Quercetin induces hepatic γ-glutamyl hydrolase expression in rats by suppressing hepatic microRNA rno-miR-125b-3p

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ScienceDirect Journal of Nutritional Biochemistry xx (2015) xxx – xxx

Quercetin induces hepatic γ-glutamyl hydrolase expression in rats by suppressing hepatic microRNA rno-miR-125b-3p Silvia Anette Wein a,⁎, Alessandro Laviano b , Siegfried Wolffram a a

Institute of Animal Nutrition & Physiology, Christian-Albrechts-University of Kiel, Hermann-Rodewald-Str. 9, 24118 Kiel, Germany b Department of Clinical Medicine, Sapienza University, Viale del Policlinico 155, 00161 Rome, Italy

Received 9 April 2015; received in revised form 5 August 2015; accepted 6 August 2015

Abstract Exogenous factors such as food components including the flavonoid quercetin are suspected to influence micro RNA (miRNA) concentrations and thus possibly target enzymes involved in xenobiotic metabolism. This study therefore investigates the influence of orally administered quercetin on hepatic miRNA and the identification of enzyme target mRNAs relevant in drug metabolism. Male Wistar rats (n=16) were fed either a diet without (C) or with (Q) the addition of 100-ppm quercetin for 7 weeks and subsequently euthanized at the end of the dark phase. To avoid strong effects of food deprivation on hepatic metabolism, food was not removed until 5 h prior to the procedure. Liver was immediately dissected and snap-frozen in liquid nitrogen. Concentrations of 352 hepatic miRNA were measured in pool samples of each dietary group (n=8) using the RT2 miRNA PCR Array System. Differential expression of miRNAs was assumed with fold changes ≥3. Target genes of differentially expressed miRNAs were identified using the database TargetScan. Because rno-miR-125b-3p showed the most prominent fold-change (−9) we further analyzed the expression of its top predicted target gene gamma-glutamyl hydrolase (GGH) by quantitative real-time PCR using hypoxanthine phosphoribosyltransferase 1 (hprt1) as endogenous control. Compared to controls, 23 miRNAs were differentially expressed in rats fed quercetin. A ninefold reduction in hepatic miRNA rno-miR-125b-3p was paralleled by significant induction of GGH mRNA in liver of quercetin fed rats. Because increased GGH expressions were repeatedly associated with resistance to methotrexate, concomitant intake with quercetin should be monitored carefully. © 2015 Elsevier Inc. All rights reserved. Keywords: Quercetin; MicroRNA; Gamma-glutamyl hydrolase; Quantitative real-time PCR; Rat; Methotrexate

1. Introduction One of the most abundant flavonoids present in food plants is the flavonol quercetin [1], whereby plants mainly contain various quercetin glycosides [2]. Oral bioavailability of quercetin has so far been confirmed in rats [3], pigs [4], dogs [5], cows [6], horses [7] and men [8]. Several health-promoting effects of the polyphenol have long been recognized [9,10], and epidemiological studies indicate an association between flavonoid intake and a reduced risk for certain chronic diseases, including type 2 diabetes mellitus and coronary heart diseases [11]. The use of plants or plant extracts for the prevention and treatment of diseases in addition to synthetic pharmaceuticals has greatly increased [12]. Some vendors of quercetin supplements recommend daily intakes of up to 2 g of this flavonol Abbreviations: GGH, gamma-glutamyl hydrolase; PPC, positive PCR controls; RIN, RNA integrity number; RTC, reverse transcription controls; TCS, total context score ⁎ Corresponding author at: Institute of Animal Nutrition & Physiology, Christian-Albrechts-University of Kiel, Hermann-Rodewald-Str. 9, 24118 Kiel. Tel.: +49-431-880-1987. E-mail addresses: [email protected] (S.A. Wein), [email protected] (A. Laviano), [email protected] (S. Wolffram). http://dx.doi.org/10.1016/j.jnutbio.2015.08.010 0955-2863/© 2015 Elsevier Inc. All rights reserved.

