Study On The Effects Of MiR-1-3p/206/613 On OATP1B1 Regulated By LXR? And Its Mechanism

Background:Our research group have found that miR-206 and miR-613 could bind to the 3'-UTR of OATP1B1 mRNA,thereby inhibiting the expression of OATP1B1 protein.It has been reported that nuclear receptors such as PXR,CAR,FXR and LXR? could mediate the transcriptional regulation of OATP1B1.MiRWalk and other bioinformatic softwares were used to predict and screen miRNAs that targeted to the 3?-UTR of LXR? and OATP1B1 mRNA.The results showed that miR-1-3p/206/613 were highly complementary with LXR? and OATP1B1 mRNA 3?-UTR.Whether mi R-1-3p/206/ 613 can regulate the expression of OATP1B1 through targeting LXR? mRNA 3?-UTR is indeed worth considering;it is also of concern that miR-1-3p/206/613 may have a direct effect on OATP1B1 mRNA 3?-UTR;the effect of miR-1-3p/206/613 on OATP1B1 regulated by LXR? and study of its mechanism can add new content to the study of the regulation of drug transporter OATP1B1.Objectives:1.To study the effect of nuclear receptor LXR? on the transcriptional regulation of the transporter OATP1B1 and its mechanism.2.To investigate the effects of miR-1-3p/206/613 on the expression of LXR?,OATP1B1 genes and proteins,and the effects on the transporting function of OATP1B1,and to further explore the molecular mechanisms underlying the above effects deeply.Methods:1.Bioinformatics databases including miRWalk,TargetScan and miRanda were used to predict miRNAs that can target at PXR,CAR,FXR,LXR? and OATP1B1 mRNA 3'-UTR.2.HLXR? eukaryotic expression plasmid(pTracer-hLXR?)and OATP1B1 promoter reporter plasmid(pGL3-OATP1B1)were constructed,double luciferase reporter assays were used to investigate the effect of LXR? on p GL3-OATP1B1 luciferase activity to explore the mechanism of LXR? in regulating OATP1B1.3.We constructed the Hep G2 cell models overexpressing or inhibitingmiR-1-3p /206/613 by transiently transfecting miR-1-3p,miR-206,miR-613 mimics or inhibitors into HepG2 cells.Finally using RT-qPCR to verify whether the HepG2 cell models were successful.4.To investigate the effects of miR-1-3p/206/613 on the expression of LXR? and OATP1B1 in HepG2 cells,RT-qPCR method and Western blotting were used to detect the mRNA and protein levels of LXR? and OATP1B1 respectively.5.The content of rosuvastatin in HepG2 cells was detected by LC-MS/MS method to investigate the effects of miR-1-3p/206/613 on the transporting function of OATP1B1.6.Constructing LXR? mRNA 3?UTR wild type reporter plasmid(pGL/LXR?-WT)and miR-1-3p/206/613 binding site mutant reporter plasmid(p GL/LXR?-Mut)and then double luciferase reporter assays to compare the difference in luciferase activity before and after mutation to find out the exact site of miR-1-3p/206/613 binding to LXR? mRNA 3'-UTR;as above,the exact site of miR-1-3p/206/613 acting on OATP1B1 mRNA 3'-UTR was ascertained;the molecular mechanisms of miR-1-3p /206/613 regulating LXR? and OATP1B1 expression were elucidated.7.Data processing and statistical analysis:the data obtained from the experiments were expressed as mean�SD,and using Graphpad Prisms 5.0,Microsoft Excel and Image J softwares to gragh and SPSS23.0 software was used for statistical analysis.The two groups were compared using Student?s t-test and one-way ANOVA was used between groups,P<0.05 indicates that there were statistical significances between groups.Result:1.MiRWalk,TargetScan and miRanda,the three bioinformatics databases,predicted that miR-1-3p/206/613 bind to LXR? and OATP1B1 mRNA 3?