Dihydropyrimidine dehydrogenase (DPD) expression is negatively regulated by certain microRNAs in human lung tissues

Lung Cancer 77 (2012) 16–23

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Dihydropyrimidine dehydrogenase (DPD) expression is negatively regulated by certain microRNAs in human lung tissues Takeshi Hirota a , Yuko Date a , Yu Nishibatake a , Hiroshi Takane b , Yasushi Fukuoka c , Yuuji Taniguchi d , Naoto Burioka e , Eiji Shimizu c , Hiroshige Nakamura d , Kenji Otsubo b , Ichiro Ieiri a,∗ a

Department of Clinical Pharmacokinetics, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan Department of Pharmacy, Tottori University Hospital, 36-1 Nishi-machi, Yonago 683-8504, Japan c Division of Medical Oncology and Molecular Respirology, Faculty of Medicine, Tottori University, 36-1 Nishi-machi, Yonago 683-8504, Japan d Division of General Thoracic Surgery, Tottori University Hospital, 36-1 Nishi-machi, Yonago 683-8504, Japan e Division of School of Health Science, Department of Pathobiological Science and Technology, Faculty of Medicine, Tottori University, 36-1 Nishi-machi, Yonago 683-8504, Japan b

a r t i c l e

i n f o

Article history: Received 15 July 2011 Received in revised form 13 December 2011 Accepted 18 December 2011 Keywords: Posttranscriptional and translational control MicroRNA Dihydropyrimidine dehydrogenase 5-FU Pharmacogenetics Non-small cell lung cancer

a b s t r a c t Dihydropyrimidine dehydrogenase (DPD) is important to the antitumor effect of 5-fluorouracil (5-FU). DPD gene (DPYD) expression in tumors is correlated with sensitivity to 5-FU. Because the 5-FU accumulated in cancer cells is also rapidly converted into inactivated metabolites through catabolic pathways mediated by DPD, high DPD activity in cancer cells is an important determinant of the response to 5-FU. DPD activity is highly variable and reduced activity causes a high risk of 5-FU toxicity. Genetic variation in DPYD has been proposed as the main factor responsible for the variation in DPD activity. However, only a small proportion of the activity of DPD can be explained by DPYD mutations. In this study, we found that DPYD is a target of the following microRNAs (miRNA): miR-27a, miR-27b, miR-134, and miR-582-5p. In luciferase assays with HepG2 cells, the overexpression of these miRNAs was associated with significantly decreased reporter activity in a plasmid containing the 3 -UTR of DYPD mRNA. The level of DPD protein in MIAPaca-2 cells was also significantly decreased by the overexpression of these four miRNAs. The results suggest that miR-27a, miR-27b, miR-134, and miR-582-5p post-transcriptionally regulate DPD protein expression. The levels of miRNAs in normal lung tissue and lung tumors were compared; miR-27b and miR-134 levels were significantly lower in the tumors than normal tissue (3.64 ± 4.02 versus 9.75 ± 6.58 and 0.64 ± 0.75 versus 1.48 ± 1.39). DPD protein levels were significantly higher in the tumors. Thus, the decreased expression of miR-27b would be responsible for the high levels of DPD protein. This study is the first to show that miRNAs regulate the DPD protein, and provides new insight into 5-FU-based chemotherapy. © 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in the catabolism of pyrimidine. Since DPD is responsible for the elimination of approximately 80% of an administered dose of 5-fluorouracil (5-FU), its activity is particularly important in determining a patient’s response to 5-FU [1]. The pharmacokinetics of 5-FU exhibits large inter-individual differences; for example, a half-life ranging from 4 to 25 min [2]. Because of DPD’s critical position in the metabolic pathway, the variation in its activity is likely responsible for the variation in the pharmacokinetics of 5-FU. DPD activity is highly variable in a population, with an estimated 3–5% of cancer patients showing low DPD

