miR-34a suppresses mutagenesis by inducing apoptosis in human lymphoblastoid TK6 cells

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Mutation Research 758 (2013) 35– 40

Contents lists available at ScienceDirect

Mutation Research/Genetic Toxicology andEnvironmental Mutagenesis

jou rn al h om ep age: www.elsev ier .com/ locate /gentoxC om mu n i ty add ress : www.elsev ier .com/ locate /mutres

iR-34a suppresses mutagenesis by inducing apoptosis in humanymphoblastoid TK6 cells�

inrong Chena, Yongbin Zhangb, Jian Yana, Rakhshinda Sadiqa, Tao Chena,∗

Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR 72079, USAOffice of Scientific Coordination, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR 72079, USA

r t i c l e i n f o

rticle history:eceived 6 December 2012eceived in revised form 27 August 2013ccepted 30 August 2013vailable online 8 September 2013

eywords:

a b s t r a c t

miR-34a, a tumor suppressor miRNA, has been identified as a direct transcriptional target of P53. miRNAprecursors and inhibitors have been used to modulate the expression of their targeted mRNA and therebystudy miRNA functions. We indicated in our previous work that X-ray induces miR-34a expression in atime and dose dependent manner. The objective of this study was to elucidate the role of miR-34a inX-ray-induced mutations in human lymphoblast TK6 cells. Neither over-expression of miR-34a by lipidtransfection of miR-34a precursor nor down regulation of endogenous miR-34a by miR-34a inhibitor

iR-34autagenesis

poptosis

had any effect on X-ray-induced micronucleus frequency in TK6 cells. Over-expression of miR-34a in TK6cells significantly reduced X-ray induced mutant frequency (MF) in the Thymidine Kinase (TK) locus whilesuppression of endogenous miR-34a can increase the background level MF in TK6 cells. Furthermore,over-expression of miR-34a promoted and down-regulation of miR-34a inhibited background and X-ray-induced apoptosis in TK6 cells. Our study suggests miR-34a is an important negative regulator of

. Introduction

MicroRNAs (miRNAs) are endogenous small non-coding RNAolecules that regulate gene expression at the post-transcriptional

evel by paring with one or multiple seeding sequences in the 3′

ntranslated region (UTR) of their target genes [1–3]. Microar-ay and bioinformatics analysis indicate that more than 30% ofll human genes are regulated by miRNAs [4]. miR-34a is a directranscriptional target of P53 [5–8]. P53 protein up-regulates miR-4a expression through binding to its promoter region [7,8]. Ineveral type of cancer cells, the promoter region of miR-34a is sub-ect to aberrant CpG methylation which leads to inactivation ofhe gene [9,10]. miR-34a functions as a tumor suppressor in P53etwork [9,11–15]. Similar to the P53 tumor suppressor gene, miR-4a inhibits cell proliferation, triggers cell cycle arrest, and induces

miR-34a was reported as a strong predictor for tumor prognosis.igh expression of miR-34a in the tumor cells of Ewing’s sarcoma

� The views presented in this article do not necessarily reflect those of the U.S.ood and Drug Administration.∗ Corresponding author at: HFT 130, NCTR, 3900 NCTR Road, Jefferson, AR 72079,SA. Tel.: +1 870 543 7954; fax: +1 870 543 7682.

E-mail addresses: xinrong.Chen@fda.hhs.gov (X. Chen),ongbin.Zhang@fda.hhs.gov (Y. Zhang), Jian.yan@fda.hhs.gov (J. Yan),akhshinda.Sadiq@fda.hhs.gov (R. Sadiq), tao.chen@fda.hhs.gov (T. Chen).

