MicroRNA hsa-miR-29a-3p modulates CYP2C19 in human liver cells

Abstract

Cytochrome P450 2C19 (CYP2C19) is involved in the metabolism of many drugs. Extensive studies have demonstrated that genetic variants and endogenous and environmental factors play important roles in the expression of CYP2C19. However, the role of microRNAs (miRNAs) in controlling CYP2C19 expression has not been investigated completely. In the present study, we performed in silico analysis to rank putative miRNA/CYP2C19 hybrids with regards to the predicted stabilities of their duplexes and then we applied a series of biochemical and molecular assays to elucidate the underlying functional mechanisms for the regulation of CYP2C19 by miRNAs. In silico analysis indicated that hsa-miR-23a-3p and hsa-miR-29a-3p target the coding region of CYP2C19 with hybrid stabilities of -27.5kcal/mol and -23.3kcal/mol, respectively. RNA electrophoresis mobility shift assays showed that both hsa-miR-23a-3p and hsa-miR-29a-3p miRNAs were able to bind directly to their cognate targets in the CYP2C19 transcript. Further, a significant inverse correlation was found between chemically-induced up-regulation of hsa-miR-29a-3p and CYP2C19 expression in HepaRG cells. In addition, inverse correlations were also observed in human liver tissue samples between the level of CYP2C19 mRNA expression and both hsa-miR-23a-3p and hsa-miR-29a-3p levels. All these results demonstrated the suppressing role of hsa-miR-29a-3p on CYP2C19 expression.

Keywords: CYP2C19; Drug metabolizing enzymes; Inter-individual variability; Pharmacogenomics; hsa-miR-29a-3p; microRNA.

Fig. 1

The miRNA hsa-miR-29a-3p inhibited exogenous CYP2C19 expression. The pCMV6-CYP2C19 (A), CYP2C19-M1 (B) or CYP2C19-M2 construct (C), was transiently transfected into 293T cells, together with 50 nmol/L miRNA negative control, CYP2C19-specific siRNA, hsa-miR-23a-3p mimic, or hsa-miR-29a-3p mimic, respectively. Cells were harvested 48 h after transfection, and the relative RNA levels and protein levels of CYP2C19 were tested by quantitative real-time PCR and western blot assays, respectively. Three independent transfection experiments were performed with a triplicate manner. Data were shown as percentage relative to CYP2C19 expression by the pCMV6-CYP2C19 vector transfection together with miRNA negative control. *P < 0.05; **P < 0.001; NC, miRNA negative control.

Fig. 2

The miRNA hsa-miR-29a-3p inhibited endogenous CYP2C19 expression in HepaRG cells. The 25 nmol/L miRNA negative control, CYP2C19-specific siRNA, hsa-miR-23a-3p mimic, hsa-miR-29a-3p mimic, inhibitor negative control, or hsa-miR-29a-3p inhibitor were transiently transfected into the differentiated HepaRG cells, respectively. Each assay was carried out in at least 3 independent experiments. *P < 0.05; **P < 0.001; miR-NC, miRNA negative control; Inhibitor-NC, inhibitor negative control. (A) Unregulated expression of hsa-miR-29-3p or hsa-miR-23-3p. Data were shown as the relative miRNA levels versus U6 snRNA. (B) Down-regulated CYP2C19 levels resulted from CYP2C19-specific siRNA or hsa-miR-29a-3p mimic transfection. Data were shown as relative CYP2C19 mRNA or protein levels versus GAPDH reference. (C) Up-regulated CYP2C19 levels resulted from hsa-miR-29a-3p inhibitor transfection. Data were shown as relative CYP2C19 mRNA or protein levels versus GAPDH reference.

Fig. 3

Chemical compounds NSC-156306 and NSC-606170 upregulated endogenous hsa-miR-29a-3p levels and down-regulated CYP2C19 expression in HepaRG cells. Differentiated HepaRG cells were treated with 0, 10 nmol/L, or 100 nmol/L NSC-156306 or NSC-606170 and cells were harvested 48 h after treatments. Each assay was carried out using at least 3 independent experiments. *P <n0.05; **P < 0.001; DMSO, no chemical compound in DMSO reagent. (A) Unregulated expression of hsa-miR-29-3p or hsa-miR-23-3p caused by NSC-156306 or NSC-606170 treatment. Data were shown as the relative miRNA levels versus U6 snRNA. (B) Down-regulated mRNA levels of CYP2C19 caused by NSC-156306 or NSC-606170 treatment. Data were shown as relative CYP2C19 mRNA levels versus GAPDH reference. (C) Down-regulated CYP2C19 protein levels caused by NSC-156306 or NSC-606170 treatment. Data were shown as relative CYP2C19 levels versus GAPDH reference.

Fig. 4

RNA EMSA with mRNA or miRNA oligonucleotides and cytoplasmic extracts from HepaRG cells. * and † indicated the CYP2C19 mRNA oligonucleotides retaining the hsa-miR-23a-3p and hsa-miR-29a-3p targeting sites, respectively. (A) The cy5.5™-labeled hsa-miR-23a-3p or hsa-miR-29a-3p oligonucleotides were incubated with 2′ O-methyl modified and IRDye®800-labeled CYP2C19 mRNA oligonucleotides. Lanes 1, 2, 4, and 5 indicated the mobility of each type of oligonucleotides, respectively; Lanes 3 and 6 indicated the mobility status of the miRNA oligonucleotides incubated with corresponding CYP2C19 mRNA oligonucleotides, respectively; Lanes 7 and 8 indicated the mobility status of the miRNA oligonucleotides incubated with unmatched CYP2C9 mRNA oligonucleotides, respectively. Arrows indicated the oligonucleotide complex formed by miRNA oligonucleotides and mRNA oligonucleotides in Lane 3 and 6. (B) Cytoplasmic extracts from HepaRG cells were incubated with hsa-miR-23a-3p or hsa-miR-29a-3p oligonucleotides, and CYP2C19 mRNA oligonucleotides. Lanes 1 and 5 indicated the mobility status of the miRNA oligonucleotides incubated with corresponding CYP2C19 mRNA oligonucleotides, respectively; Lanes 2 and 6 indicated the mobility status of the miRNA and mRNA oligonucleotides incubated with cell extracts, respectively; Lanes 3, 4, 7, and 8 indicated the mobility status of the oligonucleotide-cytoplasmic extracts complex with Ago1 or Ago2 antibody, respectively. Complex A indicated the supershift complex formed by oligonucleotides, cytoplasmic proteins and antibodies in Lane 6. (C) RNA EMSA in the presence of non-labelled various unlabeled competitors. Arrows indicated the oligonucleotide complexes formed by miRNA oligonucleotides and mRNA oligonucleotides in Lane 7, 8 and 9.

Fig. 5

Relationship between CYP2C19 mRNA expression and hsa-miR-23a-3p (A) or hsa-miR-29a-3p levels (B) in 96 non-tumor tissues in GSE22058 public dataset using Spearman correlation analysis.