∗ Corresponding author. Tel.: +81 92 642 6656; fax: +81 92 642 6660. E-mail address: [email protected] (I. Ieiri). 0169-5002/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2011.12.018

activity [3]. Part of this variability is explained by genetic variation in the DPD gene (DPYD), where over 40 polymorphisms have been described so far. Several of these gene variants have been associated with reduced activity and severe toxic side effects of 5-FU [3–6], the most prominent being a point mutation in the splice site of intron 14 (IVS 14 + 1G>A) resulting in the deletion of exon 14 and thus a non-functional enzyme [7,8]. However, the clinical consequences of this and other mutations in the DPYD gene remain unclear, and a large proportion of the observed variation in DPD activity is still unexplained. Many reports showed that intratumoral DPD activity affects resistance to 5-FU [9–11]. Especially, nonsmall-cell lung cancers (NSCLCs) may be considered to be a tumor type that commonly displays a high level of DPD expression, which in turn is associated with decreased 5-FU activity [12,13]. In fact, S-1 (composed of tegafur, 2-chloro-2,4-dihydroxypyridine, and potassium oxonate in the molar ratio 1:0.4:1), which inhibits DPD activity, has been

T. Hirota et al. / Lung Cancer 77 (2012) 16–23

developed for cancer therapy including the treatment of NSCLCs. It is important to investigate the mechanisms of high DPD expression in NSCLCs from a viewpoint of finding more appropriate therapeutic opportunities. Some reports have indicated no strong link between DPYD mRNA levels and DPD protein levels. Thus, post-transcriptional regulation is suggested to be important for differences in DPD expression in lung tumor tissue. MicroRNA (miRNA) is a class of endogenous, noncoding RNA whose final product is a functional RNA molecule approximately 22 nucleotides long [14]. The miRNAs bind with the 3 untranslated region (3 -UTR) and negatively regulate target mRNAs [15]. They are widely expressed in all tissues and at all stages of development. Greater than one third of all human genes have been predicted to be miRNA targets [16] and therefore miRNAs are an abundant and important class of regulatory molecules in post-transcriptional regulation. Recently, it has been shown that miRNA expression profiles and specific miRNAs in lung tissue are correlated with clinical outcome [17]. Hu et al. reported that miRNAs may serve as an indicator of overall survival in cases of NSCLC [18]. Among pharmacokineticrelated genes, the cytochrome P450 (CYP) 1B1 gene was identified as regulated by miRNA [19], indicating miRNAs to play an important role in the regulation of drug-metabolizing enzymes [20]. In this study, we investigated whether the human DPYD gene was post-transcriptionally regulated by miRNA, and the contribution of miRNAs to DPD protein levels in human lung tumors. 2. Materials and methods 2.1. Subjects Blood samples were obtained from unrelated Caucasian and Japanese volunteers (48 subjects each, Tennessee Blood Services, Memphis, TN). Samples of lung tumors and normal lung tissue from 16 Japanese donors were used for the quantification of DPD protein and mRNA expression. This study was approved by the relevant University Ethics Committees. Written informed consent was obtained from all subjects before their participation.

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vector (Promega, Madison, WI). The pGL3-promoter vectors combined with the amplified DPYD 3 -UTR haplotypes were termed pGL3/Hap1–5. Sequences matching perfectly with the mature miR27a, miR-27b, miR-134, and miR-582-5p were cloned into the XbaI site of the pGL3-promoter vector; these plasmids were termed pGL3/miR-27a, pGL3/miR-27b, pGL3/miR-134, and pGL3/miR-5825p, respectively. HepG2 cells were temporarily transfected with the plasmids by the LipofectAMINE method, and cultured in Dulbecco’s modified Eagle’s medium (Sigma–Aldrich, St. Louis, MO) with 10% fetal bovine serum. Briefly, cells were transfected with a mixture of 1.0 ␮l of LipofectAMINE 2000 reagent (Invitrogen, Carlsbad, CA), 1 ␮g of reporter plasmid, and a precursor for miRNA (miR-27a, miR-27b, miR-134, or miR-582-5p) (Ambion, Austin, TX) or nontargeting control miRNA (control miRNA; Ambion), and kept for 48 h. As a control, 0.25 ␮g of pRL-CMV (Promega) containing Renilla luciferase driven by a CMV (cytomegarovirus) promoter was co-transfected. Luciferase activity was measured using the Dual Luciferase assay system (Promega) and values are given as the mean ± S.D. of three determinations. 2.4. Real-time RT-PCR for DPYD mRNA Total RNA extraction and reverse-transcription polymerase chain reaction (RT-PCR) procedures for human tissue samples were previously described [21]. Briefly, total RNA was extracted with an RNeasy Kit (Qiagen, Valencia, CA) and treated with RNasefree DNase I. The RNA samples were then reverse-transcribed into first strand cDNA with 1 ␮g of total RNA, 4 ␮l of 5× first strand buffer, 4 ␮l of 0.1 mM DTT, 1 ␮l of 500 ␮g/ml random primer (Promega), 4 ␮l of 10 mM dNTP mixture, and 200 units of SuperScript II RNase H- reverse transcriptase (Life Technologies, Rockville, MD). The reaction mixture was incubated at 42 ◦ C for 60 min. The mRNA level was measured with a real-time PCR system (Applied Biosystems). The following primers were used: for the DPYD mRNA, 5 -GTAAGGACTCGGCGGACATC-3 (forward) and 5 -GAGTTGCATGAGTTTGTGTTCGA-3 (reverse), and for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, 5 -GCTGAGAACGGGAAGCTTGTC-3 (forward) and 5 -TCTCGCTCCTGGAAGATGGT-3 (reverse).