383-5718/$ – see front matter. Published by Elsevier B.V.ttp://dx.doi.org/10.1016/j.mrgentox.2013.08.010

m is possibly mediated through apoptosis.Published by Elsevier B.V.

patients indicates a low possibility of an adverse event while lowexpression predicts an increased risk for disease recurrence [17].Up-regulation of miR-34a in the tumor cells of non-small-cell lungcancer patients had a positive association with longer survival[9]. The therapeutic effect of miR-34a was explored in a recentstudy. The expression of miR-34a was down-regulated in CD44+

prostate cancer stem cells and forced expression of miR-34a inthese cells inhibited clonogenic expansion, tumor regeneration,and metastasis. Conversely, suppression of miR-34a activity inCD44− prostate cancer cells promoted tumor development andmetastasis. Of note, systemically delivered miR-34a significantlyinhibited prostate cancer metastasis and increased the survival oftumor-bearing mice [18].

The somatic mutation theory of carcinogenesis has been the pre-vailing paradigm in cancer research for the last 50 years [19,20]. Itis hypothesized that cancer is derived from somatic cells that haveaccumulated DNA mutations over time. Considering the close rela-tionship of DNA mutation and carcinogenesis, there is a good reasonto believe that miR-34a may exert its tumor suppressor functionby inhibiting DNA mutation. Oral administration of benzopyrene,a classic genotoxic carcinogen, to MutaMice led to elevated DNAadduct formation and increased mutant frequency (MF) in theLacZ transgene. Interestingly, miR-34a was the only hepatic miRNA

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iR-34a was required for normal DNA damage response in humanreast cancer cells [26]. Low expression of miR-34a in chronic lym-hocytic leukemia patients was associated with an impaired DNAamage response [12]. In particular, Caenorhabditis elegans with

oss-of-function mutations in the miR-34 gene had an abnormalellular survival response to radiation [26]. The expression levelsf miR-34a in TK6, WTK1, and NH32 cells were inversely correlatedith their mutability: higher levels of miR-34a correspond with lessutable cells [22]. Thus, miR-34a could play an important role in

uppression of mutations induced by different mutagens.In the present study, we explored the role of miR-34a in cytotox-

city, genotoxicity and apoptosis in human lymphoblast TK6 cells.ur results showed that miR-34a has a suppressive effect on spon-

aneous and X-ray-induced mutations in TK6 cells. The inhibitionf mutation by miR-34a could contribute to its tumor suppressionunction.

. Materials and methods

.1. Reagents

siPORTTM NeoFXTM transfection agent, miRNA isolation kit, cDNA synthesis kit,CR amplification reagent, and miRNAs, including Pre-miR negative #1 control,iR-34a precursor, and miR-34a inhibitor, were obtained from Ambion (Carlsbad,

A). Cell culture medium and horse serum were obtained from Gibco (Carlsbad,A). Deoxycytidine, hypoxanthine, aminopterin, thymidine, and trifluorothymidineTFT) were obtained from Sigma (St. Louis, MO). The in vitro micronucleus assayit was purchased from Litron Laboratories (Rochester, New York). The 6 � fluo-escent latex microspheres (counting beads) used in the micronucleus assay wasurchased from Invitrogen (Carlsbad, CA). The caspase 3/7 assay kit was purchasedrom Promega (Madison, WI). Tunnel assay kit was purchased from R&D systemsMinneapolis, MN).

.2. Cell culture and X-ray treatment

TK6 cells were used for the micronucleus assay, TK mutation assay, and apopto-is assay. TK6 cell line (CRL-8015) was purchased from ATCC (Manassas, VA). Theells were maintained in RPMI 1640 medium supplemented with 10% horse serum,% l-glutamine, 1% penicillin, and 1% streptomycin. X-ray treatment was conducedith a RS-2000 Biological Irradiator (Rad Source, Suwanee, GA) at a cell density of

× 105 cells/ml. Relative increase in cell count (RICC) was used for the evaluation ofytotoxicity using the following formula [27]:

ICC = Increase in number of cells in treated cultures (final − starting)Increase in number of cells in control cultures (final − starting)

