Suppression of CYP2C9 by MicroRNA hsa-miR-128-3p in Human Liver Cells and Association with Hepatocellular Carcinoma

Published studies have identified genetic variants, somatic mutations, and changes in gene expression profiles that are associated with hepatocellular carcinoma (HCC), particularly involving genes that encode drug metabolizing enzymes (DMEs). CYP2C9, one of the most abundant and important DMEs, is involved in the metabolism of many carcinogens and drugs and is down-regulated in HCC. To investigate the molecular mechanisms that control CYP2C9 expression, we applied integrative approaches including in silico, in vitro, and in vivo analyses to elucidate the role of microRNA hsa-miR-128-3p in the regulation of CYP2C9 expression and translation. RNA electrophoresis mobility shift assays demonstrated a direct interaction between hsa-miR-128-3p and its cognate target, the CYP2C9 transcript. Furthermore, the expression of a luciferase reporter gene containing the 3′-UTR of CYP2C9 and the endogenous expression of CYP2C9 were suppressed by transfection of hsa-miR-128-3p. Importantly, chemically-induced up- or down-regulation of hsa-miR-128-3p correlated inversely with the expression of CYP2C9. Finally, an association analysis revealed that the expression of hsa-miR-128-3p is inversely correlated with the expression of CYP2C9 in HCC tumor tissues. Altogether, the study helped to elucidate the mechanism of CYP2C9 regulation by hsa-miR-128-3p, and the inverse association in HCC.

and environmental toxins by metabolizing them into inactive compounds. However, CYP450 enzyme activity also catalyzes the formation of reactive metabolites that cause DNA, RNA or protein damage 11 . Alteration in the expression CYP genes can affect the efficiency of xenobiotic detoxification and also the production of messenger molecules that regulate downstream signal-transduction pathways, thus having a paradoxical impact in carcinogenesis 11 . Therefore, it is reasonable to speculate that the dysregulation of CYP family genes might be involved in hepatocarcinogenesis. CYP2C9, one of the most abundant CYP2C proteins, accounts for ,20% of hepatic CYP content and contributes to the metabolism of many carcinogens and drugs 12,13 . Suppression of CYP2C9 expression has been reported as a biomarker of HCC [14][15][16][17] ; however, the mechanism of CYP2C9 dysregulation through microRNA (miRNAs) modulation has not been investigated in HCC.
The miRNAs are small RNA molecules that modulate gene expression through translational repression of cognate mRNA targets and thus are important mediators in gene regulatory networks. Consequently, miRNA-dependent modulation of the expression of DMEs among individuals could lead to substantial changes in phenotypes. Such changes may have a significant influence on quantitative traits, including the development of cancer and other diseases 18 . In the last decade, many human DMEs and nuclear receptors have been reported to be regulated by miRNAs, such as CYP1B1, CYP2E1, CYP3A4, CYP 24A1, SUL1A1, PXR and VDR 19 . The miRNA-dependent regulation of DMEs and transporters should have potential effects in modulation of carcinogen metallizationeither activation or detoxification, thus may be associated with cancer development. For example, evidences demonstrated that CYP1B1 is regulated by miR-27b, and a decreased miR-27b expression is inversely associated with an increased protein level of CYP1B1 in breast cancer patients 20 . Pathogenically, although the high level of CYP1B1 may decrease the estrogen activity by CYP1B1-mediated 4hydroxylation, the increased metabolite 4-hydroxyestadiol level may contribute more significantly to breast carcinogenesis 19 .
There are several hundred miRNAs expressed in human tissues, and characterization of miRNA expression patterns in human tumor tissues reveal miRNA signatures that are associated with tumor initiation, progression metastasis, diagnosis, prognosis, response to treatment and survival 21 ; however, there is a lack of conclusive information on the role for miRNA modulation of drug metabolizing genes in the pathogenesis of HCC.
