Abstract
Mouse cytochrome P450 2b9 (Cyp2b9) is a testosterone 16α-hydroxylase enzyme showing female-specific expression in many inbred mouse strains, including C57BL/6J. Previous studies have recognized that some sex-dependently secreted endogenous modulating factors were involved in the sexually dimorphic expression of Cyp2b9 through transcriptional regulation. In this study, we found evidence that some microRNAs contributed to the sexually biased expression of Cyp2b9 via post-transcriptional regulation. Cyp2b9 was upregulated in livers of hepatocyte-specific Dicer1 knockout mice at 3 weeks. The age-dependent downregulation of Cyp2b9 in the livers of male mice was diminished when Dicer1 was specifically knocked out in hepatocytes. When these data were combined with bioinformatics analysis and microRNA profiles of male and female mice, we found that 18 microRNAs were associated with the sexually dimorphic expression of Cyp2b9, which showed higher expression levels in male C57BL/6J mice when compared with females. Luciferase assays revealed that approximate half of these microRNAs repressed luciferase activity in a reporter system containing the 3′-untranslated region (3′-UTR) of Cyp2b9, and also inhibited Cyp2b9 protein expression. MicroRNA seed region mutation or mutations in putative binding sites of the microRNAs in Cyp2b9 3′-UTR led to the loss of the suppression of luciferase activity. There was also a negative correlation between the levels of these microRNAs and Cyp2b9. Our results suggested that multiple microRNAs participated in the regulation of Cyp2b9 expression, and that the lower expression levels of these microRNAs potentially contributed to the female-specific expression of Cyp2b9 in the livers of C57BL/6J mice.
Introduction
The cytochrome P450 (P450) superfamily is a large and diverse group of enzymes that catalyze the metabolism of drugs and carcinogens. Many studies have demonstrated that gender plays an important role in the pharmacologic and toxicologic responses to drugs. In humans, the expression of CYP1A2, CYP2E1, CYP3A4, CYP2A6, and CYP2B6, which influences risk factors for clinically relevant adverse drug reactions (Anderson, 2008), has been shown to have gender bias. In animal models, such as mice, there are gender differences in the expression of Cyp2a5, Cyp2b9/10/13, Cyp2d9, and Cyp3a41/44 (Hrycay and Bandiera, 2009). Cyp2b9 is a testosterone 16α-hydroxylase enzyme showing female-specific expression in many inbred mouse strains, including C57BL/6J (Noshiro et al., 1988). Previous studies have identified some mechanisms involved in the sexually dimorphic expression of Cyp2b9 at the transcriptional level. Sex-dependent secretion of endogenous modulating factors, especially growth hormone, glucocorticoid hormone, and sex hormones, are involved in this regulatory pathway (Jarukamjorn et al., 1999, 2001, 2002).
MicroRNAs are a large family of endogenous noncoding RNAs that regulate gene expression primarily by binding to the 3′-untranslated region (3′-UTR) of target genes, resulting in suppressed translation or decreased mRNA stability. MicroRNAs may regulate about 60% of all genes in humans (Bartel, 2009). Dicer1 is an RNase III endonuclease that is essential for the biogenesis of microRNAs, and multiple Dicer1 knockout (KO) animal models show significant down-regulation of microRNAs and upregulation of microRNA targeting genes in these models (Hand et al., 2009; Sekine et al., 2009).
Recently, it has been demonstrated that microRNAs regulate cell processes necessary for sexual differentiation (Morgan and Bale, 2012). MiR-23a contributes to sex differences in the response to cerebral ischemia by regulating the expression of X-linked inhibitor of apoptosis (Siegel et al., 2011). Sex-based differences in miR-1 may underlie the sex-based differences in Cx43 expression in cardiomyocytes in pathologic conditions (Stauffer et al., 2011).
In this study, we used a hepatocyte-specific Dicer1 KO mouse model, bioinformatics analysis, and in vitro studies to investigate the role of microRNAs in the regulation of sexually dimorphic Cyp2b9.
Materials and Methods
Animals.
All animal treatments were approved by the Institutional Animal Care and Use Committee of the Shanghai Institute of Materia Medica (Shanghai, China). The hepatocyte-specific Dicer1 KO C57BL/6 mouse strain was a kind gift from Professor Yizheng Wang (Institute of Neuroscience, Chinese Academy of Sciences, China). In this study, 3- and 6-week-old male hepatocyte-specific Dicer1 KO C57BL/6 mice were used (Hand et al., 2009; Sekine et al., 2009).
Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction.
