Green tea catechins increase cholesterol 7a-hydroxylase mRNA and promoter activity in HepG2 cell

Abstract

본 실험에서는 녹차의 카테킨에 의하여 cholesterol 7α-hydroxyase (CYP7A1) 유전자의 전사가 조절되는 기전을 규명하기 위하여 human hepatocarcinoma cell line인 HepG2 cell을 이용해서 녹차 카테킨 처리 후 human CYP7A1 유전자 발현 및 promoter의 활성을 측정하였다. HepG2 cell에 카테킨 종류별로 5μM 농도로 처리한 후 40시간 배양하여 총 RNA를 추출하여 CYP7A1의 mRNA 발현율을 Real time PCR을 이용해서 측정한 결과 대조군에 비해서 ECG (5배) > EGCG (4배) > EGC (3배) > EC (2배) 순으로 발현이 증가되었다. EGCG를 0, 0.1, 1, 5, 10, 20μM 농도별로 처리한 결과 EGCG 5, 10μM에서 발현율이 약 4배가 증가하였다. CYP7A1 promoter를 HepG2 cell의 genomic DNA를 주형으로 하여 PCR로 증폭시킨 후 PGL3-basic vector에 재조합 하였다. 재조합된 DNA를 HepG2 cell에 transfection 시킨 후 catechin의 종류별로 처리하여 40시간 배양시킨 후 promoter 활성을 측정한 결과 대조군에 비해 ECG (6배) > EGCG (4배) > EGC (3배) > EC (2배) 순으로 promoter 활성을 증가시키는 것으로 나타났다. EGCG를 0, 0.1, 1, 5, 10, 20μM 농도별로 처리한 결과EGCG 5~10μM 처리군에서 promoter 활성이 약 4~5배 증가하였다. Promoter의 활성과 mRNA 발현이 비슷한 정도로 증가함을 나타내 CYP7A1은 녹차의 카테킨에 의해서 전사가 조절되며 이는 promoter 활성에 영향을 주기 때문으로 추측된다. -1312 bp, -311 bp, -92 bp CYP7A1 promoter를 제작하여 deletion study를 실시해 본 결과 -1312 CYP7A1 promoter만이 유일하게 EGCG에 의한 활성의 증가를 보여 EGCG에 의해서 전사 조절의 영향을 받는 위치는 -1312 bp와 -311 bp 사이에 위치하는 것으로 사료된다. -311 bp CYP7A1 promoter의 활성은 -1312 CYP7A1 promoter에 비하여 3~4배 높게 나타났는데 이것은 -311 bp의 upstream에 repressor sequence가 존재하고 downstream에는 activator sequence가 존재하기 때문으로 사료된다. 결론적으로 본 연구를 통해서 EGCG는 CYP7A1 promoter의 -1312 bp 와 -311 bp 사이에 작용하며 CYP7A1의 mRNA의 발현율을 증가시켜줌으로써 콜레스테롤 강하효과를 보여주는 것으로 사료된다.;In the present study, to find molecular mechanism of CYP7A1 by catechins of green tea, we investigated the effect of catechins on the CYP7A1 mRNA level and promoter activity. For this purpose we used human hepatocellular blastoma cells called HepG2 in vitro system. After EGCG, EGC, ECG or EC treatment of 5μM concentration in HepG2 cells were incubated for 40 hr, total RNA was extracted. mRNA expression of CYP7A1 was estimated by using Real time PCR. The result of EGCG, EGC, ECG or EC treatment was shown in the order ECG (5-folds) > EGCG (4-folds) > EGC (3-folds) > EC (2-folds) compared to control. HepG2 cells were treated with EGCG of 0, 0.1, 1, 5, 10, 20 μM concentration. 5, 10 μM EGCG treatment increased the mRNA expression superlatively by approximately 4-fold compared to untreated with EGCG. The human CYP7A1 promoter was made by polymerase chain reaction(PCR) using genomic DNA isolated from HepG2 cells as a template. The human CYP7A1 promoter was recombinated with pGL3-basic vector. To analysis promoter activity, HepG2 cells were treated with EGCG, EGC, ECG or EC of 5μM concentration for 40 hr after transfected with the recombinated DNA. The data were shown in the order ECG (6-folds) > EGCG (4-folds) > EGC (3-folds) > EC (2-folds) compared to control. HepG2 cells were treated with EGCG of 0, 0.1, 1, 5, 10, 20 μM concentration. 5, 10 μM EGCG treatment increased promoter activity superlatively by approximately 4, 5-folds compared to untreated with EGCG. The activity of promoter was elevated by EGCG treatment similarly with mRNA. From these results, it can be guessed that the regulation of CYP7A1 gene expression involved the regulation of promoter activity. In order to define the promoter region which mediated the hypocholesterolemic effect by EGCG, the CYP7A1 promoter (-1312 bp, -311 bp, -92 bp) was recombinated with reporter gene. HepG2 cell transfected with CYP7A1 promoter was treated 5μM concentration of EGCG. The effect of EGCG on the activity of -1312 bp CYP7A1 promoter was significant but -311 bp, -92 bp CYP7A1 promoters were not affected by EGCG treatment. Therefore it can be postulated that the promoter region involved in the EGCG action may be located between -1312 bp and -311 bp of the CYP7A1 promoter. As compared to -1312 CYP7A1 promoter, regardless of EGCG treatment, the activity of -311 bp CYP7A1 promoter was increased by approximately 3~4 fold. It can be thought that the repressor region may be located upstream of -311 bp and activator region downstream of -311bp. In conculusion, EGCG may stimulate CYP7A1 promoter between -1312 bp and -311 bp and induce up-regulation of CYP7A1 mRNA. Through this mechanism, EGCG may have hypocholesterolemic effect finally.