(e.g. vitanet1) resulting in approximately 20 mg/kg body weight. However, aside from promising health-promoting actions, one must not neglect possible negative side effects under certain circumstances. Flavonoids have been shown to modulate the expression and activity of enzymes involved in drug [13–18] and folate metabolism [19–21] and, thus, might also interact with chemotherapeutic drugs used in cancer treatment [22]. These actions might well be explained by an impact of quercetin on micro RNAs (miRNA). These small (typically composed of 22 nucleotides) noncoding single-stranded miRNAs are transcribed from endogenous DNA. By the year 2013, more than 1500 miRNA have been described in humans which seem to be involved in key cellular processes [23], and a single miRNA can target hundreds of distinct messenger RNA (mRNA) through seed-matched sites [24]. Mature miRNAs interact with transcripts by binding to mRNA resulting in target degradation or translational blocking [25]. The aim of the present study was to investigate the impact of dietary quercetin on hepatic miRNA expression and the identification of target mRNAs of enzymes relevant in drug metabolism of healthy Wistar rats.

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http://vitanetonline.com/description/SN1690/vitamins/Activated-QuercetinCapsule).

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2. Material and methods

2.4. Real-time quantitative RT-PCR

2.1. Animals and diets

Two-step real-time quantitative RT-PCR with SYBR green detection was performed for each liver sample (0.4-μg total RNA per reaction) using the iCycler® device (BIORAD, Munich, Germany). All reagents necessary were purchased from peqlab (peqlab Biotechnologie, Erlangen, Germany). Validated primer sets were purchased from Qiagen (Hilden, Germany): hypoxanthine phosphoribosyltransferase 1 (endogenous control; primer assay for rat hprt1; Cat. PPR42247F-200), γ-glutamyl hydrolase (target gene; primer assay for rat GGH; Cat. PPR44755A-200). Reaction parameters were: 1 cycle: 3 min at 95 °C (initial denaturation); 40 cycles: 3 s at 95 °C (denaturation), 30 s at 60 °C (annealing), and 45 s at 72 °C (extension). All measurements were performed in triplicate. Relative quantity of mRNA was calculated with the iCycler® software. The efficiency of the PCR was between 90 and 100%.

Male Wistar rats [n=16, initial body weight 81.9±3.2 g [mean±standard deviation (S.D.)], Charles River Laboratories, Kißleg, Germany] were group housed (n=4) in cages with sawdust-covered solid flooring in a controlled environment (22±2 °C, humidity 55%) with a 12-h light/dark cycle. Animals had free access to food and tap water. During the first 2 weeks, all animals were fed a purified flavonoid-poor maintenance diet (C1090, Altromin, Lage, Germany); thereafter, animals received the fat-enriched flavonoid-poor diet containing (mg/kg) 211240 crude protein, 226300 crude fat, 55707 crude fibre, 39113 crude ash and 39510 moisture (C1090-45, Altromin, Lage, Germany) either with (Q) or without (C) the addition of 100-ppm quercetin (n=8 in each group) for 7 weeks. Concentration of quercetin in the diet is corresponding to an average daily intake of 10-mg/kg body weight. Fresh food was offered daily at the beginning of the dark phase, and food residuals were removed and weighed concomitantly. After the 7-week feeding period, animals were sacrificed by blood removal under anaesthesia (Isofluran, Baxter, Unterschleißheim, Germany) at the end of the dark phase. To avoid strong effects of food deprivation on hepatic metabolism, food was not removed until 5 h prior to the procedure. Liver and fat pads (epididymal, retroperitoneal) were immediately dissected, and fresh weights were determined before liver was snap-frozen in liquid nitrogen and stored at −86 °C for further analyses. The animal experiments were approved by the Ministry of Energy, Agriculture, the Environment and Rural Areas of Schleswig-Holstein, Germany (V 312-72241.121-25).