Total RNA was isolated from the liver by UNIQ-10/Trizol total RNA extraction kit (Sangon, Shanghai, China) and reverse-transcribed to cDNA with Primescript RT Reagent Kit (Takara, Otus, Shiga, Japan). The primers for Cyp2b9 mRNA were 5′- CCTCCACTATGGAGTCCTGC-3′ (forward) and 5′- ACTTGGACTGTTGGGA GGAAGA-3′ (reverse), and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) analysis was performed using SYBR Premix Ex Taq (Takara). Cyp2b9 mRNA levels were normalized to mouse Gapdh mRNA detected by primers 5′- GGCTACACTGAGGACCAGGTT-3′ (forward) and 5′- TGCTGTAGCCGTATTCATTGTC-3′ (reverse).
MicroRNAs were isolated with the mirVana miRNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer’s instructions. Reverse transcription and detection of microRNAs were carried out using NCode VILO miRNA cDNA Synthesis Kit and EXPRESS SYBR GreenER miRNA qRT-PCR Kit, respectively (Invitrogen, Carlsbad, CA). MicroRNA-specific forward primers were designed according to the manufacturer’s instructions. The microRNA expression levels detected were normalized to mRnu6 levels.
Western Blot.
Liver tissues were lysed in radioimmunoprecipitation assay lysis buffer (Sangon) with phenylmethanesulfonyl fluoride. Cell samples were lysed in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 1% nonidet-P40, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl, and 100 mM dithiothreitol. Proteins (20μg) were separated by 8% SDS-PAGE. Anti-mouse Cyp2b9 polyclonal antibody was generated by immunizing rabbits with a peptide of amino acids 462-473 from Cyp2b9 by Abmart (Shanghai, China). Verification for the specificity of this antibody was performed (Supplemental Fig. 1). Anti- glyceraldehyde-3-phosphate dehydrogenase (Sigma-Aldrich, St. Louis, MO) (1:5000) served as a loading control.
Construction of Luciferase Reporter Plasmids.
The 3′-untranslated region (3′-UTR, 376bp) of mouse Cyp2b9 (NM_010000, GeneBank) was amplified by polymerase chain reaction (PCR) using cDNA from the liver of a female C57BL/6J mouse. The primers are 5′-TAGGCGATCGCTCGAGTTGGGGTGAGGGAGCCAGGTGTC-3′ (forward) and 5′-TTGCGGCCAGCGGCCGCCACACACTATGTGATTCTGT-3′ (reverse). This PCR fragment was cloned into the psiCHECK-2 vector (Promega, Madison, WI) with the In-fusion Advantage PCR Cloning Kit (Clontech, Mountain View, CA).
Site-DMutation of Luciferase Reporter Plasmids.
The mutated plasmids were cloned using the KOD-Plus-Mutagenesis Kit (Toyobo, Osaka, Japan) with standard primers (Supplemental Table 1). DNA sequencing confirmed the nucleotide sequence of these plasmids.
Luciferase Assays.
For luciferase reporter assays, various luciferase reporter plasmids were co-transfected with 100nM microRNA mimics and their seed region mutants (GenePharma, Shanghai, China) into Huh7 cells using Lipofectamine 2000 (Invitrogen). The entire sequence of microRNA mimics is summarized in Supplemental Table 2. Luciferase activity was analyzed after 72 hours by the Dual-Luciferase Reporter Assay System according to the manufacturer’s protocol (Promega).
Establishment of Huh7 Cell Line Stably Expressing.
Full-length of the mouse Cyp2b9 (NM_010000) cDNA (1877bp, including 3′-UTR) was amplified by PCR using the primers of 5′-GGACTCAGATCTCGAGAAGCAGACTCTTGTTAG ACC-3′ (forward) and 5′-GTCGACTGCAGAATTCCACACACTATGTGATTCTG T-3′ (reverse). The fragment was cloned into pLVX-AcGFP-N1 lentiviral expression vector with In-fusion Advantage PCR Cloning Kit (Clontech). The nucleotide sequence of the plasmids was confirmed by DNA sequencing. The plasmid was transfected into the HEK 293T packaging cell line using the Lenti-X HT Packaging System (Clontech), and 48 hours after transfection, viral supernatant was collected. Huh7 cells were transfected with pLVX-AcGFP-N1-Cypb9 virus-containing media and were selected in 4 μg/ml puromycin (Sigma) for 7 days, and the pooled cell population was used for subsequent experiments. The expression of Cyp2b9 by was confirmed by Western blot (unpublished data).
Statistical Analyses.
Statistical significance was determined by unpaired, two-tailed Student’s t test. Correlation coefficients (R) were determined by Pearson’s correlation test.
Results
Effects of Dicer1 Disruption on the Expression of Cyp2b9 in the Livers of Male and Female Mice.