In the current study, we hypothesized that hsa-miR-128-3p 22-24 and hsa-miR-143-3p 25,26 , which play important roles in cancer, could influence CYP2C9 expression by targeting its mRNA transcripts based on in silico analyses of putative mRNA/miRNA complexes. To test this hypothesis, we employed a series of biochemical assays to investigate the interaction of these two miRNAs with the CYP2C9 transcript and identified hsa-miR-128-3p as an efficient suppressor of CYP2C9 expression.
Compared to the related hsa-miR-143-3p complex, the enhanced thermodynamic stability predicted for the hsa-miR-128-3p complex with its CYP2C9 39-UTR target sequence correlated with results from RNA EMSA experiments, confirming the in vitro stability of the latter complex only. To explore the applicability of this predictive strategy further, we calculated the free energy of binding between hsa-miR-128-3p and 3 other putative target sequences (2 probes from the PAIP2 39-UTR and 1 probe from the PFKFB4 39-UTR). The predicted free energies of binding were 220.5 kcal/mol, 218.0 kcal/mol, and 215.8 kcal/mol for miR-128-PAIP2-target2, miR-128-PAIP2-target1 and miR-128-PFKFB4-target, respectively. EMSA assays were then conducted with these miRNAs and their targets. The hsa-miR-128-3p mimic was able to bind the probe with the free energy of 220.5 kcal/mol (Fig. 2b, lane 8), but not the ones with free energy of 218.0 kcal/mol, or 215.8 kcal/mol (Fig. 2b, lane 7 or 9), showing a correlation between the predicted free energy of binding directly and the observed interaction between miRNAs and their counterparts, which we propose should also correlate with the efficiencies of gene regulation by these miRNAs.
Suppression of endogenous CYP2C9 expression/translation by exogenous hsa-miR-128-3p. It was reported that HepaRG cells express CYP2C9 at a level similar to that found in primary hepatocytes, while the HepG2 cells barely express CYP2C9 owing to the lack of nuclear factors and other transcription factors for the www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 8534 | DOI: 10.1038/srep08534 expression of DMEs 27 . We did observed that the inherent CYP2C9 mRNA level in HepaRG cells was ,10-fold and ,20-fold greater than those in 293T cells and HepG2 cells, respectively, while the hsa-miR-128-3p was expressed in a similar level among these cell lines( Supplementary Fig. 3a). To investigate the effects of hsa-miR-128-3p on endogenous CYP2C9 transcripts and protein levels, hsa-miR-128-3p mimics, miRNA negative control or a CYP2C9-specific siRNA that served as the positive control, were transiently transfected into HepaRG cells. Fig. 3a shows that the level of hsa-miR-128-3p was dramatically increased by more than 200-fold after transfection with hsa-miR-128-3p mimics at concentrations of 25 nmol/L or 50 nmol/ L. Consequently, CYP2C9 mRNA expression was significantly decreased compared to that in cells transfected with the miRNA negative control (53.7% at 25 nmol/L and 81.1% at 50 nmol/L; all P , 0.05) (Fig. 3b). In addition, the CYP2C9 protein level decreased significantly (76.7%) when the cells were transfected with 50 nmol/L hsa-miR-128-3p mimics in comparison with the transfection of the miRNA negative control ( Fig. 3c and 3d). Notably, transfection of hsa-miR-128-3p was more efficient at suppressing CYP2C9 protein expression than transfection of the CYP2C9-specific siRNA positive control (Fig. 3b, 3c and 3d).
PTEN was proved to be negatively associated with hsa-miR-128-3p level in pituitary cells, probably due to hsa-miR-128-3p targeting BMI1, one transcriptional suppressor of PTEN 28 . As a positive control, we observed that the PTEN mRNA level was decreased significantly after hsa-miR-128-3p transfection, compared with that in cells transfected with the miRNA negative control (40.1% at 25 nmol/ L and 46.6% at 50 nmol/L; all P , 0.05)(Supplementary Fig.  S4a), suggesting that our transfection experimental system is validated.
