Multiple microRNAs may regulate the DNA repair enzyme uracil-DNA glycosylase

DNA Repair 12 (2013) 80–86

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Brief report

Multiple microRNAs may regulate the DNA repair enzyme uracil-DNA glycosylase Siv A. Hegre a , Pål Sætrom a,b , Per A. Aas a , Henrik S. Pettersen a , Marit Otterlei a , Hans E. Krokan a,∗ a b

Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, NO-7489 Trondheim, Norway Department of Computer and Information Science, Norwegian University of Science and Technology, NO-7491 Trondheim, Norway

a r t i c l e

i n f o

Article history: Received 11 September 2012 Received in revised form 25 October 2012 Accepted 25 October 2012 Available online 8 December 2012 Keywords: DNA repair Uracil-DNA glycosylase MicroRNA

a b s t r a c t Human nuclear uracil-DNA glycosylase UNG2 is essential for post-replicative repair of uracil in DNA, and UNG2 protein and mRNA levels rapidly decline in G2/M phase. Previous work has demonstrated regulation of UNG2 at the transcriptional level, as well as by protein phosphorylation and ubiquitylation. UNG2 mRNA, encoded by the UNG gene, contains a long 3 untranslated region (3 UTR) of previously unknown function. Here, we demonstrate that several conserved regions in the 3 UTR are potential seed sites for microRNAs (miRNAs), such as miR-16, miR-34c, and miR-199a. Our results show that these miRNAs down-regulate UNG activity, UNG mRNA, and UNG protein levels. Down-regulation was dependent on the 3 UTR, indicating that the miRNAs directly target the conserved seed sites in the 3 UTR. These results add miRNAs as a new modality to UNG’s increasing list of complex regulatory mechanisms. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Uracil in DNA may result from misincorporation of dUMP during replication or from deamination of cytosine, generating pre-mutagenic U:A pairs or directly mutagenic U:G mismatches [1,2]. In most cells, deamination of cytosine occurs spontaneously [3]. However, in B-cells, uracil may be actively generated by the enzyme activation-induced cytidine deaminase (AID). This deamination initiates somatic hypermutation (SHM) and class-switch recombination (CSR) in immunoglobulin genes that is required for the production of mature, high affinity antibodies [4]. In other cells, U:G mismatches are corrected by base excision repair (BER) initiated by a uracil-DNA glycosylase (UDG) [5]. In humans, UNG2 is by far the most efficient uracil-DNA glycosylase in the nucleus in terms of catalytic turnover [6,7], and it is the major enzyme for removal of both misincorporated uracils and deaminated cytosines [6,8]. Moreover, UNG2 is essential in antibody diversification, by removing uracil from AID-induced U:G mismatches in B-cells [9,10]. The human UNG gene encodes two mRNAs generated by alternative splicing and the use of two distinct promoters [11,12]. The mitochondrial form UNG1, and the nuclear form UNG2, share a

Abbreviations: 3 UTR, 3 untranslated region; AID, activation-induced cytidine deaminase; ATM, mutated in ataxia teleangiectasia; BER, base excision repair; CLL, chronic lymphocytic leukemia; CSR, class-switch recombination; miRNA, microRNA; MMR, mismatch repair; NER, nucleotide excision repair; qRT-PCR, quantitative real-time PCR; SHM, somatic hypermutation; TDG, thymine-DNA glycosylase; UDG, uracil-DNA glycosylase; UNG1, mitochondrial uracil-DNA glycosylase 1; UNG2, nuclear uracil-DNA glycosylase 2. ∗ Corresponding author. Tel.: +47 72573074; fax: +47 72576400. E-mail address: [email protected] (H.E. Krokan). 1568-7864/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.dnarep.2012.10.007

