- Research article
- Open Access
- Open Peer Review
MicroRNA-433 negatively regulates the expression of thymidylate synthase (TYMS) responsible for 5-fluorouracil sensitivity in HeLa cells
© Gotanda et al.; licensee BioMed Central Ltd. 2013
- Received: 9 April 2013
- Accepted: 1 August 2013
- Published: 2 August 2013
Thymidylate synthase (TYMS) is an important folate-dependent enzyme in DNA synthesis and an important target for cancer chemotherapy. High TYMS expression levels in tumors are generally associated with resistance to 5-fluorouracil (5-FU). The cause of the variability in TYMS expression is still not fully understood, however, only a small proportion of the TYMS expression can be explained by TYMS genetic polymorphisms. The purpose of this study is to identify novel microRNAs (miRNAs) which regulate the expression of TYMS and to determine whether miRNAs binding to the 3′-untranslated region (UTR) of TYMS mRNA affect the proliferation of HeLa cells treated with 5-FU.
An in silico search was performed to find potential binding sites of miRNAs in TYMS mRNA. The efficacy of predicted miRNAs at the 3′-UTR of TYMS mRNA was evaluated using a dual-luciferase reporter assay. TYMS mRNA and protein expression in HeLa cells was quantified with real-time RT-PCR and Western blotting, respectively. The effects of miR-433 on cell proliferative activity were determined by WST-8 assay.
The overexpression of miR-433 was associated with significantly decreased reporter activity in the plasmid containing the 3′-UTR of TYMS mRNA (P < 0.01). The levels of TYMS mRNA and protein in HeLa cells were significantly decreased by the overexpression of miR-433 (P < 0.05). Furthermore, miR-433 increased inhibition of cell proliferation in HeLa cells treated with 5-FU at over 2.0 μM.
The results indicate that miR-433 post-transcriptionally regulates the expression of TYMS mRNA and protein, and increases sensitivity to 5-FU in HeLa cells. This is the first report showing that a miRNA regulating TYMS expression has a significant impact on sensitivity to 5-FU treatment.
- Thymidylate synthase
Thymidylate synthase (TYMS) is an intracellular enzyme critical for de novo synthesis of thymidine monophosphate (dTMP), a precursor of the DNA metabolite thymidine triphosphate. Inhibition of TYMS suppresses cellular growth and leads to cell death. Due to this critical function, TYMS has been a major target of anti-cancer drugs for the past 50 years . Five-fluorouracil (5-FU), one of several TYMS inhibitors, is widely used to induce temporary tumor regression and improve survival, especially for gastrointestinal cancers . The level of TYMS expression is known to be related to the response of tumor cells to 5-FU. Overexpression of TYMS in human colon cancer cells induced resistance to fluoropyrimidine . In addition, higher TYMS levels in tumor tissues in cancer patients were associated with resistance to 5-FU-based chemotherapy [4–7].
There is considerable interindividual variability in the clinical response to 5-FU . The 5-FU dose in cancer patients has yet to be well standardized, due to high variability in plasma 5-FU levels, up to 100-fold , leading to undesired side effects. This variability may be the major contributor to toxicity and subsequent treatment failure [10, 11]. Dose management of 5-FU could therefore prove essential to reducing 5-FU toxicity in patients. Interindividual differences in TYMS expression may be responsible for the different clinical responses to 5-FU. Indeed, a large difference in TYMS mRNA expression was observed between colorectal tumor and normal tissues . Three functionally important polymorphisms were identified in the TYMS gene: (i) rs34743033: a variable number of 28-bp tandem repeat polymorphisms in the promoter region of the 5′-untranslated enhancer region (UTR) ; (ii) rs16430: a 6-bp deletion in the 3′-UTR ; and (iii) rs2853542: a G/C polymorphism in the 5′-UTR enhancer region . These polymorphisms showed a significant association with poor outcome in 5-FU-treated patients [16–18]. However, some reports have indicated that TYMS polymorphisms did not affect the response to 5-FU therapy [12, 19].