Modulation of CYP2C9 expression/translation through chemicallyinduced alteration of hsa-miR-128-3p levels. According to the CellMiner TM database (version 1.5, http://discover.nci.nih.gov/ cellminer), chemical compounds NSC-156306 and NSC-606170 appear to inhibit or induce, respectively, the expression of hsa-miR-128 in human liver tissue. Therefore, we treated HepaRG cells with these compounds to determine their impact on CYP2C9 transcription and translation. Fig. 4 shows that treatment of the HepaRG cells with 100 nmol/L NSC-156306 resulted in a significant decrease in the expression of hsa-miR-128-3p and that its level was markedly increased after treating cells with NSC-606170 (Fig. 4a). The treatment-induced hsa-miR-128-3p expression changes were accompanied by inverse alterations in the expression of CYP2C9 gene expression (Fig. 4b) and protein production ( Fig. 4c and 4d). Specifically, the levels CYP2C9 mRNA and protein in the NSC-156306-treated HepaRG cells were 3-fold and ,2-fold greater (Fig. 4b, 4c and 4d). In contrast, the levels of CYP2C9 mRNA and protein were dramatically decreased, by 81.1% and 79.6%, in cells treated with NSC-606170 (Fig. 4b, 4c and 4d). In addition, we also observed that the PTEN mRNA level was decreased significantly after NSC-606170 treatment in HepaRG cells (67.6% at 10 nmol/L    Fig. S4b), suggesting that the modulation of hsa-miR-128-3p by NSC-606170 was obtained.
Inverse correlation between hsa-miR-128-3p and CYP2C9 expression in HCC. The expression levels of hsa-miR-128-3p and CYP2C9 mRNA in human HCC tumor tissue samples and adjacent normal liver samples were extracted from The Cancer Genome Atlas (TCGA) database. Levels of hsa-miR-128-3p measured in tumor tissues were significantly higher than those measured in matched non-tumor tissues; whereas, levels of CYP2C9 were significantly reduced in tumor tissues compared to surrounding non-tumor tissues (Fig. 5a). The relationship between hsa-miR-128-3p and CYP2C9 expression was evaluated by the Spearman Rank Order Correlation analysis in patient matched tumor and non-tumor tissues. In tumor tissues, there is a negative correlation between hsa-miR-128-3p and CYP2C9 (r 5 20.424, P 5 0.025, Fig. 5b), but in non-tumor tissues there is no significant correlation (r 5 20.204, P 5 0.304, Fig. 5b). Interestingly, no statistically significant correlation between PTEN mRNA (as a known miR-128 target) levels and hsa-miR-128-3p levels was observed in those tissues ( Supplementary Fig. S5), probably due to the complexity and high heterogeneity of gene expression in tumor tissue. When hsa-miR-143-3p was examined, no statistically significant results were obtained (Data not shown).

Discussion
Inter-individual variability in the expression of DMEs in humans is an important phenotypic trait that may contribute significantly to disparities in disease susceptibility and drug efficacy [29][30][31] . The mechanism underlying the phenotype is due, to a certain extent, to genetic polymorphisms and epigenetic variation among human populations. Genetic polymorphism(s) could alter gene transcription or change an enzyme's catalytic activity, while miRNAs regulate gene expression by targeting the mRNAs, repressing protein translation or accelerating mRNA degradation. In this study, we demonstrated that hsa-miR-128-3p plays an important role in the suppression of CYP2C9 expression and translation in human liver cells by a series of in silico analyses and in vitro and in vivo experiments. This study helped to elucidate the functional mechanism by which miRNA regulates CYP2C9 expression and translation. In addition, a modified RNA EMSA method provided direct evidence for the interaction between miRNAs and their cognate mRNA sequences that was dependent on the predicted free energy of binding.
CYP2C9 is one of the most abundant and important xenobiotic metabolizing enzymes, with substrates including commonly prescribed drugs such as warfarin, NSAIDs (non-steroidal anti-inflammatory drugs), tolbutamide, phenytoin, and torasemide 13 . CYP2C9 is also reported to participate in the bioactivation of carcinogens. For example, metabolism of benzo[a]pyrene by CYP2C9 results in the formation of 9-hydroxybenzo[a]pyrene-4,5-oxide and benzo[a]pyrene-7,8-diol-9,10-epoxide, which are reactive species involved in DNA adduct formation [32][33][34] . In addition, a high activity CYP2C9 genotype (CYP2C9*1) is associated with increased risk of colorectal cancer 35 while a low activity CYP2C9 genotype (CYP2C9*2) is associated with increased risk of colorectal adenoma 36 and lung cancer 37 . Furthermore, decreased CYP2C9 expression was reported in HCC tissue by several studies [14][15][16][17] , suggesting the role of CYP2C9 in detoxification may be involved in the etiology of HCC; however, the mechanisms controlling CYP2C9 expression are not fully understood.