2.2. Tissue preparation, miRNA- and RNA-analysis Frozen livers were ground in a cryo-mill (A11 basic, IKA, Staufen, Germany) cooled with liquid nitrogen. Total miRNA was isolated from each liver according to manufacturer's (SABioscience, USA) protocol using the RT2 qPCR-grade miRNA isolation kit. Total RNA was isolated from each liver according to manufacturer's (peqlab Biotechnologie, Erlangen, Germany) protocol using pegGOLD HP Total RNA Kit. Qualitative analyses were performed on each sample using either the Agilent small RNA kit or Agilent RNA 6000 Nano Kit for miRNA and RNA, respectively, on the Agilent 2100 Bioanalyzer (Agilent Technologie, Böblingen, Germany). For miRNA, quality proof the electropherogram was checked visually, and only samples showing distinct peaks in the range of 20 to 40 nucleotides were used. For total RNA quality assessment, the RNA integrity number (RIN) was used. Only samples bearing RIN values N8 were used for further analysis. Total RNA and miRNA was quantified spectrophotometrically and stored at −86 °C until use.

2.3. miRNA analyses by PCR array Hepatic miRNA of all animals within each dietary group (n=8) was pooled (400 ng/μL) and converted into complementary DNA using an RT2 miRNA First Strand Kit (SABiosciences, Hamburg, Germany). The PCR templates were mixed with the instrument-specific RT2 SYBR Green/Fluorescein qPCR Mastermixes (SABiosciences, Hamburg, Germany) to obtain an amount of 100 ng of small RNA per well and dispersed (10 μL) into a 96-well Genome miRNA PCR Array (RT2 miRNA PCR Array System, Rat Genome, Cat No. MAR-100A-4, SABiosciences, Hamburg, Germany) containing the predispensed miRNAspecific RT2 miRNA qPCR Assays (SABiosciences, Hamburg, Germany). The PCR array contained a panel of primer sets for 352 miRNAs, four blanks, four small RNAs as the internal controls (housekeeping gene, HG), two reverse transcription controls (RTCs) and two positive PCR controls (PPCs). The real-time qRT-PCR was performed on a iCycler® (Bio-Rad, Munich, Germany) with the following cycling parameters: 95 °C for 10 min, then 40 cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s. SYBR Green fluorescence was recorded from every well during the annealing step of each cycle. The threshold cycle (Ct) value of each sample was calculated with iCycler® (Bio-Rad, Munich, Germany). Mean PPC Ct values were 20±2, and differences in CTPPC across arrays was below two. The difference between the average CTRTC and the average CTPPC was below 5. All samples passed the quality controls based on the Ct values of RTC and PPC. Finally, relative expression was analysed using the PCR Array Data Analysis Web Portal (http://www.sabiosciences. com/pcrarraydataanalsis.php), where any Ct value less than 35 was considered a positive call. Relative miRNA expression levels were determined with the ΔCt method, normalized with the HG Rnu6, rno-U87 (endogenous controls), which were geometrically averaged and reported as 2-ΔCt, where ΔCt=(miRNA of interest)–Ct (endogenous controls). Differences in miRNA-expression between groups were assumed (A) if they were expressed in both feeding groups and (B) if fold change was N3. We performed in silico analysis to find target genes of the identified miRNAs using the software TargetScan (Release 6.2, June 2012) [26,27]. TargetScan predictions are scored by a “context score” which combines contributions from a wide range of features known to affect target site efficacy, including seed type, compensatory binding in the 3’ region of the miRNA, AU content and position on the 3’UTR. Lower values indicate stronger downregulation of expression. Total context score (TCS) for an miRNA:gene pair is calculated by adding scores for the individual target sites on the gene. The top predicted target gene according to the TCS was then further investigated [28].

2.5. Statistical analysis Because we have used one pooled sample per feeding group in the present miRNAarray analysis, it was not possible to statistically evaluate group differences. Pooling was performed on basis of each single miRNA concentration, and determined Ct values refer to the average of the results of a single determination of the eight samples. Thus, the results well mirror probable biological effects, provided a method of analysis with high sensitivity (e.g. PCR) [29–31]. Differences between feeding groups in relative gene expression of the identified target mRNA were analyzed in tissue samples (triplicates) of each animal and were analyzed using the software REST© [32]. Zootechnical parameters are presented as group means±S.D. Statistical analysis was performed using unpaired samples t test using Graph Pad Prism (GraphPad Software, Inc., Version 4.03, 2005; San Diego, CA, USA); level for significance was set, Pb0.001.

