| Background and Objective Cytochrome P450 3A4(cytochrome P450 3A4,CYP3A4)is the most important subtype of CYPs in the liver,and more than 50% of clinical drugs are metabolized by it.Due to the individual differences of CYP3A4 up to dozens of times,it leads to serious clinical adverse drug reactions and drug interactions.The differential expression of CYP3A4 is affected by both genetic and environmental factors.Previous studies have focused on the effect of genetic polymorphisms on CYP3A4.Due to its low frequency of distribution and ethnic differences,individual differences in CYP3A4 cannot be fully explained and predicted.Long non-coding RNAs(lncRNAs),as an important part of epigenetic regulation,play a key role in the regulation of gene expression.It has been reported that lnc RNAs can act as coactivators in the nucleus to interact with transcription factors to regulate the expression of target genes.The previous research of the research group confirmed that the lnc RNA HNF1A-AS1(the antisense lnc RNA of HNF1A)is involved in regulating the expression of CYP3A4 and transcription factors(HNF1A,HNF4 A,etc.)still not clear.The main research contents of this project: Firstly,the role of HNF1A-AS1 in regulating the expression of CYPs in hepatoma carcinoma cells;Secondly,the molecular mechanism of HNF1A-AS1 regulating the expression of CYP3A4 in hepatoma carcinoma cells.This study provides a new idea for establishing molecular markers for predicting and evaluating individual differences and drug effects of CYP3A4,and also provides a new direction for guiding clinical rational drug use.Methods 1.To explore the role of HNF1A-AS1 in regulating the expression of HNF1A-AS1,transcription factors and CYPs in hepatocytesThe loss-of-function and gain-of-function experiments of HNF1A-AS1,HNF1 A and PXR were used to detect the basal and rifampicin(Rif,10 μM)-induced expression levels of HNF1A-AS1,transcription factors and CYPs by RT-q PCR and Western Blot methods.2.To explore the molecular mechanism of HNF1A-AS1 regulating CYP3A4 expression in hepatocytes(1)RNA in situ hybridization(RNAscope)and nucleocytoplasmic separation experiments were used to clarify the cellular localization of HNF1A-AS1The slides were prepared,and then the RNAscope experiment was carried out;The nuclear and cytoplasmic separation experiments were used to detect the localization of HNF1A-AS1 by RT-q PCR and Western Blot methods.(2)RNA pull down-mass spectrometry and RNA co-immunoprecipitation(RIP)detection of HNF1A-AS1 binding proteinBiotin-labeled RNA probes(sense/antisense)were incubated with Streptavidin magnetic beads and whole cell lysate to form HNF1A-AS1-magnetic bead-protein complexes,which were detected by mass spectrometry to screen HNF1A-AS1 binding proteins.The cell lysate was co-incubated with target protein-specific antibody and A/G beads to form protein-beads-HNF1A-AS1 complex,and the enrichment levels of HNF1A-AS1 was detected by RT-q PCR.(3)Immunofluorescence(IF),RNA in situ hybridization(RNAscope)and nucleocytoplasmic separation experiments were used to clarify the cellular localization of HNF1A-AS1 and PRMT1The slides were prepared,PRMT1 localization was carried out according to the immunofluorescence procedure,and then the RNAscope experiment was carried out;the co-localization of PXR and PRMT1,and the effect of HNF1A-AS1 on the subcellular localization of PRMT1 were observed by laser confocal.The nuclear and cytoplasmic separation experiments were used to detect the localization of HNF1AAS1 and PRMT1 by RT-q PCR and Western Blot methods.(4)Co-immunoprecipitation(Co-IP)assay for protein interactionsProtein interactions were analyzed by Western Blot by performing Co-IP experiments using antibodies specific for PXR/arginine methyltransferase 1(PRMT1)and HNF1A/tri-motif protein 25(TRIM25).Overexpression or knockdown of HNF1AAS1 was used to perform Co-IP experiments to detect the binding between proteins.(5)Chromatin immunoprecipitation(Ch IP)analysis of the effect of HNF1A-AS1 on histone modification levels in the promoter region of CYP3A4The cells were cross-linked 48 h after transfection,and the DNA fragments were sonicated,immunoprecipitated and purified.Ch IP-q PCR was used to detect the H3K4 trimethylation of the PXR binding site(PXRE)in the promoter region of the CYP3A4 gene by HNF1A-AS1(H3K4me3),H4R3 dimethylation(H4R3me2),H3K27 trimethylation(H3K27me3)and H4 acetylation(H4ace)levels and effects on PXR binding.