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The International Journal of Biochemistry & Cell Biology 45 (2013) 2519– 2529

Contents lists available at ScienceDirect

The International Journal of Biochemistry& Cell Biology

journa l h om epage: www.elsev ier .com/ locate /b ioce l

iR-320a regulates erythroid differentiation through MAR bindingrotein SMAR1

mriti P.K. Mittala,b, Jinumary Mathaib, Abhijeet P. Kulkarnia,1,ayanta K. Pala,∗∗, Samit Chattopadhyayb,∗

Department of Biotechnology, University of Pune, Pune 411007, IndiaNational Centre for Cell Science, Pune University Campus, Ganeshkhind, Pune 411007, India

r t i c l e i n f o

rticle history:eceived 8 February 2013eceived in revised form 7 July 2013ccepted 12 July 2013vailable online 20 July 2013

a b s t r a c t

Erythropoiesis is controlled by a complex interplay of several signaling pathways and key transcriptionfactors, as well as microRNAs (miRNAs). MicroRNAs function as critical modulators of gene expressionfor cellular processes. In the present study, we found that miR-320a inhibits erythroid differentiation bytargeting Matrix Attachment Region binding protein SMAR1. miR-320a negatively regulates the expres-sion of SMAR1 by directly binding to its 3′UTR. In response to mild DNA damage, miR-320a expression is

eywords:iR-320aiR-221/222

MAR1rythroid differentiationemin

decreased resulting in enhanced expression of SMAR1 protein, which in turn, reduces its targets, Bax andPuma inhibiting apoptosis. Our data demonstrate that during hemin-induced erythroid differentiation,enhanced expression of SMAR1 negatively correlates with miR-320a expression. Further analysis revealsthat SMAR1 regulates erythroid differentiation, by binding to the promoter of miR-221/222, which playa crucial role in early erythropoiesis. Overall, our studies provide an insight into the regulation of heminmediated erythroid differentiation of K562 cells through post-transcriptional regulation of SMAR1.

. Introduction

Chromatin architecture is maintained by nuclear matrix andatrix binding proteins present in the nucleus. Scaffold/Matrix

ttachment regions (S/MARs) are present mostly upstream of pro-oter sequences and play a role in regulating gene expression.

/MAR binding proteins (MARBPs) bind to these S/MARs and thuslter the chromatin structure and transcription of nearby genesn response to cellular stresses. SMAR1 (Scaffold/Matrix attach-

ent region binding protein) is one such protein identified fromouse double positive thymocytes that affects V(D)J (Variable-iversity-Junctional) recombination (Chattopadhyay et al., 2000;aul-Ghanekar et al., 2005). It interacts with histone deacety-

ase 1 (HDAC1)-associated repressor complex and allows histoneeacetylation to cause Cyclin D1 transcriptional repressionRampalli et al., 2005). SMAR1 leads to retention of p53 in the

ucleus leading to cell cycle arrest by inducing gene expres-ion of cyclin dependent kinase inhibitor p21 and phospho cdc2Jalota et al., 2005; Jalota-Badhwar et al., 2007; Kaul et al.,

∗ Corresponding author. Tel.: +91 20 2570 8064; fax: +91 20 2569 2259.∗∗ Corresponding author. Tel.: +91 20 25692248; fax: +91 20 25691821.

E-mail addresses: jkpal@unipune.ac.in, jkpal@hotmail.com (J.K. Pal),amit@nccs.res.in (S. Chattopadhyay).

1 Present address: Bioinformatics Centre, University of Pune, Pune 411007, India.

357-2725/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.biocel.2013.07.006

© 2013 Elsevier Ltd. All rights reserved.

2003). The crosstalk between SMAR1 and promyelocytic leukemia(PML) nuclear bodies decides the cell fate (survival/death) throughBax and Puma during mild DNA damage (Sinha et al., 2010).Transforming growth factor � (TGF-�) plays a major role intumor cell migration, invasiveness and epithelial to mesenchy-mal transition. SMAR1 acts as an anti-tumorigenic protein byregulating cell growth and metastasis in breast cancer and alsoacts as a connecting link between p53 and TGF-� pathwaysleading to tumor regression (Singh et al., 2007). Thus, SMAR1has multifaceted functions by which it regulates many cellu-lar gene targets that are involved in cell growth, apoptosis andtumorigenesis.

A gene can be regulated at many levels i.e., transcriptional,post-transcriptional, translational and post-translational levels. Ithas been demonstrated that microRNAs (miRNAs) regulate geneexpression mostly at the post-transcriptional level (Bartel, 2004).miRNAs (22-nucleotide long RNA) regulate mRNA translationmainly by binding to the 3′UTR of the mRNA by partial com-plementary base pairing (Guarnieri and DiLeone, 2008) leadingto translational inhibition or degradation of mRNAs; in timeand location specific manner. As miRNAs target multiple genes,they are involved in the regulation of many biological processes

including apoptosis (miR-218, miR-17–92a) (Guo et al., 2012; Heet al., 2012), metastasis and epithelial to mesenchymal transition(miR-124, miR-708, miR-200, let-7) (Guo et al., 2013; Liang et al.,2013; Ryu et al., 2013), and hematopoietic lineage differentiation

2 Bioch

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520 S.P.K. Mittal et al. / The International Journal of

miR-125b, miR-142-3p, miR-142-5p, miR-146a, miR-451) etc.Chaudhuri et al., 2012; Jin et al., 2012).

miRNA mediated regulation might be an additional mechanismf controlling SMAR1 expression and function. Therefore, we inves-igated such possibility by in silico database searches and usedifferent computational tools to predict binding sites for possibleiRNAs in its 3′UTR. Through this approach, we observed that miR-

20a can target SMAR1 by binding to its 3′UTR. miR-320 gene familyonsists of 5 miRNAs, namely miR-320a, miR-320b, miR-320c, miR-20d and miR-320e. As the seed sequence of all these microRNAs

s same, they might function in a similar manner. Hence, we havesed miR-320a specific overexpression plasmids and primers in ourtudy. Induction of SMAR1 upon mild DNA damage prevents cellu-ar apoptosis. Therefore, SMAR1 has been proved to be an importantheckpoint regulator of apoptosis (Sinha et al., 2010). We furtheralidated this phenomenon through miR-320a. miR-320 is knowno fine tune the translational activities of CD-71 during differentia-ion to reticulocytes (Chen et al., 2008). Therefore, we hypothesized

role of SMAR1 in erythropoietic differentiation and indeed webserved its role by regulating miR-221/222 during erythroid dif-erentiation. Our results demonstrate that ectopic expression of

iR-320a reduces SMAR1 protein level and also reduces its func-ion by upregulating SMAR1 target genes involved in apoptosis andrythropoietic differentiation.

