| Background:Organic anion transfer peptide 1B1(OATP1B1)is an important uptake transporter located in the liver cell basement membrane,mediating most commonly used clinical drugs across the liver plasma membrane.Due to the lack of understanding of the regulatory mechanism,the drug individual difference based on OATP1B1 has always not been well explained.Therefore,in-depth study of the molecular mechanism of OATP1B1 regulation is of great significance for clinical individualized medication.OATP1B1 regulatory mechanism is relatively complicated,and the pregnane X receptor(PXR)play an important role.PXR is a kind of ligand dependent transcription factors,when it is activated by the ligand,conformation change and go to the nucleus and combined with retinol X receptor(RXR)to form different source dimers,acting on the DNA response elements of downstream target gene promoter,triggered a series of regulation effect.In addition to the ligand activated way,the mechanism of after-transcription level regulation of the miRNA PXR activity cannot be ignored.However,whether the miRNA acts on the PXR that participate in the molecular mechanism of OATP1B1 regulation remains to be further proved.Hence,the exact mechanism of the miRNAs regulate "PXR-OATP1B1" is intended to proven in this study,on this basis,the influence of various factors of the "miRNA-PXR-OATP1B1" transport regulation shaft on that the OATP1B1 substrates fluvastatin intervention the pharmacokinetic and pharmacodynamics of hyperlipidemia patients.Using quantitative pharmacology research method to establish fluorine laval statin PK/PD model of intervention in patients with hyperlipidemia.The PK/PD model of fluvastatin intervention hyperlipidemia patients is intended to build by quantitative pharmacology research method.And various factors ?s affect weight will be analysised.population pharmacokinetic and concentration effect relationship of fluvastatin on the hyperlipidemia patients will be discussed.The scientific basis of clinical individualized medication of fluvastatin will be expect to provided at last.Methods and results:1.The influence and mechanism research of miR148a/192 post-transcriptional control on PXR-OATP1B1 pathwaysMethods:1.Using online software predicted target genes such as Target Scan,miRanda,predicate miRNAs that has potential regulation ability of PXR m RNA 3 ’UTR and high expression in liver cells.2.Build wild type(pmir GLO-PXR-Wt)and mutant(pmir GLO-PXR-Mut)reporter gene plasmid,and using the method of dual luciferase report gene confirm the regulation function of miR148a/192 on PXR m RNA 3‘UTR.3.By TR-QPCR and Western blotting method,further investigate that miR148 a /192 in Hep G2 cells influence on the expression of PXR,OATP1B1.4.Set up PXR silent Hep G2 cell model,to investigate the influence of miR148a/192 on the expression of OATP1B1 m RNA in the model,and disscuse the mechanism of the influence of miR148a/192 on OATP1B1.5.Statistical methods: the measurement data was showed in the form of mean ±standard deviation(mean±SD),using single factor variance analysis to comparate differences between groups,the tests were all two-sided test,and the difference is regarded as statistically significant when P < 0.05.Results:1.Target Scan and miRanda can predicate that complementary pairing combination of miR-148a/192 and PXR m RNA 3’-UTR,With high specificity,conservative,and combine stability.2.The results of dual luciferase report gene show that,over expression of miR-148a/192 can significantly inhibit pmir GLO-PXR-Wt report gene luciferase activity,and compared with the control group luciferase activity fell 42% and 36%respectively,the difference is statistically significant.Suppressing expression of miR-148a/192 can significantly activate pmir GLO-PXR-Wt report gene luciferase activity,and compared with the control group luciferase activity rise 170% and 120%respectively,the difference is statistically significant.However,in reporter gene system(pmir GLO-PXR-Mut)with PXR m RNA 3’-UTR miR-148a/192 binding site mutation,Over or Suppressing expression of miR-148a/192 has no influence on the report gene luciferase activity.3.Compared with the control group,miR-148a/192 mimic works on Hep G2 cells after 48 h can obviously increase the expression level of corresponding miRNA in the cell.The expression level of miR-148 a was increased by 14.2 times after 20 n M miR-148 a mimic treatment.The expression level of miR-192 was increased by 17.2times after 20 n M miR-192 mimic treatment.Mi R-148a/192 inhibitor works on Hep G2 cells after 48 h can obviously suppress the expression level of corresponding miRNA in the cell.The expression level of miR-148 a was reduced by 82.6% after 100 n M miR-148 a inhibitor treatment.The expression level of miR-192 was reduced by85.2% after 100 n M miR-192 inhibitor treatment.There was statistically significant difference in the above results,prompting that the cell model that over or suppressing expression of miR-148a/192 was build successfully.4.miR-148 a mimic and inhibitor works on Hep G2 cells after 48 h can obviously regulate the expression level of OATP1B1 m RNA and the influence on PXR m RNA is weak.Compared with the control group,20 n M miR-148 a mimic can reduce the expression level