因表达和蛋白质合成的抑制作用。该siRNA特异性地抑制MMP - 1表达，因为它不影响内源性甘油醛 - 3 - 磷酸脱氢酶（GAPDH）的表达，也不影响其他胶原酶的表达。最重要的是，siRNA介导的MMP - 1表达降低与软骨肉瘤细胞侵袭I型胶原基质的能力下降相关。用MMP - 1 siRNA转染的人类软骨肉瘤细胞所表现出的侵袭行为减少以及这种抑制作用的特异性，支持了这一金属蛋白酶分子参与软骨肉瘤转移起始的假说。