MicroRNAs (miRNAs) constitute a class of endogenous, small (19–25 nucleotides), non-coding single-stranded RNAs, and negatively regulate the translation of multiple mRNAs by binding to their 3′-UTRs and inhibiting mRNA translation or breaking down mRNA . It has been reported that miRNAs play an important role as either oncogenes or tumor suppressors, therefore, miRNAs have been increasingly recognized as useful biomarkers, as well as therapeutic tools [21, 22]. Moreover, recent evidence suggests that miRNA expression influences chemosensitivity in human cancer cells [23–26].
TYMS expression is reported to be regulated by miR-192 and miR-215 in colorectal cancer cell lines . However, down regulation of TYMS expression by these miRNAs did not sensitize cells to 5-FU in colorectal cancers. Although high TYMS levels are associated with resistance to 5-FU-based chemotherapy [4–7], no miRNAs which repress TYMS expression leading to an increase in sensitivity to 5-FU, have been identified. Interestingly, the 6-bp deletion (rs16430) in the TYMS 3′-UTR is reported to decrease mRNA stability in vitro and gene expression in vivo. These results suggest that certain miRNAs, which target sites proximal to the 6-bp deletion resulting in lower TYMS expression, exist.
In the current study, we identified a novel miRNA regulating the expression of TYMS and evaluated its effect on the proliferation of a human cervical cancer cell line exposed to 5-FU.
The human cervical cancer HeLa cell line was obtained from RIKEN Cell Bank. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Nichirei Biosciences Inc., Tokyo, Japan) and incubated at 37°C in 5% carbon dioxide.
Dual luciferase reporter assays
The full-length 3′-UTR of TYMS gene with and without the 6-bp deletion was amplified from human genomic DNA using forward and reverse primers (Forward 5′- TCATCTAGAACCCAGACCTT -3′, Reverse 5′- CCAATCTAGAATACAGCACA -3′). XbaI (TaKaRa Inc., Otsu, Japan) restriction sites were created in the primers for cloning. The products were cloned into the pGL3 Promoter vector (Promega, Madison, WI, USA) and named the pGL3 + reference allele or pGL3 + 6 bp deletion allele. Control luciferase constructs were made by ligating oligonucleotides containing a perfect complementary sequence to miR-433 into the XbaI site of the pGL3 Promoter vector (pGL3 + miR-433). HeLa cells were plated in a 24-well plate at a density of 6.0 × 104 cells/well and maintained in DMEM with 10% FBS at 37°C in 5% carbon dioxide. After 24 hours, 380 ng of reporter plasmid, 20 ng of pRL-TK (Promega), and negative control precursor miRNA (pre-miR-negative control) (Applied Biosystems Life Technologies, Carlsbad, CA, USA) or pre-miR-433 miRNA precursor (pre-miR-433) (Applied Biosystems) were transfected by using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA). Reporter assays were performed at 48 hours post-transfection using the Dual luciferase assay system (Promega), and normalized for transfection efficiency by co-transfected Renilla-luciferase.
Transfections of miRNA and small interfering RNA (siRNA)
For Western blotting and quantification of mRNA expression, 0.75, 1.5, and 3.0 nM of pre-miR-negative control, pre-miR-433, TYMS siRNA (On-Target plus SMART pool L-004717-00-0005, Dharmacon, Denver, CO, USA) and 1.0 μL/well Lipofectamine 2000 were mixed with Opti-MEM I medium (Invitrogen), then introduced into HeLa cells (1.0 × 105 cells/well) in a 24-well plate.
