| Background:Micro RNAs(miRNAs) are 19-25 nucleotides of small non-coding RNA molecules that post-transcriptionally regulate the expression of target genes by sequence specific base pairing on the 3'-untranslated region(3'-UTR) of the target mRNAs, resulting in degradation of target mRNAs or inhibition of protein translation. Our previous study which carried out around “micro RNA-PXR-OATP1B1” pathway shows that miR-148 a post-transcriptionally regulate the expression of PXR and indirectly regulate the expression of OATP1B1. With the aid of Bioinformatic analysis such as Targetscan, miRBase, miRanda and RNA Hybrid, it was shown that the seed sequences of miR-206/miR-613 has perfect complementary with 3'-UTR of OATP1B1 mRNA which has relatively high specificity and the secondary structure between miR-206/miR-613 and 3'-UTR of OATP1B1 mRNA was rather stable. Then, the phenomenon has attracted our attention about whether miR-206/miR-613 can regulate the expression of OATP1B1 protein and the mechanism whether are associated with post-transcriptionally regulation. Therefore, this project aims to clarify the effect of miR-206/miR-613 on the expression of OATP1B1 mRNA and protein and its mechanism by using molecular biology techniques, and provide new ideas for research of individual differences in drug response based on the change of OATP1B1 transport activity. Objectives:This project aims to investigate the effect of miR-206/miR-613 on the expression of OATP1B1 mRNA and protein in Hep G2 cells; and deeply explore the molecular mechanism of the effect of miR-206/miR-613 on the expression of OATP1B1 mRNA and protein based on miR-206/miR-613 targeting the 3'-UTR of OATP1B1 mRNA. Methods:1.According to the mode and the minimum free energy of miRNA seed sequence complementarity with 3'-UTR of OATP1B1 mRNA, bioinformatic analysis was used to predict and screen the miRNAs that can target the 3'-untranslated region(3'-UTR) of OATP1B1 mRNA.2.Hep G2 cells were transfected with miR-206/miR-613 mimic or inhibitor respectively. The expression level of miR-206/miR-613 in Hep G2 cells were detected by RT-qPCR and verified whether the Hep G2 cell model with overexpression or inhibition of miR-206/miR-613 was constructed successfully.3.Hep G2 cells were transfected with miR-206/miR-613 mimic or inhibitor respectively. The expression level of OATP1B1 mRNA and protein were detected by RT-PCR and Western blotting and explored the effect of overexpression or inhibition of miR-206/miR-613 on the expression of OATP1B1 mRNA and protein.4. Luciferase assay was used to explore the exact mechanism of the effect of miR-206/miR-613 on the expression of OATP1B1 mRNA and protein. Firstly, the sequence of OATP1B1 mRNA 3'-UTR was cloned into the pMIR-REPORT expression reporter vector as pMIR/OATP1B1-WT plasmids. The pMIR/OATP1B1-WT plasmids were cotransfected with miR-206/miR-613 mimic or inhibitor into HEK293 T cells or Hep G2 cells to detect the effect of miR-206/miR-613 on luciferase activity. Secondly, the site-directed mutagenesis was employed to mutate the binding sites of miR-206/miR-613 conplementary with 3'-UTR of OATP1B1 mRNA and subsequently, to clone, transfect plasmids and to detect the luciferase activity. At last, the differences of the luciferase activity before and after mutation were compared to further analyze the binding site of miR-206/miR-613 to OATP1B15. Data processing and statistical analysis: All values are presented as the mean ± SD. Statistical analyses were carried out using Graph Pad Prism version 5.00. Comparisions between experimental group and control group were carried out using one-way analysis of variance. A value of P<0.05 was considered statistically significant. A value of P>0.05 has no significant difference. Results:1.With the aid of Bioinformatic analysis such as Targetscan, miRBase, miRanda and RNA Hybrid, it was shown that miR-206/miR-613 has high complementarity with 3'-UTR of OATP1B1 mRNA which has relatively high specificity and the secondary structure between miR-206/miR-613 and 3'-UTR of OATP1B1 mRNA was rather stable.2. Hep G2 cells were transfected and treated with miR-206 mimic for 48 h. The relative miR-206 expression level of control group and miR-206 group were 1.00±0.00?1227.85±142.42 respectively and was up-regulated by 1227.85 fold compared with control group. Hep G2 cells were transfected and treated with miR-206 inhibitor for 48 h. The relative miR-206 expression level of control group and miR-206 inhibitor group were 1.00±0.00?0.48±0.13 respectively and was just 0.48 fold compared with control group. In the same way, Hep G2 cells were transfected and treated with miR-613 mimic for 48 h. The relative miR-613 expression level was up-regulated by 935.38 fold compared with control group. Hep G2 cells were transfected and treated with miR-613 inhibitor for 48 h. The relative miR-613 expression level was just 0.67 fold compared with control group. Thus, we have constructed Hep G2 cell model successfully with overexpression or inhibition of miR-206/miR-613.3. Hep G2 cells were transfected with miR-206/miR-613 mimic, the relative expression level of OATP1B1 mRNA was 1.00±0.00?1.29±0.41?1.05±0.32,and there was no significant differences(P>0.05), whereas the relative expression level of OATPB1 protein was 1.00±0.02 ? 0.75±0.02 ? 0.61±0.01 respectively and was down-regulated by 24.7%?38.8% compared with control group and there was statistically significant differences(P<0.05). Hep G2 cells were transfected with miR-206 /miR-613 inhibitor, the relative expression level of OATP1B1 mRNA was 1.0±0.00 ? 1.16±0.16 ? 1.17±0.21 respectively and there was no significant differences(P>0.05), whereas the relative expression level of OATPB1 protein was 1.00±0.06?1.25±0.07?1.38±0.08 respectively and was up-regulated by 25%?38.2% compared with control group respectively and there was statistically significant differences(P<0.05).4.The results of luciferase assay are as follows: The luciferase activity of pMIR/ OATP1B1-WT luciferase reporter gene was significantly decreased when pMIR/ OATP1B1-WT plasmids was cotransfected with miR-206/miR-613 mimic. Relative luciferase activity of each group was 1.00±0.03, 0.65±0.05, 0.70±0.03 respectively and the luciferase activity was decreased by 35%, 30% compared with control group, and there was statistically significant differences(P<0.05). The luciferase activity of pMIR/ OATP1B1-WT luciferase reporter gene was significantly increased when pMIR/ OATP1B1-WT plasmids was cotransfected with miR-206/miR-613 inhibitor. Relative luciferase activity of each group was 1.00±0.04, 1.33±0.12, 1.33±0.12 respectively and the luciferase activity was increased by 33.1%?32.5% compared with control group, and there was statistically significant differences(P<0.05). Conclusions:1.The Hep G2 cell model with overexpression or inhibition of miR-206/miR-613 was constructed by this research. The model has good repeatability for the research from gene and protein levels.2.miR-206/miR-613 exactly regulated the expression of OATP1B1 protein, whereas miR-206/miR-613 did not affect the expression of OATP1B1 mRNA. The results suggested that miR-206/miR-613 may post-transcriptionally regulate the expression of OATP1B1.3.miR-206/miR-613 mimic or inhibitor complementary with 3'-UTR of OATP1B1 mRNA inhibited or induced the luciferase activity of pMIR/ OATP1B1-WT reporter gene.When the binding sites were mutated, miR-206/miR-613 mimic or inhibitor had no effect on the luciferase activity of reporter gene.This suggested that miR-206/miR-613 post-transcriptionally regulate the expression of OATP1B1 through targeting the 3'-UTR of OATP1B1 mRNA. |
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