| Research background and purposeDrug metabolism and disposal are mediated by a series of drug-metabolizing enzymes(DMEs)and drug transporters expressed in the liver and intestine.Their tissue expression abundance is regulated transcriptionally by specific nuclear receptors(NRs).These nuclear receptors are Ligand-activated transcription factors.Cytochrome P450 is the most important drug metabolizing enzyme in humans,and more than 90%of clinical drug interactions are caused by changes in the activity of cytochrome P450(CYP450 enzyme).Studies have shown that there are significant individual differences in the expression and activity of metabolic enzymes,and this huge difference of up to several dozen times causes severe drug interactions and adverse reactions in the clinic.Therefore,elucidating the regulatory mechanisms of CYPs expression and identifying the main causes of the individual differences in CYPs activity have important implications for the promotion of personalized medicine and precise medical treatment.The expression of CYPs is influenced by both genetic and environmental factors.Previous studies have focused on genetic factors,but CYPs polymorphisms,due to their low frequency of mutation and apparent racial differences,do not fully explain and predict individual differences in CYPs expression and activity.In recent years,with the deepening of research,epigenetic factors have attracted more and more attention to the regulation of drug metabolism enzymes.Epigenetics refers to genetic changes that occur when DNA sequences do not change,that is,genetic material other than DNA sequences changes.Epigenetic regulation mainly includes histone modifications,DNA methylation,and non-coding RNAs(ncRNAs).Studies have shown that histone modifications,DNA methylation and microRNAs can regulate the expression of CYPs.However,studies on the regulation of CYPs expression by long-noncoding RNAs(lncRNAs)have not been reported.To investigate whether lncRNAs are involved in the regulation of CYPs expression,this study was intended to:(1)investigate the genetic conservation of HNF1A and HNF1A-AS1.2 HNF1A was correlated with the expression of nuclear receptors and CYPs.3 Based on the lncRNAs that can be regulated by HNF1A screened by previous chips of this group,and further correlates the expression of lncRNAs with nuclear receptors and CYPs in liver tissue samples;this study first described individuals expressing CYPs from the perspective of lncRNA regulation.The differential epigenetic regulation mechanism provides a basis for the establishment of molecular markers that can evaluate and predict CYPs individual differences in order to achieve the goal of guiding clinical safe and rational drug use.Research methods1.Forty-nine human liver tissue specimens were collected from patients admitted to the First Affiliated Hospital of Zhengzhou University.The main source of liver tissue is liver normal tissue obtained by partial hepatectomy in patients with common bile duct stones,gallbladder cancer,hepatic hemangiomas,and liver transplantation liver repair.Extracts total liver RNA and protein.2.The expression levels of HNF1A,HNF1A-AS1,PXR,CAR,AHR and CYPs in human liver tissue were detected by Quantitive Real-time PCR.3.Western Blot was used to detect the expression of HNF1A and CYPs in human liver tissue.Correlation analysis of HNF1A-AS1 expression with CYPs mRNA expression,CYPs protein expression,and PXR,CAR,AHR,HNF1A and other nuclear receptor mRNAs to verify the chip results.4.Statistical analysis:Statistical analysis was performed using IBM SPSS21.0software.All data were expressed as mean±standard deviation(Mean±SD).An independent sample t-test was used to analyze the differences between the two groups;one-way analysis of variance was used to test the differences between the groups,Dunnett’s test was used to analyze the differences between the two groups,and the Spearman test was used for correlation analysis.The difference was considered statistically significant at P<0.05.Results1.Conservative analysis of HNF1A and HNF1A-AS1The HNF1A gene is conserved among chimpanzees,macaques,dogs,cattle,mice,rats,chickens,zebrafish and frogs.The HNF1A-AS1 gene sequence is conserved only in primates and there are no conserved sequences in rodents,possums,platypus,chickens,zebrafish and other species.2.There was a positive correlation between mRNA expression of HNF1A and nuclear receptors in human liver tissues,and there was a highly positive correlation between mRNA expression of HNF1A and PXR(r=0.403,p<0.001)and AHR(r=0.641,p<0.001).There was a low positive correlation between m RNA expression and CAR(R=0.342,p<0.01).3.There was a positive correlation between HNF1A expression and mRNA expression of metabolic enzymes in human liver tissues,with CYP2C8(r=0.368,p<0.001),CYP2C9(r=0.544,p<0.001),CYP2E1(r=0.507).,p<0.001),CYP2C19(r=0.780,p<0.001),CYP3A4(r=0.546,p<0.001)m RNA expression was highly positively correlated with CYP1A2(r=0.369,p<0.05),CYP2B6(The mRNA expression of r=0.332,p<0.05)showed a moderate positive correlation and a low positive correlation with the mRNA expression of CYP2D6(r=0.383,p<0.01).The protein expression of HNF1A was positively correlated with the expression of CYP1A2(r=0.537,p<0.01)and CYP2E1(r=0.337,p<0.01),with CYP2C9(r=0.023,p=0.875)and CYP3A4(Protein expression of r=-0.211,p=0.154)was not correlated.HNF1A-AS1 was positively correlated with CYP1A2,CYP2C9,CYP2E1,CYP2C8,CYP2C19,CYP,CYP3A4 mRNA expressions,which was highly positively correlated with CYP2E1(r=0.486,p<0.001)mRNA expression,and CYP1A2(r).=0.425,p<0.05),CYP2D6(r=0.364,p<0.05),CYP2C19(r=0.350,p<0.05)mRNA expression was positively correlated with CYP2C8(r=0.390,p<0.01),The mRNA expressions of CYP2C9(r=0.415,p<0.01)and CYP3A4(r=0.461,p<0.01)were low correlated,and were not correlated with the mRNA expression of CYP2B6(r=0.098,p=0.516).The expression of HNF1A-AS1 was positively correlated with that of CYP3A4(r=0.425,p<0.05),but the protein expression of CYP1A2(r=-0.150,p=0.307)and CYP2C9(r=-0.299,p=0.992)was negative.Correlation was negatively correlated with protein expression of CYP2E1(r=0.425,p<0.05).5.In human liver tissue samples,the expression of HNF1A-AS1 was highly correlated with the expression of CAR(r=0.596,p<0.001)and PXR(r=0.528,p<0.001),with HNF1A(r=0.451 p<The mRNA of 0.01)showed a low correlation and was not correlated with the mRNA expression of AHR(r=-0.148,p=0.311).ConclusionsThe Lnc-HNF1A-AS1/HNF1A pathway is involved in the regulation of expression of related nuclear receptors and CYPs. |
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