. Materials and methods

.1. Cell culture

Various cell lines namely, K562, HCT116, HCT116 p53−/−, MCF7nd HEK293 were cultured in DMEM or RPMI with 10% FBS at 37 ◦Cnd 5% CO2 with penicillin-streptomycin solution (1X, Gibco).

.2. Plasmids and transfections

Plasmids pMIR-REPORT SMAR1 3′UTR (intact), pMIR-REPORTMAR1 3′UTR (mutant) constructs were prepared. To generate theicroRNA target site deletion mutant, PCR based site directedutagenesis was performed by using pMIR-REPORT 3′UTR (intact)

s template described by Nassal and Rieger, 1990 to delete 17ases from 1963 to 1979 that constitute microRNA target bind-

ng sequence. The plasmids used for transfection experiment wererepared using Qiagen Midiprep kit. Transfection was carriedut using Lipofectamine-2000 in DMEM without FBS. Cells wereo-transfected with pMIR-REPORT plasmid (Applied Biosystems)ontaining SMAR1 3′UTR and CMV-miR-320a plasmid (Origene).fter 5 h of transfection, 10% FBS (final conc.) was added to theedium. After 24 h, it was replaced by fresh medium. After 48 h,

ells were harvested and luciferase assay or protein extraction orNA extraction was done.

.3. RNA extraction and quantitative real-time PCR

Total cellular RNA was extracted from the cells using TRIZOLeagent as per the manufacturer’s (Sigma, USA) protocol. The RNAas quantified by spectrophotometric method, which was then

everse transcribed to cDNA. For quantitative analysis of SMAR1xpression, Real-time PCR was performed by Mastercycler gradi-nt (Eppendorf) using double stranded DNA specific fluorophoreYBR Green I. In a 10 �l PCR reaction, cDNA was amplified using× iQTN SYBR Green Supermix (Bio-Rad) and 50 pmol of forwardnd reverse primer mix. Annealing temperature of 58 ◦C and 55 ◦C

as used for SMAR1 and GAPDH, respectively for 40 cycles. Confir-ation of single amplicon was determined by melt curve analysis.uantification of SMAR1 mRNA, described as fold increase overontrol was done by the comparative Ct method, where target

emistry & Cell Biology 45 (2013) 2519– 2529

(SMAR1) was normalized to the endogenous reference (GAPDH).The �Ct was determined by subtracting the Ct of GAPDH fromthat of SMAR1. The fold increase over control = 2−��Ct where��Ct = �Ct(control) − �Ct(treatment).

2.4. miRNA isolation and quantification

miRNA was isolated using mirVana miRNA isolation kit(Ambion). In brief, the cells were lysed in a denaturing bufferand then RNA was extracted using acid phenol: chloroform toremove most of the DNA. Quantitative real-time PCR was done toquantify the levels of specific miRNAs during various treatments.Quantitative real-time PCR was performed in Mastercycler gradi-ent (Eppendorf) using SYBR-green PCR master mix. The relativeabundance of the specific miRNAs was normalized to U6snRNA.

2.5. Western blot analysis

�-Actin, SMAR1, Bax, Puma and GATA-1 protein levels weredetermined by immunodetection. In brief, the proteins wereextracted using protein extraction buffer (20 mM Tris–HCl pH 7.8,1 mM EDTA, 1 mM PMSF, 0.1% (v/v) Triton X-100, PI cocktail), quan-tified using Bradford’s microestimation assay and were analyzedby SDS PAGE. Proteins were electrotransferred to nitrocellulose orPVDF membranes at 70 mA constant current. The blots were thensaturated with BSA/non-fat dry milk and reacted with differentprimary antibodies and appropriate secondary antibodies taggedwith horseradish peroxidase. Signals were detected by chemi-luminescence using luminol as a substrate.

2.6. Cell cycle analysis

The control and treated cells were washed with 1× PBS con-taining 2% FBS, and fixed at 4 ◦C for 2 h using 70% ethanol. Afterfixation, cells were rinsed with 1× PBS and passed through a nylonmesh for getting single cell suspension. Such cells were then treatedwith RNase A (100 �g/ml) at 37 ◦C for 30 min followed by stainingwith 50 �g/ml propidium iodide (Liu et al., 2005). The stained cellswere then analyzed with a FACS Calibur fluorescence activated cellsorter using cell quest software (Becton Dickinson, USA). The cellcycle profile was obtained by analysing 10,000 cells per sample.

2.7. Luciferase reporter assay

Luciferase reporter assays were performed as previouslydescribed (Sinha et al., 2010). In brief, firefly pMIR-luciferasereporter vectors and pMIR-3′UTR SMAR1 (intact) and pMIR-3′UTR SMAR1 (mut)-luc vector (100 ng) were transfected intocells in 6-well plates together with CMV-miR-320a expressionvector (1 �g) or miR-320a inhibitor (50 nM) using Lipofectamine2000. GFP vector/pMIR-beta-Gal (0.5 �g) that expresses GFP/beta-Galactosidase was co-transfected to normalize the transfectionefficiency. Luciferase activities were measured at 24 h after trans-fection by using the luciferase assay kit (Promega). Firefly luciferaseactivities were normalized to GFP fluorescence/beta-Galactosidaseactivity.

2.8. Chromatin immunoprecipitation-sequencing (ChIP-seq)

Chromatin immunoprecipitation (ChIP) was done using HCT116and HCT116 p53−/− cells as described earlier (Rampalli et al.,

2005). The eluted DNA fragments were subjected to single endsequencing using Illumina GAIIx Analyzer. After initial QC analy-sis using SeqQC V1.1 program, the sequence reads were alignedagainst the Homo sapiens reference genome (Hg19 UCSC Build)

Biochemistry & Cell Biology 45 (2013) 2519– 2529 2521

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Table 1Predicted miRNAs that can target SMAR1.

Computational tools used Predicted miRNAs that can target SMAR1

miRanda (2008 release)hsa-miR-320hsa-miR-183

TargetScan 4.2

hsa-miR-320hsa-miR-129-5phsa-miR-129

S.P.K. Mittal et al. / The International Journal of

sing Bowtie 0.12.6 program. On the basis of fold enrichment val-es; the enriched region sequences were extracted in FASTA formatnd annotated.

.9. Chromatin immunoprecipitation

ChIP assays were performed as stated above using ChIP assayit (Upstate Biotechnology) following manufacturer’s instructions.CR was performed using different promoter specific primers.

.10. RNA immunoprecipitation

RNA immunoprecipitation was carried out to confirm the bind-ng of miR320 a to the SMAR1 mRNA. In MCF-7 cells, miR-320a

as overexpressed and the control and treated cells were fixedith formaldehyde, quenched and then lysed. DNA and RNA were

ragmented by sonication and precleared with protein A/G agaroseeads. Protein A/G beads were then coated with control IgG andntibody against Argonaute 2. The lysates were then incubated withntibody coated protein A/G agarose beads. The bound complexas then reverse crosslinked and eluted. The RNA was extracted

rom the eluate using TRIZOL. cDNA was prepared and PCR waserformed using miR-320a and SMAR1 UTR specific primers.

