Regulation And Mechanism Of MiR-23B In BDE47-induced The Expression Of CYP3A1

Objective:This study aims to determine the critical role and related molecular mechanism of miR-23b in BDE47-induced the expression, activity and function of CYP3A1, which will help to further elaborate the biological toxic effects and adverse health effects. The study will also provide a theoretical basis and technical support for the health risk assessment of environmental pollutants.Methods:To determine the toxic dose of BDE47and its induction of CYP3A1, rat H4ⅡE cell lines which express CYP3A1was used by CCK-8, Western Blot, siRNA interference, CYP3A1inducers (dexamethasone, DEX) treatment and immunofluorescence test. Then through Bioinformatics prediction method and real-time PCR, the target microRNA (miR-23b) was further confirmed. Dual luciferase reporter gene assay was used to clearly the targeting relationships between miR-23b and CYP3A1(rat)/CYP3A4(human). Then microRNA function test (miR-23b overexpression and inhibition test) was used for verification the role of miR-23b on regulation CYP3A1/CYP3A4’s. Finally rats were exposed to Lentiviral-anti-miR-23b and BDE47. Animal samples were detected to determine the level of miR-23b, CYP3A1expression levels and activity changes, and GC-MS was used for detection BDE47level for further validating the role of miR-23b in BDE47-induced the expression and activity of CYP3A1.Results:(1) BDE47dose-dependently decreased the cell viability of H4IIE cells, which was aggravated by DEX (dexamethasone, a typical inducer of CYP3A1). Besides, inhibition of CYP3A1by specific siRNA could block BDE47-induced cytotoxicity. Immunofluorescence and Western Blot experiments results showed that BDE47dose-dependently induced the expression of CYP3A1in either rat liver tissue or H4ⅡE cells, which was enhanced by DEX.(2) With bio informatics software (miRanda-mirSVR, miRBase19, RNAhybrid, miRecords and PITA), targeting relationship between miR-23b and CYP3A1was initially identified. Dual luciferase reporter gene assay results show that, miR-23b are significantly decreased in BDE47-treated cells and liver tissue of rats exposed to BDE47.(3) A series of reporter constructs results showed that miR-23b could reduce the expressions of CYP3A1(rat) or CYP3A4(human) by binding to their3’UTR or CDS region respectively. And by further determination, several specific action sites in CYP3A4CDS region were confirmed, which were located at (+450-+750)、(+1150-+1400)、(+1490-+1710)(4) CYP3A1expression and cytotoxicity of BDE47were decreased24h after transfected with miR-23b mimic in H4ⅡE cells, and were increased24h after transfected with miR-23b inhibitor in H4ⅡE cells.(5) CYP3A1expression and cytotoxicity were decreased24h after transfected with miR-23b mimic in H4IIE cells, and were increased24h after transfected with miR-23b inhibitor in H4ⅡE cells.(6) Rats were treated with BDE47and micro-down lentivirus of specific inhibition expression of miR-23b. By real time PCR detection of rats liver samples, the results showed that miR-23b expression was significantly reduced, and expression and activity of CYP3A1was significantly increased. BDE47distribution and its metabolites were detected in serum and liver samples of treated rats by GC-MS. The results showed that BDE47level was decreased.Three (3-OH-BDE47,4’-OH-BDE49and4-OH-BDE42) of BDE47metabolites levels in serum of treated rats were increased significantly, and BDE47metabolites levels did not change significantly in liver samples of treated rats.Conclusions:(1) BDE47dose-dependently induced the expression of CYP3A1in H4ⅡE cells. CYP3A1could metabolize BDE47to metabolites with more toxic, which in turn affected the cytotoxicity of BDE47.(2) With bioinformatics software and luciferase reporter gene assay, miR-23b could inhibit CYP3A expression though matched with rat or human CYP3A4CDS/CYP3A13’UTR region, suggesting that the important role of miR-23b in regulation the expression of CYP3A.(3) In vitro and in vivo experiments, miR-23b could regulate the expression and activity of CYP3A1, and subsequently affect the BDE47metabolism and the toxic effects as well.