Regulatory Mechanism Of Organic Anion Transport Peptide SLCO1B3 Gene

Objective:Organic Anion Transporting Polypeptide 1B3(OATP1B3)is one of the members of the human organic anion transporting polypeptide family.It is mainly distributed on the basal side of hepatocytes and is an important transmembrane uptake transporter.The expression level and function of OATP1B3 are related to the uptake and distribution of drugs in liver cells,and are also related to the occurrence and development of many diseases.This study aims to study the gene regulation mechanism of SLCO1B3(the OATP1B3 encoding gene),and further detect the protein transport function,so as to provide experimental basis for further understanding of drug uptake and distribution by liver transporters and ensuring the effectiveness and safety of clinical medication..Methods:We first constructed the recombinant vectors of SLCO1B3 gene promoter full-length and various segments,and analyzed the key active region of SLCO1B3gene promoter by double luciferase reporter assay.Potential transcription factors are then predicted by bioinformatics.Double luciferase reporter gene assay,EMSA and ChIP assay were used to verify the specific binding of transcription factor to SLCO1B3 gene promoter.Through the construction of transcription factor overexpression vector,chemical synthesis of siRNAs to silence gene expression,Western blot and real time RT-PCR were used to detect the mRNA and protein expression levels of SLCO1B3,and to verify the effect of the overall level of transcription factors on SLCO1B3/OATP1B3.After confirming that c-Jun regulates the transcriptional expression of SLCO1B3,we further treated the cells with overexpression vectors,phosphorylation activators and inhibitors to detect the expression levels of c-jun,p-cjun and OATP1B3 by Western blot.Ibuprofen has been reported to promote the phosphorylation of c-Jun,we further treated cells with ibuprofen to study whether ibuprofen can regulate the expression of OATP1B3 protein by promoting the phosphorylation of c-jun,and then affect the uptake and transport function of OATP1B3(Cefalexin,a specific substrate of OATP1B3,was used as the test object).CCK-8 method was used to detect the half inhibitory concentration(IC₅₀)of ibuprofen and Cefalexin on HepG2 and Huh7 cells.The expression of OATP1B3 protein was detected by Western blot,and the uptake function of OATP1B3 was detected by HPLC-MS.Results:1.The key region of SLCO1B3 promoter-198nt?+40nt was determined by double luciferase reporter gene experiment.2.Specific binding sites of c-jun and HNF1?in-198nt?+40nt region was predicted by Bioinformatics analysis.3.EMSA assay confirmed that c-Jun could specifically bind to the specific recognition sequence of SLCO1B3 promoter in vitro,while HNF1?could not specifically bind to the specific recognition sequence of SLCO1B3 promoter in vitro.4.ChIP assay confirmed that c-jun can interact with the specific sequence of the SLCO1B3 promoter in vivo,while the interaction of HNF1?with the specific sequence of the SLCO1B3 promoter in vivo was not detected.5.Dual-Luciferase Reporter Assay confirmed that overexpression of c-jun increased the activity of the SLCO1B3 gene promoter.On the contrary,silencing the endogenous c-jun reduced the activity of the SLCO1B3 gene promoter.6.Realtime RT-PCR and Western blot confirmed that overexpression of c-jun increased the the expression level of SLCO1B3/OATP1B3 mRNA and protein.On the contrary,silencing endogenous c-jun reduced the level of SLCO1B3/OATP1B3mRNA and protein.The increased phosphorylation of c-jun could significantly up regulate the expression of OATP1B3 protein.7.High-performance liquid chromatography-mass spectrometry assay confirmed that ibuprofen can promote the phosphorylation of c-jun in cells,increase OATP1B3protein level,and further increase the uptake of Cefalexin,a specific substrate of OATP1B3.Conclusion:1.Through the construction the full-length and various segment recombinant vector of SLCO1B3 promoter,confirmed that-198nt?+40nt is the key region of promoter regulation.2.Transcription factor c-jun can specifically bind to the specific binding sequence in the-198nt?+40nt region of the SLCO1B3 promoter in vivo and in vitro.C-jun phosphorylation can significantly up regulate the expression level of OATP1B3 and enhance the transport function.