In this study, we demonstrated that hsa-miR128-3p plays a pivotal role in the regulation of CYP2C9 expression in human liver using series of in silico, in vitro, and in vivo analyses. It is well known that the interaction between miRNAs and their cognate mRNA targets is complicated, and there are more than ten algorithms for predicting miRNA targets 38 . Therefore, for a given miRNA or transcript, many ''putative'' or ''potential'' interactions are predicted by in silico approaches. To reduce false positive predictions and validate true interactions, we first screened the miRNAs that could potentially target the 39-UTR of CYP2C9 mRNA using the microRNA.org database, and then evaluated the resultant candidate miRNAs in the PITA and TargetScan databases. The candidate miRNAs that were predicted by all three database algorithms to target CYP2C9 mRNA were then experimentally examined by both in vivo and in vitro approaches for functional interaction with CYP2C9 mRNA. The selection of hsa-miR-128-3p and hsa-miR-143-3p as candidate miRNAs for further biochemical characterization was based on their reported biological significance in tumorigenesis and metastasis [22][23][24][25][26] . For example, hsa-miR-128 is aberrantly expressed in many types of tumors, including acute lymphoblastic leukemia, glioblastoma, and breast cancer. By targeting EGFR, Bim-1, ABCC5 and other genes, hsa-miR-128 is involved in tumor differentiation, proliferation, inva-sion, apoptosis and resistance to drugs 39 . The miRNA hsa-miR-143 was reported as a tumor suppressor in cervical cancer 40 and prostate cancer 41 , by suppressing KRAS, ERK5, and other genes.
We first transfected the hsa-miR-128-3p or hsa-miR-143-3p mimics into liver HepG2 and kidney 293T cells, together with a reporter gene (luciferase) plasmid containing the core region of CYP2C9 39-UTR, and found that hsa-miR-128-3p suppressed luciferase activity in both liver cells and kidney cells, while hsa-miR-143-3p exhibited a relatively smaller suppression effect only in kidney cells. RNA EMSA assays revealed that hsa-miR-128-3p bound CYP2C9 mRNA, while an interaction between hsa-miR-143-3p and the CYP2C9 39-UTR was not detected. Further, we used RNA EMSA to test the binding efficiencies between hsa-miR-128-3p and three other target sequences with different free energies of binding predicted by the RNAhybrid software. It was observed that only the probes with free energy of less than 220 kcal/mol could bind hsa-miR-128-3p under our experimental conditions. Therefore, we postulate that the binding efficiency between miRNAs and their cognate mRNA targets is mainly dependent on the free energy state of the binding. Althogh further studies on the precise mechanisms of miRNA targeting mRNA sequences are under way, our current results provide evidence that the free energy of binding is important for accurate predictions of miRNA targeting sites.
Because of the low expression of DMEs and transporters by HepaG2 and other hepatocellular carcinoma cell lines 30,42 , we used HepaRG cells, which express DMEs and transporters at levels similar www.nature.com/scientificreports to primary hepatocytes, to investigate the suppression effects of hsa-miR-128-3p on endogenous CYP2C9 expression and translation. Our results showed that enforced up-regulattion of hsa-miR-128-3p reduced CYP2C9 production at both the protein and mRNA levels, indicating that hsa-miR-128-3p is at least involved in CYP2C9 mRNA degradation. Two chemicals, with strong negative or positive effect on hsa-miR-128 expression, were used to produce CYP2C9 alterations through the modulation of hsa-miR-128 expression. The results confirmed that the dramatic changes in hsa-miR-128-3p expression caused by these two compounds (Fig. 4a) indeed altered the CYP2C9 expression/production inversely (Fig. 4b, 4c and 4d), which is consistent with the transfection assays. Altogether, our results indicated that hsa-miR-128-3p is able to suppress CYP2C9 expression/production in human hepatic cells by specifically targeting the 39-UTR of CYP2C9 mRNA molecules.