2.2. Genotyping and haplotype of DPYD 2.5. Real-time RT-PCR for mature miRNA Genomic DNA was isolated from the blood samples with a Toyobo blood kit on a Toyobo HMX-2000 robot (Toyobo, Osaka, Japan). Genetic variations of the DPYD sequence including the 3 -UTR were examined. DPYD-specific primers for genotyping were designed based on a reference sequence of the DPYD gene (Genbank accession no. NG 008807.1). PCR was performed for 35 cycles at 95 ◦ C for 40 s (denaturing), 52–65 ◦ C for 45 s (annealing), and 72 ◦ C for 1 min (extension). PCR products were screened by a single strand conformation polymorphism analysis to assess the genetic variations. Sequencing was performed either directly or after subcloning on an ABI 3100 automatic sequencer (Applied Biosystems, Foster City, CA) using a Big-Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). Haplotypes in the DPYD 3 -UTR were estimated using the EM algorithm embedded in the program Arlequin Ver. 3.1. 2.3. Luciferase reporter assay Based on the estimated haplotype patterns in DPYD 3 UTR, 5 haplotype fragments, approximately 1.3 kb (+3186 bp to +4525 bp downstream from the ATG codon) in size, were amplified. The primers, containing a XbaI restriction site, were 5 -GCTCTAGAGTACCCTTATCTGTGAA-3 (forward) and 5 GCTCTAGAACCAAAAACTGCTCTATCTC-3 (reverse). The PCR product was ligated into the XbaI site of the pGL3-promoter

Small RNA was isolated by using the mirVana miRNA Isolation kit (Ambion). The TaqMan miRNA Assay (Applied Biosystems) was used to detect and quantify mature miRNAs (miR-27a, miR27b, miR-134, and miR-582-5p) on a real-time PCR instrument according to the manufacturer’s instructions. The miRNA levels were normalized to the small nuclear RNA U6 (RNU6B, Applied Biosystems). Comparative real-time PCR was performed in triplicate. Relative expression was calculated using the comparative Ct method. 2.6. Western blotting Western blotting was done at 48 h after each transfection. MIAPaca-2 cells were homogenized with a homogenization buffer [10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, and 1% Nonidet P-40] containing protease inhibitors (0.5 mM (pamidinophenyl) methanesulfonyl fluoride, 2 ␮g/ml aprotinin, and 2 ␮g/ml leupeptin). Lysate from tumor tissue and from normal tissue was prepared using an AllPrep DNA/RNA/Protein Mini Kit (QIAGEN Science) according to the manufacturer’s instructions. Protein concentrations were determined with a BCA Protein Assay kit (Pierce, Rockford, IL). The lysate samples were separated on 8.5% SDS-polyacrylamide gels and transferred to polyvinylidene membranes. The membranes were reacted with a rabbit polyclonal

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Table 1 Genotype and haplotype configurations in the DPYD gene in Caucasian and Japanese subjects (n = 48). Mutation