3. Results Daily food intake (C: 22±3.0; Q: 21±2.5 g/day), fresh liver weight (C: 15±2.0: Q: 14±1.3 g) and final body weight (C: 489±35: Q: 455± 28 g) were not different between groups. Using the miRNA array, 352 hepatic miRNA were investigated simultaneously. Some miRNA were not detectable, and additionally, we excluded specific miRNA from further analysis if Ct levels were ≥35. In the liver of quercetin-supplemented animals, the expression of 19 miRNA was reduced (Table 1), and that of four miRNA was enhanced compared to controls (Table 2). Each respective top predicted target gene is listed with its corresponding miRNA and TCS (Tables 1 and 2). With a fold change of −9, the hepatic miRNA rno-miR-125b-3p was the most affected miRNA investigated, and its expression was suppressed. According to the TCS of −0.61, the top predicted target mRNA of the miRNA rno-miR-125b-3p encodes GGH. To verify the quality of this calculated prediction, we additionally measured the mRNA concentration of hepatic GGH in each individual animal by quantitative real-time PCR. The mRNA concentration of this hepatic enzyme was significantly (Pb0.001) induced by a mean factor of 1.843 (S.E. range is 0.997–3.505) in the quercetin-supplemented group in comparison to controls.

Table 1 Suppressed hepatic miRNA with their top predicted target gene in quercetin-supplemented rats compared to controls after a 7-week feeding period. miRNA

Fold change

Top predicted target gene according to TCS

TCS

rno-miR-125b-3p rno-miR-133b rno-miR-505 rno-miR-1 rno-miR-342-3p rno-miR-298 rno-miR-503 rno-miR-206 rno-miR-33 rno-miR-216a rno-miR-301a

−9 −7 −7 −6 −5 −5 −4 −4 −4 −4 −3

−0.61 −0.73 −0.8 −0.59 −0.92 −1.0 −0.64 −0.49 −0.73 −0.6 −0.84

rno-miR-21 rno-miR-205

−3 −3

Gamma-glutamyl hydrolase GA binding protein transcription factor OTU domain containing 4 Solute carrier family 44, member 1 Suppressor of cytokine signaling 6 C-type lectin domain family 2 Kinesin family member 1C Coronin, actin binding protein, 1C ATP-binding cassette, sub-family A 1 Adenylosuccinate synthase Ligand-dependent nuclear receptor corepressor-like Chromosome 6 open reading frame 105 Chimerin 1

−0.57 −0.67

rno=rattus norvegicus; miR: mature micro ribonucleic acid; TCS (efficiency of prediction; TCS=0: no efficiency of prediction; TCS≤−0,2: sufficient efficiency for prediction; TCS=−1: maximum efficiency of prediction.

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Table 2 Induced hepatic miRNA with their top predicted target gene in quercetin-supplemented rats compared to controls after a 7-week feeding period. miRNA

Fold change

Top predicted target gene according to TCS

TCS

rno-miR-125a-3p rno-miR-132 rno-miR-411

5 5 4

−0.76 −0.61 −0.85

rno-miR-484

3

Endomucin High mobility group AT-hook 2 Regulator of G protein signaling 9 binding protein F-box and leucine-rich repeat protein 18

−0.88

TCS (efficiency of prediction; TCS=0: no efficiency of prediction; TCS≤−0,2: sufficient efficiency for prediction; TCS=−1: maximum efficiency of prediction.

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repair and are required for normal cellular proliferation and for replication. Antifolates (e.g. methotrexate) are essential inhibitors of folate-dependent enzymes and, thus, are commonly used in the treatment of various cancer forms including acute lymphoblastic leukemia, lymphoma and breast cancer [47]. The concomitant intake of quercetin together with antifolates should be monitored carefully because quercetin induced hepatic GGH mRNA concentration most likely by the inhibition of the miRNA rno-miR-125b-3p in rats. Conflict of Interest Authors have no competing financial interests or any other conflicts of interest.