.11. Erythroid differentiation of K562 cells

K562 cells were treated with 25 �M hemin for 7 days, whicheads to the production of hemoglobin in these cells.

.12. Benzidine staining

In order to determine percent cell differentiation during var-ous treatments, benzidine staining was done using the protocolescribed elsewhere (Nagy et al., 1995). Benzidine dihydrochlo-ide (2 mg/ml) was prepared in 3% acetic acid. H2O2 (1%) wasdded immediately before use. Control and treated cell suspen-ions were mixed with benzidine solution in 1:1 ratio and incubatedor 5 min. Preparations were either counted in hemocytometer orhotographed using bright field microscope on a plane glass slide.

.13. Statistical analysis

All the experiments were performed at least three times andhe data are expressed as mean ± standard deviation. Student’s test was performed to determine significant differences betweenreatment and control. Differences at p ≤ 0.05/0.005 level were con-idered statistically significant.

. Results

.1. In silico identification of miRNA binding sites

To predict the potential miRNA that can target SMAR1, in silicoearch was performed. Tools available in public domain were usedor such prediction which determine the miRNA targets by lookingt the conserved region in the query 3′UTR and then find its homol-gy to the known miRNA sequences. The computational tools usedere PicTar (http://pictar.bio.nyu.edu/), miRanda (2008 release)

nd Targetscan 4.2 (http://www.targetscan.org/). Using SMAR1′ UTR as query sequence, miRanda predicted hsa-miR-320 andsa-miR-183, Targetscan predicted hsa-miR-320, hsa-miR-129-5p,sa-miR-129 and hsa-miR-579, and PicTar predicted hsa-miR-320

s the regulator of SMAR1 (Table 1). Therefore, the miRNA that wasonsistently predicted by all these tools to target SMAR1 was hsa-iR-320. The miR-320 binding region in SMAR1 UTR was found

o be conserved across the species, as determined by multiple

hsa-miR-579

PicTar hsa-miR-320

sequence alignment of the UTRs of SMAR1 from various speciesnamely human, mice, rat and dog. The predicted binding of hsa-miR-320a had a 7-mer-m8 seed, WC base-pairing from positions 2to 8. The average free energy of mRNA-miRNA hybrid structure wasfound to be −18.3 kcal/mol, suggesting a stable binding of miR-320with SMAR1 UTR.

3.2. miR-320a negatively regulates SMAR1

To examine whether miR-320a regulates SMAR1 protein levels,miR-320a overexpression plasmid was transfected into cell linesexpressing endogenous SMAR1. Scrambled miRNA was used as anegative control. Four human cell lines namely MCF-7, HEK293,HCT116 and HCT116 p53−/− were employed for this experiment toavoid the effects caused by cell- and tissue-type specificity. HEK293is human embryonic kidney cell line resembling non-transformedcells, MCF-7 is a human breast cancer cell line, HCT116 is a humancolon cancer cell line and HCT116 p53−/− is the cell line lackingp53. We determined the endogenous levels of miR-320a in thesecell lines and observed that HCT116 p53−/− has the maximumlevel of endogenous miR-320a (Fig. 1A). Further, the amount ofendogenous miR-320a was found in decreasing order in HCT116p53−/−, HCT116, HEK293 and MCF-7 respectively. Similarly, West-ern blot analysis was performed to observe SMAR1 protein levelsand an inverse correlation was seen in endogenous miR-320a leveland SMAR1 protein level (Fig. 1B). To further confirm this neg-ative correlation, miR-320a was ectopically overexpressed in adose-dependent manner in these four different cell lines. SMAR1expression level was checked in all these lines. The overexpressionof miRNA in these cell lines was confirmed by real-time PCR andmore than 3-fold increase was observed in all these lines (Fig. 1C).The results showed decrease in SMAR1 protein level with dose-dependent increase in miR-320a level in all the four cell lines(Fig. 1D). These results thus indicate that miR-320a might regulateSMAR1 protein level.

3.3. miR-320a negatively regulates SMAR1 by binding to its 3′UTR

To confirm the binding of miR-320a to SMAR1 3′UTR, RNAimmunoprecipitation was performed. Argonaute 2 (Ago-2) proteinhelps in the binding of miRNAs to their target sequence. In MCF-7cells, we overexpressed miR-320a and immunoprecipitated Ago-2 protein with RNA bound to it. IgG was used as a control. Afterprecipitation of the immunoprecipitated complex, RNA was iso-lated using TRIZOL and then DNaseI treatment was given to removeany traces of DNA. Further, cDNA was prepared followed by RT-PCR analysis. We observed that the miR-320a overexpressed, Ago-2immunoprecipitated sample, was more enriched with SMAR1 UTRthan the control sample (Fig. 2A). In the same sample, the immuno-precipitation of miR-320a along with Ago-2 and SMAR1 UTR was

confirmed by real-time PCR (Fig. 2B). This confirms that miR-320abinds to the 3′UTR of SMAR1. The sequence of SMAR1 UTR bind-ing to miR-320a and mutated UTR (Fig. 2C and D) was cloned inthe pMIR-REPORT vector and the four cell lines were transfected

2522 S.P.K. Mittal et al. / The International Journal of Biochemistry & Cell Biology 45 (2013) 2519– 2529

Fig. 1. Hsa-miR-320a negatively regulates the protein expression of SMAR1. (A) RNA was extracted from the four cell lines namely MCF-7, HEK293, HCT116 and HCT116p53−/− . Quantitative real-time PCR was performed to detect the endogenous expression of miR-320a. (B) The same four cell lines as in (A) were used for protein extractionand Western blot analysis was performed to determine the endogenous level of SMAR1 protein. The graph is densitometric analysis of blots. (C) miR-320a was overexpressedin a dose-dependent (1-2 microgram) manner in the four cell lines and after 60 h of transfection, the over-expression of miR-320a was determined by real-time PCR in thet t anals

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ransfected cells. (D) Protein was extracted from the samples of (C) and Western bloignificant difference at P ≤ 0.05/0.005, respectively).

ith the control pMIR-REPORT and SMAR1 UTR pMIR-REPORT.uciferase assay was performed to determine the binding of miR-20a to the SMAR1 UTR. We found that the luciferase activityecreased as the endogenous miR-320a levels increased in theour cell lines (Fig. 2E). Luciferase assay was also performed usingMIR-REPORT, pMIR-REPORT SMAR1 UTR, pMIR-REPORT SMAR1TR-mut with miR-320 overexpression constructs and inhibitor in

hree cell lines namely HCT116, MCF-7 and HEK293. We observedhat in the deletion mutant, miR-320a overexpression had no effectn luciferase activity. Whereas, the control pMIR-REPORT SMAR1TR with intact miR-320a binding site showed significant reduc-

ion in luciferase activity upon miR-320a overexpression. Similarly,ransfection with miR-320a inhibitor showed increased luciferasectivity (Fig. 2F). The overexpression and downregulation of miR-20a (by inhibitor) was also confirmed by quantitative real-timeCR which correlated well with the transfection and luciferasessay results (Fig. 2G). Thus, these results confirm the binding ofiR-320a to SMAR1 UTR.