-UTR with high specificity,conservation and binding stability.2.The relative luciferase activity of the experimental group was about 2.6 times that of the control group.3.miR-1-3p/206/613 mimics could upregulate their miRNA expression levels by 1069.04,1228.99 and 1157.48 folds respectively;miR-1-3p/206/613 inhibitors significantly reduced their miRNA expression levels to 0.31,0.28 and 0.28 times of the control group with a statistically significant difference(P<0.05).It was demonstrated that the HepG2 cell models overexpressing or inhibiting the expression of miR-1-3p/206/613 were successfully constructed.4.The results showed that miR-1-3p/206/613 mimics significantly downregulated the mRNA levels of LXR? to 0.40,0.45 and 0.51 times of the control group,miR-1-3p/206/613 inhibitors significantly up-regulated the mRNA levels of LXR? to 5.26,4.51,3.97 fold of the control group respectively with a statistically significant difference(P<0.05).The mRNA levels of OATP1B1 in mi R-1-3p/206/613 mimics groups were 1.04,1.10 and 1.03 times of the control group,that in miR-1-3p/206/613 inhibitors groups were 1.09,0.98 and 1.01 times of the control group,therefore whether miR-1-3p/206/613 mimics or inhibitors had no direct effects on the expression of OATP1B1 mRNA and the differences were not statistically significant(P>0.05).5.MiR-1-3p/206/613 mimics significantly down-regulated the levels of LXR? and OATP1B1 proteins,compared with the control group,the levels of LXR? proteins were downregulated by 16.5%,16.0% and 25.1% respectively and OATP1B1 proteins were down-regulated by 30.4%,30.5% and 44.4%.MiR-1-3p/206/613 inhibitors significantly up-regulated the expression of LXR? and OATP1B1 proteins,LXR? proteins were upregulated by 13.3%,13.3%,and 16.0%,OATP1B1 proteins were upregulated by 25.0%,25.6% and 30.4%,respectively.The differences above were statistically significant(P<0.05).6.When the concentration of RSV was 5?M,miR-1-3p/206/613 mimics down-regulated the uptakes of RSV to 0.50,0.19 and 0.30 times of the control group respectively in Hep G2 cells;when the concentration was 60?M,the uptakes of RSV were down-regulated to 0.49,0.24 and 0.23 times of the control group;when the concentration of RSV was 125?M,the uptakes of RSV were down-regulated to 0.64,0.48 and 0.31 times of the control group.In contrast,miR-1-3p/206/613 inhibitors up-regulated the uptake of RSV in HepG2 cells to 1.26,1.59 and 2.07 times of the control group when the concentration of RSV was 5?M;when the concentration was 60?M,the uptakes of RSV were increased to 1.97,2.44 and 2.63 times of the control group;at a concentration of 125?M,that were up-regulated to 2.22,2.86 and 2.93 times of the control group,and the differences were statistically significant(p<0.05).7.MiR-1-3p/206/613 mimics significantly inhibited the luciferase activities of pGL/LXR?-WT reporter gene and that activities were decreased by 38.5%,32.5% and 32.8% compared with the control group respectively;miR-1-3p/206/613 inhibitors significantly activated the luciferase activities of the pGL/LXR?-WT reporter gene by 137%,75.8% and 55.7% compared with the control group respectively and the differences were statistically significant(P<0.05);while in the pGL/LXR?-Mut reporter gene system where the binding sites were mutated,miR-1-3p/206/613 mimics or inhibitors had no obviously effect on that luciferase activity(P>0.05).It suggested that miR-1-3p/206/613 can bind to LXR? mRNA 3?-UTR to inhibit the luciferase activity.8.As above,miR-1-3p/206/613 mimics significantly inhibited the luciferase activities of the pGL/OATP1B1-WT reporter gene by 24.3%,28.9% and 32.1% respectively when compared with the control group;miR-1-3p/206/613 inhibitors significantly activated the luciferase activities of the pGL/OATP1B1-WT reporter gene by 33.5%,48.3% and 61.6% with statistically significant differences(P<0.05);whereas in the pGL/OATP1B1-Mut reporter gene system where the binding sites were mutated,miR-1-3p/206/613 mimics or inhibitors had no obvious effects on that luciferase activity either(P>0.05).It was suggested that miR-1-3p/206/613 can bind to OATP1B1 mRNA 3'-UTR to inhibit the luciferase activity.Conclusion:1.The HepG2 cell models over-expressing or inhibiting the expression of miR-1-3p/206/613 in this study had good reproducibility.2.Nuclear receptor LXR? could participate in the transcriptional regulation of the transporter OATP1B1.3.MiR-1-3p/206/613 could regulate the expression and transporting function of OATP1B1 by directly targeting the OATP1B1 mRNA 3'-UTR.4.MiR-1-3p/206/613 could indirectly regulates the expression and transporting function of OATP1B1 by targeting the LXR? mRNA 3?-UTR.