common catalytic domain and 3 untranslated region (3 UTR), but have unique N-terminal sequences required for sub-cellular sorting and protein interactions [11]. Both mRNAs are cell cycle regulated with the highest levels just prior to and during S phase [13,14]. In late S phase and early G2/M phase, there is a rapid decrease in UNG2 protein activity and mRNA level, such that UNG2 mRNA is depleted within a period of three hours [12]. During S phase, UNG2 is sequentially triple-phosphorylated, which directs ubiquitylation and apparent proteolytic breakdown of the protein [13,15,16]. However, the molecular mechanisms behind the rapid UNG2 mRNA clearance in G2/M phase have not yet been revealed. MicroRNAs (miRNAs) are small non-coding RNA molecules that negatively regulate gene expression through sequence-specific targeting primarily in the 3 UTR of protein coding genes. It is predicted that >60% of human protein coding genes contain potential miRNA target sites in their 3 UTRs and may be regulated by one or several miRNAs [17]. Animal miRNAs down-regulate gene expression by cleavage, degradation, or translational repression of target mRNA [18–23]. Importantly, miRNAs are commonly dysregulated in human cancers [24] and they can function both as tumor suppressor genes and oncogenes [25–28]. Additionally, miRNAs are found to regulate key components in the cell cycle control machinery [29–31]. Several miRNAs are also shown to be involved in DNA damage response and repair [32,33], and have regulatory roles in double-strand break repair [34–36], nucleotide excision repair (NER) [35], and mismatch repair (MMR) [37,38]. Here, we report miRNA-regulation of the BER protein UNG2. We have identified multiple conserved miRNA target sites in the UNG 3 UTR mRNA and show that miR-16, miR-34c, and miR-199a downregulate UNG activity, mRNA transcript level, and protein level in a 3 UTR-dependent process.

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2. Materials and methods

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Membranes were scanned on the LI-COR Biosciences detection system.

2.1. Bioinformatic analyses 2.4. RNA isolation and quantitative real-time PCR (qRT-PCR) To predict miRNA target sites in the UNG 3 UTR, we used the following three-step procedure. First, using the UCSC 17 species Vertebrate Multiz Alignment and conservation annotation data [39], we identified within the annotated UNG 3 UTR (RefSeq ID NM 080911) sequence blocks consisting of at least six consecutive nucleotides conserved between human, chimp, macaque, dog, cow, and rabbit. Second, we determined whether any of the resulting sequence blocks had perfect sequence complementarity to the seed sequence (nucleotides 2–7 from the 5 end) of known human mature miRNAs (miRBase release 10.0; [40]). Third, we used RNAhybrid [41] to predict the binding pattern between UNG 3 UTR and the miRNAs identified in step two. Specifically, we searched the complete annotated human UNG 3 UTR and used RNAhybrid’s helix constraints to identify additional putative binding sites for the miRNA candidates.

Total RNA for mRNA analysis was prepared using the mirVana miRNA isolation kit (Ambion) according to the manufacturer’s instructions. RNA concentration and quality was measured on a NanoDrop ND-1000 UV-Vis spectrophotometer. Total RNA (770–1000 ng) was reverse transcribed for gene expression analysis in a 20 ␮l reaction using TaqMan reverse transcription reagents (Applied Biosystems). The TaqMan gene expression assay Hs00422172 m1 (Applied Biosystems) was used to quantify the expression of UNG mRNA. Quantitative PCR was carried out on a Chromo4 real-time PCR detection system (BioRad). The relative expression of UNG mRNA was analyzed by the Ct method [43] using GAPDH as endogenous control. 2.5. Plasmid construction and Luciferase assay

2.2. Cell culture and miRNA mimic transfections The human cell lines HeLa (cervical adenocarcinoma), HaCaT (keratinocyte), AGS (gastric adenocarcinoma), SW480 (colon adenocarcinoma), and T-47D (breast carcinoma) were cultured in DMEM (4.5 g/l glucose) supplemented with 10% FBS, 2 mM glutamine, 0.1 mg/ml gentamicin, and 1.25 ␮g/ml fungizone at 37 ◦ C and 5% CO2 . Cells were seeded in six-well dishes at 40–50% confluency in 2 ml antibiotic-free DMEM and cultured overnight. Then, cells were transfected using 4 ␮l/well of DharmaFECT 1 transfection reagent (Dharmacon) and miRNA mimic precursors (diluted in OptiMEM, Invitrogen) at a final concentration of 30 nM. MicroRNA mimics for hsa-miR-16 (Assay ID: PM10339), hsa-miR-34c (Assay ID: PM11039), and hsa-miR-199a (Assay ID: PM10893), and miRNA negative control 1 (AM17110), were all purchased from Ambion. Validated siRNA targeting the UNG gene (Assay ID: 36376) was used as control for the transfection efficiency. Cells were harvested by trypsination for mRNA (24 h after transfection) and protein (48 h after transfection) analysis. Cell pellets for mRNA analysis were resuspended in 100 ␮l RNAlater (Ambion), kept at 4 ◦ C overnight, and stored at −80 ◦ C. Cell pellets for protein analysis were washed once in ice-cold PBS, snap-frozen in liquid nitrogen and stored at −80 ◦ C.