Population

3352T>C (rs56160474) 3620G>A

Caucasian Japanese Caucasian Japanese Caucasian Japanese Caucasian Japanese Caucasian Japanese Caucasian Japanese Caucasian Japanese Caucasian Japanese Caucasian Japanese

3651G>A (rs1042482) 3846G>A (rs291592) 3858C>T (rs291593) 3907G>A 3978T>C (rs17470762) 4124C>A (rs55992536) 4140T>G (rs41285690)

Genotype

Allele frequency

r/r

r/v

v/v

r

v

38 48 48 47 41 37 17 0 25 10 48 47 46 48 48 46 47 48

9 0 0 1 6 10 24 2 19 27 0 1 2 0 0 2 1 0

1 0 0 0 1 1 7 46 4 11 0 0 0 0 0 0 0 0

0.885 1.000 1.000 0.990 0.917 0.875 0.604 0.021 0.719 0.490 1.000 0.990 0.979 1.000 1.000 0.979 0.990 1.000

0.114 0.000 0.000 0.010 0.083 0.125 0.396 0.979 0.281 0.510 0.000 0.010 0.021 0.000 0.000 0.021 0.010 0.000

Frequency

Position

Haplotype 1 Haplotype 2 Haplotype 3 Haplotype 4 Haplotype 5

3352

3651

3846

3858

Caucasian

Japanese

C T T T T

G A G G G

G A A A G

C T C T C

0.125 0.083 0.115 0.188 0.490

0.000 0.125 0.469 0.385 0.021

Reference sequence: GenBank accession number NG 008807.1. The positions of these mutations were assigned using the start codon ATG as base 1. Abbreviations: r, reference allele; v, variant allele.

antibody against human DPD (kindly provided by Taiho Pharmachemical Co Ltd., Saitama, Japan) and ␤-actin (SIGMA, St Louis, MO). The immunocomplexes were additionally reacted with anti-rabbit IgG, peroxidase-linked species-specific whole antibody (Amersham Biosciences, Piscataway, NJ). Membranes were then washed three times in TBS-Tween, and specific bands were visualized using the ECL system (GE Healthcare, Little Chalfont, Buckinghamshire, UK) according to the manufacturer’s instructions.

Fig. 1. Analysis of miRNA-binding sites in the DPYD gene. (A) The locations of SNPs and miRNAs in the DPYD 3 -UTR. The locations of the SNPs are indicated by arrows. (B) Homology between the 3 -UTR of DPYD and the predicted target sequence of miRNAs. Numbers indicate distance from the stop codon.

3.2. miRNAs complementary to the 3 -UTR of human DPYD mRNA We searched for potential target sites within the 3 -UTR of DPYD mRNA using bioinformatic logarithms available on-line (miRANDA, TargetScan, miRBase and Pictar), and selected the following miRNAs based on matching sites; miR-27a, miR-27b, miR-134, and miR-582-5p (Fig. 1B). Among the four SNPs on which haplotypes 1–5 were based, 3352T>C was located 26 bp downstream from the miR-582-5p-binding site. 3846G>A and 3858C>T were located 15 and 3 bp upstream of the miR-134-binding site, respectively (Fig. 1A).

2.7. Statistical analysis

3.3. Effect of overexpression of miRNAs on luciferase activity

The statistical significance of differences between the control miRNA- and precursor miRNA-transfected cells was determined by an analysis of variance followed by Dunett’s multiple comparison. Comparisons of two groups were made with the Wilcoxon signedrank test. A 5% level of probability was considered to be significant.

We generated reporter constructs containing each haplotype of the full-length human DPYD 3 -UTR downstream of the luciferase gene. Various luciferase reporter plasmids were co-transfected with precursor miRNAs into HepG2 cells. HepG2 cells showing undetectable DPD expression are appropriate for analyzing interaction between miRNAs and DPYD mRNA [22]. We measured the effect of the haplotypes in the 3 -UTR on post-transcriptional regulation of DPYD expression. There is no remarkable difference among the five haplotypes (Fig. 2A), indicating that these polymorphisms are non-functional SNPs for the regulation of DPYD expression. Irrespective of the DPYD 3 -UTR genotype, all the miRNAs significantly decreased luciferase activity to approximately 80% of the control values (Fig. 2B). To evaluate the synergistic effect of miRNA, 60 nM of precursor miRNA (15 nM each for miR-27a, miR-27b, miR-134, and miR-582-5p) was transiently transfected into HepG2 cells. As shown in Fig. 2C, reporter activities were significantly decreased by 50–60% of the control values in the major haplotype for Japanese (Haplotype 3) and Caucasians (Haplotype 5). These results suggest that miR-27a, miR-27b, miR-134, and miR-582-5p recognize the 3 -UTR of DPYD mRNA and cooperate to repress the expression of the DPD protein irrespective of haplotype patterns in the 3 -UTR of DPYD.