RNA isolation and quantitative reverse transcription-PCR analysis of TYMSmRNA
Total RNA from HeLa cells was extracted using the miRVana PARIS kit (Ambion Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions at 48 hours after transfection. The RNA samples were then reverse-transcribed into first strand cDNA with 200 ng of total RNA, 2.0 μl of 5 × first strand buffer, 2.0 μl of 0.1 mM DTT, 0.5 μl of 500 μg/ml random primer (Promega), 2.0 μl of 10 mM dNTP mixture and 100 units of SuperScript II RNase H-reverse transcriptase (Invitrogen). The reaction mixture was incubated at 42°C for 60 minutes. The mRNA level was measured with a real-time PCR system (Applied Biosystems). The following primers were used: for the TYMS mRNA, 5′-TCTGGAAGGGTGTTTTGGAG-3′ (forward) and 5′-CCTCCACTGGAAGCCATAAA-3′ (reverse), and for the β-actin (ACTB) mRNA, 5′-ATGTGGCCGAGGACTTTGATT-3′ (forward) and 5′-AGTGGGGTGGCTTTTAGGATG-3′ (reverse).
HeLa cells were harvested and homogenized with CelLytic M cell Lysis Reagent (Sigma-Aldrich) and Protease Inhibitor Cocktail (Sigma-Aldrich) at 48 hours after transfection. The lysate samples were separated on 12% SDS-polyacrylamide gels and transferred to polyvinylidene membranes. The membranes were hybridized with a mouse monoclonal antibody against human TYMS (Abcam Inc., Cambridge, MA, USA) and β-actin (Sigma-Aldrich). The immunocomplexes were hybridized with anti-mouse IgG horseradish peroxidase-linked whole antibody (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Membranes were washed two times in PBS-Tween, and specific bands were visualized using the ECL system (GE Healthcare) according to the manufacturer’s instructions.
HeLa cells were plated onto a 96-well plate and incubated for 24 hours (4.0 × 103 cells/well). They were then transfected with Pre-miR-negative control (3.0 nM), pre-miR-433 (3.0 nM) or TYMS siRNA (3.0 nM) and treated in 5-FU (Wako, Osaka, Japan) at a final concentration of 0.1, 0.5, 1.0, 2.0, 3.0, 4.0, or 5.0 μM. Cell proliferation was assessed at 120 hours post-transfection using Cell Counting Kit-8 (Dojindo Laboratories Co., Ltd., Kumamoto, Japan) following the manufacturer’s directions.
Statistical analyses were performed with the statistical software program R version 2.13.1 (R Development Core Team, 2011). Means for two groups were compared with an unpaired Student’s t-test (two-tailed). Comparisons of means for multiple groups against the pre-miR-negative control transfected cells were analyzed with Dunnett’s multiple comparison tests. A 5% level of probability was considered to be significant.
Prediction of miRNAs targeting the 3′-UTR of TYMSmRNA
Repressive regulation in the 3’-UTR of TYMSmRNA by miR-433
Effects of overexpression of miR-433 on TYMSmRNA levels in HeLa cells
Effects of overexpression of miR-433 on TYMS protein levels in HeLa cells
Effects of overexpression of miR-433 on sensitivity to 5-FU in HeLa cells
In the present study, we tried to identify a novel miRNA which regulates the expression of TYMS, and evaluate its effects on 5-FU treatment using HeLa cells. After the screening of miRNAs targeting the 3′-UTR of TYMS mRNA, miR-433 was predicted to be located in the 3′-UTR proximal to the 6-bp deletion (rs16430). Interestingly, the 6-bp deletion has been associated with decreased mRNA stability and lower gene expression. To demonstrate that miR-433 directly targets the 3′-UTR with or without 6-bp deletion allele, we analyzed the luciferase activity using the vector containing the two alleles. The luciferase vector with the 6-bp deletion allele showed significantly less activity than that with the wild-type vector, suggesting that the 6-bp deletion in the 3′-UTR was involved in the post-transcriptional regulation of TYMS expression. Mandola et al. reported that the 6-bp deletion influenced TYMS mRNA stability . As shown in Figure 2, overexpression of miR-433 decreased the luciferase activity, suggesting that miR-433 directly repressed TYMS expression, but it was independently observed for the 6-bp deletion allele. Transfection with the miR-433 precursor significantly decreased TYMS mRNA and protein levels in a precursor miRNA concentration-dependent manner (Figures 3 and 4). It was suggested that miR-433 suppresses the expression of the endogenous TYMS protein by controlling the stability of TYMS mRNA transcripts. Taking these observations into consideration, miR-433 acts as a repressive regulator, but is not responsible for the decreased mRNA stability due to the 6-bp deletion.