× 100

.3. miRNA transfection

A reverse transfection assay was used to introduce the miRNAs into TK6ells using the lipid-based siPORTTM NeoFXTM transfection agent. The transfectionrocedure was adapted from previous studies [28–30]. Briefly, transfection wasonducted in serum-free, antibiotics-free RPMI 1640 medium, at a cell density of

× 105 cells/ml and the miRNA concentration is 20 nM. Ten microliter of lipid trans-ection agent were first diluted with 240 �l of 1640 medium and equilibrated atoom temperature for 10 min. miRNA was then diluted with 1640 medium to 250 �l,nd the lipid solution was combined with the miRNA solution, and the mixture wasncubated at room temperature for 10 min. The nucleotide acid/lipid transfectionomplex was dispensed into 6 well tissue culture plates. TK6 cells were added andhe final transfection volume was 2 ml, and the transfection was conducted at 37 ◦Cor 6 h. Horse serum was added to a final concentration of 10% to stop the trans-ection without washing the cells. To obtain maximum transfection efficiency, theransfection procedure was repeated once 2 or 3 days after the first transfection29]. Cells were collected for X-ray treatment 5 days after the first transfection. For

ock transfection, a non-targeting miRNA was used as a negative control. RT-PCRas performed on transfected cells to evaluate the transfection efficiency.

.4. miRNA isolation

miRNA was isolated using the mirVanaTM miRNA isolation kit in accordanceith manufacturer’s instructions. Briefly, cultured cells were washed with phos-hate buffered saline (PBS) and then Lysis/Binding buffer was added to lyse theells. A ten percent volume of miRNA homogenate additive was added to the cells

rch 758 (2013) 35– 40

1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE) and the qual-ity of the RNA was evaluated with an Agilent BioAnalyzer (Agilent Technologies,Santa Clara, CA).

2.5. Quantitative real time RT-PCR

cDNA was reverse transcribed from 80 ng of total RNA with a gene specificmiRNA primer and MultiScribeTM Reverse Transcriptase. The reverse transcriptionwas conducted in a total volume of 15 �l at 16 ◦C for 30 min and 42 ◦C for 30 min,followed by 85 ◦C for 5 min. The cDNA was diluted 6.7 times and then quantitativereal-time PCR was conducted. The 20 �l real-time PCR amplification mixture con-tained 10 �l of TaqMan PCR master mix, 9 �l of diluted cDNA, and 1 �l of TaqManPCR primers. Real-time PCR was performed on an ABI PRISM 7500 (Applied Biosys-tems, Carlsbad, CA) at 95 ◦C for 10 min, followed by 40 cycles of 95 ◦C for 15 sec and60 ◦C for 1 min. Relative gene expression was calculated as 2−�CT with U6 miRNAused as an endogenous control for miRNA expression. Three replicate assays wererun for each sample, and each experiment was repeated three times.

2.6. Caspase 3/7 assay

Caspase 3/7 is activated in apoptotic cells and its activity reflects the degreeof apoptosis. The tetrapeptide sequence DEVD (Promega, Madison, WI) is cleavedby caspase 3/7 and generates a luminescence signal. Caspase 3/7 activity is pro-portional to the luminescence generated. The assay was conducted as previouslydescribed [31]. Briefly, miRNA transfected lymphoblast cells were washed withPBS and transferred to 96-well plates (2 × 104 cells/well). One hundred microliterof Caspase-Glo3/7 reagent was added to each well, followed by 1 h incubation atroom temperature. The luminescence was recorded by a microplate reader (BioTek,Winooski, VT). PBS was used as blank control for the assay. All values were normal-ized and expressed as fold changes relative to the negative control.

2.7. TUNEL assay

Tunnel assay was conducted with TiterTACS In Situ Detection Kit (R&D systems4822-96-K) to measure apoptosis. miRNA transfected TK6 cells were permeabilizedfirst with formaldehyde and then methanol, the fixed cells were pre-treated withproteinase K. DNA fragmentation induced by apoptotic agent was labeled with biotinconjugated dNTP using Terminal Deoxynucleotidyl Transferase (TdT) enzyme. Biotinlabeling is then detected and quantified by streptavidin conjugated horseradishperoxidase and a colorimetric substrate.

