RILES a novel method for temporal analysis of thein vivo regulation of miRNA expressionSafia Ezzine1 Georges Vassaux2 Bruno Pitard3 Benoit Barteau4 Jean-Marc Malinge1

Patrick Midoux1 Chantal Pichon1 and Patrick Baril1

1Centre de Biophysique Moleculaire CNRS UPR4301 Universite drsquoOrleans and Inserm Orleans France2UMRE 4320 Faculte de Medecine Universite de Nice-Sophia-Antipolis Nice France 3Inserm UMR1087CNRS UMR 6291 Universite de Nantes Faculte de medecine Lrsquoinstitut du Thorax Nantes F-44000 and4In-Cell-Art Nantes F44200 France

Received March 26 2013 Revised July 16 2013 Accepted August 13 2013

ABSTRACT

Novel methods are required to investigate thecomplexity of microRNA (miRNA) biology andparticularly their dynamic regulation under phys-iopathological conditions Herein a novel plasmid-based RNAi-Inducible Luciferase ExpressionSystem (RILES) was engineered to monitor theactivity of endogenous RNAi machinery WhenRILES is transfected in a target cell the miRNA ofinterest suppresses the expression of a transcrip-tional repressor and consequently switch-ON theexpression of the luciferase reporter gene HencemiRNA expression in cells is signed by theemission of bioluminescence signals that can bemonitored using standard bioluminescence equip-ment We validated this approach by monitoringin mice the expression of myomiRs-133 206and 1 in skeletal muscles and miRNA-122 in liverBioluminescence experiments demonstrated ro-bust qualitative and quantitative data that correl-ate with the miRNA expression pattern detectedby quantitative RT-PCR (qPCR) We furtherdemonstrated that the regulation of miRNA-206 ex-pression during the development of muscularatrophy is individual-dependent time-regulatedand more complex than the information generatedby qPCR As RILES is simple and versatile webelieve that this methodology will contribute toa better understanding of miRNA biology andcould serve as a rationale for the developmentof a novel generation of regulatable gene ex-pression systems with potential therapeuticapplications

INTRODUCTION

MicroRNAs (miRNAs) are a class of endogenousnoncoding RNAs 18ndash25 nt in length that posttranscrip-tionally regulate the expression of eukaryotic genes in asequence-specific manner miRNAs act by bindingto mRNA targets preferentially to the 30-untranslatedregion (30UTR) by a base-pairing mechanismDepending on the degree of complementarity miRNAseither inhibit translation or induce degradation of thetarget mRNA (1) To date gt1000 miRNAs have beenidentified in the human genome and they are predictedto regulate 60 of the whole transcriptome (2)MiRNAs are implicated in most if not all cellularprocesses from proliferation apoptosis and differenti-ation to hematopoiesis developmental timing and or-ganogenesis (1) Therefore it is not surprising thatderegulation of miRNAs has also been associated with anumber of diseases and that RNAi-based therapeuticagents show promise as therapeutic drugs (3)Current methods used to determine the expression of

miRNAs have strongly impacted our knowledge of thebiological roles that miRNAs play under physiologicaland pathophysiological conditions While the datagenerated cannot be disputed they lack spatial andmore importantly temporal resolution Methods such asPCR(-based approaches) microarrays northern blot andELISA are fully invasive and require complex tissuesampling and processing (45) making these proceduresunsuitable for monitoring miRNA regulation during lon-gitudinal studies This is particularly problematic asmiRNAs are spatiotemporally regulated and subject toconsiderable interindividual variation (67) This sourceof complexity is even more pronounced when the expres-sion of miRNAs needs to be investigated at the whole-organism level For instance it is well established thatmiRNAs are finely regulated during embryonic

To whom correspondence should be addressed Tel +33 2 38 25 56 40 Fax +33 2 38 63 15 17 Email patrickbarilcnrs-orleansfr

The authors wish it to be known that in their opinion the first two authors should be regarded as joint First Authors

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The Author(s) 2013 Published by Oxford University PressThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (httpcreativecommonsorglicensesby30) whichpermits unrestricted reuse distribution and reproduction in any medium provided the original work is properly cited