TYMS is considered the primary target of 5-FU [29, 30]. It has been suggested that high TYMS expression is correlated with low sensitivity to 5-FU [31, 32]. In addition to TYMS genetic polymorphisms, the contribution of miRNAs to sensitivity to 5-FU treatment has recently been studied. MiR-192/215 decreased TYMS mRNA and protein levels, but significantly reduced sensitivity to 5-FU by targeting cell cycle progression . To date, no miRNA which represses TYMS expression leading to an increase in sensitivity to 5-FU has been identified. In addition to TYMS, miR-433 also targets tumor-associated protein growth factor receptor-bound protein 2 (GRB2) involved in the molecular pathogenesis of gastric cancer . MiR-433 expression was reported to be down-regulated in gastric carcinoma . Furthermore, gastric cancer patients with low expression of miR-433 showed lower survival rates than patients with high expression . Previous reports indicated that miR-433 acts as an anti-oncogenic miRNA (anti-oncomir). However, as shown in Figure 5A, overexpression of miR-433 did not affect cell proliferation in HeLa cells without 5-FU treatment. Our experimental concentration for transfection (at 3.0 nM) was approximately 7-fold lower than that of the previous report (at 25–50 nM) . It is possible that miR-433 does not act as an anti-oncomir at lower concentrations.
Conversely, we showed that miR-433 decreased the proliferation of HeLa cells treated with over 2.0 μM of 5-FU. A concentration of 2.0 μM, approximately 260 μg/L, is close to the clinical target range of 5-FU; indeed, 450–550 μg/L is recommended for FOLFOX6 in metastatic colorectal cancer patients with grade 0–1 toxicity . Hence, miR-433 may improve the efficiency of 5-FU chemotherapy in keeping with Luo’s report  that overexpression of miR-433 acts as an anti-oncomir.
This study showed that miR-433 had a significant impact on sensitivity to 5-FU by regulating TYMS expression in vitro. Overexpression of TYMS has been linked to drug resistance . Since miR-433 regulates TYMS expression, it is expected to influence the effect of some TYMS-related drugs (e.g., methotrexate). If this is true, miR-433 may have potential as an adjuvant therapeutic to overcome drug resistance.
TYMS, thymidylate synthase; dTMP, thymidine monophosphate; 5-FU, 5-fluorouracil; UTR, untranslated enhancer region; miRNAs, microRNAs.
We appreciate the technical support from the Research Support Center, Graduate School of Medical Sciences, Kyushu University.
- Di Cresce C, Figueredo R, Ferguson PJ, Vincent MD, Koropatnick J: Combining small interfering RNAs targeting thymidylate synthase and thymidine kinase 1 or 2 sensitizes human tumor cells to 5-fluorodeoxyuridine and pemetrexed. J Pharmacol Exp Ther. 2011, 338: 952-963. 10.1124/jpet.111.183178.View ArticlePubMedGoogle Scholar
- Longley DB, Harkin DP, Johnston PG: 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer. 2003, 3: 330-338. 10.1038/nrc1074.View ArticlePubMedGoogle Scholar
- Copur S, Aiba K, Drake JC, Allegra CJ, Chu E: Thymidylate synthase gene amplification in human colon cancer cell lines resistant to 5-fluorouracil. Biochem Pharmacol. 1995, 49: 1419-1426. 10.1016/0006-2952(95)00067-A.View ArticlePubMedGoogle Scholar