3. Results 3.1. Mutations in the 3 -UTR of the human DPYD Nine single nucleotide polymorphisms (SNPs) were identified in the 3 -UTR of DPYD; 3352T>C (rs56160474), 3620G>A, 3651G>A (rs1042482), 3846G>A (rs291592), 3858C>T (rs291593), 3907G>A, 3978T>C (rs17470762), 4124C>A (rs55992536) and 4140T>G (rs41285690) (Table 1). 3620G>A and 3907G>A were novel SNPs. 3846G>A and 3858C>T were more common in the Japanese population. Table 1 summarizes estimated haplotype patterns based on the four SNPs, 3352T>C, 3651G>A, 3846G>A, and 3858C>T, which are located near miRNA target sites (Fig. 1A). The haplotype patterns and frequencies showed clear ethnic-related differences. Haplotype 5 was most common in Caucasians, while haplotypes 3 and 4 were dominant in Japanese.

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Fig. 2. Repressive regulation in the human DPYD haplotypes by miR-27a, miR-27b, miR-134, and miR-582-5p. (A) Relative luciferase activity of the five haplotypes in the 3 -UTR of DPYD gene in transient transfected in HepG2 cells. Luciferase activity was normalized to Renilla luciferase activity. Relative luciferase activity is expressed as a percentage of the relative luciferase activity of the pGL3-Hap 1 plasmid. Each column represents the mean ± S.D. for three independent experiments. (B) Reporter constructs containing the full-length 3 -UTR of DPYD were transiently transfected with 15 nM of precursor for miR-27a, miR-27b, miR-134, miR-582-5p, or control miRNA in HepG2 cells. Relative luciferase activity is expressed as a percentage of the relative luciferase activity of the precursor for the control. Each column represents the mean ± S.D. for three independent experiments. *, P < 0.05, compared with the precursor for the control. (C) The reporter constructs were transiently transfected with 60 nM of precursor for miRNAs (15 nM each for miR-27a, miR-27b, miR-134, and miR-582-5p) or the control in HepG2 cells. Each column represents the mean ± S.D. for three independent experiments. *, P < 0.05, compared with the precursor for the control.

3.4. Effect of overexpression of miRNAs on DPD protein levels We investigated the effect of the miRNAs on DPD protein using MIAPaca-2 cells (Fig. 3A). In contrast to HepG2 cells, MIAPaca-2 cells with relatively high DPYD expression were generally used for

analyzing DPD activity [23]. All four miRNAs significantly decreased the protein levels compared to the control values (Fig. 3B). Among the miRNAs tested, miR-134 was associated with a remarkable change, an approximately 70% decrease, in the DPD protein level. In contrast to the luciferase assay, no clear synergistic effect of miRNAs

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Fig. 3. Effects of overexpression of miR-27a, miR-27b, miR-134, and miR-582-5p on DPD protein levels in MIAPaCa-2 cells. (A) The precursor for each miRNA or control (50 nM or 200 nM) was transfected into MIAPaCa-2 cells. DPD protein levels were determined by Western blotting. (B) Relative DPD protein levels represent the mean ± S.D. for three independent experiments. The DPD level was normalized to the ␤-actin level. Relative DPD protein levels were expressed as percentages of the relative DPD protein of the precursor for the control. *, P < 0.05, compared with the control.