Full-length UNG 3 UTR was amplified by PCR from HeLa genomic DNA and cloned into the unique XhoI-NotI restriction site downstream of the Renilla luciferase gene in the psi-CHECK2.2 vector (Promega). Primers used for amplification were UNG 3 UTR+ (5 -GGAATTCCTCGAGTCATCAGCTGAGGGGTGGCCTTTGAG-3 , the XhoI site is underlined) and UNG 3 UTR(5 -GGAATTCCGCGGCCGCAAACTTTTAACAAACTTTTATTAACAAACCTCGC-3 , the NotI site is underlined). The construct was verified by sequencing. HeLa cells were cotransfected with the UNG 3 UTR psiCheck2.2 reporter construct (25 ng) and one of the following miRNA mimic precursors: hsamiR-16, hsa-miR-34c, or hsa-miR-199a, and the miRNA negative control (75 nM each). Lipofectamine 2000 (Invitrogen) was used as transfection reagent and cells were transfected according to the manufacturer’s instructions. After 48 h, cells were lysed with passive lysis buffer (Promega) and Renilla and firefly luciferase levels were analyzed on a fluorescence plate reader (FLUOstar OPTIMA, BMG labtech) using the Dual luciferase reporter assay (Promega). Changes in Renilla luciferase levels were calculated relative to the firefly luciferase levels (internal control) and normalized to the miRNA negative control. 3. Results

2.3. Preparation of whole cell extracts, UDG activity assay and Western blot analysis Whole cell extracts for UDG activity assays were prepared by dissolving cell pellets in 500 ␮l UDG buffer [20 mM Tris–HCl pH 7.5, 60 mM NaCl, 1 mM EDTA, 1 mM DTT, and 1 x Complete protease inhibitor (Roche)] followed by sonication for 2 × 45 s at 4 ◦ C. Protein extracts were cleared by centrifugation and protein concentrations were measured using the Bradford method (Bio-Rad). UDG activity was assayed in a standard UDG assay as described [6] and the amount of released uracil was measured by scintillation counting. Whole cell extracts for western blot analysis were prepared as described [42] and proteins were separated by electrophoresis on NuPAGE 10% Bis–Tris gels (Invitrogen) and blotted onto PVDF membranes (Immobilon, Millipore) by standard procedures. Primary antibodies were diluted in 5% dry milk in PBS containing 0.1% Tween and membranes were incubated overnight at 4 ◦ C. We used the PU59 antibody [7] [0.5 ␮g/ml] for UNG2 detection (36 kDa). The mouse monoclonal anti-GAPDH [1:10,000] (6C5, Santa Cruz) was used as loading control. Secondary antibodies used were the Odyssey infrared imaging antibodies; IRDye 800CW goat anti-rabbit and goat anti-mouse [1:15,000] (LI-COR Biosciences).

3.1. Multiple human miRNAs may regulate UNG expression by targeting highly conserved sites in UNG 3 UTR We hypothesized that miRNAs could be involved in regulating UNG2 mRNA through the long 3 UTR. To investigate this hypothesis, we used miRNA target predictions to identify miRNAs that could be involved in UNG-regulation. Most animal miRNAs target their sites via imperfect sequence complementarity in the 3 UTR of protein coding genes [44]. Target sites are characterized by complementarity between the 3 UTR and nucleotides 2–7 of the miRNA 5 end, called seed sites [45]. Requiring that seed sites are phylogenetically conserved is assumed to reduce the number of false-positives when predicting miRNA target sites [45–47]. To predict miRNA target sites in the common UNG1 and UNG2 3 UTR [11,12], we therefore identified five sequence blocks (B1-B5) consisting of at least six consecutive nucleotides conserved between human, chimp, macaque, dog, cow, and rabbit (Fig. 1). Several annotated human miRNAs (miRBase release 10.0; [40]) had perfect six or seven-nucleotide seed-complementarity to the sequence blocks B1, B3, and B4 (Fig. 1 and Table 1). As multiple target sites in the same 3 UTR characterise miRNA targeting and can potentially increase the effectiveness

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S.A. Hegre et al. / DNA Repair 12 (2013) 80–86 chr12:

5 kb

108,020,000

hg18

108,025,000

108,030,000

3´UTR

UNG2 UNG1 1A

1B

2

108,032,000 Conserved blocks ≥ 6nt

3

4

5

108,032,500

6

108,033,000

B1: miR-15/16/195

B3: miR-34 B5 B2 B4: miR-199

Fig. 1. The UCSC Genome Browser (March 2006, NCBI36/hg18) illustrating location of UNG1 and UNG2 at chromosome 12. The seven exons in the UNG gene are marked with numbers 1A, 1B, and 2–6. Five conserved sequence blocks (B1–B5) were identified in UNG 3 UTR. Several miRNAs were found to have perfect six or seven-nucleotide seed complementarity to the sequence blocks B1, B3, and B4.

of translational repression [48], we aligned the candidate miRNAs to the human UNG 3 UTR [41]. We found that each miRNA had several potential target sites in UNG 3 UTR (Table 1). These findings indicate that some of the miRNAs identified may regulate the UNG gene by targeting one or several sites in the UNG 3 UTR.