- Kaira K, Serizawa M, Koh Y, Miura S, Kaira R, Abe M, Nakagawa K, Ohde Y, Okumura T, Murakami H, Tsuya A, Nakamura Y, Naito T, Takahashi T, Kondo H, Nakajima T, Endo M, Yamamoto N: Expression of thymidylatesynthase, orotate phosphoribosyltransferase and dihydropyrimidine dehydrogenase in thymic epithelial tumors. Lung Cancer. 2011, 74: 419-425. 10.1016/j.lungcan.2011.04.001.View ArticlePubMedGoogle Scholar
- Nakano J, Huang C, Liu D, Masuya D, Nakashima T, Yokomise H, Ueno M, Wada H, Fukushima M: Evaluations of biomarkers associated with 5-FU sensitivity for non-small-cell lung cancer patients postoperatively treated with UFT. Br J Cancer. 2006, 95: 607-615. 10.1038/sj.bjc.6603297.View ArticlePubMedPubMed CentralGoogle Scholar
- Yeh KH, Shun CT, Chen CL, Lin JT, Lee WJ, Lee PH, Chen YC, Cheng AL: High expression of thymidylate synthase is associated with the drug resistance of gastric carcinoma to high dose 5-fluorouracil-based systemic chemotherapy. Cancer. 1998, 82: 1626-1631. 10.1002/(SICI)1097-0142(19980501)82:9<1626::AID-CNCR5>3.0.CO;2-8.View ArticlePubMedGoogle Scholar
- Johnston PG, Lenz HJ, Leichman CG, Danenberg KD, Allegra CJ, Danenberg PV, Leichman L: Thymidylate synthase gene and protein expression correlate and are associated with response to 5-fluorouracil in human colorectal and gastric tumors. Cancer Res. 1995, 55: 1407-1412.PubMedGoogle Scholar
- Gamelin EC, Danquechin-Dorval EM, Dumesnil YF, Maillart PJ, Goudier MJ, Burtin PC, Delva RG, Lortholary AH, Gesta PH, Larra FG: Relationship between 5-fluorouracil (5-FU) dose intensity and therapeutic response in patients with advanced colorectal cancer receiving infusional therapy containing 5-FU. Cancer. 1996, 77: 441-451. 10.1002/(SICI)1097-0142(19960201)77:3<441::AID-CNCR4>3.0.CO;2-N.View ArticlePubMedGoogle Scholar
- Saif MW, Choma A, Salamone SJ, Chu E: Pharmacokinetically guided dose adjustment of 5-fluorouracil: a rational approach to improving therapeutic outcomes. J Natl Cancer Inst. 2009, 101: 1543-1552. 10.1093/jnci/djp328.View ArticlePubMedGoogle Scholar
- Kline CL, Sheikh HS, Scicchitano A, Gingrich R, Beachler C, Finnberg NK, Liao J, Sivik J, El-Deiry WS: Preliminary observations indicate variable patterns of plasma 5-fluorouracil (5-FU) levels during dose optimization of infusional 5-FU in colorectal cancer patients. Cancer Biol Ther. 2011, 12: 557-568. 10.4161/cbt.12.7.18059.View ArticlePubMedGoogle Scholar
- Gamelin E, Delva R, Jacob J, Merrouche Y, Raoul JL, Pezet D, Dorval E, Piot G, Morel A, Boisdron-Celle M: Individual fluorouracil dose adjustment based on pharmacokinetic follow-up compared with conventional dosage: results of a multicenter randomized trial of patients with metastatic colorectal cancer. J Clin Oncol. 2008, 26: 2099-2105. 10.1200/JCO.2007.13.3934.View ArticlePubMedGoogle Scholar
- Vignoli M, Nobili S, Napoli C, Putignano AL, Morganti M, Papi L, Valanzano R, Cianchi F, Tonelli F, Mazzei T, Mini E, Genuardi M: Thymidylate synthase expression and genotype have no major impact on the clinical outcome of colorectal cancer patients treated with 5-fluorouracil. Pharmacol Res. 2011, 64: 242-248. 10.1016/j.phrs.2011.04.006.View ArticlePubMedGoogle Scholar