on the DPD protein level was observed with 200 nM of precursor (50 nM each for miR-27a, miR-27b, miR-134, and miR-582-5p) (Fig. 3A). 3.5. Endogenous expression of miRNAs and DPD protein in HepG2 and MIAPaca-2 cells We measured endogenous miRNAs expression in HepG2 and MIAPaca-2 cells (Fig. 4A). The expression levels of miR-27a and miR-27b were significantly higher in HepG2 cells. The levels of miR-134 and miR-582-5p were extremely low in both cell lines. Endogenous DPD protein level was much lower in HepG2 cells (Fig. 4B). Interestingly, HepG2 cells had significantly lower translational efficiency compared to MIAPaca-2 cells (Fig. 4C). 3.6. Expression of DPD protein, DPYD mRNA and miRNAs in human lung tissue To assess the effect of miRNA in vivo, levels of DPD protein and DPYD mRNA in samples of lung tumors and normal tissue obtained from 16 patients were measured (Fig. 5). The mean mRNA level was significantly lower in the tumors than normal tissue (Fig. 5A), whereas the protein level was significantly higher in tumor tissue (Fig. 5B). These results suggest that post-transcriptional regulation is responsible for the high expression of DPD protein in tumor tissues. Next, we compared miRNA levels between tumors and normal tissue. The levels of miR-134 and miR-27b were significantly lower, and the expression of miR-27a tended to be lower in tumor tissue than in normal tissue (Fig. 5C). 4. Discussion Recently, it has been reported that some SNPs located at miRNA-binding sites influence the expression of target genes [24–26]. So, we first analyzed the 3 -UTR sequence and identified nine SNPs. Two of the SNPs, 3620G>A and 3907G>A, were novel,

Fig. 4. Endogenous expression levels of miRNAs and DPD protein, and translational efficiency in HepG2 and MIAPaca-2 cell lines. (A) Expression of miRNAs in cells was determined by real-time RT-PCR. Values are the miRNAs level normalized with the U6 RNA. (B) Relative DPD protein levels were determined by Western blotting, and normalized to ␤-actin levels. (C) Relative translational efficiencies were the ratio of relative DPD protein levels (/␤-actin) versus relative mRNA expression levels (/GAPDH), and normalized to the efficiency in HepG2 cells. Each column represents the mean ± S.D. for three independent experiments. *, P < 0.05, compared with HepG2 cells.

and the frequencies of two others, 3648G>A and 3858C>T, differed between Japanese and Caucasian populations (Table 1). These results were consistent with a previous report [27]. To determine whether candidate miRNAs, predicted using bioinformatic logarithms, repress the DPYD gene, and SNPs in the 3 -UTR of DPYD are associated with inter-individual differences in the regulation of DPD activity by miRNA binding, we estimated haplotype patterns for the 3 -UTR (Table 1). The polymorphisms at 3352T, 3846G, and 3858C are located near the miRNA-binding

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Fig. 5. Relative DPD mRNA, protein, and miRNA levels in normal and tumor tissues. (A) Relative DPD mRNA levels were determined by RT-PCR. The data was normalized with GAPDH. (B) Relative DPD protein levels were determined by Western blotting. Relative DPD protein levels were normalized to ␤-actin levels. (C) Relative miRNA expression was determined by RT-PCR. The data was normalized to the U6 snRNA level. *, P < 0.05, compared with normal tissue.

sites (Fig. 1A). The upstream and downstream regions of the miRNA-binding site may interact with RNA-induced silencing complex (RISC) mediating miRNA-mRNA binding [28]. Next, we evaluated the efficacy of precursor miRNA using a luciferase reporter bearing each haplotype sequence cloned into its 3 -UTR. It was reported that the accessibility of mRNA is important in determining the efficacy of miRNA-mediated translational repression [29]. Thus, the luciferase activity was measured using a luciferase reporter vector containing a full-length 3 -UTR of DPYD (1230 bp).