of PXR m RNA by 25%(P <0.05),and reduce the expression level of OATP1B1 m RNA by 65%(P <0.05).However,100 n M miR-148 a inhibitor can increase the expression level of PXR m RNA by 10%(P >0.05),and increase the expression level of OATP1B1 m RNA by 110%,and the difference is statistically significant(P <0.05).Similar to the regulation of miR-148 a,compared with the control group,20 n M miR-192 mimic can increase the expression level of PXR m RNA by 10%(P >0.05),and reduce the expression level of OATP1B1 m RNA by55%(P<0.05).100 n M miR-192 inhibitor can increase the expression level of PXR m RNA by 14%(P >0.05),and increase the expression level of OATP1B1 m RNA by50%,and the difference is statistically significant(P <0.05).5.miR-148 a mimic and inhibitor works on Hep G2 cells after 48 h can obviously regulate the expression level of PXR and OATP1B1 protein.Compared with the control group,20 n M miR-148 a mimic can reduce the expression level of PXR protein by 29%,and reduce the expression level of OATP1B1 protein by 24%.100 n M miR-148 a inhibitor can increase the expression level of PXR protein by 45%,and increase the expression level of OATP1B1 protein by 31%,and the difference is statistically significant(P <0.05).Compared with the control group,20 n M miR-192 mimic can reduce the expression level of PXR protein by 29%,and reduce the expression level of OATP1B1 protein by 26%.100 n M miR-192 inhibitor can increase the expression level of PXR protein by 50%,and increase the expression level of OATP1B1 protein by 30%,and the difference is statistically significant(P <0.05).6.In PXR silent Hep G2 cell model,compared with the mimic control,the expression level of OATP1B1 m RNA changed obviously by miR-148a/192,prompting that miR-148a/192 works on OATP1B1 may mediated by PXR.2.The distribution study of PXR,OATP1B1 genetic polymorphism and miR-148a/192 in Chinese han populationMethods:1.Subjects:Healthy subjects were screen from the second affiliated hospital of nanchang university Ⅰperiod clinical trial center during January 2016 to June 2017.Hyperlipidemia patients were screen from nanchang first hospital and the jiangxi province people’s hospital during December 2014 to November 2015.2.Build up the detection method of the expression level of miR-148a/192 in blood plasma,then investigate and compare the distribution of the expression level of miR-148a/192 in blood plasma in the healthy subjects and hyperlipidemia patients,and analyze the connection between the expression level of miR-148a/192 and hyperlipidemia.3.Build up PXR,OATP1B1 genotyping method,then investigate and compare the gene polymorphism of PXR 7635G>A,PXR 24381C>A,OATP1B1 388A>G,OATP1B1 521T>C in the healthy subjects and hyperlipidemia patients,and analyze the connection between the PXR,OATP1B1 genetic polymorphism and hyperlipidemia.4.Statistical methods: The data were analyzed by Shapiro-Wilk normality test.Measurement data comparing with independent samples t test,single factor analysis of variance,or nonparametric rank sum test.Count data,such as genotype frequency and allele frequency compared with chi-square test,the distribution of genotype frequency in the general population with Hardy Weinberg equilibrium test.Results:1.The expression level of miR-148 a and miR-192 in healthy subjects‘ blood plasma were separately 11.78±4.65,14.01±4.19,and the miR-148 a expression level was significantly higher than miR-192,that the difference was statistically significant;The two groups of data grouped by quartile,respectively,miR-148 a quarterback groups relative expression level of △Ct were respectively 5.58±1.99,10.51±0.86,13.48±0.81 and 17.56±2.12.miR-192 quarterback groups relative expression level of△Ct were respectively 8.70±1.43,12.30±1.12,15.73±0.89 and 19.31±1.58.Each quarterback groups was statistically significant difference(P<0.05).2.The expression level of miR-148 a and miR-192 in hyperlipidemia patients‘blood plasma were separately 10.18±4.27,13.71±4.09,and the miR-148 a expression level was significantly higher than miR-192,that the difference was statistically significant;Compared with healthy subjects,the expression level of miR-148 a in hyperlipidemia patients‘ blood plasma was Significantly improved,and the difference was statistically significant;the expression level of miR-192 was slightly higher than the healthy subjects,and there was no statistically significant difference.3.Four SNP sites of the experiment test were still in the Hardy Weinberg equilibrium(P > 0.05),and sample genetic was homeostasis,suitabled for the group correlation analysis.4.PXR 7635 G>A genotype and allele distribution: The detection rate of G/G,G/A,A/A in hyperlipidemia patients was respectively 19.2%,36.5% and 44.2%,and the detection rate in healthy subjects was respectively 44.8%,44.0% and 11.2%,and the difference was statistically significant;The distribution frequency of allele G and A in hyperlipidemia patients was respectively 37.5% and 62.5%,and the distribution frequency in healthy subjects was respectively 61.5% and 33.2%,and the difference was statistically significant.5.PXR 24381 C>A genotype and allele distribution: The detection rate of C/C,C/A,A/A in hyperlipidemia patients was respectively 9.6%,46.2% and 44.2%,and