- Horie N, Aiba H, Oguro K, Hojo H, Takeishi K: Functional analysis and DNA polymorphism of the tandemly repeated sequences in the 5′-terminal regulatory region of the human gene for thymidylate synthase. Cell Struct Funct. 1995, 20: 191-197. 10.1247/csf.20.191.View ArticlePubMedGoogle Scholar
- Ulrich CM, Bigler J, Velicer CM, Greene EA, Farin FM, Potter JD: Searching expressed sequence tag databases: discovery and confirmation of a common polymorphism in the thymidylate synthase gene. Cancer Epidemiol Biomarkers Prev. 2000, 9: 1381-1385.PubMedGoogle Scholar
- Mandola MV, Stoehlmacher J, Muller-Weeks S, Cesarone G, Yu MC, Lenz HJ, Ladner RD: A novel single nucleotide polymorphism within the 5′ tandem repeat polymorphism of the thymidylate synthase gene abolishes USF-1 binding and alters transcriptional activity. Cancer Res. 2003, 63: 2898-2904.PubMedGoogle Scholar
- Afzal S, Gusella M, Jensen SA, Vainer B, Vogel U, Andersen JT, Brødbæk K, Petersen M, Jimenez-Solem E, Adleff V, Budai B, Hitre E, Láng I, Orosz E, Bertolaso L, Barile C, Padrini R, Kralovánszky J, Pasini F, Poulsen HE: The association of polymorphisms in 5-fluorouracil metabolism genes with outcome in adjuvant treatment of colorectal cancer [abstract]. Pharmacogenomics. 2011, 12: 1257-1267. 10.2217/pgs.11.83.View ArticlePubMedGoogle Scholar
- Cho HJ, Park YS, Kang WK, Kim JW, Lee SY: Thymidylate synthase (TYMS) and dihydropyrimidine dehydrogenase (DPYD) polymorphisms in the Korean population for prediction of 5-fluorouracil-associated toxicity [abstract]. Ther Drug Monit. 2007, 29: 190-196. 10.1097/FTD.0b013e318040b1fe.View ArticlePubMedGoogle Scholar
- Jennings BA, Kwok CS, Willis G, Matthews V, Wawruch P, Loke YK: Functional polymorphisms of folate metabolism and response to chemotherapy for colorectal cancer, a systematic review and meta-analysis [abstract]. Pharmacogenet Genomics. 2012, 22: 290-304. 10.1097/FPC.0b013e328351875d.View ArticlePubMedGoogle Scholar
- Fariña-Sarasqueta A, Gosens MJ, Moerland E, Van Lijnschoten I, Lemmens VE, Slooter GD, Rutten HJ, van den Brule AJ: TS gene polymorphisms are not good markers of response to 5-FU therapy in stage III colon cancer patients. Cell Oncol (Dordr). 2011, 34: 327-335. 10.1007/s13402-011-0030-z.View ArticleGoogle Scholar
- Lee RC, Feinbaum RL, Ambros V: The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993, 75: 843-854. 10.1016/0092-8674(93)90529-Y.View ArticlePubMedGoogle Scholar
- Spizzo R, Nicoloso MS, Croce CM, Calin GA: SnapShot: MicroRNAs in Cancer. Cell. 2009, 137: 586-586.e1. 10.1016/j.cell.2009.04.040.View ArticlePubMedGoogle Scholar
- Nicoloso MS, Spizzo R, Shimizu M, Rossi S, Calin GA: MicroRNAs-the micro steering wheel of tumour metastases. Nat Rev Cancer. 2009, 9: 293-302.View ArticlePubMedGoogle Scholar
- Liu H, D’Andrade P, Fulmer-Smentek S, Lorenzi P, Kohn KW, Weinstein JN, Pommier Y, Reinhold WC: mRNA and microRNA expression profiles of the NCI-60 integrated with drug activities. Mol Cancer Ther. 2010, 9: 1080-1091. 10.1158/1535-7163.MCT-09-0965.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhou M, Liu Z, Zhao Y, Ding Y, Liu H, Xi Y, Xiong W, Li G, Lu J, Fodstad O, Riker AI, Tan M: MicroRNA-125b confers the resistance of breast cancer cells to paclitaxel through suppression of pro-apoptotic Bcl-2 antagonist killer 1 (Bak1) expression. J Biol Chem. 2010, 285: 21496-21507. 10.1074/jbc.M109.083337.View ArticlePubMedPubMed CentralGoogle Scholar