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development and control complex regulatory networks ofgene expression involved in cell-lineage decisions and sub-sequently morphogenesis (8ndash10) Similarly in cancersome miRNAs are implicated in the early phases oftumor development while they can at later stagesinhibit the formation of metastases (1112) Thereforethe average measurement of miRNAs from a heteroge-neous population at a specific time point underestimatesthe biological relevance of the time-dependent nature ofmiRNA regulation as well as the heterogeneity of miRNAexpression at the individual level Consequently thesedata could result in the loss of important information con-necting miRNA expression and cell function Addressingthese limitations can impact directly on basic and thera-peutic research fields Noninvasive molecular imagingmethods have the potential to overcome these limitations(13) and to provide an alternative method to studymiRNA expression under physiological conditions (14)However the monitoring of miRNAs in real time in acomplex organism is challenging primarily owing to theshort length of miRNAs This could explain the limitednumber of reports in the literature The first reportedmethod (1516) is based on the use of the luciferasereporter gene carrying complementary block sequencesto a specific miRNA in the 30UTR of the luciferasegene Therefore when a miRNA of interest is expressedin the cell it binds to the luciferase transcript and inhibitsthe production of luciferase In this way miRNA expres-sion in cells is signed by a decrease in the bioluminescencesignal (Off-System) However such a lsquonegativersquo imagingmodality is not adequate as the loss of the biolumines-cence signal may reflect nonspecific regulations of theluciferase promoter or even cell death More recentlypositive molecular imaging systems (ON-systems) havebeen developed to overcome this limitation Some ofthese systems are based on the use of oligonucleotide mo-lecular beacons labelled both with a fluorophore at oneend and a quencher at the other end (16ndash22) In presenceof a specific miRNA the stem-loop structure of thebeacons is linearized separating the fluorophore fromthe quencher As a result the fluorescence signal emittedin cells was found to be proportional to the concentrationof miRNAs While this advance represents a more rationaland in vitro fully validated approach (18) this method-ology does have limitations principally the weak sensitiv-ity of fluorogenic probes in small animals and therestricted application to miRNA analysis expressed fromcells implanted in vivo (202123) Moreover majorcomplex normalization procedures are required becauseof the necessity of repeating administration of theprobes in the course of longitudinal studiesWe developed a RNAi-Inducible Luciferase Expression

System (RILES) with the aim of generating positive bio-luminescence signals in mice that will allow the qualitativeand quantitative measurement of endogenous expressedmiRNAs with sufficient sensitivity to monitor thedynamic regulation of miRNA expression during thedevelopment of a chronic disease For this purpose wecustomized the recently characterized Cumate gene-switch inducible expression system (24) This system likeother repressor-based inducible expression systems

(2526) uses an inducer that when bound to a repressorprotein changes its conformation impeding the bindingto an operator sequence located downstream to the trans-lation start codon within a constitutive promoterConsequently the expression of the transgene isswitched-ON by the presence of the exogenous inducerWe reasoned that placing expression of the repressormolecule directly under the control of the endogenousRNAi machinery rather than an exogenous moleculewould be an alternative way to switch-ON expression ofthe transgene Consequently if the luciferase reporter geneis used as a transgene the system will generate biolumin-escence signals that will qualitatively and quantitativelyreflect the expression pattern of miRNAs

Here we report a complete proof of principle study anddemonstrate that RILES provides specific and relevantbiological information about the expression pattern andthe temporal regulation of endogenous miRNA underphysiological and pathological conditions

