인간 배아 피부 섬유모세포주에서 Matrix Metalloproteinase 2와 9의 발현에 대한 HPV 16 E6 단백질의 역할
(The) Role of HPV16 E6 Protein on MMP 2 and MMP 9 Expression in Human Embryo Skin Fibroblast Cell
인간 배아 피부 섬유모세포주 Matrix Metalloproteinase 2;
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Objective : The purpose of this study was to evaluate the role of HPV16 E6 protein on the expression of MMP 2 and 9, important proteins associated with tumor invasion and metastasis, in human embryo skin fibroblast cell. Materials and methods : HPV16 E6 ORF was obtained by PCR from CaSki cell and was cloned into mammalian expression vector, pT_(ARGE)T. Negative control vector was constructed with self ligated pT_(ARGE)T. After confirmation of plasmid with color selection and restriction enzyme digestion, pT_(ARGE)T-E6 and negative control vector were transfected into Detroit 551. On post-transfection third day, supernatant of both transfected cell were harvested and quantitative analysis was carried out with bradford method. Zymographic analyses were run in parallel. Samples corresponding to 11㎍ of total proteins were migrated on 8% SDS-PAGE copolymerized with 8mg/mL of gelatin as substerate under non-reducing condition. After renaturation and development, gel was fixed and stained in 0.5% Coomassie Brilliant Blue R250 for 3 hours and then destained for 24 hours. Areas of gelatinolytic activity were detected as clear bands on blue background and analyzed with computerized program. Results : PCR product of HPV16 E6 ORF, 477 bp, was confirmed on 2% agarose gel electrophoresis. After restriction enzyme digestion of palsmid with EcoR Ⅰ and Puv Ⅱ, correctly cloned vectors were confirmed on 1% agarose gel electrophoresis by desired bands. Zymogram followed by densitometric analysis was revealed the increased expression of MMP 2 zymogen(72kDa) and active form(67kDa) by 10.5% and 36.3%, respectively. On the contrary, MMP 9 expression was decreased by 0.21% on densitometric analysis. Conclusion : HPV16 E6 protein has influenced on MMP 2 over-expression.