4. Discussion MiRNAs play an important role in the regulation of gene expression, and although the underlying mechanism has not been elucidated yet, miRNAs might well be involved in the broad biological effects of quercetin [33]. One major finding of the present study is that the addition of 100 ppm quercetin to the diet, corresponding to an average daily intake of approximately 10 mg/kg body weight, modifies the expression of hepatic miRNAs in vivo. It is remarkable that the number of suppressed miRNA is about five times higher than the number of induced miRNAs. Because the induction of miRNA expression normally results in an inhibited translation and/or degradation of the target mRNA [34] and the suppression of miRNA expression results in increased translation of the target mRNA [34,35], we conclude that quercetin more often enhances the translation of target mRNAs, at least with respect to miRNA-dependent gene expression. For further analysis of the differentially expressed miRNA, we performed a TargetScan database search. The TargetScan software predicts biological targets of miRNAs by searching for the presence of conserved 8mer and 7mer sites that match the seed region of each miRNA [36]. In mammals, predictions are ranked based on the predicted efficacy of targeting as calculated using the basic TargetScan algorithms [36]. In addition thereto, another parameter to the TCS has been taken into account for the analysis in the present study. By including more binding characteristics, this parameter shall evaluate the efficiency of the prediction [28]. However, one general problem in assessing the biological impact of miRNA on protein biosynthesis is the lack of inverse linear correlation between the miRNA expression and protein concentration [34]. Interestingly, in the quercetin-supplemented group, the top predicted target mRNA of the ninefold suppressed hepatic miRNA rno-miR-125b-3p is encoding GGH, an enzyme well known to be involved in folate metabolism [37]. This is of particular interest, because the antifolate methotrexate is a widely used chemotherapeutic drug [38], and quercetin has been found beneficial by exerting antioxidative effects [39], inducing cell death/cell cycle arrest preferentially in cancer cells [40] and by reducing antineoplastic drug-induced cyto-genotoxicity [41]. It can therefore be assumed that methotrexate treatment could occur concomitantly with an intensified quercetin intake. To assess the impact of quercetin at the level of mRNA and, thus, to verify data from miRNA analysis, we have additionally analyzed GGH mRNA concentration using quantitative real-time RT PCR. The PCR data confirmed the results from miRNA-array analysis and subsequent target gene prediction. The GGH mRNA concentrations were significantly higher in the quercetin-supplemented group compared to controls. The quercetin-induced up-regulation of GGH mRNA concentration might be of importance because increased expression levels of GGH were repeatedly reported to be associated with resistance to methotrexate [42–46]. GGH catalyzes the hydrolysis of (anti)folylpolygamma-glutamates by the removal of gamma-linked polyglutamates [37]. Intracellular folates are essential co-factors in DNA synthesis and

Authors' contributions to manuscript SAW, SW and AL designed research including project conception, development of overall research plan and study oversight; SAW conducted research and analyzed data and performed statistical analysis; SAW wrote the paper; SAW, SW and AL had primary responsibility for final content. Acknowledgements The authors would like to thank Wiebke Kühl for excellent technical assistance. References