Finally, the correlation between the expression of CYP2C9 and hsa-miR-128-3p in HCC tissues was evaluated using the GSE22058 and TCGA datasets. We observed that CYP2C9 mRNA was significantly down-regulated in HCCs, consistent with findings reported by others [14][15][16][17] . Most importantly, this study revealed an up-regulation of hsa-miR-128-3p expression and a significant inverse correlation between CYP2C9 expression and hsa-miR-128-3p expression in HCC tissues, indicating that hsa-miR-128-3p combined with CYP2C9 are potential biomarkers for HCC diagnosis.
In summary, our study identified and experimentally confirmed that hsa-miR-128-3p is a suppressor for CYP2C9 expression in HCC, and revealed a direct interaction between a miRNA and its target mRNA sequence in vitro, which demonstrated a molecular mechanism of miRNA mediated CYP2C9 suppression.

Methods
Cell lines. HepG2 and 293T cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and HepaRG cells were obtained from Biopredic International (Overland Park, HS). The cells were maintained according to ATCC and Biopredic International's recommendations.
Luciferase reporter gene assay. The pGL3-Control vector (Promega, Madison, WI) was modified by adding the Universal USER Cassette (New England Biolabs, Ipswich, MA), similar to the modification of the pGL3-Promoter 44 . Briefly, the Xba I site was digested and blunted by using the Quick Blunting Kit (New England Biolabs). The USER Cassette sequence was then inserted, resulting in the pGL3-CU vector. Cloning primers CYP2C9-F and CYP2C9-R (All primer or oligo sequences used in this study were listed in Supplementary Table S1), with extension oligonucleotides 59-GGA GAC AU-39 or 59-GGG AAA GU-39 in their 59 end, were designed to amplify the core region of CYP2C9 39-UTR that harbors the putative binding sites for hsa-miR-128-3p and hsa-miR-143-3p. PCR products were digested with USER enzyme (New England Biolabs) and cloned into the linearized nicked pGL3-CU vector that was prepared following the Universal USER Cassette protocol. The DNA sequence of the resultant plasmid, designated CYP2C9-CU, was determined to confirm its identity. Besides, CYP2C9-MUT1-CU or CYP2C9-MUT2-CU construct, which with mutated hsa-miR-128-3p target sequences in the CYP2C9 39UTR, was created by site-directed mutagenesis using CYP2C9-MUT1-F and CYP2C9-MUT1-R primers, or CYP2C9-MUT2-F and CYP2C9-MUT2-R primers, respectively.
HepG2, a human hepatoma cell line, and 293T, a human embryonic kidney line, were used for luciferase assays. HepG2 or 293T cells were cultured in Rosewell Park Memorial Institute 1640 or Dulbecco's Modified Eagle medium with 10% fetal bovine serum. Cells were seeded in 96-multiwell plates. When the cells reached 70%-80% confluence, they were transfected with the CYP2C9-CU plasmid (100 ng/well) that contains the 39-UTR of CYP2C9 together with 50 nmol/L (final concentration) hsa-miR-128-3p mimic, hsa-miR-143-3p mimic, or miRNA negative control (Thermo Scientific, Tewksbury, MA) using the Lipofectamine reagent 2000 (Life Technologies, Carlsbad, CA). The pRL-SV40 (1 ng/well; Promega) plasmid, which expresses Renilla reniformis luciferase, was co-transfected to standardize transfection efficiency. The CYP2C9-CU plasmid co-transfected with pRL-SV40 plasmid and miRNA negative control served as a reference. Three independent transfection experiments were carried out, and each transfection was performed in triplicate.