- Bai H, Xu R, Cao Z, Wei D, Wang C: Involvement of miR-21 in resistance to daunorubicin by regulating PTEN expression in the leukaemia K562 cell line. FEBS Lett. 2011, 585: 402-408. 10.1016/j.febslet.2010.12.027.View ArticlePubMedGoogle Scholar
- Tomimaru Y, Eguchi H, Nagano H, Wada H, Tomokuni A, Kobayashi S, Marubashi S, Takeda Y, Tanemura M, Umeshita K, Doki Y, Mori M: MicroRNA-21 induces resistance to the anti-tumour effect of interferon-α/5-fluorouracil in hepatocellular carcinoma cells. Br J Cancer. 2010, 103: 1617-1626. 10.1038/sj.bjc.6605958.View ArticlePubMedPubMed CentralGoogle Scholar
- Boni V, Bitarte N, Cristobal I, Zarate R, Rodriguez J, Maiello E, Garcia-Foncillas J, Bandres E: miR-192/miR-215 influence 5-fluorouracil resistance through cell cycle-mediated mechanisms complementary to its post-transcriptional thymidilate synthase regulation. Mol Cancer Ther. 2010, 9: 2265-2275. 10.1158/1535-7163.MCT-10-0061.View ArticlePubMedGoogle Scholar
- Mandola MV, Stoehlmacher J, Zhang W, Groshen S, Yu MC, Iqbal S, Lenz HJ, Ladner RD: A 6 bp polymorphism in the thymidylate synthase gene causes message instability and is associated with decreased intratumoral TS mRNA levels. Pharmacogenetics. 2004, 14: 319-327. 10.1097/00008571-200405000-00007.View ArticlePubMedGoogle Scholar
- Danenberg PV: Thymidylate synthetase - a target enzyme in cancer chemotherapy. Biochim Biophys Acta. 1977, 473: 73-92.PubMedGoogle Scholar
- Radparvar S, Houghton PJ, Houghton JA: Characteristics of thymidylate synthase purified from a human colon adenocarcinoma. Arch Biochem Biophys. 1988, 260: 342-350. 10.1016/0003-9861(88)90458-4.View ArticlePubMedGoogle Scholar
- Leichman CG, Lenz HJ, Leichman L, Danenberg K, Baranda J, Groshen S, Boswell W, Metzger R, Tan M, Danenberg PV: Quantitation of intratumoral thymidylate synthase expression predicts for disseminated colorectal cancer response and resistance to protracted-infusion fluorouracil and weekly leucovorin. J Clin Oncol. 1997, 15: 3223-3229.PubMedGoogle Scholar
- Lenz HJ, Leichman CG, Danenberg KD, Danenberg PV, Groshen S, Cohen H, Laine L, Crookes P, Silberman H, Baranda J, Garcia Y, Li J, Leichman L: Thymidylate synthase mRNA level in adenocarcinoma of the stomach: a predictor for primary tumor response and overall survival. J Clin Oncol. 1996, 14: 176-182.PubMedGoogle Scholar
- Luo H, Zhang H, Zhang Z, Zhang X, Ning B, Guo J, Nie N, Liu B, Wu X: Down-regulated miR-9 and miR-433 in human gastric carcinoma. J Exp Clin Cancer Res. 2009, 28: 82-10.1186/1756-9966-28-82.View ArticlePubMedPubMed CentralGoogle Scholar
- Ueda T, Volinia S, Okumura H, Shimizu M, Taccioli C, Rossi S, Alder H, Liu CG, Oue N, Yasui W, Yoshida K, Sasaki H, Nomura S, Seto Y, Kaminishi M, Calin GA, Croce CM: Relation between microRNA expression and progression and prognosis of gastric cancer: a microRNA expression analysis. Lancet Oncol. 2010, 11: 136-146. 10.1016/S1470-2045(09)70343-2.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://0-www.biomedcentral.com.brum.beds.ac.uk/1471-2407/13/369/prepub
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