Analysis of human CYP7A1 mRNA decay in HepG2 cells by reverse transcription-polymerase chain reaction.
The conversion of cholesterol to bile acids is the major pathway through which cholesterol is removed from the body. The initial and rate-limiting step in this catabolic pathway is catalyzed by the liver-specific enzyme cholesterol 7alpha-hydroxylase (CYP7A1). The HepG2 cell line has been used as a model to study human CYP7A1. The levels of CYP7A1 mRNA, however, are quite low in this cell line and require the use of poly(A)+ mRNA for detection using standard Northern analysis. As an alternative, we established a reverse transcription-polymerase chain reaction (RT-PCR) assay that can be used to study CYP7A1 mRNA in HepG2 cells. Using RT-PCR, we analyzed the influence of cell culture conditions on CYP7A1 mRNA levels. We observed an increase in CYP7A1 mRNA levels as the density of the cell culture increased. This rise in CYP7A1 was accompanied by a reciprocal drop in the levels of the proto-oncogene c-myc. Since c-myc expression is strongly associated with cell growth status, this inverse relationship suggests that conditions that favor reduced cell proliferation result in higher levels of CYP7A1 expression. We also established the validity of using RT-PCR for the measurement of mRNA decay rates using c-myc and glyceraldehyde-3-phosphate dehydrogenase mRNAs as a model: The same half-life value was obtained for the c-myc mRNA using either Northern analysis or RT-PCR. Using our RT-PCR method we determined that human CYP7A1 mRNA decays with a half-life of 4.6 +/- 0.9 h (n = 8) in HepG2 cells. We show that the protein synthesis inhibitor cycloheximide prolonged the CYP7A1 mRNA half-life, suggesting that translation is required for mRNA decay. Dexamethasone treatment, however, did not alter CYP7A1 mRNA decay rate but it increased CYP7A1 steady-state mRNA levels, suggesting that the effect of this glucocorticoid in HepG2 cells may be transcriptional.
Department of Pharmacology, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
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Andreou
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Prokipcak
D006528:Carcinoma, Hepatocellular; D018929:Cell Culture Techniques; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects / Q000235:genetics*; D003907:Dexamethasone / Q000494:pharmacology; D006207:Half-Life; D006801:Humans; D010940:Plant Proteins; D016133:Polymerase Chain Reaction / Q000379:methods*; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured
C000715470:MAS1 protein, human; D010940:Plant Proteins; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System