Chen Gen-Hung, Huang Chun-Hua, Luo Huang-Yao, Jiang Shann-Tzong
Department of Cosmetic Science, Providence University, 200, Chung-Chi Rd., Taichung 43301, Taiwan.
Department of Food & Nutrition, Providence University, Taiwan.
Biotechnol Rep (Amst). 2014 Jul 20;4:5-13. doi: 10.1016/j.btre.2014.07.003. eCollection 2014 Dec.
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%.
根据人基质金属蛋白酶1（MMP1）的mRNA序列（NCBI，NM_002421）设计了三种小双链干扰RNA（506-MMP1、859-MMP1和891-MMP1），每种均包含25 - 26个核苷酸，对人MMP1具有高度特异性。为监测MMP1基因表达，提取了人皮肤成纤维细胞（底特律551，BCRC 60118）的总RNA。通过RT-PCR和PCR反应特异性扩增出一个包含所有三个干扰RNA靶序列的人基质金属蛋白酶1（MMP1）部分序列cDNA，并将三个合成的干扰RNA靶DNA分别克隆到带有绿色荧光蛋白（GFP）的pAcGFP1-N3中。然后将这些报告质粒分别转染到恶性黑色素瘤细胞（MeWo，BCRC 60540）中，48小时后检测GFP。荧光结果表明，859干扰RNA显示出最高的抑制能力（近90%），因此将其转染到MeWo细胞中。根据实时定量PCR和蛋白质免疫印迹法，其沉默MMP1基因表达的能力为85 - 89%。