2.8. Micronucleus assay

X-ray treated TK6 cells were transferred to fresh medium and grown for1.5–2 generations (about 24 h). The cells were collected for micronucleus assay,using a MicroFlow® kit (Litron Laboratories, Rochester, NY) according to themanufacturer’s instruction. Briefly, 5 × 105 TK6 cells were stained with the fluo-rescent dye ethidium monoazide (EMA, also called nucleic acid dye A), a reagentthat crosses the compromised outer membrane of mid/late stage apoptotic andnecrotic cells and binds to DNA through photo-activation. Cells were then washed,treated with RNase and stained with a pan-nucleic acid dye (SYTOX Green ornucleic acid dye B). Micronuclei are scored according to their forward light scatter,side light scatter, and fluorescence intensity (1/100th to 1/10th of intact nuclei).Apoptotic/necrotic cells were gated as EMA positive SYTOX Green positive cells.Flow cytometry was performed on a BD FACS Canto II flow cytometer (BD Bio-sciences, San Jose, CA) and ten thousand TK6 cells were collected from eachsample for micronuclei scoring. The data were analyzed using the BD FACSDivasoftware.

2.9. Thymidine kinase (TK) mutation assay

TK6 cells were pre-treated with CHAT (deoxycytidine, hypoxanthine,aminopterin, and thymidine) medium for 2 days to reduce the background mutantfrequency. CHAT-treated cells were recovered in THC (deoxycytidine, hypoxanthine,and thymidine) medium for one day. The recovered cells were transfected with neg-ative control miRNAs, miR-34a precursors, or miR-34a inhibitors. The transfectedcells were treated with X-rays and maintained in growth medium for 3 days to allowfor expression of the TK mutant phenotype. The TK mutation assay was performedin 96-well plates using a standard protocol [32,33]. Both trifluorothymidine (TFT)selected and non-selected colonies were grown in RPMI 1640 medium containing20% horse serum. For each treatment, two 96-well non-selected plates were seededat a density of 1.6 cells/well to determine the plating efficiency. Four 96-well selectedplates were seeded for determining TK mutant frequency. The cells in the selectedplates were seeded in the presence of 3 �g/ml TFT at a density of 4 × 104 cells per

Research 758 (2013) 35– 40 37

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Fig. 1. miR-34a induction and inhibition, and their effects on cell growth in TK6 cells.TK6 cells were transfected with negative control miRNAs (Pre-miRTM miRNA Pre-cursor Negative Control #1, AM 17110), miR-34a precursors, or miR-34a inhibitors.The transfection procedure was repeated once on day 2 or 3 to achieve maximumtransfection efficiency. Total RNA was isolated 5 days after the first transfectionand real time RT-PCR was performed to determine the transfection efficiency. miR-34a expression was calculated as fold changes relative to TK6 control cells. Relativeincrease in cell count (RICC) was used to measure cytotoxicity of treated cells.(A) Expression level of miR-34a in miR-34a precursor-transfected TK6 cells; (B)Expression level of miR-34a in miR-34a inhibitor-transfected TK6 cells; (C) RICC formiR-34a precursor-transfected TK6 cells; (D) RICC for miR-34a inhibitor-transfected

X. Chen et al. / Mutation

ormulas:

MF = plating efficiencies of mutant plate (PEM)/plating efficiencies of viable plate

(PEV). PEM = (−ln(EW/TW))/(4 × 104), PEV = (−ln(EW/TW))/1.6, where EW is

the total number of empty wells and TW is the total number of wells. RTG

= relative suspension growth(RSG) × relative plating efficiencies of viable plates

(RPEV). RSG = (SG1 (test)

×SG2 (test) × SG3 (test))/(SG1 (control) × SG2 (control) × SG3 (control));

RPEV = PEV (test)/PEV (control).