the detection rate in healthy subjects was respectively 9.5%,31.0% and 59.5%,and there was no statistically significant difference;The distribution frequency of allele C and A in hyperlipidemia patients was respectively 32.7% and 67.3%,and the distribution frequency in healthy subjects was respectively 25.0% and 75.0%,and there was no statistically significant difference.6.OATP1B1 388 A>G genotype and allele distribution: The detection rate of A/A,A/G,G/G in hyperlipidemia patients was respectively 82.5%,17.3% and 0.0%,and the detection rate in healthy subjects was respectively 66.5%,31.9% and 2.6%,and there was no statistically significant difference;The distribution frequency of allele A and G in hyperlipidemia patients was respectively 91.3% and 8.7%,,and the distribution frequency in healthy subjects was respectively 81.5% and 18.5%,and the difference was statistically significant.7.OATP1B1 521 T>C genotype and allele distribution: The detection rate of T/T,T/C,C/C in hyperlipidemia patients was respectively 84.6%,11.5% and 3.8%,and the detection rate in healthy subjects was respectively 72.4%,24.1% and 3.4%,and there was no statistically significant difference;The distribution frequency of allele T and C in hyperlipidemia patients was respectively 90.4% and 9.6%,and the distribution frequency in healthy subjects was respectively 84.5% and 15.5%,and the difference was statistically significant.8.The correlation analysis of PXR 7635 G > A and blood lipid level found that,the TG level of AA gene carriers was Slightly higher than the G allele carriers,but there was no statistically significant difference.And there was also no statistically significant difference on other blood lipid index.The blood lipid level of different OATP1B1 388 A > G genotype in hyperlipidemia patients and healthy subjects also has no statistically significant difference.3.The study of fluvastatin PK/PD in hyperlipidemia patients based on the“miRNAs-PXR-OATP1B1” pathways Methods:1.Study design: Further study the 52 hyperlipidemia patients in part 2,those patients were randomly assigned to the fluvastatin 40 mg and 80 mg group according to the proportion of 1:1.Two tubes upper limb venous blood in empty stomach patients are collected at the day before the medicine,14 th,21th,28 th day after taking this medicine,and the one used to test blood lipid,the other for test fluvastatin plasma concentration.2.Build up population pharmacokinetic model: Collect participants’ demographic information collection,data of dosage regimen,vital signs and laboratory data,and establish the PKK data set by Microsoft Office Excel 2003.Based on fluvastatin plasma concentration data,select a suitable model from one,two,and three compartment model,then comparing the objective function value and the model of goodness of fit,and choose the best performers for structural model.Build up fluvastatin PPK model using NONMEN? and PDx Pop? software,the influence of 49 factors,such as gender,age,height,weight,drug combination,dosage,creatinine clearance,on fluvastatin population pharmacokinetic are inspected.Concomitant variable is screened by forward screening and backward stripping.The final PPK model was formed after concomitant variable was bring into,model validation through sampling 500 times by Bootstrap,And further evaluate the goodness of fit of fluvastatin PPK model by visual prediction(VPC).Results:1.In population pharmacokinetic database of this study,52 hyperlipidemia patients and 112 effective blood drug concentration datas are included.2.Using one compartment model describe the pharmacokinetic after hyperlipidemia patients oral fluvastatin,and the interindividual variability of the compartment model parameter conform to multiplication model,and the idiovariation of blood drug concentration conform to scale model.There are 6 concomitant variables including the expression level of OATP1B1 388A>G,OATP1B1 521T>C,PXR 7635G>A,miR-148 a,salviae miltiorrhizae drug combination,age bring into model.The regression equation of final population pharmacokinetics model is:Conclusion:1.miR-148 a and miR-192 can be targeted on the PXR m RNA 3’-UTR,and inhibit its translation process,thus reduce its protein expression level,and reduce the expression of the OATP1B1.2.The exrpesssion of miR-148 a and miR-192 in human plasma were normal distribution and the distribution in plasma between the two exist remarkable individual difference,the mean expression level of miR-148 a was slightly higher than miR-192;the expression of miR-148 a in hyperlipidemia patients plasma was higher than healthy subjects,which indicate that miR-148 a may be associated with increased risk of hyperlipemia;The gene mutation frequency of four SNP sites including PXR7635 G>A,PXR 24381 C>A,OATP1B1 388 A>G,OATP1B1 521 T>C were all greater than 5%,and Conform to the Hardy Weinberg equilibrium,and suitabled for the group correlation analysis.The constituent ratio of PXR 7635 G>A genotype and allele were differences between hyperlipidemia patients and healthy subjects,and the constituent ratio of OATP1B1 388 A>G allele was differences,which indicated that the two site mutation may be associated with the onset of hyperlipidemia. |
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