[1] Harborne JB, Williams CA. Advances in flavonoid research since 1992. Phytochemistry 2000;55:481–504. [2] Hollman PCH, Arts ICW. Flavonols, flavones and flavanols - nature, occurrence and dietary burden. J Sci Food Agric 2000;80:1081–93. [3] Graf BA, Milbury PE, Ameho C, Dolnikowski GG, Blumberg JB. Metabolism of dietary quercetin in the gastrointestinal tract, liver and kidney of the rat. Abstr Pap Am Chem Soc 2005;229:U59. [4] Ader P, Wessmann A, Wolffram S. Bioavailability and metabolism of the flavonol quercetin in the pig. Free Radic Biol Med 2000;28:1056–67. [5] Reinboth M, Wolffram S, Abraham G, Ungemach FR, Cermak R. Oral bioavailability of quercetin from different quercetin glycosides in dogs. Br J Nutr 2010;104: 198–203. [6] Berger LM, Wein S, Blank R, Metges CC, Wolffram S. Bioavailability of the flavonol quercetin in cows after intraruminal application of quercetin aglycone and rutin. J Dairy Sci 2012;95(9):5047–55. [7] Wein S, Wolffram S. Oral bioavailability of quercetin in horses. J Equine Vet Sci 2013;33:441–5. [8] Erlund I, Kosonen T, Alfthan G, Maenpaa J, Perttunen K, Kenraali J, et al. Pharmacokinetics of quercetin from quercetin aglycone and rutin in healthy volunteers. Eur J Clin Pharmacol 2000;56:545–53. [9] Middleton E, Kandaswami C, Theoharides TC. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol Rev 2000;52:673–751. [10] Williamson G, Manach C. Bioavailability and bioefficacy of polyphenols in humans. II. Review of 93 intervention studies. Am J Clin Nutr 2005;81(1 Suppl.):243S–55S. [11] Graf BA, Milbury PE, Blumberg JB. Flavonols, flavones, flavanones, and human health: epidemiological evidence. J Med Food 2005;8:281–90. [12] Vargas-Murga L, Garcia-Alvarez A, Roman-Vinas B, Ngo J, Ribas-Barba L, van den Berg SJPL, et al. Plant food supplement (PFS) market structure in EC Member States, methods and techniques for the assessment of individual PFS intake. Food Funct 2011;2:731–9. [13] Cermak R, Wein S, Wolffram S, Langguth P. Effects of the flavonol quercetin on the bioavailability of simvastatin in pigs. Eur J Pharm Sci 2009;38:519–24. [14] Hsiu SL, Hou YC, Wang YH, Tsao CW, Su SF, Chao PDL. Quercetin significantly decreased cyclosporin oral bioavailability in pigs and rats. Life Sci 2002;72: 227–35. [15] Mandery K, Balk B, Bujok K, Schmidt I, Fromm MF, Glaeser H. Inhibition of hepatic uptake transporters by flavonoids. Eur J Pharm Sci 2012;46:79–85. [16] Mesia-Vela S, Kauffman FC. Inhibition of rat liver sulfotransferases SULT1A1 and SULT2A1 and glucuronosyltransferase by dietary flavonoids. Xenobiotica 2003; 33:1211–20. [17] Ofer M, Wolffram S, Koggel A, Spahn-Langguth H, Langguth P. Modulation of drug transport by selected flavonoids: involvement of P-gp and OCT? Eur J Pharm Sci 2005;25:263–71.

4

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[18] Wein S, Cermak R, Wolffram S, Langguth P. Chronic quercetin feeding decreases plasma concentrations of salicylamide phase II metabolites in pigs following oral administration. Xenobiotica 2012;42:477–82. [19] Alemdaroglu NC, Wolffram S, Boissel JP, Closs E, Spahn-Langguth H, Langguth P. Inhibition of folic acid uptake by catechins and tea extracts in Caco-2 cells. Planta Med 2007;73:27–32. [20] Alemdaroglu NC, Dietz U, Wolffram S, Spahn-Langguth H, Langguth P. Influence of green and black tea on folic acid pharmacokinetics in healthy volunteers: potential risk of diminished folic acid bioavailability. Biopharm Drug Dispos 2008;29:335–48. [21] Augustin K, Frank J, Augustin S, Langguth P, Ohrvik V, Witthoft CM, et al. Green tea extracts lower serum folates in rats at very high dietary concentrations only and do not affect plasma folates in a human pilot study. J Physiol Pharmacol 2009;60: 103–8. [22] Moon YJ, Wang XD, Morris ME. Dietary flavonoids: effects on xenobiotic and carcinogen metabolism. Toxicol in Vitro 2006;20:187–210. [23] Lancon A, Michaille JJ, Latruffe N. Effects of dietary phytophenols on the expression of microRNAs involved in mammalian cell homeostasis. J Sci Food Agric 2013;93:3155–64. [24] Lim LP, Lau NC, Garrett-Engele P, et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 2005;433:769–73. [25] Garcia DM, Baek D, Shin C, Bell GW, Grimson A, Bartel DP. Weak seed-pairing stability and high target-site abundance decrease the proficiency of lsy-6 and other microRNAs. Nat Struct Mol Biol 2011;18:1139-U75. [26] Fendler A, Stephan C, Yousef GM, Jung K. MicroRNAs as regulators of signal transduction in urological tumors. Clin Chem 2011;57:954–68. [27] Witkos TM, Koscianska E, Krzyzosiak WJ. Practical aspects of microRNA target prediction. Curr Mol Med 2011;11:93–109. [28] Grimson A, Farh KKH, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP. MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell 2007;27:91–105. [29] Wang WT, Zhao YN, Han BW, Hong SJ, Chen YQ. Circulating MicroRNAs identified in a genome-wide serum MicroRNA expression analysis as noninvasive biomarkers for endometriosis. J Clin Endocrinol Metab 2013;98:281–9. [30] Docherty SJ, Butcher LM, Schalkwyk LC, Plomin R. Applicability of DNA pools on 500 KSNP microarrays for cost-effective initial screens in genomewide association studies. BMC Genomics 2007;8. [31] Meaburn E, Butcher LM, Liu L, Fernandes C, Hansen V, Al-Chalabi A, et al. Genotyping DNA pools on microarrays: tackling the QTL problem of large samples and large numbers of SNPs. BMC Genomics 2005;6. [32] Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST (c)) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucl Acids Res 2002;30.