Estimated five haplotypes did not show polymorphic luciferase activities, suggesting that genetic variants in the 3 -UTR were not related to post-transcriptional regulation in DPD synthesis (Fig. 2A). Over expression of miR-27a, miR-27b, miR-134, and miR582-5p led to reduce the activity through DPYD 3 -UTR, regardless of the haplotype pattern (Fig. 2B). A region of the miRNA known as a seed (nucleotides 2–8) is important for target recognition. Most SNPs in the DPYD 3 -UTR were located several base pairs away from the sequence matching the seed of miRNA. Therefore,

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haplotype patterns may not be an important determinant for decreasing activity through miRNA overexpression. The transient transfection of 60 nM of precursor miRNA (15 nM each for miR-27a, miR-27b, miR-134, and miR-582-5p) was associated with a remarkable decrease in activity; about one-half that of the control precursor miRNA #1 (Fig. 2C). Since each miRNA would bind to a different seed sequence, the four may cooperate to repress the expression of DPD protein. miR-27a, miR-27b, miR-134, and miR-582-5p all suppressed the expression of the endogenous DPD protein in MIAPaca-2 cells, but miR-134 was most effective (Fig. 3). A total of 200 nM of precursor miRNA (50 nM each for miR-27a, miR-27b, miR-134, and miR-582-5p) repressed DPD protein levels; however, the efficiency was similar to that obtained at 50 nM of miR-134. Contrary to the results of the luciferase assay, no synergistic effect on the endogenous DPD protein was observed. This controversial result may be due to saturation of the RISC complex and competition between the transfected miRNAs [30], because the amount of transfected miRNA for evaluating DPD protein was much larger than that in the luciferase assay. To estimate the contribution of endogenous miRNA expression to the DPD protein levels, we measured the miRNA expression in the two cell lines (Fig. 4A). The miR-27a and miR-27b expression levels were significantly higher in HepG2 cells than MIAPaca-2 cells. In particularly, miR-27b showed markedly high expression in HepG2 cells. Consistent with the previous reports [22,23], we showed that HepG2 cells had extremely lower DPD protein expression than MIAPaca-2 cells. Furthermore, relative translational efficiency in HepG2 cells was significantly lower than in MIAPaca-2 cells. Higher miR-27b expression may be responsible for lower DPD expression in HepG2 cells. DPYD mRNA and DPD protein levels were measured in both normal and tumor lung tissues obtained from 16 patients. DPD protein was more abundantly expressed in the tumor tissue than normal tissue, consistent with a previous finding [13]. However, DPYD mRNA levels were significantly lower in the tumor tissue. A discrepancy between DPD protein and DPYD mRNA expression has also been reported in cases of gastrointestinal cancer and in normal mucosa [31]. Levels of miR-27b and miR-134 were significantly lower in tumor tissue than in normal tissue, suggesting that the weak expression causes high DPD protein levels, and the difference in miRNA expression between tumor and normal tissues may be responsible for the discrepancy between DPD protein and mRNA levels. Some studies reported that the protein degradation system is another important factor for DPD protein expression [32–34]. Interestingly, miR-133, which represses the caspase 9 (an enzyme for the ubiquitin-proteasome pathway), showed lower expression in lung tumor compared to normal lung tissues [35,36]. Direct effects of the miRNA on protein degradation system may be another reason for the discrepancy between DPD protein and mRNA levels. The higher levels of miR-27b and miR-134 may contribute to repression of the DPD protein in normal lung tissue, but the lower levels of miR-27b and miR-134 in tumors may be associated with higher DPD protein levels. It has been shown that DPD activity or expression in tumors differs from that in normal tissues depending on the type of carcinoma [37–41]. The expression status in each type of carcinoma would be responsible for the regulation of DPD expression by miR-27b and miR-134. The miR-27b and miR-134 expression levels in most tumor tissues were extremely low; however, a wide inter-individual difference was existed in DPD expression levels in tumor tissues. These observations suggested that inter-individual variability in DPD expression in tumor tissue is responsible for not only miR-27a and miR-134 but also other mechanisms regulating DPD expression including other miRNAs.

