Effect of small interfering RNAs on matrix metalloproteinase 1 expression

Three small double strand siRNAs (506-MMP1, 859-MMP1 and 891-MMP1), each contains 25–26 nucleotides, with high specific to human MMP1 were designed according to mRNA sequence of human MMP1 (NCBI, NM_002421). To monitor the MMP1 gene expression, the total RNAs of human skin fibroblast (Detroit 551, BCRC 60118) were extracted. One human matrix metalloproteinase 1 (MMP1) partial sequence cDNA, included all the three siRNA target sequences, amplified specifically via RT-PCR and PCR reactions, and three synthesized siRNA target DNAs were cloned individually into pAcGFP1-N3 with green fluorescent protein (GFP). These reporter plasmids were then transfected individually into malignant melanoma (MeWo, BCRC 60540) and the GFP was detected after 48 h. Fluorescence results indicated that the 859 siRNA revealed highest inhibitory ability (almost 90%), and was, accordingly, transfected into MeWo cells. According to the real-time quantitative PCR and western blot, the exhibition ability to silence MMP1 gene expression was 85–89%.