We obtained the expression profile of microRNAs and CYPs in hepatocyte-specific Dicer1 KO male mice (male-KO mice). Dicer1, miR-122 and miR-192 were significantly downregulated in male-KO mice (Supplemental Fig. 2), indicating efficient KO of Dicer1. It has been previously shown that the expression of Cyp2b9 decreased in male mice during development after birth (Hashita et al., 2008). In our study, mRNA (Fig. 1, A and B) and protein (Fig. 1, C and D) levels of Cyp2b9 at 3 weeks were higher in the livers of male-KO mice compared with wild-type (WT) male mice. The expression of Cyp2b9 in WT male mice at 6 weeks was extremely low, whereas that of KO male mice was still high (Fig. 1). However, these phenomena were not observed in female mice at 3 or 6 weeks (Supplemental Fig. 3).
Screening of MicroRNAs that May Be Involved in the Sexually Dimorphic Expression of Cyp2b9.
Using bioinformatics tools (Targetscan, Microcosm, and mirWalk) (Lewis et al., 2005; Griffiths-Jones et al., 2008; Dweep et al., 2011), we predicted microRNAs that potentially regulate the expression of Cyp2b9 and found that over 60 microRNAs might bind to the 3′-UTR of Cyp2b9. We combined this result with the microRNA profiles from male and female mice at 6 weeks and excluded the microRNAs with potential binding sites too close to the open reading frame of Cyp2b9, which were reported less likely to have regulatory function (Lewis et al., 2005). We finally found 22 microRNAs expressed in liver, thus we focused on studying the roles of these 22 microRNAs in the regulation of Cyp2b9 (Table 1). Results of qRT-PCR further revealed that 18 of 22 microRNAs had higher expression levels in male mice (Fig. 2). The screening strategy was summarized in Supplemental Fig. 4.
Multiple MicroRNAs Negatively Regulated the Expression of Cyp2b9.
We constructed a Cyp2b9 3′-UTR luciferase reporter plasmid to study the posttranscriptional regulation of microRNAs on Cyp2b9 expression. Luciferase reporter assays showed that 8 of 22 microRNAs decreased luciferase activity at the final concentration of 100nM (Fig. 3, A and B). These microRNAs also downregulated the protein level of Cyp2b9 (Fig. 3C). When mutations were introduced into the putative binding sites of microRNAs (mmu-miR-139-3p, 1b-5p, 21*, 291a-5p, -297a*, 297b-3p, 467g, -667) in Cyp2b9 3′-UTR (Fig. 3A), the inhibition of luciferase activity was partially or totally abolished (Fig. 3, A and B). MicroRNAs with mutations in their seed regions also lost the inhibition of the luciferase activity (Fig. 3D). These results suggested that eight microRNAs (mmu-miR-139-3p, -1b-5p, -21*, -291a-5p, -297a*, -297b-3p, -467g, and -667) inhibited Cyp2b9 expression posttranscriptionally through binding to the 3′-UTR region.
Correlation Analysis of the Expression of microRNAs and Cyp2b9 in Male and Female Mice.
All 8 microRNAs were found to regulate Cyp2b9 had higher expression levels in male C57BL/6J mice compared with female mice (Table 1). We performed Pearson’s correlation tests to identify potential correlations between the protein levels of Cyp2b9 protein and the levels of microRNAs in male and female WT mice at 6 weeks. Expression of mmu-miR-139-3p, 21*, 297b-3p, 467g, and 667 showed weak negative correlation with Cyp2b9 expression (Fig. 4, A, C, F–H), whereas the expression of mmu-miR-1b-5p, 291a-5p and 297a* demonstrated strong negative correlation with Cyp2b9 expression (Fig. 4, B, D, and E). Finally, the expression of mmu-miR-490-5p, which showed no regulation of Cyp2b9 by luciferase activity assay, did not correlate with Cyp2b9 expression (Fig. 4I). The correlation coefficients between the levels of 22 microRNAs and Cyp2b9 expression are listed in Table 2.
Discussion
It has been previously reported that the expression of Cyp2b9 developmentally decreased after birth in male mice (Hashita et al., 2008). Previous studies have shown that the growth hormone Stat5/constitute androgen receptor (CAR)/forkhead box A2/hepatocyte nuclear receptor α pathways influence the expression of female-specific genes, including Cyp2b9 (Lahuna et al., 2000; Wiwi et al., 2004; Mota et al., 2010; Baik et al., 2011). In our study, the expression levels of Stat5, forkhead box A2, and hepatocyte nuclear receptor α in 6-week-old male KO mice were similar to levels in WT mice, whereas CAR, a positive regulator of Cyp2b9 in male mice (Mota et al., 2010), was downregulated (unpublished data). These results suggest that other factors are involved in the upregulation of Cyp2b9 in male KO mice.