3.2. Selection of miRNA candidates for UNG-regulation For experimental validation we chose the miR-16, miR-34c, and miR-199a as these were representative miRNAs from each of the three miRNA-groups predicted to target the three conserved blocks, B1, B3, and B4 (Table 1). The four miRNAs predicted to target block B1 had similar prediction characteristics, but we chose miR-16 as candidate for validation as it is generally highly expressed in multiple tissues and cell lines [49]. Further, we chose miR-34c as it has the most target sites (nine) of the candidates (Table 1), and miR-199a as it has a conserved seven-nucleotide seed-complementarity to its target sites in the UNG 3 UTR (Table 1). The endogenous expression of miR-16, miR-34c, and miR-199a in all cell lines used in this study was quantified by qRT-PCR. Among the three miRNAs, miR-16 was expressed at high levels (Fig. S4) in all cell lines analyzed, whereas miR-34c was detected at background levels and miR-199a was not detected at all (data not shown). This is not surprising, since miR34c is damage inducible and only present in low levels in many tumor cell lines in the absence of challenge [50,51]. In addition, both miR-34c and miR-199a have been found at low levels in the cell lines and organs examined in [49]. However, this does not make miR-34c and miR-199a less relevant as potential regulators of UNG expression. Thus, we wanted to examine whether the human miRNAs miR-16, miR-34c, and miR-199a could be negatively regulating UNG expression.

3.3. UNG activity, mRNA transcript levels, and protein levels are down-regulated by miR-16, miR-34c, and miR-199a Before testing the effect of predicted miRNA candidates on UNG activity in several cell lines, we carried out initial screens in HeLa cells. Both miR-16 and miR-199a showed dose- and time-related responses with respect to UDG activity when transiently transfected in HeLa cells (Fig. S1). Subsequently, we used 30 nM miRNA mimic oligonucleotides and 48 h time of transfection, as this gave significant and reproducible effect without significant effect in the controls (Fig. S1). MTT viability assays in HeLa cells showed that transfecting the cells restrained cell survival (Fig. S2). Studies have demonstrated that up-regulation of miR-16 [52] and miR-34c [53] causes cell cycle arrest in G1 phase. A slightly lowered survival was observed after miR-16 mimic transfection as compared to the miR-34c and miR-199a transfections. However, as there were no significant differences between the transfections, we concluded that the reduced effect in viability was due to the DharmaFECT transfection reagent, and not caused by the specific miRNA mimics. To examine whether UNG expression is regulated by the predicted human miRNAs, we transfected synthetic miRNA precursor molecules into five human cell lines (T-47D, AGS, SW480, HeLa, and HaCaT) to mimic miRNA up-regulation. The cell lines were selected based on their differences in tissue origin, different UNG2 protein levels and UDG activity [54,55]. We carried out standard UDG assays using whole cell extracts from miRNA mimic transfected cell lines. Fig. 2A demonstrates that miR-16, miR-34c, and miR-199a significantly reduced UDG activity in HeLa, AGS, T-47D, and SW480 cell lines relative to the controls. Results of UDG activity for HaCaT cells are presented in Fig. S3 and indicate down-regulated activity also in this cell line. As miRNAs can down-regulate a specific target gene by affecting mRNA translation or stability [19], we tested whether UNG mRNA was affected by miR-16, miR-34c, and

Table 1 Several human miRNAs may regulate UNG expression by targeting UNG 3 UTR. Conserved blocks in UNG 3 UTR

Human miRNAs

Seed-complementarity (number of nucleotides)