5. Conclusion In the present study, we found that expression of the human DPD protein was repressed by some miRNAs, especially miR-27b and miR-134. Recently, it was reported that human serum/plasma contains large amounts of stable miRNAs and the unique expression patterns of serum miRNAs imply great potential as biomarkers to predict individual differences of phenotype [42]. High DPD expression in NSCLCs is responsible for failure in the 5-FU therapy. Since miR-27b and miR-134 can regulate DPD expression levels in NSCLCs, the clinical usefulness of monitoring of these biomarkers in human tissues should be tested. Although larger-scale clinical trials and additional studies are necessary to confirm our results and to advance appropriate therapy for NSCLCs, this study provides new insights into individualizing 5-FU dosages to reduce the risk of toxicity and improving therapeutic efficacy in patients with lung cancer. 6. Conflict of interest statement None declared. Acknowledgments The authors are grateful to the Taiho Pharmachemical Co Ltd. (Tokyo, Japan) for kindly providing the anti-human DPD antibody. They also thank the Japan Society for the Promotion of Science, and Japan Research Foundation for Clinical Pharmacology for financial support. References [1] van Kuilenburg AB, Haasjes J, Richel DJ, Zoetekouw L, Van Lenthe H, De Abreu RA, et al. Clinical implications of dihydropyrimidine dehydrogenase (DPD) deficiency in patients with severe 5-fluorouracil-associated toxicity: identification of new mutations in the DPD gene. Clin Cancer Res 2000;6:4705–12. [2] Heggie GD, Sommadossi JP, Cross DS, Huster WJ, Diasio RB. Clinical pharmacokinetics of 5-fluorouracil and its metabolites in plasma, urine, and bile. Cancer Res 1987;47:2203–6. [3] Mattison LK, Soong R, Diasio RB. Implications of dihydropyrimidine dehydrogenase on 5-fluorouracil pharmacogenetics and pharmacogenomics. Pharmacogenomics 2002;3:485–92. [4] Lazar A, Mau-Holzmann UA, Kolb H, Reichenmiller HE, Riess O, Schömig E. Multiple organ failure due to 5-fluorouracil chemotherapy in a patient with a rare dihydropyrimidine dehydrogenase gene variant. Onkologie 2004;27:559–62. [5] Raida M, Schwabe W, Häusler P, Van Kuilenburg AB, Van Gennip AH, Behnke D, et al. Prevalence of a common point mutation in the dihydropyrimidine dehydrogenase (DPD) gene within the 5 -splice donor site of intron 14 in patients with severe 5-fluorouracil (5-FU)- related toxicity compared with controls. Clin Cancer Res 2001;7:2832–9. [6] Seck K, Riemer S, Kates R, Ullrich T, Lutz V, Harbeck N, et al. Analysis of the DPYD gene implicated in 5-fluorouracil catabolism in a cohort of Caucasian individuals. Clin Cancer Res 2005;11:5886–92. [7] Wei X, McLeod HL, McMurrough J, Gonzalez FJ, Fernandez-Salguero P. Molecular basis of the human dihydropyrimidine dehydrogenase deficiency and 5-fluorouracil toxicity. J Clin Invest 1996;98:610–5. [8] Vreken P, Van Kuilenburg AB, Meinsma R, Smit GP, Bakker HD, De Abreu RA, et al. A point mutation in an invariant splice donor site leads to exon skipping in two unrelated Dutch patients with dihydropyrimidine dehydrogenase deficiency. J Inherit Metab Dis 1996;19:645–54. [9] Nakano J, Huang C, Liu D, Masuya D, Yokomise H, Ueno M, et al. The clinical significance of splice variants and subcellular localisation of survivin in nonsmall cell lung cancers. Br J Cancer 2008;95:607–15. [10] Inoue K, Takao M, Watanabe F, Tarukawa T, Shimamoto A, Kaneda M, et al. Role of dihydropyrimidine dehydrogenase inhibitory fluoropyrimidine against non-small cell lung cancer—in correlation with the tumoral expression of thymidylate synthase and dihydropyrimidine dehydrogenase. Lung Cancer 2005;49:47–54. [11] Peters GJ, Laurensse E, Leyva A, Lankelma J, Pinedo HM. Sensitivity of human, murine, and rat cells to 5-fluorouracil and 5 -deoxy-5-fluorouridine in relation to drug-metabolizing enzymes. Cancer Res 1986;46:20–8. [12] Huang CL, Yokomise H, Fukushima M, Kinoshita M. Tailor-made chemotherapy for non-small cell lung cancer patients. Future Oncol 2006;2:289–99. [13] Yano T, Koga T, Ninomiya S, Takeo S. Dihydropyrimidine dehydrogenase levels in nonsmall-cell lung cancer tissues. Int J Clin Oncol 2002;7:361–4.

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