.10. Statistical analysis

Statistical analysis was performed using SigmaStat 3.11 (San Jose, CA). In all stud-es, the differences between group means were evaluated using one-way ANOVA ortudent’s t-test. For comparisons using ANOVA, post hoc analysis was performedsing the Holm–Sidak method. All values are reported as the mean ± S.D.

. Results

.1. Transfection of miR-34a precursor and inhibitor in TK6 cells

To explore the role of miR-34a in mutagenesis, the human lym-hoblast TK6 cells were transfected with miR-34a precursor or

nhibitor. We first examined the cytotoxicity of miR-34a transfec-ion. When miR-34a precursor was delivered into TK6 cells throughwo rounds of transfection [29] over a total of 5 days, ectopicxpression of miR-34a was dramatically increased in TK6 cells.uantitative real-time RT-PCR analysis indicated the induction wasore than 1000 fold (Fig. 1A). The huge amount of exogenousiR-34a in the cells, however, produced no obvious cytotoxicity;

he RICC for miR-34a precursor-transfected cells was not differentrom the negative control miRNA-transfected TK6 cells (Fig. 1C).o exclude the possibility that negative control miRNA or lipidaused cytotoxicity, the negative control miRNA-transfected cellslso were compared with lipid transfected cells and with un-ransfected cells. Our data show no differences among these threeroups for cytotoxicity, suggesting that transfection of lipid or neg-tive control miRNA does not cause cytotoxicity.

To investigate the role of miR-34a in mutagenesis, a loss of func-ion experiment was conducted in TK6 cells. miR-34a inhibitor wasransiently transfected into TK6 cells, and quantitative real-timeT-PCR analysis indicated the endogenous miR-34a was success-

ully suppressed by the miR-34a inhibitor. miR-34a expression inhe transfected cells was suppressed by 80% compared with theoncurrent control (Fig. 1B). According to the RICC data, attenua-ion of miR-34a in TK6 cells was not cytotoxic since the RICC in

iR-34a inhibitor-transfected cells was comparable to the RICC ofhe negative control cells (Fig. 1D).

.2. Increase of miR-34a did not inhibit micronucleus inductiony X-ray

Micronuclei are cytoplasmic bodies having a portion of acen-ric chromosome or a whole chromosome that is not incorporatednto the newly formed daughter nuclei. An induction of micronucleindicates chromosome damage resulting from DNA breaks or aneu-enicity. Human lymphoblast TK6 cells were developed as modelells to investigate genotoxicity in mammal cells [35–37]. The spon-aneous micronucleated cell frequency in TK6 was very stable,pproximately 100 per 104 intact nuclei under our experimen-

Fig. 2A). Inhibition of endogenous miR-34a also had no effect on

TK6 cells. All data are reported as means ± S.D. (n = 3). **P ≤ 0.01 when comparedwith the negative controls. The results shown are mean of three independent exper-iments.

micronucleus frequency. The high level of micronuclei in the X-ray-treated TK6 cells remained the same regardless of transfection ofmiR-34a precursor or miR-34a inhibitor. Interestingly, inhibitionof endogenous miR-34a slightly increased the proliferation of TK6cells (Fig. 2B), while over-expression of miR-34a has no effect tothe proliferation of TK6 cells.

3.3. miR-34a inhibition of basal and X-ray induced mutation

It has been reported that transactivation of miR-34a can broadlyinfluence gene expression and promote cell cycle arrest, apopto-sis, and DNA repair [6,16]. To investigate the role of miR-34a inmutagenesis, we examined the effects of miR-34a on the basaland X-ray-induced mutant frequencies in the Thymidine Kinase (Tk)gene in TK6 cells.