[33] Joven J, Espinel E, Rull A, Aragones G, Rodriguez-Gallego E, Camps J, et al. Plantderived polyphenols regulate expression of miRNA paralogs miR-103/107 and miR-122 and prevent diet-induced fatty liver disease in hyperlipidemic mice. Biochim Biophys Acta Gen Subj 1820;2012:894–9. [34] Selbach M, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N. Widespread changes in protein synthesis induced by microRNAs. Nature 2008; 455:58–63. [35] Esau C, Davis S, Murray SF, Yu XX, Pandey SK, Pear M, et al. miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab 2006;3: 87–98. [36] Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005;120:15–20. [37] Galivan J, Ryan TJ, Chave K, Rhee M, Yao R, Yin DZ. Glutamyl hydrolase: pharmacological role and enzymatic characterization. Pharmacol Ther 2000;85: 207–15. [38] Celtikci B, Lawrance AK, Wu Q, Rozen R. Methotrexate-induced apoptosis is enhanced by altered expression of methylenetetrahydrofolate reductase. AntiCancer Drugs 2009;20:787–93. [39] Lamson DW, Brignall MS. Antioxidants and cancer, part 3: quercetin. Altern Med Rev 2000;5:196–208. [40] Russo M, Spagnuolo C, Tedesco I, Bilotto S, Russo GL. The flavonoid quercetin in disease prevention and therapy: facts and fancies. Biochem Pharmacol 2012;83: 6–15. [41] Sekeroglu ZA, Sekeroglu V. Effects of Viscum album L. extract and quercetin on methotrexate-induced cyto-genotoxicity in mouse bone-marrow cells. Mutat Res Genet Toxicol Environ Mutagen 2012;746:56–9. [42] Li WW, Waltham M, Tong W, Schweitzer BI, Bertino JR. Increased activity of gamma-glutamyl hydrolase in human sarcoma cell lines: a novel mechanism of intrinsic resistance to methotrexate (MTX). Adv Exp Med Biol 1993;338:635–8. [43] Mauritz R, Peters GJ, Priest DG, Assaraf YG, Drori S, Kathmann I, et al. Multiple mechanisms of resistance to methotrexate and novel antifolates in human CCRFCEM leukemia cells and their implications for folate homeostasis. Biochem Pharmacol 2002;63:105–15. [44] Rhee MS, Wang Y, Nair MG, Galivan J. Acquisition of resistance to antifolates caused by enhanced gamma-glutamyl hydrolase activity. Cancer Res 1993;53:2227–30. [45] Rhee MS, Ryan TJ, Galivan J. Glutamyl hydrolase and the multitargeted antifolate LY231514. Cancer Chemother Pharmacol 1999;44:427–32. [46] Shubbar E, Helou K, Kovacs A, Nemes S, Hajizadeh S, Enerback C, et al. High levels of gamma-glutamyl hydrolase (GGH) are associated with poor prognosis and unfavorable clinical outcomes in invasive breast cancer. BMC Cancer 2013;13:47. [47] Zhao RB, Goldman ID. Resistance to antifolates. Oncogene 2003;22:7431–57.