Study On The Relationship Between MiRNA - 660, MiRNA - 27b And CYP3A4 Expression In Human Liver Tissue

CYP3A4is one of the most important human phsae I drug metabolism enzymes, accounting for up to60%of the total hepatic cytochrome P450activities. CYP3A4is involved in the metabolism of approximately50%of the commonly prescribed drugs. However, the expression and activity of CYP3A4vary greatly among individuals, influencing both drug responses and disease susceptibility. Both environmental and genetic factors are involved in the regulation of CYP3A4expression, but it hsa been suggested that genetic variability will account for approximately90%of the inter-individual differences in hepatic CYP3A4activity. These genetic variants may contribute to but are unlikely to be the major cause of inter-individual differences in CYP3A4activity, because of the low allele frequencies and limited alterations in enzyme expression or catalytic function. The miRNAs may control posttranscriptional regulation of CYP3A4by directly targeting the3’-untranslated region (3’UTR) of CYP3A4. The miRNA-27b is involved in the negative regulation of CYP3A4expression in vitro. However, questions remain as to whether other miRNAs act directly on CYP3A43’-UTR, and whether miRNA-27b is associated with the negative regulation of CYP3A4in human liver tissues.ObjectiveTo predict the CYP3A4-target miRNAs and verify whether the candidate miRNAs, including miRNA-27b, are associated with the expression of CYP3A4.Methods and results1) Three kinds of software were used to predict the potential miRNAs that directly target the3’-UTR of CYP3A4. A total number of108miRNAs were classified as the candidates involved in the regulation of CYP3A4.2) Based on the high throughout cell-transfection and dual-luciferase reporter assay experiments, a total number of12miRNAs were found to significantly decrease the relative luciferase activity. The miRNA-660was classified as the potential miRNA among the candidates according to the abundance in liver and negative effects on the luciferase activity. Compared with the negative control, the miRNA-660could significantly down-regulate the Renilla luciferase activity of35%.3) A total number of28human liver tissues were collected to extract the total RNA. The expressions of miRNA-660and miRNA-27b were quantitated using LNA-specific primers and SYBR Green q-PCR. General linear regression was used to assess the effects of miRNAs on the expression of CYP3A4.4) The mRNA level of CYP3A4was quantitated by qPCR, western blot was used to quantitate CYP3A4protein in28liver tissues, and HPLC tested the CYP3A4enzyme acitivities. The result shows miRNA-660is negatively correlated with CYP3A4protein and enzyme activity of CYP3A4(p<0.05), but not significantly associated with the mRNA level of CYP3A4(p>0.05). But no correlation was found between miRNA-27b and the mRNA level, or protein, or the enzyme activity of CYP3A4(p>0.05).Conclusions1) The miRNA-660is most likely involved in the posttranscriptional regulation of CYP3A4in human liver by directly targeting the3’-UTR of CYP3A4, and depressing the translation.2) The miRNA-27b is not significantly associated with the regulation of CYP3A4expression in human liver tissues.