(6)Determination of the effect of HNF1A-AS1 on HNF1 A protein stability and ubiquitination levelsCycloheximide and proteasome inhibition experiments,Western Blot detection of HNF1 A protein expression changes,Co-IP analysis of HNF1 A ubiquitination levels.To clarify whether HNF1A-AS1 affects HNF1 A degradation through the proteasome pathway,and to explore the molecular mechanism of HNF1A-AS1 regulating HNF1 A ubiquitin degradation.Results 1.HNF1A-AS1 is involved in regulating the expression of CYPs and related transcription factors in hepatocytes(1)HNF1A-AS1 is involved in regulating the expression of CYPs and related transcription factorsCompared with the control group,knockdown of HNF1A-AS1 resulted in downregulated m RNA expression of PXR and most CYPs(except CYP1A2)(P<0.05),and Rif induced down-regulated expression of most CYPs(P<0.05),HNF1 A,PXR and CYP3A4 protein expression after overexpression of HNF1A-AS1,the basal expressions of PXR,CYP1A2 and 2E1 were up-regulated(P<0.05),the induced expression of CYP1A2 was increased(P<0.05),and the basic and induced expressions of other CYPs remained unchanged.After overexpression of HNF1A-AS1 in Hep G2 cells,the expression changes of transcription factors were consistent with those in Huh7 cells,and the expression of most CYPs was up-regulated(P<0.05).The results suggest that the endogenous expression of HNF1A-AS1 is required for the regulation of PXR and most CYPs.(2)HNF1A is involved in regulating the expression of HNF1A-AS1,CYPs and transcription factorsThe expression of HNF1A-AS1 was down-regulated with the knockdown of HNF1A(P<0.05).Except for Ah R,the expression of transcription factors decreased(P<0.05);except for the increase of CYP1A2 expression(P<0.05),most CYPs basal and induced The expression was down-regulated(P<0.05).In Huh7 cells overexpressed HNF1 A,the expression of HNF1A-AS1 was upregulated(P<0.05),the expression of Ah R and CYP1A2 was decreased(P<0.05),and the basal and induced expressions of other CYPs were upregulated(P<0.05);Hep G2 cells overexpressed HNF1 A,The m RNA expressions of HNF1A-AS1 and PXR were up-regulated(P<0.05),and the basal and induced expressions of most CYPs were up-regulated(P<0.05).These results suggest that HNF1 A regulates the basal and Rif-induced expression of HNF1A-AS1,transcription factors and CYPs.(3)PXR is located downstream of HNF1A-AS1/HNF1 A and participates in the regulation of CYP3A4 expression in hepatoma cellsKnockdown of PXR down-regulated the m RNA and protein expressions of CYP3A4(P<0.05),while the expressions of HNF1 A and HNF1A-AS1 were unchanged.Overexpression of PXR up-regulated both the m RNA and protein expressions of CYP3A4(P<0.05),but did not affect the expression of HNF1 A.The results suggest that PXR is located downstream of HNF1A-AS1/HNF1 A and is involved in regulating the expression of CYP3A4.2.Molecular mechanism of HNF1A-AS1 regulation of CYP3A4 expression in Huh7 and Hep G2 cells(1)HNF1A-AS1 binds to PRMT1 and affects PRMT1 subcellular localizationRNAscope and nucleocytoplasmic separation experiments showed that HNF1AAS1 was mainly localized in the nucleus;RNA pull down and RIP experiments showed that HNF1A-AS1 was combined with PRMT1;RNA-protein dual localization showed that HNF1A-AS1 and PRMT1 were co-localized in the nucleus,and HNF1A-AS1 was overexpressed promotes nuclear enrichment of PRMT1.The results suggest that HNF1A-AS1 binds PRMT1 and affects its subcellular localization.(2)HNF1A-AS1 mediates the transcriptional regulation of CYP3A4 by PRMT1/PXR pathwayIn Huh7 cells,knockdown of PRMT1 decresaed the m RNA and protein expressions of PXR and CYP3A4(P<0.05).Ch IP-q PCR analysis showed that knockdown of PRMT1 resulted in the binding of PXR in the promoter region of CYP3A4 and the enrichment of histone modifications H3K4me3,H4R3me2 and H4 ace.decreased(P<0.05),while the levels of H3K27me3 increased(P<0.05);Rescue experiments showed that PRMT1 could reverse the increase of CYP3A4 expression caused by HNF1A-AS1 overexpression(P<0.05);PRMT1 knockdown reduced HNF1A-AS1 overexpression and resulted in the levels of H4R3me2,H3K4me3 and H4 ace in the CYP3A4 promoter region were increased(P<0.05).Co-IP and immunofluorescence experiments showed that PRMT1 interacted with PXR,and HNF1A-AS1 promoted the binding of PRMT1 to PXR.The results suggest that HNF1A-AS1-mediated PRMT1 affects PXR binding and histone modification status in the CYP3A4 promoter region.(3)HNF1A-AS1 stabilizes the protein by inhibiting HNF1 A ubiquitinationHNF1A-AS1 knockdown and overexpression did not affect HNF1 A m RNA levels,but affected HNF1 A protein expression;RNA pull down and RIP experiments showed that HNF1A-AS1 binds to HNF1A;CHX experiments confirmed that HNF1A-AS1 affects HNF1 A protein degradation;Western Blot experiments showed that HNF1A-AS1 affects HNF1 A protein stability through the proteasomal degradation pathway;co-immunoprecipitation demonstrated that HNF1A-AS1 inhibits HNF1 A protein ubiquitination;HNF1A-AS1 does not affect TRIM25 expression,and HNF1 A protein levels is up-regulated after TRIM25 knockdown,and TRIM25 knockdown affects HNF1 A ubiquitination levels;Co-IP indicated that HNF1A-AS1 inhibited the binding of HNF1 A and TRIM25 in Huh7 cells.The results suggest that HNF1A-AS1 stabilizes HNF1 A protein by blocking proteasomal degradation.Conclusions 1.The HNF1A-AS1/HNF1A/PXR pathway mediates basal and inducible expression of CYPs in hepatocytes.2.HNF1A-AS1 promotes the interaction between PRMT1 and PXR and achieves PXR transactivation and CYP3A4 transcriptional regulation by affecting histone modifications.3.HNF1A-AS1 inhibits the interaction of HNF1 A with TRIM25,prevents HNF1 A ubiquitination and protein degradation,and then regulates the expression of CYP3A4. |
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