The total mutant colony numbers scored on day 22 after theplating is very similar to the colony numbers scored on day 11;we choose to present all data on day 11. As expected, X-ray causecytotoxicity to treated cells and results in a 30% RTG (Fig. 3A). Over-expression of miR-34a in TK6 cells resulted in a 75% RTG. Unlike

38 X. Chen et al. / Mutation Research 758 (2013) 35– 40

Fig. 2. miR-34a has no effect on X-ray induced micronucleus production in TK6 cells. TK6 cells were transfected with negative control miRNAs, miR-34a precursors, ormiR-34a inhibitors. The transfected cells were further treated with 0.73 Gy X-rays, cytotoxicity and the number of micronuclei were measured 24 h after the treatment. Datawere presented as micronucleus fold induction relative to negative control miRNA-transfected cells. (A) MN fold induction for miR-34a precursor and miR-34a inhibitor-transfected TK6 cells; (B) RICC for miR-34a precursor and miR-34a inhibitor-transfected TK6 cells; each biological sample was assayed three times and the experiment wasrepeated three times. The results shown are mean of three independent experiments. **P ≤ 0.01 when compared with the negative controls.

Fig. 3. miR-34a inhibits X-ray-induced mutation in TK6 cells. TK6 cells were transfected with negative control miRNAs, miR-34a precursors, or miR-34a inhibitors; transfectedcells were further treated with 0.73 Gy X-rays and the cells were assayed for TK mutant frequency. Relative total growth (RTG) (A) and mutant frequency (MF) (B) in TK locusw 0.01 cc 01 coms

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emained 30% RTG regardless of miR-34a over-expression ornder-expression.

Consistent with earlier reports [28,37,38], the spontaneousK mutant frequency in TK6 cells was about 2 × 10−6 in thistudy. Treatment of the cells with 0.73 Gy X-ray increased theutant frequency about 4-fold over the control (Fig. 3B). Over-

xpression of miR-34a did not cause a significant change in thepontaneous TK mutant frequency in TK6 cells, but the exoge-ous miR-34a significantly decreased the X-ray-induced mutant

requency (Fig. 3B). This indicates that over-expression of miR-4a protects TK6 cells from X-ray-induced mutation. Importantly,own-regulation of endogenous miR-34a by transfecting TK6 cellsith miR-34a inhibitor led to a 2-fold increase in basal mutant fre-

uency. miR-34a inhibitor-transfected TK6 cells still responded to-ray treatment. That is, the mutant frequency in miR-34a inhibitor

ransfected TK6 cells was further increased to 7 × 10−6 by X-rayreatment (Fig. 3B).

.4. miR-34a induces apoptosis in TK6 cells

miR-34a has well-documented pro-apoptotic activities inther cells types [6,8,16]. We evaluated whether or not miR-34aould promote apoptosis in TK6 cells. Apoptosis was evaluated

ompared with negative control miRNA-transfected cells. #P ≤ 0.05 and ##P ≤ 0.01pared with X-ray-treated negative control miRNA-transfected cells. The results

by caspase assay and TUNEL assay (Fig. 4). Both assays indicatethat X-ray is a good positive control for apoptosis assay. As seenin Fig. 4, both X-ray and miR-34a induce apoptosis in TK6 cells.The apoptotic activity reached to the maximum level when themiR-34a over-expressed cells were further treated with X-ray. Ofnote, the basal apoptosis of TK6 cells was significantly decreasedwhen the endogenous miR-34a expression was suppressed in TK6cells. The miR-34a under-expressed cells respond normal to X-raytreatment since X-ray can still induce apoptosis in these cells.