.4. Hsa-miR-320a reduces anti-apoptotic signal in response toild DNA damage

Studies from our lab have earlier shown that mild DNA damageauses SMAR1 to specifically repress BAX and PUMA, which leadso cell cycle arrest. SMAR1 knockdown induces apoptosis, only inhe presence of p53. Conversely, apoptotic DNA damage results in

ysis was performed using anti-SMAR1 and actin antibody (*/** indicate statistically

sequestration of SMAR1 in promyelocytic leukemia (PML) nuclearbodies. Thus, in response to DNA damage, SMAR1–PML nuclearbodies interaction dictates the commitment of the cell toward cellcycle arrest or apoptosis (Sinha et al., 2010). In order to validate theeffect of miR-320a in the regulation of apoptosis through SMAR1,we performed cell cycle analysis in HCT116 and HCT116 p53−/−

with 5 J/m2 UV stress along with SMAR1 knockdown or miR-320aoverexpression. As expected, we did not observe significant lev-els of cell death in 5 J/m2, while when SMAR1 was downregulated(sh1077) or when miR-320a was overexpressed, we detected asignificant level of apoptosis. In HCT116 p53−/− cells, there wasno significant difference between sh1077 and sh1077 + 5 J/m2 UV.But significant difference was observed in the level of apoptosisbetween miR320 and miR-320a + UV (Fig. 3A and B), which sug-gests that miR-320a has other targets as well which are involved inapoptosis. Thus, these results suggest that SMAR1 specific shRNA(sh1077) and miR-320a operate in the same way by reducingSMAR1 protein level. The quantitative representations of the FACSanalysis and Western blot of SMAR1 are shown in Fig. 3C and D.It was interesting to determine the expression level of miR-320aand SMAR1 in 5 J/m2 and 100 J/m2 UV treated cells, since SMAR1 isknown to get activated after UV stress (Sinha et al., 2010). There-

fore, HCT116 cells were treated with 5 J/m2 and 100 J/m2 UV, cellswere pelleted down after 24 and 48 h and protein was extracted.Western blot analysis was performed for SMAR1 and its targetsBax and Puma which are involved in apoptosis. We observed that

S.P.K. Mittal et al. / The International Journal of Biochemistry & Cell Biology 45 (2013) 2519– 2529 2523

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Fig. 2. miR-320a directly binds to the 3′UTR of SMAR1 mRNA. (A) miR-320a was overexpressed in MCF-7 cells, the cells were sonicated and the RNA was pulled usinganti-Argonaute-2 antibody and control IgG. The RNA from the pulled fraction was isolated and total cDNA and miR-320a specific cDNA was prepared. PCR was performedusing SMAR1 UTR specific primers. (B) Quantitative real-time PCR was performed from the samples in (A) using miR-320a specific primers. (C) Interaction of nucleotides inSMAR1 3′UTR (intact/mutated) with miR-320a. (D) SMAR1 3′ UTR intact and mutated was cloned in pMIR-report vector for luciferase assays. (E) The pMIR-Report vector andpMIR-Report SMAR1 3′UTR were transfected in four cell lines and luciferase assays were performed. (F) Luciferase assays following transfection with indicated constructsa iR32q

dpieaRPi

nd their combinations in three cell lines namely HCT116, MCF-7 and HEK293. (G) muantitative real-time PCR.

uring both high and low dose UV treatment SMAR1 was overex-ressed. However, its targets Bax and Puma were downregulated

n a time-dependent manner in 5 J/m2 UV treatment and the lev-ls increased in 100 J/m2 UV treatment suggesting occurrence of

poptosis in these cells (Fig. 3E). Further, from these treated cellsNA was extracted, cDNA was prepared and quantitative real-timeCR was performed. We observed that level of miR-320a decreasedn a time-dependent manner in UV treated cells (Fig. 3F). Thus, as

0a levels after overexpression and downregulation (by inhibitor) as determined by

expected upon UV treatment SMAR1 and miR-320a showed inversecorrelation.

SMAR1 shows its effect on apoptosis through downregulationof Bax and Puma by binding to the MAR element in their promoter.

Thus if miR-320a is overexpressed, then Bax and Puma levelsshould be upregulated. As expected, we observed an increase intheir mRNA levels in HCT116, HCT116p53−/−, MCF-7 and HEK293cells (Fig. 3G and H). If mRNA levels are upregulated, protein

2524 S.P.K. Mittal et al. / The International Journal of Biochemistry & Cell Biology 45 (2013) 2519– 2529

Fig. 3. Hsa-miR-320a reduces anti-apoptotic signal in response to mild DNA damage. (A) miR-320a overexpression construct (miR-320a), SMAR1 knockdown construct(sh1077) were transfected in HCT116 cells with or without UV treatment (5 J/m2) and cell cycle analysis was performed. The cells showed increased sensitivity to low doseUV after miR-320a overexpression and SMAR1 knockdown. (B) Cell cycle analysis was performed with similar transfections as in Fig. 3A in HCT116 p53−/− . (C) Quantitativeanalysis of the data observed in (A). Western blot image of SMAR1 and actin for the different treatments and transfections of HCT116 cells in (A). (D) Quantitative analysis ofthe data observed in (B). Western blot image of SMAR1 and actin for the different treatments and transfections of HCT116 p53−/− cells in (B). (E) HCT116 cells were treatedwith 5 J/m2 and 100 J/m2 and after indicated time points, Western blot was performed with anti-SMAR1, anti-Bax, anti-Puma and anti-actin antibody. SMAR1 was upregulatedafter 5 J/m2 as well as after 100 J/m2 UV treatment. The pro-apoptotic proteins Bax and Puma were found to decrease in low dose while the expression increased in high doseUV treatment. (F) Quantitative real-time PCR analysis was performed after low and high dose of UV treatment to determine the level of miR-320a. miR-320a levels decreasedin low and high dose of UV treatment. (G) miR-320a was overexpressed in four cell lines namely MCF-7, HEK293, HCT116 and HCT116 p53−/− and quantitative real-timePCR was performed to determine the mRNA levels of Bax and (H) Puma. (I and J) In the four cell lines, miR-320a was overexpressed, and Western blot was performed usinganti-SMAR1, anti-Bax, anti-Puma and anti-actin antibodies. On miR-320a overexpression, SMAR1 protein levels decreased, however Bax and Puma protein levels increased(*/** indicate statistically significant difference at P ≤ 0.05/0.005 respectively).