RNA EMSAs were performed according to the LightShift Chemiluminescent RNA EMSA Kit (Thermo Scientific) protocol. Briefly, in each 20 mL binding reaction containing 200 nmol synthetic miRNA or/and cognate mRNA binding oligonucleotides were mixed, heated for 5 minutes at 80uC, placed on ice to relax RNA secondary structures, and then incubated at 25uC for 20 min. The reaction mixtures were separated on a 12% PAGE by electrophoresis at 4uC, and the resultant mobility shifts were detected with an Odyssey CLx Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).
Transfection of HepaRG cells with hsa-miR-128-3p and treatments with chemical compounds. HepaRG cells (Biopredic International, Overland Park, KS) were first incubated in Williams' E medium supplemented with growth supplement (Biopredic International) for 2 weeks and then cells were differentiated by adding the differentiation supplement (Biopredic International) for 10 additional days. Differentiated cells were then seeded into 6-well plates at a density of 70,000 cells/cm 2 with 3 ml medium and incubated for another 2 days for further experiments.
The miRNA transfection was performed as previously reported 45 . Briefly, 25 nmol/ L or 50 nmol/L (final concentration) hsa-miR-128-3p mimic, miRNA negative control, or CYP2C9-specific siRNA (positive control), was transfected into the differentiated HepaRG cells using Lipofectamine transfection reagent (Life Technology), and cells were harvested 48 hours after transfection. Chemical compounds with the NSC numbers NSC-156306 (o-amsa monomethanesulfonate) and NSC-606170 (zalcitabine) were obtained from the Developmental Therapeutics Program (DTP) of the National Cancer Institute (NCI) and diluted to 20 mmol/L using dimethyl sulfoxide (DMSO). Differentiated HepaRG cells were treated with 0, 10, or 100 nmol/L (final concentration) of NSC-156306 or NSC-606170 and cells were harvested 48 hours after treatment. Each assay was conducted using at least three independent transfection experiments or chemical treatment.
RNA isolation and quantitative reverse-transcription PCR (qRT-PCR). Total RNA was extracted from HepaRG cells after transfection or chemical treatment using the miRNeasy Mini Kit (Qiagen, Valencia, CA) and cDNA was synthesized using QuantiTect Reverse Transcription Kit (Qiagen) or NCode TM miRNA First-Strand cDNA Synthesis Kit (Life Technologies). CYP2C9, PTEN and GAPDH RNA levels were measured by qRT-PCR on an ABI Prism7900 Sequence Detection System (Applied Biosystems) according to the QuantiFast SYBRH Green RT-PCR Kit (Qiagen) protocol using the CYP2C9-RT-F and CYP2C9-RT-R primers, PTEN-RT-F and PTEN-RT-R primers, or GAPDH-RT-F and GAPDH-RT-R primers, respectively. The miR-128-RT-F and U6-F primers, together with the Universal Reverse Primer supplied with the NCode TM miRNA First-Strand cDNA Synthesis Kit, were used to detect the hsa-miR-128-3p and U6 levels. The RNA expression levels of CYP2C9 or hsa-miR-128-3p were calculated relative to expression of GAPDH or U6, respectively.
Western blot analysis. Proteins were isolated from HepaRG cells that were harvested after transfection or chemical treatment. Quantitative Western blotting was performed following the Odyssey TM Western Blotting Kit (LI-COR Biosciences) protocol. Antibodies against CYP2C9 or GAPDH (Abcam, Cambridge, MA) were used to detect CYP2C9 or GAPDH protein levels and an Odyssey CLx Infrared Imaging System was used to perform quantitative analyses, with infrared labeled secondary antibodies.
Retrieval of data from online databases. RNA expression levels of CYP2C9 and hsa-miR-128-3p were obtained from The Cancer Genome Atlas database (TCGA, http:// cancergenome.nih.gov/). Statistical analyses. The rank sum test was used to evaluate the difference in the expression of CYP2C9 or hsa-miR-128-3p in HCCs, with P , 0.05 as the significant criterion. Spearman Rank Order Correlation analysis was used to test the correlation between CYP2C9 levels and hsa-miR-128-3p expression. Student's t-test was also used to compare results from luciferase reporter gene assays and to compare CYP2C9 or hsa-miR-128-3p protein or RNA levels between subgroups.