Number of target sites

B1

miR-15a miR-15b miR-16 miR-195

6 6 6 6

3 3 3 3

B2

None

B3

miR-34a miR-34b miR-34c

6 6 6

8 6 9

B4

miR-199a miR-199b

7 6

4 4

B5

None

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Fig. 2. UNG activity, mRNA transcript level, and protein levels are down-regulated by miR-16, miR-34c, and miR-199a. Cells were transiently transfected with miRNA mimics, miR-negative control, or siR-UNG. Significant differences were determined by Student’s t-test assuming equal variances (*P < 0.05; **P < 0.001). (A) The effect on UNG activity was assayed in a standard UDG assay. Total UDG activity is presented as mean ±SD (n ≥ 3) relative to the activity obtained for the miR-negative control transfection. (B) The effect on UNG mRNA transcript levels was measured by qRT-PCR. UNG mRNA levels in HeLa cells are presented as mean ±SD (n ≥ 3) relative to GAPDH expression (endogenous control) and miR-negative control transfection (reference sample), calculated by the Ct method [43]. (C) Whole cell extracts from transfected cell lines were analyzed by Western blot.

miR-199a mimic transfections in HeLa cells. Compared to the control transfections, the candidate miRNAs significantly decreased the UNG mRNA level as measured by qRT-PCR (Fig. 2B). Furthermore, by western blot analysis we found that all three miRNA candidates markedly reduced UNG2 protein levels when compared to the control transfection (Fig. 2C). In summary, the human miR-16, miR-34c, and miR-199a negatively regulate UNG activity and UNG mRNA and protein levels in five human cell lines.

3.4. UNG expression is directly regulated by miR-16, miR-34c, and miR-199a The observed down-regulation of UNG expression could either be caused by a direct interaction between miRNAs and UNG mRNA, or by an indirect regulation of unknown targets, which in turn negatively regulate UNG expression. To test whether UNG is a direct target of the three miRNAs miR-16, miR-34c, and miR-199a, we performed transient miRNA mimic transfections in HeLa cells stably expressing an UNG2-construct without the UNG 3 UTR (pProA3xFLAG-UNG2-EYFP construct) [56]. This construct uses the UNG2 PA -promoter to regulate the expression of a 3xFLAG-UNG2-EYFP fusion protein, thus it has a low level of over-expression (∼1.5fold) and is normally regulated throughout the cell cycle [56]. As positive controls for the experiments, we used two different siRNAs (Ambion) targeting UNG (Fig. 3A); siR-UNG (Assay ID: 36376) targeting UNG in the UNG 3 UTR and siR-UNG Select (Assay ID: s14678) targeting UNG in the coding region. Consequently, these two siRNAs served as separate controls for knockdown of endogenous UNG (both siRNAs) and knockdown of the UNG2-EYFP fusion protein (siR-UNG Select only). Western blot analysis after miRNA

mimic transfections revealed that the endogenous level of UNG2, containing the 3 UTR, was down-regulated by all miRNAs tested. The UNG2 level in the UNG2-EYFP construct without the 3 UTR, however, remained constant after miRNA over-expression (Fig. 3B). These results indicate that UNG is down-regulated by the predicted miRNAs directly in a process requiring the UNG 3 UTR. To further investigate if UNG is directly regulated by the predicted miRNAs, the full-length UNG 3 UTR with putative miRNA binding sites was cloned into the psi-CHECK2.2 dual luciferase (Renilla and firefly) vector downstream of the Renilla luciferase reporter gene. Using this system, a direct miRNA-mRNA interaction would result in reduced Renilla luciferase signal, while the firefly reporter gene serves as an internal control. Co-transfecting the psi-CHECK2.2 reporter vector and each of the three miRNA mimics resulted in significant reduction of luciferase activity for all miRNAs compared to control transfection (Fig. 3C). Collectively, these results strongly suggest that miR-16, miR-34c, and miR-199a directly target the UNG 3 UTR mRNA and down-regulates UNG expression.

4. Discussion Expression of uracil-DNA glycosylase UNG2 is up-regulated in the S phase and rapidly drops towards the end of the S phase/early G2, remaining low in G1 phase [12–14]. Here we report that UNG2 mRNA may be regulated by miRNAs that target seed sites in the long 3 UTR of UNG. Transfection of miRNA mimics for miR-16, miR34c, and miR-199a significantly reduces UNG activity and mRNA and protein levels. MicroRNAs in the miR-16 and miR-34 families are known to induce G1 arrest by negatively regulating cell cycle

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Fig. 3. Down-regulation of UNG expression after miR-16, miR-34c, and miR-199a mimic transfection is caused by a direct targeting of the miRNAs in UNG 3 UTR. (A) Transient transfection experiments of miRNA mimics were performed in stable transfected HeLa cells that in addition to expressing the endogenous UNG2 (top), also expressed a UNG2-construct without the 3 UTR (pProA-3xFLAG-UNG2-EYFP, bottom). The 3xFLAG and EYFP are separated by the UNG2 coding region at each side by linker (L) regions. Positions of the siRNAs targeting UNG are illustrated; siRUNG Select (Assay ID: s14678) targets the UNG coding region and siR-UNG (Assay ID: 36376) targets the 3 UTR of UNG. (B) Whole cell extracts from the transfected stable HeLa pProA-3xFLAG-UNG2-EYFP cell line were analyzed by Western blot. (C) HeLa cells were transiently transfected with the UNG 3 UTR psiCheck2.2 reporter construct in combination with miRNA mimics or miR-negative control. Luciferase activity is presented as mean ±SD (n = 3) relative to the activity obtained for the miRnegative control transfection. Significant differences were determined by Student’s t-test assuming unequal variances (*P < 0.05).

progression [52,53,57,58]. Interestingly, DNA damage-inducible p53 is an important activator of the miR-34 family, which reduces cell proliferation by targeting genes required for proliferation, e.g. CDK4/6, cyclin E and E2F, and anti-apoptotic proteins like BCL2 [53,59–61]. Furthermore, miR-199a causes apoptosis in several cancer cell lines through targeting the MET proto-oncogene [62]. Although the reduced UNG2 levels observed in our experiments could in principle be due to cell cycle arrest or apoptosis, viability data from MTT assays (Fig. S2) indicated no significant difference in survival after miRNA mimic transfections, as compared to control transfection. We suggest that miRNAs targeting the UNG 3 UTR may explain down-regulation of UNG2 outside of the S phase. In the cell lines used in this study, miR-16 was expressed at high levels (Fig. S4), whereas miR-34c and miR-199a had low or nonexistent expression, respectively (data not shown). This is in accordance with the findings by Landgraf et al. [49]. There was no correlation between miR-16 expression levels in the different cell lines and the level of UNG2 expression (data not shown). In general, tumor cell lines express widely variable levels of UNG-proteins [54,55] and it is unknown which factors are responsible for this variation. However, the expression of transcription factor AP-2 may be important to regulate the UNG2 level [63]. When UNG2 mRNA transcription is down-regulated during S phase [12], the constantly expressed miR16 could act in rapid clearance of UNG2 mRNA levels in G2/M phase. Thus, we propose a model stating that a high and constant miR-16 level is consistent with the rapid UNG2 mRNA clearance outside S phase. We investigated the effect of antagomirs to miR-16 on miR-16 and UDG activity. Although we achieved approximately 90% decrease in miR-16 levels (data not shown), a substantial amount of miR-16 remained in the cell, due to the high starting levels. A decrease in UDG-activity was not observed during the treatment period (data not shown). Thymine-DNA glycosylase (TDG) is another human DNA glycosylase capable of excising uracil in DNA. The activity of TDG is also cell cycle regulated, but TDG is up-regulated in G2/M, strictly inverse to that of UNG2. Whereas TDG presence in S phase prevents cell cycle progression and proliferation [16], it is not obvious that UNG2 needs to be down-regulated after S phase, since UNG2 is also likely to have a physiological role in repairing deaminated cytosine outside of S phase [6]. However, in fission yeast, over-expression of UNG2 causes DNA damage and cytotoxicity [64]. There are also some indications that UNG2 may have functions possibly not directly related to DNA repair, as UNG2 is required for assembly of histone 3 variant CENP-A in chromatin, although the mechanism remains elusive [65]. Several recent reports indicate important roles of miRNAs in regulating the DNA damage response, apoptosis, and cell cycle check points, as recently reviewed [32]. The mutated in ataxia teleangiectasia (ATM) kinase is important in coordination of these responses and is the major sensor of double-strand breaks. ATM is apparently regulated by at least two miRNAs, miR101 [66] and miR-421 [67]. MicroRNA-regulation of NER [35,68] and MMR have also been described [37,38], but miRNA-regulation of BER proteins has not previously been reported. Our study demonstrates that miRNAs miR-16, miR-34c, and miR-199a may be important regulators of the BER protein UNG2, either alone or in a concerted action by two or more. This regulation is clearly dependent on the 3 UTR of UNG2 mRNA. However, it remains to be documented whether the corresponding endogenous miRNAs are important in regulation of UNG2, including cell cycle-regulation.

Conflict of interest statement There are no conflicts of interest.

S.A. Hegre et al. / DNA Repair 12 (2013) 80–86

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