S.P.K. Mittal et al. / The International Journal of Biochemistry & Cell Biology 45 (2013) 2519– 2529 2525

Fig. 4. miR-320a inhibits erythroid differentiation through regulating SMAR1. (A) K562 cells were treated with 25 �M hemin for 7 days to induce differentiation in vitro,such cell pellet showed red color. The control and differentiated cells were stained with benzidine. Differentiated cells turned blue coloured upon staining which werephotographed under light microscope (magnification – 40×). (B) The expressions of molecular markers of erythroid differentiation: GATA-1, (C) GATA-2 and (D) GlycophorinA were determined using real-time PCR. During differentiation GATA-1 and Glycophorin A were found to increase while GATA-2 was found to decrease. (E) Western blotanalysis was performed to determine the levels of GATA-1 and SMAR1 protein during erythroid differentiation in K562 cells. (F and G) Quantitative real-time PCR showedapproximately 2.5 fold increase in SMAR1 while 0.5 fold decrease in miR-320a. (H) Quantitative real-time PCR depicting the variation in GATA-1, GATA-2, Glycophorin A ina time-dependent manner during erythroid differentiation. (I) Quantitative real-time PCR depicting the levels of SMAR1 and miR-320a levels in a time-dependent mannerduring erythroid differentiation in K562 cells. (J) K562 cells were transfected with sh1077 or GFP and after 60 h of transfection, 25 �M hemin treatment was given for 7 days.After the treatment, GFP positive and negative cells were sorted and benzidine staining of GFP positive, GFP negative, sh1077 positive, sh1077 negative transfected cells andcontrol cells was performed. Stained cells were then observed under light microscope (magnification – 40×) (*/** indicate statistically significant difference at P ≤ 0.05/0.005,respectively).

2526 S.P.K. Mittal et al. / The International Journal of Biochemistry & Cell Biology 45 (2013) 2519– 2529

Fig. 5. SMAR1 regulates miR-221/222 during erythroid differentiation. (A) K562 cells were treated with 25 �M hemin for 3, 5 and 7 days. Quantitative real-time PCR wasperformed to detect the levels of SMAR1, miR-320a and miR-222 during erythroid differentiation. (B) In HCT116 and HCT116 p53−/− cells, SMAR1 was overexpressed, miR-221and miR-222 levels were detected in control and SMAR1 overexpressed (C3-SMAR1) samples through real-time PCR. (C) In HCT116 and HCT116 p53−/− cells, SMAR1 wasknocked down, miR-221 and miR-222 levels were detected in control and SMAR1 knocked down samples through real-time PCR (D) MAR-Wiz analysis for the miR-222promoter, where ChIP-seq data detected the binding of SMAR1. This showed presence of MAR in the promoter. (E) Chromatin immunoprecipitation was performed in HCT116c sed asM d miR

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ells by SMAR1 antibody using miR-222 promoter specific primers. Rabbit IgG was uCF-7, HCT116p53−/− and HCT116. In these samples the expression of miR-221 an

evels should also increase after miR-320a overexpression. Toonfirm this, Western blot analysis was performed in all the fourell lines after miR-320a overexpression. We observed that afteriR-320a overexpression, the protein levels of Bax and Puma were

lso upregulated (Fig. 3I and J). These results thus suggest thatiR-320a reduces anti-apoptotic signal generated by UV stress

hrough SMAR1 regulation.

.5. miR-320a inhibits erythroid differentiation throughegulating SMAR1

Vertebrate animal’s blood cells have defined life span; therefore,ematopoiesis takes place to meet the continuous requirementf mature cells. Entry of pluripotent hematopoietic stem cell inny blood cell lineage is characterized by rejuvenation of pluripo-ent hematopoietic stem cells and determination, commitment andifferentiation to particular cell types. Mammalian erythropoiesis

s an intricate process involving several stages, such as the dif-erentiation of early erythroid progenitors, BFU-E (burst-formingnits-erythroid) through late erythroid progenitors, CFU-E (colony-

orming units-erythroid), and finally erythroid precursors (Ney,006). Formation of mature erythrocytes is primarily character-

zed by nuclear condensation and later by enucleation. Althoughhe molecular and cellular mechanisms involved in these processes

a control. (F and G) miR-320a was overexpressed in four cell lines, namely HEK293,-222 were determined respectively.

are poorly understood, we know that cells regulate erythropoiesisat molecular level by regulating gene expression at the levels oftranscription, post-transcription and translation.

Lineage-specific transcription factors and matrix associatedregion binding proteins play essential roles in red blood cell devel-opment as the nuclear material condenses during the process.SATB1, a MAR binding protein is known to play a role in regula-tion of hemoglobin genes (Wen et al., 2005). miR-320 is reportedto mediate the translational activities of CD-71 in differentiationto reticulocytes (Chen et al., 2008). miR-320 level does not changeduring the transition of reticulocytes to erythrocytes, since loss ofCD-71 surface expression contributes to the exosome release inerythrocytes. Therefore, we were interested in understanding therole of SMAR1 in erythroid differentiation. To achieve this objec-tive, we used human erythroleukemic K562 cells as a model. K562cells were treated with 25 �M hemin for 7 days. The cells startedproducing hemoglobin after hemin treatment and the cell pelletshowed red color. The hemoglobinisation was further confirmedby benzidine staining. The differentiated cells stained blue whilecontrol cells remained colorless after staining (Fig. 4A). This sug-

gests that after 25 �M hemin treatment for 7 days, K562 cellsshowed erythroid differentiation. To confirm the differentiation atthe molecular level, GATA-1, Glycophorin A and GATA-2 mRNA lev-els were determined. GATA-1 and Glycophorin A are markers of

S.P.K. Mittal et al. / The International Journal of Biochemistry & Cell Biology 45 (2013) 2519– 2529 2527

tein S

eoutwdbtaTtsAwdiFdmG6pdcbbpc

3m

ttma

Fig. 6. Model depicting the negative regulation of MAR binding pro

rythropoietic lineage. Therefore as expected, these markers werebserved to increase during differentiation. GATA-2 is a marker forndifferentiated cells, therefore as expected GATA-2 was observedo decrease during differentiation (Fig. 4B–D). These results alongith the benzidine staining suggest that hemin-induced in vitroifferentiation can mimic in vivo erythroid differentiation and cane used as a model. In differentiated K562 cells, we determinedhe expression of SMAR1 and GATA-1 at the protein level. SMAR1nd GATA-1 were found to increase during differentiation (Fig. 4E).hen we detected the expression of SMAR1 and miR-320a by quan-itative real-time PCR and observed that SMAR1 and miR-320a levelhowed negative correlation during differentiation (Fig. 4F and G).fter confirming the increase in SMAR1 during differentiation, weere interested in understanding the levels of these in a time-ependent manner. We observed that GATA-1 and Glycophorin A

ncreased while GATA-2 decreased during differentiation (Fig. 4H).urther, we observed the levels of SMAR1 and miR-320a duringifferentiation and determined that SMAR1 levels increase whileiR-320a decreases as the cells differentiate (Fig. 4I). In K562 cells,FP and shRNA plasmid of SMAR1 (sh1077) was transfected. After0 h of transfection, hemin treatment was given for 7 days, then GFPositive and negative cells were sorted using FACS ARIA and benzi-ine staining was performed. We determined that sh1077 negativeells get stained while sh1077 positive cells did not get stainedy benzidine, whereas both GFP+ve and GFP−ve cells stained withenzidine (Fig. 4J). After SMAR1 knockdown, K562 cells could notroceed for erythroid differentiation suggesting that SMAR1 is cru-ial in the process.

.6. SMAR1 regulates erythroid differentiation throughiR-221/222

As it was confirmed that SMAR1 is a vital protein during ery-

hroid differentiation, we were then interested in understandinghe regulation of miRNAs by SMAR1 during this process. Chro-

atin immunoprecipitation (ChIP) was carried out using SMAR1ntibody in HCT116 and HCT116 p53−/− cells. The eluted DNA

MAR1 by miR-320 and its implication in erythroid differentiation.

fragments were subjected to single end sequencing using IlluminaGAIIx Analyzer. After initial QC analysis using SeqQC V1.1 program,the sequence reads were aligned against the Homo sapiens referencegenome (Hg19 UCSC Build) using Bowtie 0.12.6 program. The peakswere filtered on the basis of fold enrichment values; the enrichedregions’ sequences were extracted in FASTA format and annotated.Using ChIP-seq data from HCT116 and HCT116 p53−/− cells, miR-NAs were isolated and a graph was prepared for the potentialSMAR1 target miRNAs and their fold enrichment values (Supple-mentary Fig. 1A and B). We found many SMAR1 miRNA targetsthat are involved in erythropoiesis and other cellular processes. Ofthem miR-221/222 plays a crucial role in early hematopoiesis anderythropoietic differentiation via unblocking of kit receptor mRNAtranslation (Felli et al., 2005). Therefore, we determined the expres-sion of miR-222 during K562 differentiation. We noticed that after3rd day of differentiation, SMAR1 level goes up and the levels ofmiR-222 go down till 7th day of differentiation (Fig. 5A). This sug-gests that SMAR1 and miR-222 showed inverse correlation. Further,it was necessary to validate the ChIP-seq data. Therefore, SMAR1was overexpressed and knocked down in HCT116 and HCT116p53−/− cells and quantitative real-time PCR analysis was performedto determine the levels of miR-221 and miR-222. We observedthat on SMAR1 overexpression and knock down, these miRNAsare downregulated and upregulated, respectively in both the celllines. Therefore, this regulation was p53 independent (Fig. 5B andC). This indicates that SMAR1 might regulate miR-221/222 expres-sion. To further confirm the binding of SMAR1 on miR-221/222promoter, MARWiz software was used to determine the MAR inmiR-221/222 promoter. Indeed, MAR was observed in the promoter(Fig. 5D) and thus primers were designed from the promoter regionof these miRs and chromatin immunoprecipitation was performedin HCT116 cells. We detected the binding of SMAR1 on miR-221/222promoter while promoter pull down was not observed with rab-

bit IgG (Fig. 5E). miR-221 and miR-222 are located close to eachother and share a common promoter. To understand the regula-tion of miR-221/222 through miR-320a, ectopic overexpression ofmiR-320a was carried out in four cell lines namely HEK293, MCF-7,

2 Bioch

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528 S.P.K. Mittal et al. / The International Journal of

CT116 and HCT116 p53−/− cells. Following transfection, miR-221Fig. 5F) and miR-222 (Fig. 5G) expression levels were determinedy real-time PCR analysis. As expected, we observed an increase inhe levels of these miRNAs upon miR-320a overexpression. Theseesults thus suggest that miR-320a regulates erythroid differentia-ion through SMAR1 and miR-221/222.

Supplementary data associated with this article can be found,n the online version, at http://dx.doi.org/10.1016/j.biocel.2013.7.006.

. Discussion

The miRNAs have implications in many fundamental biologi-al processes. Regulation of many miRNAs is not yet known, andany miRNA-target interactions are also not experimentally vali-

ated. miRNAs are very important post-transcriptional regulators,nd they regulate critical cellular processes like apoptosis and dif-erentiation. Many transcription factors are known to be regulatedy miRNAs. On the other hand, several of these transcription factorslso control many miRNAs. One of the transcription factors p53 isnown to regulate miR-34a, miR-34b and miR-34c (Corney et al.,007). It is also known to be regulated by miR-504 and 125b byinding to its 3′UTR (Le et al., 2009; Hu et al., 2010). Therefore, weypothesized that SMAR1 being a MAR binding protein might beegulated by miRNAs and might also regulate many miRNAs.

In this study, we have elucidated the regulation of apoptosis andrythroid differentiation by miR-320a, mediated through SMAR1.e observed that miR-320a regulates SMAR1 through binding to

ts 3′UTR. Further, we observed that miR-320a regulates apopto-is through SMAR1. Upon miR-320a overexpression, HCT116 cellshowed increased apoptotic cell population and this effect wasnhanced on UV treatment (5 J/m2). Similar condition was observedith cells when SMAR1 was knocked down. miR-320a overexpress-

on led to decrease in SMAR1 protein as expected and thus increasen Bax and Puma mRNA and protein levels. This suggests that miR-20a regulates apoptosis mediated through SMAR1, Bax and Puma.ole of miR-320 in regulating cardiac ischemia/reperfusion injuryas been studied. miR-320 overexpression significantly increasedeath and apoptosis of cardiomyocyte (Ren et al., 2009).

miR-320 levels negatively correlate with higher CD71 surfacearker expression and is underexpressed in HbSS reticulocytes

Chen et al., 2008; Schaar et al., 2009). miR-320 is also downreg-lated by hoxa9 in bone marrow cells (Hu et al., 2010). Therefore,ole of SMAR1 in erythroid differentiation was determined. Hemin-nduced differentiation of K562 cells led to increase in hemoglobinroduction. Enhanced SMAR1 level and reduced miR-320a levelccompanies erythroid differentiation. SMAR1 knockdown led tonhibition in erythroid differentiation of K562 cells. To understandhe downstream target of SMAR1 during erythroid differentiation,hIP-seq was performed. We observed miR-221/222 as the targetsf SMAR1 and critical in erythropoiesis. miR-221/222 inhibit ery-hropoiesis via kit receptor repression (Felli et al., 2005). Thus, weonfirmed that SMAR1 regulates erythroid differentiation throughiR-221/222. miR-320a thus regulates erythroid differentiation

hrough SMAR1, miR-221/222 and kit receptor (Fig. 6). In K562ells cultured in the presence of IL-3 and Epo for 22 days, miR-320s known to be downregulated (Lawrie, 2009). SMAR1 is known toranslocate from nucleus to cytoplasm on Phorbol 12-myristate 13-cetate (PMA) treatment (unpublished data). Previously, we haveeported that SMAR1 regulates miR-34a in a p53-dependent man-er (Sinha et al., 2012). Similar regulation of miR-34a, miR-221

nd miR-222 has been shown upon PMA treatment in K562 cellsIchimura et al., 2010). Similar to our results, miR-320 was foundo decrease during erythroid differentiation in MEL cells (Zhant al., 2007). Role of miR-221/222 has been well studied during

emistry & Cell Biology 45 (2013) 2519– 2529

erythropoiesis. SMAR1 has important roles in various biologicalprocesses including apoptosis and cancer, therefore we hypothe-size the role of these miRNAs (miR-221, miR-222 and miR-320a)in apoptosis and cancer as well. There are a few reports depictingthe role of miR-221 and miR-222 in cancer. miR-221 and 222 arefound to be upregulated in papillary thyroid carcinoma by regulat-ing kit expression (He et al., 2005). miR-221 is also known to beupregulated in brain cancer (Ciafre et al., 2005). miR-221/222 actdownstream of the RAS-RAF-MEK pathway in breast cancer cells.These mediate increased metastases and invasion as a consequenceof repression of tricho-rhino-phalangeal syndrome type 1 protein(TRPS1), that leads to an increase in the EMT-promoting proteinzinc finger E-box-binding homeobox 2 (ZEB2) (Stinson et al., 2011).miR-221/222 are also known to be upregulated in hepatocellularcarcinoma (Karakatsanis et al., 2011). Upregulation of miR-221 hasbeen observed in multiple cancers, such as colorectal (Volinia et al.,2006), gastric (Kim et al., 2009; Song et al., 2012), prostate (Galardiet al., 2007) and breast cancer (Miller et al., 2008). There is differ-ential expression of miR-221 in Nodal Marginal Zone Lymphoma(NMZL) against Reactive Lymphoid Hyperplasia (RLN) suggestingits potential with diagnostic value (Arribas et al., 2012).

To summarize, we have identified miR-320a as a regulator ofSMAR1. Our study adds to the present understanding of miRNAs byrevealing the role of miR-320a in apoptosis and erythroid differen-tiation.

Acknowledgements

We thank Dr. Shekhar C. Mande, Director, National Centre forCell Science, for generous support. The work is financially sup-ported by NCCS, Department of Biotechnology (DBT), India. S.P.K.M.is supported by CSIR-SPM fellowship, J.M. is supported by UGC-SRFfellowship.

References

Arribas AJ, Campos-Martine Y, Gomez-Abad C, Algara P, Sanchez-Beato M,Rodriguez-Pinilla MS, et al. Nodal marginal zone lymphoma: gene expressionand miRNA profiling identify diagnostic markers and potential therapeutic tar-gets. Blood 2012;119:e9–21.

Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell2004;116:281–97.

Chattopadhyay S, Kaul R, Charest A, Housman D, Chen J. SMAR1, a novel, alternativelyspliced gene product, binds the Scaffold/Matrix-associated region at the T cellreceptor beta locus. Genomics 2000;68:93–6.

Chaudhuri AA, So AY, Mehta A, Minisandram A, Sinha N, Jonsson VD, et al.Oncomir miR-125b regulates hematopoiesis by targeting the gene Lin28A.Proceedings of the National Academy of Sciences of the United States of America2012;109:4233–8.

Chen S-Y, Wang Y, Telen MJ, Chi J-T. The genomic analysis of erythrocyte microRNAexpression in sickle cell diseases. PLoS ONE 2008;3:e2360.

Ciafre SA, Galardi S, Mangiola A, Ferracin M, Liu CG, Sabatino G, et al. Extensivemodulation of a set of microRNAs in primary glioblastoma. Biochemical andBiophysical Research Communications 2005;334:1351–8.

Corney DC, Flesken-Nikitin A, Godwin AK, Wang W, Nikitin AY. MicroRNA-34b andMicroRNA-34c are targets of p53 and cooperate in control of cell proliferationand adhesion-independent growth. Cancer Research 2007;67:8433–8.

Felli N, Fontana L, Pelosi E, Botta R, Bonci D, Facchiano F, et al. MicroRNAs 221 and222 inhibit normal erythropoiesis and erythroleukemic cell growth via kit recep-tor down-modulation. Proceedings of the National Academy of Sciences of theUnited States of America 2005;102:18081–6.

Galardi S, Mercatelli N, Giorda E, Massalini S, Frajese GV, Ciafrè SA, et al. miR-221and miR-222 expression affects the proliferation potential of human prostatecarcinoma cell lines by targeting p27Kip1. Journal of Biological Chemistry2007;282:23716–24.

Guarnieri DJ, DiLeone RJ. MicroRNAs: a new class of gene regulators. Annals ofMedicine 2008;40:197–208.

Guo L, Chen C, Shi M, Wang F, Chen X, Diao D, et al. Stat3-coordinatedLin-28-let-7-HMGA2 and miR-200-ZEB1 circuits initiate and maintain

oncostatin M-driven epithelial-mesenchymal transition. Oncogene 2013.,http://dx.doi.org/10.1038/onc.2012.573.

Guo L, Xu J, Qi J, Zhang L, Wang J, Liang J, et al. MicroRNA-17∼92a upregulation byestrogen leads to Bim targeting and inhibition of osteoblasts apoptosis. Journalof Cell Science 2012., http://dx.doi.org/10.1242/jcs.117515.

Bioch

H

H

H

I

J

J

J

K

K

K

K

L

L

L

L

S.P.K. Mittal et al. / The International Journal of

e H, Jazdzewski K, Li W, Liyanarachchi S, Nagy R, Volinia S, et al. The role ofmicroRNA genes in papillary thyroid carcinoma. Proceedings of the NationalAcademy of Sciences of the United States of America 2005;102:19075–80.

e X, Dong Y, Wu CW, Zhao Z, Ng SS, Chan FK, et al. MicroRNA-218 inhibits cell cycle progression and promotes apoptosis in coloncancer by downregulating oncogene BMI-1. Molecular Medicine 2012.,http://dx.doi.org/10.2119/molmed.2012.00304.

u W, Chan CS, Wu R, Zhang C, Sun Y, Song JS, et al. Negative regulation of tumorsuppressor p53 by microRNA miR-504. Molecular Cell 2010;38:689–99.

chimura A, Ruike Y, Terasawa K, Shimizu K, Tsujimoto G. MicroRNA-34a inhibitscell proliferation by repressing mitogen-activated protein kinase kinase 1during megakaryocytic differentiation of K562 cells. Molecular Pharmacology2010;77:1016–24.

alota A, Singh K, Pavithra L, Kaul-Ghanekar R, Jameel S, Chattopadhyay S. Tumorsuppressor SMAR1 activates and stabilizes p53 through its arginine–serine-richmotif. Journal of Biological Chemistry 2005;280:16019–29.

alota-Badhwar A, Kaul-Ghanekar R, Mogare D, Boppana R, Paknikar KM, Chat-topadhyay S. SMAR1-derived P44 peptide retains its tumor suppressor functionthrough modulation of p53. Journal of Biological Chemistry 2007;282:9902–13.

in HL, Kim JS, Kim YJ, Kim SJ, Broxmeyer HE, Kim KS. Dynamic expression of spe-cific miRNAs during erythroid differentiation of human embryonic stem cells.Molecules and Cells 2012;34:177–83.

arakatsanis A, Papaconstantinou I, Gazouli M, Lyberopoulou A, Polymeneas G,Voros D. Expression of microRNAs, miR-21, miR-31, miR-122, miR-145, miR-146a, miR-200c, miR-221, miR-222, and miR-223 in patients with hepatocellularcarcinoma or intrahepatic cholangiocarcinoma and its prognostic significance.Molecular Carcinogenesis 2011., http://dx.doi.org/10.1002/mc.21864.

aul R, Mukherjee S, Ahmed F, Bhat MK, Chhipa R, Galande S, et al. Direct inter-action with and activation of p53 by SMAR1 retards cell-cycle progression atG2/M phase and delays tumor growth in mice. International Journal of Cancer2003;103:606–15.

aul-Ghanekar R, Majumdar S, Jalota A, Gulati N, Dubey N, Saha B, et al. AbnormalV(D)J recombination of T cell receptor beta locus in SMAR1 transgenic mice.Journal of Biological Chemistry 2005;280:9450–9.

im Y-K, Yu J, Han TS, Park S-Y, Namkoong B, Kim DH, et al. Functional links betweenclustered microRNAs: suppression of cell-cycle inhibitors by microRNA clustersin gastric cancer. Nucleic Acids Research 2009;37:1672–81.

awrie CH. microRNA expression in erythropoiesis and erythroid disorders. BritishJournal of Hematology 2009;150:144–51.

e MTN, Teh C, Shyh-Chang N, Xie H, Zhou B, Korzh V, et al. MicroRNA-125bis a novel negative regulator of p53. Genes and Development 2009;23:862–76.

iang YJ, Wang QY, Zhou CX, Yin QQ, He M, Yu XT, et al. MiR-124 targets Slug to

regulate epithelial–mesenchymal transition and metastasis of breast cancer.Carcinogenesis 2013 [Epub ahead of print].

iu MJ, Yue PY, Wang Z, Wong RN. Methyl protodioscin induces G2/M arrest and apo-ptosis in K562 cells with the hyperpolarization of mitochondria. Cancer Letters2005;224:229–41.

emistry & Cell Biology 45 (2013) 2519– 2529 2529

Miller TE, Ghoshal K, Ramaswamy B, Roy S, Datta J, Shapiro CL, et al. MicroRNA-221/222 confers tamoxifen resistance in breast cancer by targeting p27Kip1.Journal of Biological Chemistry 2008;283:29897–903.

Nagy K, Pasti G, Bene L, Nagy IZ. Involvement of Fenton reaction products indifferentiation induction of K562 human leukemia cells. Leukemia Research1995;19:203–12.

Nassal M, Rieger A. PCR-based site-directed mutagenesis using primers with mis-matched 3′ ends. Nucleic Acids Research 1990;18:3077–8.

Ney PA. Gene expression during terminal erythroid differentiation. Current Opinionin Hematology 2006;13:203–8.

Rampalli S, Pavithra L, Bhatt A, Kundu TK, Chattopadhyay S. Tumor suppres-sor SMAR1 mediates cyclin D1 repression by recruitment of the SIN3/histonedeacetylase 1 complex. Molecular and Cellular Biology 2005;25:8415–29.

Ren XP, Wu J, Wang X, Sartor MA, Jones K, Qian J, et al. MicroRNA-320 is involvedin the regulation of cardiac ischemia/reperfusion injury by targeting Hsp20.Circulation 2009;119:2357–66.

Ryu S, McDonnell K, Choi H, Gao D, Hahn M, Joshi N, et al. Suppression of miRNA-708by polycomb group promotes metastases by calcium-induced cell migration.Cancer Cell 2013;23:63–76.

Schaar DG, Medina DJ, Moore DF, Strair RK, Ting Y. miR-320 targets transferrinreceptor 1 (CD71) and inhibits cell proliferation. Experimental Hematology2009;37:245–55.

Singh K, Mogare D, Giridharagopalan RO, Gogiraju R, Pande G, Chattopadhyay S.p53 target gene SMAR1 is dysregulated in breast cancer: its role in cancer cellmigration and invasion. PLoS ONE 2007;2:e660.

Sinha S, Malonia S, Mittal S, Singh K, Kadreppa S, Kamat R, et al. Coordinated reg-ulation of p53 apoptotic targets BAX and PUMA by SMAR1 through an identicalMAR element. EMBO Journal 2010;29:830–42.

Sinha S, Malonia SK, Mittal SPK, Mathai J, Pal JK, Chattopadhyay S. Chromatinremodelling protein SMAR1 inhibits p53 dependent transactivation by regu-lating acetyl transferase p300. International Journal of Biochemistry and CellBiology 2012;44:46–52.

Song M-Y, Pan K-F, Su H-J, Zhang L, Ma J-L, Li J-Y, et al. Identification of serummicrornas as novel non-invasive biomarkers for early detection of gastric cancer.PLoS ONE 2012;7:e33608.

Stinson S, Lackner MR, Adai AT, Yu N, Kim HJ, O’Brien C, et al. TRPS1 targeting by miR-221/222 promotes the epithelial-to-mesenchymal transition in breast cancer.Science Signaling 2011;4:ra41.

Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, et al. A microRNAexpression signature of human solid tumors defines cancer gene targets.Proceedings of the National Academy of Sciences of the United States of America2006;103:2257–61.

Wen J, Huang S, Rogers H, Dickinson LA, Kohwi-Shigematsu T, Noguchi CT. SATB1

family proteins expressed during early erythroid differentiation modifies globingene expression. Blood 2005;105:3330–9.

Zhan M, Miller CP, Papayannopoulou T, Stamatoyannopoulos G, Song C-Z. MicroRNAexpression dynamics during murine and human erythroid differentiation.Experimental Hematology 2007;35:1015–25.