4. Discussion

miR-34a is a component of the P53 network and its expressionis under the rigorous control of P53 protein. miR-34a expression islow in different types of tumors, consistent with miR-34a function-ing as tumor suppressor. However, little is known about the role ofmiR-34a in mutagenesis. In this study, both loss of function and gainof function experiments in human lymphoblast TK6 cells demon-strated that endogenous miR-34a can inhibit spontaneous mutantfrequency, while over-expression of miR-34a significantly inhibits

X. Chen et al. / Mutation Research 758 (2013) 35– 40 39

Fig. 4. miR-34a induces apoptosis in TK6 cells. TK6 cells were transfected with negative control miRNAs, miR-34a precursors, or miR-34a inhibitors; the transfected cells weref panel)a controc

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urther treated with 0.73 Gy X-rays. Apoptosis was evaluated by caspase assay (left

s means ± S.D. (n = 3). *P ≤ 0.05 and **P ≤ 0.01, when compared with the negative

ells. The results shown are mean of three independent experiments.

Thymidine Kinase (TK) is one of the most commonly used geneticoci for mammalian cell mutation studies and the mutations mea-ured in this gene reflect those in genes related to tumorigenesis32]. The TK assay detects many types of mutations, includingransitions, transversions, tandem base substitutions, frameshifts,mall deletions, and a small duplication [39]. Inhibition of miR-4a in TK6 cells leads to a significant increase in the spontaneousutant frequency. This indicates that under normal physiological

onditions miR-34a protects the cells from high levels of sponta-eous mutations. Noticeably, X-ray treatment did not induce theiR-34a inhibitor transfected TK6 cells to a higher level as expected

Fig. 3B). This may be because TK6 cells have other protection mech-nisms to compensate the reduced miR-34a level and prevent theutant frequency goes too high under X-ray treatment.Furthermore, over-expression of miR-34a in TK6 cells signifi-

antly reduced X-ray-induced mutant frequency, suggesting thatnder genotoxic stress such as X-ray treatment, when miR-34axpression is usually get induced, the up-regulated miR-34a canrotect cells from X-ray-induced mutations and as a result, pro-otes genomic stability.In contrast to its ability to inhibit gene mutation, miR-34a had no

ffect on micronucleus induction in TK6 cells. The reason for this isnknown. Although both the TK mutation assay and micronucleusssay are genotoxicity tests, they measure different end points.he micronucleus assay measures chromosome damage [40–42]uch as chromosome fragments or lagging whole chromosomesnd detects clastogens and aneugens, while the TK assay assesses

whole range of mutations that occur in the TK gene. It might behat miR-34a does not interfere with micronucleus formation butoes suppress mutations.

Mutation induction and apoptosis are two closely related cellu-ar processes [43,44]. Apoptosis removes cells with pre-mutagenicesions and eliminates passage of any damaged DNA. Therefore,igher levels of apoptosis will result in less mutation induction.ver-expression of miR-34a led to a higher level of apoptosis andecreased the mutant frequency (MFs) in TK6 cells. Conversely,

nhibition of miR-34a led to a lower basal level of apoptosis andigh MFs in TK6 cells. These results suggest that miR-34a mayegulate mutational response through apoptosis. miR-34a couldxert its pro-apoptotic effect through regulating proteins involvedn apoptosis. One of the example is BCL-2, a well characterized anti-poptotic factor and a direct transcriptional target of miR-34a, was

and TUNEL assay (right panel) 24 h after the X-ray treatment. All data are reportedls. ##P ≤ 0.01 compared with miR-34a precursor or miR-34a inhibitor-transfected

repair, so that it can affect MFs through cell cycle arrest to repairdamaged DNA. Several proteins in this category are CDK4/6, CyclinE2, and MET [16,45].

In summary, this is the first study directly demonstrating a roleof miR-34a in suppression of mutation induction. Expression levelof miR-34a is directly related to spontaneous and X-ray-inducedmutations in human lymphoblast TK6 cells. The mechanism of themutation inhibition could be mediated through apoptosis. There-fore, miR-34a may function as an important tumor suppressor bymaintaining low levels of mutations.

Conflict of interest

Acknowledgement

This work was supported by the commissioner’s fellowship pro-gram in U.S. Food and Drug Administration.

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miR-34a suppresses mutagenesis by inducing apoptosis in human lymphoblastoid TK6 cells

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