We investigated the possibility that microRNAs may be involved in the regulation of Cyp2b9 and identified multiple microRNAs involved in the sexually dimorphic expression of Cyp2b9. Several lines of evidence supported our conclusion. First, Cyp2b9 upregulation in the liver of hepatocyte-specific Dicer1 KO male mice indicated the potential role of microRNAs in gender differences of Cyp2b9 expression; this was further supported by the differential expression of microRNAs with binding sites in the 3′-UTR of Cyp2b9 between male and female mice. Second, multiple microRNA exerted inhibitory effects on Cyp2b9 protein expression and luciferase activity of reporters containing Cyp2b9 3′-UTR; however, when mutations were introduced into Cyp2b9 3′-UTR or seed region of the microRNAs, the inhibition was reversed, which suggested multiple microRNAs regulated Cyp2b9 at posttranscriptional levels via their seed region complementary to Cyp2b9 3′-UTR. Finally, correlation analyses showed negative correlation between the expression of microRNAs and Cyp2b9. Altogether, these data strongly suggested that microRNAs posttranscriptionally regulate the expression of Cyp2b9 and participate in the gender differences of Cyp2b9 expression.
Our work suggested that some male-predominant microRNAs may participate in the negative regulation of Cyp2b9 in male mice. A previous study showed that CAR knockout caused upregulation of Cyp2b9 expression in female mice, suggesting a negative correlation between CAR and Cyp2b9 in female mice (Mota et al., 2010). In contrast, Cyp2b9 was down-regulated in 6-week-old Dicer1 KO female mice (Supplemental Fig. 3), which also showed a great increase of CAR (unpublished data). Here, we suggested that the downregulation of Cyp2b9 in female mice may be due to the combination of upregulation of CAR and the low levels of predicted microRNAs (Figs. 2 and 4) in female mice. Future studies are needed to clarify the mechanisms involved in the downregulation of Cyp2b9 in Dicer1 KO female mice.
Human CYP2B6 expression shows gender bias; in the mouse, Cyp2b9 and Cyp2b10 expression has also been found to be differ by gender. In our study, knockdown of DICER1 increased the mRNA levels of CYP2B6 (Supplemental Fig. 5, A and B). Dicer1 knockout also increased the protein levels of Cyp2b10 in the liver of male-KO mice (Supplemental Fig. 5C). The luciferase activity of the reporter containing the 3′UTR of Cyp2b10 or CYP2B6 was also repressed by some microRNAs (Supplemental Fig. 5, D–E). Altogether, these results suggested that microRNAs may also play a role in the sexual dimorphic expression of CYP2B6.
Gender and individual differences in the expression of CYPs are universal phenomena that are driven by multiple mechanisms including genetic variation, transcriptional factors, and posttranscriptional regulation (Hrycay and Bandiera, 2009). Sex differences in the expression of P450 enzymes lead to sex differences in the pharmacokinetics and pharmacodynamics of many drugs; this is a primary cause of sex-related pharmacokinetics and side effects (Tanaka, 1999). Approximately 7% of new drug applications with sex analyses show at least a 40% differential in pharmacokinetics between men and women (Anderson, 2005). In this study, we demonstrated a microRNA-directed posttranscriptional regulatory mechanism that may participate in sex-biased expression of P450 enzymes. Our results suggest a novel potential mechanism for sex-based differences in the pharmacokinetics and pharmacodynamics of drugs, and indicate that microRNAs may be important factors in sex-biased drug administration.
Acknowledgments
The authors thank the staff at the Center for Drug Safety Evaluation and Research for their assistance with the preparation of this article.
Authorship Contributions
Participated in research design: Ren, Wang, Qi.
Conducted experiments: Xie, Miao, Yao, Li, Feng, Gao, Liu.
Performed data analysis: Xie, Miao, Qi, Gong.
Wrote or contributed to the writing of the manuscript: Xie, Miao, Qi.
Footnotes
- Received April 1, 2013.
- Accepted May 22, 2013.
Xiaofeng Xie and Lingling Miao contributed equally to this work.
This work was supported by the National Science and Technology Major Project [Grants 2012ZX09302-003 and 2012ZX09301-001-006].
This work has been submitted as part of Xiaofeng Xie’s Master thesis: Xiaofeng X (2012) Role of Dicer and microRNAs in the Development of Hepatocellular Carcinoma. M.Sc. thesis, Chinese Academy of Sciences, Beijing, China.
↵This article has supplemental material available at dmd.aspetjournals.org.
Abbreviations
- 3′-UTR
- 3′-untranslated region
- CAR
- constitute androgen receptor
- KO
- knockout
- P450
- cytochrome P450
- PCR
- polymerase chain reaction
- qRT-PCR
- quantitative real-time reverse transcription-polymerase chain reaction
- WT
- wild type
- Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics