microrna 299-3p modulates replicative senescence in … · 2017. 7. 11. · microrna 299-3p...

MicroRNA 299-3p modulates replicative senescence in endothelial cells

Hui-Lan Jong,1 Mohd Rais Mustafa,1 Paul M. Vanhoutte,2 Sazaly AbuBakar,3 and Pooi-Fong Wong1

1Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; 2Li Ka Shing Faculty ofMedicine, The University of Hong Kong, Hong Kong; and 3Department of Microbiology, Faculty of Medicine, University ofMalaya, Kuala Lumpur, Malaysia

Submitted 29 May 2012; accepted in final form 25 January 2013

Jong HL, Mustafa MR, Vanhoutte PM, AbuBakar S, Wong PF.MicroRNA 299-3p modulates replicative senescence in endothelialcells. Physiol Genomics 45: 256–267, 2013. First published January29, 2013; doi:10.1152/physiolgenomics.00071.2012.—MicroRNAs(miRNAs) regulate various cellular processes. While several genesassociated with replicative senescence have been described in endo-thelial cells, miRNAs that regulate these genes remain largely un-known. The present study was designed to identify miRNAs associ-ated with replicative senescence and their target genes in humanumbilical vein endothelial cells (HUVECs). An integrated miRNAand gene profiling approach revealed that hsa-miR-299-3p is upregu-lated in senescent HUVECs compared with the young cells, and oneof its target genes could be IGF1. IGF1 was upregulated in senescentcompared with young HUVECs, and knockdown of hsa-miR-299-3pdose-dependently increased the mRNA expression of IGF1, moresignificantly observed in the presenescent cells (passage 19) com-pared with the senescent cells (passage 25). Knockdown of hsa-miR-299-3p also resulted in significant reduction in the percentage of cellspositively stained for senescence-associated �-galactosidase and in-creases in cell viability measured by MTT assay but marginal in-creases in cell proliferation and cell migration capacity measured byreal-time growth kinetics analysis. Moreover, knockdown of hsa-miR-299-3p also increased proliferation of cells treated with H2O2 toinduce senescence. These findings suggest that hsa-miR-299-3p maydelay or protect against replicative senescence by improving themetabolic activity of the senesced cells but does not stimulate growthof the remaining cells in senescent cultures. Hence, these findingsprovide an early insight into the role of hsa-miR-299-3p in themodulation of replicative senescence in HUVECs.

microRNA; aging; hsa-miR-299-3p; IGF1

NORMAL CELLS HAVE A LIMITED lifespan in which they cease toproliferate after a finite number of population doublings (55).Replicative senescence is achieved when the cells enter theirreversible growth arrest phase (G0/G1 arrest) of the cell cycle(40). Senescent cells do not divide but remain viable andmetabolically active. These cells have enlarged and flattenedmorphology, increased granularity and vacuolization, alteredexpressions of genes and proteins, and shortened telomerelength; they characteristically express senescence-associated�-galactosidase (SA-�-gal) activity at pH 6.0 (22). Besidesreplicative senescence, numerous factors can contribute tostress-induced premature senescence. They include exposure tosublethal dose of DNA damaging agents (e.g., certain antican-cer drugs), gamma irradiation, oxidative stress, overexpressionof activated oncogenic Ras (or of its downstream effectors suchas Raf), activation of tumor suppressor genes (e.g., p53, p16,

and pRB), deprivation of nutrients or growth factors, andimproper cell contacts (4, 12).

MicroRNAs (miRNAs) are a well-recognized group ofshort (�22 nt), noncoding RNAs (27). Currently, themiRNA database [Sanger miRBase (http://www.mirbase.org/)] lists �18,000 entries. miRNAs regulate gene expressionby targeting 3=-untranslated region (UTR) of mRNA tran-scripts that in turn results in translational inhibition or degra-dation of mRNA. The majority of miRNAs repress geneexpression, but a minority group activates it by upregulatingtranslation (39, 54). Unique alterations in miRNA expressioncontribute to the regulation and induction of diseases such asvarious types of cancers, diabetes, and autoimmune diseases(31). In particular, the miRNA profile is altered in heartdisease, and the pattern of miRNA expression is distinct indifferent forms of cardiac disorders (19). A unique miRNAsignature is associated with the involvement of specific signaltransduction pathway and thus with the prognosis and patho-genesis of the disease. For instance, miRNA (miR)-21 con-tributes to myocardial disease by stimulating MAP kinasesignaling in fibroblasts, and miR-126 accelerates the devel-opment of atherosclerosis (3, 51). In addition, overexpres-sion of miR-34a induces cell cycle arrest and senescence inhuman umbilical vein endothelial cells (HUVECs), an effectmediated by the SIRT1-p53 pathway (59).

Endothelial cells are specialized cells that line the vascularlumen. They play an important role in modulating vasculartone, exchanges of molecules between blood and tissues, pre-vention of coagulation, and formation of new blood vessels.Senescent endothelial cells contribute to age-related vasculardisorders such as atherosclerosis and cardiovascular diseases(36). Alterations in miRNA expression have been reported insenescent endothelial cells, suggesting that miRNAs play rolesin the regulation of senescence in these cells (35, 53). HUVECshave been widely used in numerous in vitro studies onhuman endothelial cells. Despite discrepancies noted be-tween HUVECs and the different parts of vasculature, they doshare many common functional and morphological features(50, 53). Hence, results obtained from HUVECs will providepreliminary observations leading to subsequent physiologi-cally relevant in depth investigations.

In earlier studies in senescent HUVECs, miRNA and geneexpression profiling have been performed independently, mak-ing direct identification of miRNA target genes challenging (7,23, 35, 40, 47). The present study, hence, was designed toperform an integrated study of miRNA and gene expression inHUVECs, in the hope to provide new insights into the role ofmiRNAs in cellular senescence and age-related disorders andto define their potential as diagnostic markers and/or therapeu-tic targets.

Address for reprint requests and other correspondence: Pooi-Fong Wong,Dept. of Pharmacology, Faculty of Medicine, Univ. of Malaya, Kuala Lumpur,Malaysia (e-mail: [email protected]).

Physiol Genomics 45: 256–267, 2013.First published January 29, 2013; doi:10.1152/physiolgenomics.00071.2012.

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MATERIALS AND METHODS

Cell culture. HUVECs were purchased from ScienCell ResearchLaboratories (Carlsbad, CA) and maintained in endothelial cell me-dium (ECM) supplemented with 5% FBS, 1% endothelial cell growthsupplement, and 1% of penicillin-streptomycin (ScienCell ResearchLaboratories) at 37°C in a humidified atmosphere of 95% air/5% CO2.The number of population doublings (PD) was calculated as described(8, 25) using the formula:

PD � [In (number of cells harvested)� In (number of cells seeded)] ⁄ In 2

Cells were seeded at a constant initial density of 5 � 105 cells perT75 flask and passaged with 0.25% trypsin EDTA every 2–3 days tomaintain the cultures in constant log phase. Replicative senescent cellswere obtained by consecutively passaging the young cells until thecells proliferation became nearly arrested, approximately at passage25 (PD �45).

Young and senescent cells used in experiments are cells with PD �10 and PD � 45, respectively, unless otherwise mentioned. Morpho-logical changes were monitored and evaluated for every subculture.The images of young and senescent cells were captured, examined andrecorded with light microscopy at a magnification of �200.

SA-�-gal. Cells were stained using a SA-�-gal staining kit (CellSignaling Technology, Beverly, MA) following the manufacturer’sinstructions. In brief, cells were washed once in one time (1�) PBS,fixed for 15 min in 1� fixative solution, washed twice in 1� PBSbefore being incubated in fresh SA-�-gal staining solution at 37°Covernight. The percentage of positively stained cell (blue cells) versustotal cells was counted by randomly choosing 10 microscopic fieldsunder �20 objective magnification.

MTT cell viability assay. Briefly, cells were seeded in 96-wellplates at optimum cell density. After 18–24 h, cells were transfectedwith 31.25, 62.5, 125, 250, and 500 nM of miRIDIAN hairpininhibitor of hsa-miR-299-3p (A299-3p) (Dharmacon, Lafayette, CO)for 24 h. Control wells were transfected with equal concentrations ofcontrol antisense oligonucleotide (CA) i.e., miRIDIAN hairpin inhib-itor negative control #2 (Dharmacon). Following transfection, cellswere incubated in the dark with 2 mg/ml MTT at 37°C for 2 h, thenthe medium was carefully removed, and 100 �l of DMSO was addedto dissolve the formazan crystals formed. Absorbance was measuredat 570 nm in a plate reader. Cell viability was calculated by thefollowing formula: (mean absorbance in test wells)/(mean absorbancein control well) � 100%.

Hydrogen peroxide study. Hydrogen peroxide (H2O2, BDHAnalaR) was used to induce premature senescence. In brief, youngcells were seeded at optimum cell density and incubated overnight.The cells were subjected to two successive sublethal treatments of 200�M H2O2 in ECM containing 5% FBS for 2 h, with one treatment perday for 2 consecutive days. At the end of each treatment, the treatmentmedia were removed and the cells were washed twice with 1� PBSand maintained in ECM with 5% FBS.

Real-time growth profile. Cells were seeded in a 16-well E-plate,and the growth kinetic of the cells was monitored continuously usinga Roche xCELLigence Real Time Cell Analyzer (RTCA; RocheDiagnostic, Mannheim, Germany) DP instrument at 37°C in a humid-ified atmosphere of 95% air/5% CO2. The xCELLigence systemutilizes E-plate (Roche Diagnostic), which contains interdigitatedmicroelectrodes on the bottom of the plate that detect local ionicchanges as cells proliferate, and the proliferation rate is measured aselectrode impedance. Cell sensor impedance was expressed as anarbitrary unit termed cell index (CI). The CI at each time point wasdefined as (Rn � Rb)/15, where Rn is the cell electrode impedance ofthe well and the Rb is the background impedance of the well with themedia alone.

Telomere length measurement. Relative telomere (T) length wasmeasured by a modified quantitative real-time polymerase chain reaction

(qRT-PCR) method (6). Genomic DNAs were extracted from cells usingQIAmp DNA blood mini kit (Qiagen, Hilden, Germany) in accordancewith the manufacturer’s protocol. Telomeric hexamer repeats were quan-titated using T and single copy gene (S) PCR. T and S PCR wereperformed using Power SYBR green PCR master mix (Applied Biosys-tems, Foster, CA) on ABI StepOne system. T and �-globin primer pairs(primer sequences shown below) were used for T and S PCR, respec-tively: telg, ACACTAAGGTTTGGGTTTGGGTTTGGGTTTGGGT-TAGTGT; telc, TGTTAGGTATCCCTATCCCTATCCCTATC-CCTATCCCTAACA; hbgu, CGGCGGCGGGCGGCGCGGGCT-GGGCGGCTTCATCCACGTTCACCTTG; hbgd, GCCCGGCCCGC-CGCGCCCGTCCCGCCGGAGGAGAAGTCTGCCGTT.

The thermal cycling profile for both T and S PCR was as follows:Holding stage: 15 min at 95°C; Cycling stage I: 2 cycles of 15 s at94°C, 15 s at 49°C; Cycling stage II: 32 cycles of 15 s at 94°C, 10 sat 62°C, 15 s at 74°C; Melt curve stage: 15 s at 94°C, 1 min at 60°C,15 s at 95°C with a gradual increment of 0.3°C.

Standard curves [a plot of Ct vs. log (amount of input DNA)] wereestablished for both T and S PCR with randomly chosen genomicDNA of young cells. Relative T lengths of samples were calculatedbased on the T/S ratio obtained from standard curves.

miRNA and gene expression microarray. Once the RS cells wereestablished, analyses of the expressions of 866 human miRNAs and89 human viral miRNAs, based on Sanger miRNA database release12.0, and 19,596 human genes were performed using Agilent HumanMicroRNAs and Whole Human Genome microarray platforms, re-spectively. Total RNAs were isolated from cells using miRNeasy minikit (Qiagen) according to manufacturer’s protocol. RNA integrity ofthe total RNA was accessed using Agilent 2100 Bioanalyzer (AgilentTechnologies, Palo Alto, CA). For miRNA microarrays, total RNAs(100 ng) were labeled with Cyanine 3-pCp and hybridized ontoHuman MicroRNA Array version 3.0 (8X15K) with 40–60 meroligonucleotides probe spotted on the slide by SurePrint Inkjet tech-nology (Agilent Technologies). For gene expression microarrays,cRNA was synthesized from 50 ng of total RNAs, labeled withCyanine 3-CTP, and hybridized onto Whole Human Genome Array(4 � 44k), which was spotted by the 60-mer SurePrint technology(Agilent Technologies). The microarray slides were scanned on mi-croarray Scanner B, and data were extracted with Feature Extractionsoftware (Agilent Technologies). Microarray data have been depos-ited in the National Center for Biotechnology Information GeneExpression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) andare accessible through GEO series accession no. GSE37093.

Microarray data analysis. Integrated analysis of microRNA andgene expression microarray data was performed using GeneSpringGX version 11.05 (Agilent Technologies). The threshold of rawsignals was set at 1.0. The normalization algorithm employed wasquantile, and baseline transformation to median of all samples wasperformed. Differential miRNA and gene expression profiles of youngvs. senescent cells were generated with Volcano Plot in GeneSpringGX, which consists of the significance analysis and fold change filter.Significance analysis was performed by Student’s t-test for unpairedobservations with asymptotic corrected P value � 0.05 and foldchange filter of � 2.0. The multiple testing correction employed wasBenjamini-Hochberg. The TargetScan program integrated in Gene-Spring was used to identify the biological targets of the differentiallyexpressed miRNAs. In addition, homology regions between miRNAsof interest and 3=-UTR of the target gene of interest were identifiedwith miRanda (http://www.microrna.org/microrna/home.do). ThemiRNA profile was then translated to the gene expression data. Thejoint miRNA and gene expression profile was then subjected topathway analysis. The pathway map generated from pathway analysiswas analyzed to select miRNAs of interest. The miRNAs of interestreported have not been previously associated with cellular senescencebut showed association with known cellular senescence-associatedgenes in the pathway maps.

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qRT-PCR validation of miRNA and gene expression. Two-stepqRT-PCR was performed to quantitatively measure the expression ofmiRNA and gene to validate the microarray data. For validation ofmiRNA expression, cDNA was synthesized from 10 ng of total RNAusing a Taqman microRNA Reverse Transcription Kit according tomanufacturer’s protocol (Applied Biosystems). qRT-PCR was per-formed using Taqman MicroRNA Assay and Taqman Universal PCRMaster Mix (Applied Biosystems). Taqman MicroRNA Assay ID areas follows: hsa-miR-130b (assay ID: 000456), hsa-miR-18b (assayID: 002217), hsa-miR-299-3p (assay ID: 001015), hsa-miR-338-3p(assay ID: 002252), hsa-miR-134 (assay ID: 001186), hsa-miR-17(assay ID: 002308), hsa-miR-188-5p (assay ID: 002320), RNU6B(control miRNA assay) (assay ID: 001093). RNU6B (human) wasused as endogenous reference RNA for normalization of data. Forvalidation of gene expression, cDNA was synthesized from 250 ngtotal RNA using the High Capacity cDNA Reverse Transcription Kit(Applied Biosystems) according to the manufacturer’s protocol. qRT-PCR was performed using Taqman Gene Expression Assay and TaqmanFast Advanced PCR Master Mix. Taqman Gene Expression assay ID areas follow: IGF1 (assay ID: Hs_01547656_m1), SLFN13 (assay ID:Hs_00431187_m1), HDAC9 (assay ID: Hs_00206843_m1). GAPDH(assay ID: 4333764F) was used as endogenous reference RNA fornormalization of data. Analysis was performed by the 2�Ct method.

miRNA knockdown study. Transfection of HUVECs was performedusing DharmaFECT 1 according to the manufacturer’s instructions(Dharmacon). In brief, cells were seeded in growth medium without

antibiotics at a density of 10,000 cells per well and incubated over-night. The cells were then transfected with 7.8, 15.6, 31.25, 62.5, 125,250 and 500 nM of A299-3p (Dharmacon) for 24 h. After theknockdown of miRNA, Taqman MicroRNA Assay was performed asdescribed above to measure the expression of hsa-miR-299-3p. Theexpression of hsa-miR-299-3p was compared with growth mediumcontrol.

For mRNA expression measurement, the cells were transfectedwith 31.25, 62.5, 125, 250, and 500 nM of A299-3p (Dharmacon) for24 h. Control wells were transfected with equal concentrations of CA,i.e., miRIDIAN hairpin inhibitor negative control #2 (Dharmacon).After the knockdown of miRNA, Taqman Gene Expression Assaywas performed as described above to measure the expression of IGF1mRNA.

For SA-�-gal staining, real-time growth profile and cell migrationassay, the cells were transfected with 31.25, 62.5, 125, 250, and 500nM of A299-3p (Dharmacon) for 48 h. Control wells were transfectedwith equal concentrations of CA i.e., miRIDIAN hairpin inhibitornegative control #2 (Dharmacon). SA-�-gal staining, real-time growthstudy, cell cycle analysis, and cell migration assay were performed todetermine the percentage of SA-�-gal positively stained cells, growthand migration capacity after transfection.

For concurrent knockdown of multiple miRNAs, cells were trans-fected with a total concentration of 31.25, 62.5, 125, 250, and 500 nMof combination of miRIDIAN miRNA hairpin inhibitor for hsa-miR-134, -299-3p, and -338-3p. Control wells were transfected with equal

Fig. 1. Characterization of senescence in human umbilical vein endothelial cells (HUVECs). A: morphological changes, B: senescence-associated �-galactosidase(SA-�-gal) staining, C: percentage of SA-�-gal-positive cells, D: real-time growth profiles, E: cell cycle analysis, F: measurement of relative telomere lengthsin young and replicative senescent HUVECs. Y, young cells; RS, replicative senescent cells. Data are means SE; ***P � 0.001, **P � 0.01.

258 microRNA 299-3p AND ENDOTHELIAL SENESCENCE




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concentrations of CA, i.e., miRIDIAN hairpin inhibitor negativecontrol #2 (Dharmacon). Total RNA were harvested after transfection,and Taqman Gene Expression Assay was performed as describedabove to check the expression of IGF1 mRNA.

Cell migration assay. Cell migration assay was performed withCIM plates (Roche Diagnostic) according to the standard protocolsuggested by the manufacturer. In brief, after 48 h transfection, cellswere starved using 0.5% FBS containing growth media for 5 h.Following starvation, they were trypsinized and seeded onto upperchamber of CIM plates at cell density of 14,000 cells per well. Thelower chamber of CIM plates were filled up with 10% FBS containinggrowth media acting as chemoattractant. The CIM plates were loadedonto RTCA DP instrument (Roche Diagnostic) to monitor real-timecell migration. Microelectronic sensors are integrated onto the under-side of the microporous polyethylene terephthalate membrane of theCIM plate (Boyden-like chamber). Cells migrate from the upperchamber through the membrane into the bottom chamber, contact, andadhere to the electronic sensors on the underside of the membrane andalter impedance. Cell sensor impedance was expressed as CI.

Statistical analysis. All data are presented as means SE. Statis-tical analysis was performed with Student’s t-test using GraphPadPrism software (version 5.0; GraphPad Software, San Diego, CA). Pvalues � 0.05 were accepted to indicate statistically significant differ-ences. Statistical significance is expressed as ***P � 0.001, **P � 0.01,*P � 0.05.

RESULTS

Characterization of replicative senescence. Replicative se-nescence (RS) was confirmed in HUVECs with �45 PD by thefollowing observations: 1) RS cells were relatively enlargedand flattened and showed an increase in granulation and vac-uolization compared with their younger counterpart (Fig. 1A);2) RS showed a significant increase (�60%) in the percentageof SA-�-gal-positive cells compared with young cells (Fig. 1,B and C); 3) the real-time growth kinetics of RS showed arelatively lower CI and remained viable in culture over a longperiod of time, while young cells showed a relatively higher CIand were eventually eliminated from the culture (Fig. 1D);4) cell cycle analysis revealed that RS cells had a significantincrease in G0/G1 population (�40%) compared with theyoung cells, suggesting that RS cells stopped proliferationthrough G0/G1 arrest in the cell cycle (Fig. 1E); and 5) the Tlength of RS cells was also shorter compared with the youngcells (Fig. 1F).

miRNA and gene expression profile. The comparison of themiRNA profile in young and RS cells showed that 29 miRNAswere expressed differentially in the latter, including upregula-tion of 19 miRNAs and downregulation of 10 others (Table 1). Inthe gene expression analysis, 251 genes were differentially regu-lated in RS, with 130 down- and 121 upregulations (Fig. 2A).Microarray datasets are available for viewing at GEO throughaccession no. GSE37093. To validate the microarray findings,we performed qRT-PCR for seven miRNAs. Overall, theqRT-PCR analysis showed satisfactory correlation with themicroarray results, although smaller fold-changes were foundby qRT-PCR (Fig. 3A). In particular, the qRT-PCR resultsshowed that hsa-miR-130b, -18b, and -17 were downregulated,while hsa-miR-299-3p, -338-3p, -134, and -188-5p were up-regulated in RS compared with young cells (Fig. 3A). Compu-tational simulation of biological interactions of differentially ex-pressed target genes of hsa-miR-299-3p in senescent HUVECswere also identified (Fig. 2C). In additional, TargetScan anal-ysis suggested that IGF1 is a target of hsa-miR-299-3p, -134,

and -338-3p. Homology region between hsa-miR-299-3p and3=-UTR of IGF1 was also identified by miRanda, where thescore of homology match (mirSVR score) was �0.4165 [Fig.2B(i)]. miRNA targets with mirSVR score ��0.1 is consid-ered as “good,” and miRNA targets with ��0.1 mirSVR scoreindicates “nongood” (46). This further confirmed the potentialregulation of IGF1 by hsa-miR-299-3p. Prior to subsequentknockdown studies, the inherent mRNA expression of IGF1was measured. The qRT-PCR results showed that IGF1 wasupregulated by approximately sevenfold in RS compared withyoung cells (Fig. 3B).

IGF1 as potential target of hsa-miR-299-3p. hsa-miR-299-3p was knocked down transiently in RS cells by transfec-tion with a range of concentrations (7.8–500 nM) of A299-3por CA. Target gene expression, percentage of SA-�-gal posi-tive, cell growth, and migration were determined after theknockdown. Transfection with the hsa-miR-299-3p inhibitorA299-3p resulted in a significant concentration-dependentknockdown, ranging from a 45% (7.8 nM) to 85% (500 nM)decrease in the expression of hsa-miR-299-3p as quantified byqRT-PCR (Fig. 3C).

To investigate if hsa-miR-299-3p targets IGF1, we measuredthe mRNA expression level of IGF1 following the knockdownof hsa-miR-299-3p. Transient inhibition of hsa-miR-299-3presulted in a dose-dependent increase in the mRNA expressionof IGF1, with the level significantly surpassing that of the

Table 1. miRNA expression profile of young controlvs. RS cells

Systematic NameCorrectedP Value P Value FC Absolute Regulation

hsa-miR-146a 1.48E-02 3.51E-04 2.93E�01 downhsa-miR-19b-1* 2.45E-02 1.90E-03 1.34E�01 downhsa-miR-218 1.48E-02 2.65E-04 1.25E�01 downhsa-miR-29b-1* 3.49E-02 4.16E-03 4.48E�00 downhsa-miR-18b 4.89E-02 8.38E-03 3.09E�00 downhsa-miR-20b 3.49E-02 3.91E-03 3.00E�00 downhsa-miR-17 4.89E-02 8.21E-03 2.65E�00 downhsa-miR-130b 1.73E-02 7.89E-04 2.16E�00 downhsa-miR-15b 4.16E-02 6.14E-03 2.04E�00 downhsa-miR-92b 4.16E-02 6.05E-03 2.01E�00 downhsa-miR-150* 1.48E-02 4.91E-04 3.62E�01 uphsa-miR-134 7.05E-03 5.21E-05 2.32E�01 uphsa-miR-1915 2.45E-02 1.83E-03 9.83E�00 uphsa-miR-338-3p 3.49E-02 4.18E-03 5.96E�00 uphsa-miR-572 4.81E-02 7.55E-03 5.67E�00 uphsa-miR-874 2.42E-02 1.60E-03 5.53E�00 uphsa-miR-34a* 1.48E-02 3.78E-04 5.19E�00 uphsa-miR-1207-5p 3.22E-02 2.87E-03 4.01E�00 uphsa-miR-1225-5p 2.43E-02 1.70E-03 3.68E�00 uphsa-miR-136* 3.49E-02 3.65E-03 3.33E�00 uphsa-miR-188-5p 2.78E-02 2.26E-03 3.31E�00 uphsa-miR-135a* 2.42E-02 1.61E-03 3.19E�00 uphsa-miR-34a 4.18E-02 6.32E-03 3.05E�00 uphcmv-miR-UL70-3p 3.49E-02 4.33E-03 2.71E�00 uphsa-miR-939 1.20E-02 1.33E-04 2.53E�00 uphsa-miR-638 4.89E-02 8.48E-03 2.49E�00 uphsa-miR-31 1.73E-02 7.09E-04 2.48E�00 uphsa-miR-299-3p 1.48E-02 4.66E-04 2.13E�00 uphsa-miR-186 3.73E-02 4.96E-03 2.06E�00 up

Fold change cut-off: 2.0; P value computation by Student’s t-test; Multipletesting correction: Benjamini-Hochberg false discovery rate; Corrected P valuecut-off: 0.05. *Less predominantly expressed miRNA (from the opposite armof the precursor); RS, replicative senescence; FC Absolute, fold changeabsolute.





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antisense oligonucleotide at 250 nM (Fig. 4A, P � 0.01) by�30% compared with the CA. Since the knockdown wasperformed on near-arrested cells at passage 25 (PD �45), thetrue effect of hsa-miR-299-3p inhibition may not be apparent.

Hence, we used cells that are at the presenescence stage at cellpassage 19 for subsequent hsa-miR-299-3p knockdown. Inpassage 19 cells, knockdown of hsa-miR-299-3p resulted in adose-dependent (except at 500 nM) increase in the mRNA

Fig. 2. A: gene expression profile of young control vs. RS cells. Fold-change: negative values indicate downregulation, positive values indicate upregulation.Fold-change cut-off: 2.0. P value computation by Student t-test. Multiple testing correction: Benjamini-Hochberg false discovery rate; Corrected P value cut-off:0.05. B: homology region between hsa-miR-299-3p, -134, -338-3p, and 3=-untranslated region of IGF1 mRNA sequence. C: computational simulation ofbiological interactions of differentially expressed target genes of hsa-miR-299-3p in senescent HUVECs as revealed by GeneSpring GX version 11.5.1 PathwayAnalysis. IGF-1 is boxed in red.





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expression of IGF1 (Fig. 4B). A significant increase wasobserved at many concentrations of A299-3p (62.5 nM on-ward). Hence, this result shows that knockdown of hsa-miR-299-3p affects IGF1 mRNA expression.

Because TargetScan analysis identified that hsa-miR-134and -338-3p also target IGF1, we hypothesize that knockdownof hsa-miR-299-3p in combination with hsa-miR-134 and-338-3p may enhance the elevation in IGF1 mRNA expression.Knockdown of hsa-miR-299-3p in combination with hsa-miR-

134 and -338-3p resulted in a significant increase in the mRNAexpression of IGF1 compared with the CA. Concurrent inhibi-tion of hsa-miR-134, -299-3p, and -338-3p at 31.25 and 62.5nM resulted in �130 and �170% increase in the mRNAexpression of IGF1, respectively (Fig. 4C; P � 0.01 and P �0.05, Student’s t-test). These data show that knockdown ofhsa-miR-299-3p together with other miRNAs that target IGF1can release the suppression of IGF1 with greater efficacy at lowconcentrations of the inhibitors used.

Inhibition of hsa-miR-299-3p and replicative senescence. Tofurther confirm the role of hsa-miR-299-3p in modulatingsenescence, SA-�-gal pH 6.0 staining, MTT cell viabilityassay, real-time growth studies, and cell migration assay wereperformed after inhibition of hsa-miR-299-3p at a range ofconcentration of the hsa-miR-299-3p inhibitor, A299-3p. Asignificant dose-dependent decrease in the percentage of SA-�-gal-positive cells was observed at all concentrations ofinhibition of hsa-miR-299-3p compared with CA except for500 nM (Fig. 5, A and B). On average, RS cultures transfectedwith CA contained �40% SA-�-gal-positive cells. The greatestreduction in the percentage of SA-�-gal-positive cells (reducedfrom �43 to 22%) was observed at 31.25 nM of inhibition of

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Fig. 3. qRT-PCR validation of miRNAs (A) and target genes (B) of interest. C: theexpression of hsa-miR-299-3p following transfection with A299-3p and growthmedium control. A299-3p, inhibitor of hsa-miR-299-3p; a.u., Arbitrary units. Dataare means SE; ***P � 0.001, **P � 0.01, *P � 0.05.

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Fig. 4. mRNA expression of IGF1 following the knockdown of hsa-miR-299-3p in passage 25 (A) and passage 19 (B) cells and a combination ofmiRNA (C) (simultaneous inhibition of hsa-miR-134, -299-3p, and -338-3p) at arange of concentrations. CA, control antisense oligonucleotide; Comb, combina-tion of miRNA inhibitors. Data are means SE; **P � 0.01, *P � 0.05.





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hsa-miR-299-3p (Fig. 5B), while the lowest reduction in per-centage of SA-�-gal-positive cells (reduced from �40 to 30%)was observed at 500 nM of inhibition of hsa-miR-299-3p,which did not reach statistical significance (Fig. 5B). Cellviability as measured by MTT assay also showed a significantdose-dependent increase in cell viability following the inhibi-tion of hsa-miR-299-3p at all tested concentrations comparedwith CA except for 500 nM (Fig. 5C). Real-time growthkinetics of RS cells was examined following knockdown ofhsa-miR-299-3p (Fig. 6, A–E). Normalized CI of RS cellsincreased after knockdown of hsa-miR-299-3p, particularly at250 nM (Fig. 6D) and 500 nM (Fig. 6E), though this did notreach statistical significance. This suggests that knockdown ofhsa-miR-299-3p marginally improved the proliferative capac-ity of RS cells. Since knockdown of hsa-miR-299-3p couldonly marginally improve the proliferative capacity of RS cells,we investigated whether knockdown of hsa-miR-299-3p couldprotect cells from H2O2-induced senescence by treating thecells with H2O2 followed by transfection with hsa-miR-299-3pinhibitor A299-3p. Real-time growth kinetics analysis showedthat transfection with 62.5 and 125 nM of hsa-miR-299-3pinhibitor A299-3p resulted in significant increase in the prolif-eration of the transfected cells compared with those transfectedwith negative control inhibitor, CA (Fig. 7, B and C; P � 0.01and P � 0.05, Student’s t-test). This finding suggests thatinhibition of hsa-miR-299-3p can overcome H2O2-inducedsenescence and stimulate the proliferation of the senescingcells.

To evaluate the role of hsa-miR-299-3p in modulating cellmigration of RS cells, we performed cell migration assayfollowing knockdown of hsa-miR-299-3p (Fig. 8, A–E). TheCI of RS cells increased at 62.5 nM (Fig. 8B) and 125 nM (Fig.8C) of A299-3p, though not statistically significantly. Thisindicates that knockdown of hsa-miR-299-3p at 62.5 and 125nM slightly increases the cell migration capacity of RS cells.

DISCUSSION

The involvement of several miRNAs is established in theregulation of replicative senescence. These include hsa-miR-146a [targeting NOX4 (53)], hsa-miR-34a [acting on theSIRT1-p53 pathway (59)], and hsa-miR-17 [targeting the cdkinhibitor p21/CDKN1A (15)]. Because of the rapid expansionof the field of miRNAs and the complexity of the interaction ofindividual miRNAs with targeting several genes, there is aneed to delineate the role of specific miRNAs in replicativesenescence. Hence, in the present study we undertook anintegrated profiling analysis of miRNAs and genes in senescentHUVECs to identify other potential senescence-associatedmiRNAs. Changes in gene expression profiles associated withsenescence have been reported in several cell types (12, 32, 41,47). In the present study, integrative analysis of miRNA andgene expression profiling of RS in HUVECs suggests that 29miRNAs in RS may regulate at least 251 genes. In particular,the miRNA profiling results reveal that hsa-miR-299-3p isupregulated in RS HUVECs, and TargetScan analysis on gene

CA A299-3p

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expression profiles derived from the same RNA samples showsthat one of its potential targets is IGF1. Other highly deregu-lated miRNAs shown in the miRNA profile were not chosenfor further investigation because these have been reported fortheir association with senescence. For instance, Watanabe et al.showed that hsa-miR-150 induce senescence in NK/T-celllymphoma lines (57). hsa-miR-146a was reported to modulateendothelial cell aging via NOX4. (53). hsa-miR-299-3p waschosen for further investigation due to its relevant target genei.e., IGF1, which was reported to be highly associated withsenescence or cell proliferation and growth.

To evaluate the effect of inhibition of hsa-miR-299-3p onreplicative senescence, we used an SA-�-gal assay. SA-�-galactivity has been widely used as biomarker of senescence invitro and in vivo since its first description by Dimri et al. (10).It has been described as the “gold standard” marker for cellularsenescence, despite the fact that its function and origin re-mained unknown (9, 14, 38, 56). Studies have shown that theincrease in SA-�-gal activity in cellular senescence reflects anincrease in lysosomal mass in senescent cells and that SA-�-gal is not required for senescence (24, 26). The MTT assay is

commonly used to screen compounds for effects on viability,and reduction of MTT to formazan is due to cellular enzymaticactivity in mitochondria, endosomes, and lysosomes (5, 30).Factors in the cell culture environment such as pH and glucose,NADH, or NADPH supply also influence MTT reduction toformazan (17, 34), and thus MTT data also reflect changes incell metabolism. Knockdown of hsa-miR-299-3p reduced thepercentage of SA-�-gal-positive cells in the senesced cultureand increased cell viability as measured with MTT assay, butthe real-time growth kinetics data did not show a significantincrease in cell proliferation. As both SA-�-gal and MTT assayare dependent on cellular enzymatic activities that are reflec-tive of cell metabolism, it is suggestive that knockdown ofhsa-miR-299-3p may have improved the metabolic activity ofthe senescent cells, without stimulating cell proliferation andincrease cell numbers. Cell cycle analysis supports this obser-vation as inhibition of hsa-miR-299-3p did not stimulate theG0/G1 population to reenter the cell cycle (data not shown).However, we can ascertain the role of hsa-miR-299-3p inHUVEC senescence when knockdown of hsa-miR-299-3p iscapable of stimulating proliferation of cells treated with H2O2

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to induce senescence. The fact that inhibition of hsa-miR-299-3p can overcome H2O2-induced senescence and stimulatethe proliferation of the senescing cells suggests that hsa-miR-299-3p has a role in senescence and its inhibition can delaypremature senescence in HUVECs.

mRNA expression of IGF1 that was upregulated in RSHUVECs but not in their young counterpart was also elevatedfollowing the inhibition of hsa-miR-299-3p in senescent andpresenescent HUVECs. Moreover, in presenescent cells, adose-dependent increase in IGF-1 expression was observed atlow concentrations of anti-miR used, suggesting that hsa-miR-299-3p may, in part, mediate its actions through IGF1. Inaddition, knockdown in combination with two other miRNAs,i.e., hsa-miR-134 and -338-3p, that target IGF-1 further in-creased the elevation of IGF1 mRNA expression at low con-centrations of anti-miR used. IGF1 signaling has been de-scribed as an evolutionarily conserved mechanism of longevityin nematodes, fruit flies, yeast, rodents, and humans (2).However, a recent contrasting review suggests that IGF1 sig-

naling is not part of an evolutionarily conserved mechanism oflongevity in humans. Human lifespan does not increase corre-spondingly with a decline in IGF1 signaling, contrary toevidence found in nematodes, fruit flies, and rodents (reviewedin Ref. 49). IGF1 plays an essential role in stimulating DNAsynthesis, amino acid uptake, protein synthesis, and glucosetransport in various cells (2). It also acts as a growth factor inmany tissues and tumors (1). Excess of IGF1 predisposehumans to risk of neoplastic diseases (49). In contrast, IGF1deficiency increases the risk for cardiovascular complicationsin aged individuals, particularly atherosclerosis (18, 52). De-spite the fact that IGF1 has been largely associated with aging,the molecular mechanism underlying its action remains con-troversial. Senescent cells do not respond to IGF1 (in combi-nation with other growth factors) with DNA synthesis andmitosis, despite possessing a number of IGF1 receptors com-parable to that of young cells (45). Treatment with IGF1extends longevity in a mouse model of premature aging byrestoring somatotrophic function (33), and localized IGF1

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transgene expression sustains hypertrophy and regeneration insenescent skeletal muscle (37). There is limited information onthe role of miRNAs in modulating IGF1 and in turn regulatingsenescence. Thus far, it is only reported that miR-71 andmiR-239 promote longevity of Caenorhabditis elegans via theinsulin/IGF-1-like pathway (42). In addition, the findings of thepresent study indicate the potential of hsa-miR-299-3p inmodulating IGF1 and thus regulating senescence.

In terms of cell migration, although in general we do not seea significant improvement in cell migration following hsa-miR-299-3p knockdown, the knockdown improved migration ca-pacity. Previous reports demonstrate that IGF1 stimulatedendothelial cell migration (20, 29, 48). Those findings supportthe suggestion that cell migration improvement is due to theoptimal IGF1 expression modulated by hsa-miR-299-3p.

miRNA expression profiles have been demonstrated to behighly tissue, organ, disease, or developmental stage specific.Numerous studies have been performed to investigate theage-related alteration of miRNA signature in various tissues ororgans, aimed at identifying a unique miRNA signature that

could be useful for the development of tissue- or organ-specificbiomarkers, as well as therapeutic strategies for age-relatedcomplications (13, 28, 31, 43, 44, 58). The miRNA signatureobtained in the present study is in agreement with findingsfrom earlier studies. For instance, hsa-miR-134 (senescentfibroblast) (11), hsa-miR-17 (senescent human diploid fibro-blast, HUVEC, renal proximal tubular epithelial cells, T-cells,bone marrow derived mesenchymal stem cells, human fore-skin) (15), and hsa-miR-146a (senescent HUVEC) (53) werefound to be differentially expressed in RS HUVECs in thepresent study. The similarities suggest that those miRNAs arepotential biomarker of cellular senescence due to the highreproducibility across various independent studies. Further-more, the similarities strengthen the reliability of current find-ings. On the other hand, differences in miRNA expressionprofile between the current study and previous studies werealso noticed. hsa-miR-217 (35) and -34a (21), which werefound to be differentially expressed in senescent HUVECs,were not present in the current study. The discrepancies foundare most likely due to the HUVECs, which originate from

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different donors and possess different sensitivity to the detec-tion methods used.

In summary, the present study reveals that hsa-miR-299-3pplays a role in cellular senescence in HUVECs. Its ability todelay premature senescence is mediated, at least in part,through IGF1. Further investigations are necessary to confirmits role in IGF1 signaling and to determine whether and howhsa-miR-299-3p acts in tandem with other miRNAs identifiedby the present experiments.

GRANTS

This study was supported by University Malaya Research Grant (RG097/09HTM) and Postgraduate Research Fund (PV044/2011B).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: H.-L.J. performed experiments; H.-L.J. and P.-F.W.analyzed data; H.-L.J. and P.-F.W. interpreted results of experiments; H.-L.J.prepared figures; H.-L.J. drafted manuscript; H.-L.J., P.M.V., and P.-F.W.edited and revised manuscript; H.-L.J., M.R.M., P.M.V., S.A., and P.-F.W.approved final version of manuscript; M.R.M., S.A., and P.-F.W. conceptionand design of research.

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