MATERIALS AND METHODS

Plasmid construction

For the construction of RILES plasmids we first restric-tion-digested and subcloned the firefly luciferase cDNAinto the pCMV5Cuo plasmid (CumateTM inducible ex-pression plasmid Qbiogene CA USA) CymR cDNAfrom the pSV40CymR plasmid (Qbiogene) was thenPCR amplified subcloned into pcDNA3Topo TA(Invitrogen) and its functionality was tested on transfec-tion of HEK 293 cells cultured in the presence of cumateas a gene-switch agent (24) (data not shown) The puro-mycin cDNA fragment driven by the SV40 promoter wasremoved from the pCMV5CuoFluc plasmid (Qbiogene)and replaced by CymR cDNA excised from pCMVTopoCymR A multiple cloning site containing Mlu I and Not Irestriction sites was inserted into the 30UTR of CymRcDNA before the polyadenylation site The sequenceand the functionality of the expression plasmid generatedwere verified and tested functionally (24) (data notshown) To place the expression plasmid under controlof a specific miRNA or a siRNA we designed a 120-bpdouble-stranded oligonucleotide containing four blocks ofcomplementary sequences to the miRNA or the siRNAThe oligonucleotides were flanked at the 30-end by a com-patible overhanging phosphorylated Not I sequenceand at the 50-end by a compatible overhangingphosphorylated Nhe I sequence The upper single-stranded oligonucleotide and the lower single-strandedoligonucleotide were synthesized separately (EurogentecSeraing Belgium) annealed at 94C for 5min and then at37C for 60min The annealed oligonucleotides wereligated to the purified expression plasmid previouslydouble-digested with Not I and Mlu I All the vectorsgenerated (Supplementary Table S1) were sequenced andamplified using Endofree plasmid kits (Qiagen)

Cell lines reagents and transfection

HEK 293 C2C12 and HuH7 cell lines were obtained fromATCC The HLE cell line was kindly provided by Pascal

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Pineau (Institut Pasteur Paris France) Cells werecultured in 4 gl Dulbeccorsquos modified Eaglersquos medium sup-plemented with 10 SVF and with penicillin and strepto-mycin C2C12 cells were differentiated into myoblasts byculturing subconfluent cell monolayers in 2 horse serumfor 4 days Synthetic precursor miRNAs were obtainedfrom Life technology pU6shRNA turbo GFP and thepU6shRNA control plasmids were obtained fromSigma and the pQE30 expression plasmid was fromQiagen Icafectin 441 (Eurogentec) and His-lPEI (27)were used as in vitro transfection reagents For transfec-tion 1 105 cellswell in 24-well plates were transfectedwith 2 mgwell of plasmid DNA and synthetic precursormiRNA (Invitrogen) The pQE30 empty expressionplasmid was used to normalize transfection conditionsRelative luciferase units (RLU) were determined 48(HEK 293 cells) and 72 h (HuH7 HLE differentiatedC2C12) after transfection using a luminometer(Berthold) Luciferase activities were normalized toprotein content (RLUmg protein) and expressed as foldinduction relative to control cells transfected with the ex-pression plasmid alone set to the arbitral value of 1

Immunohistochemistry

Immunodetection of luciferase protein in muscle tissueswas performed using a specific luciferase antibody(Promega) A solution composed of 10mM TrisEDTApH 9 was used to unmask antigen sites from paraffin-embedded tissues

Quantitative reverse transcriptase-polymerase chainreaction and genomic polymerase chain reaction

Extraction of total RNA was performed by adding 120(wvol) volume of lysis binding buffer (mirVanamicroRNA isolation kit Ambion) followed by tissuehomogenization and gridding using CKMix ceramic-bead tubes (Ozyme Paris France) and the Precelyss 24Unit (Precelyss Bertin France) RNA integrity wasdetermined by calculating the RNA integrity numberusing a BioAnalyzer 2100 (Agilent technologies)Samples with an RNA integrity number superior orequal to 8 were considered for further analysis FormiRNA analysis cDNA was synthesized using theNCode VILO miRNA cDNA synthesis kit according tothe manufacturerrsquos instructions (Invitrogen) This stepadds a polyadenylate tail to the miRNA populationwithin the total RNA samples For mRNA analysis100 ng of total RNA samples was also used but re-verse transcripted using the SuperScript II ReverseTranscriptase Kit (Invitrogen) as previously described(28) The real time quantitative PCR products weregenerated from 50 ng of cDNA template (used in tripli-cate) with QuantiFast SYBR Green master mix (Qiagen)with specific forward and reverse primers of the gene ofinterest for mRNA analysis and with a mix of forwardspecific primers of the mature miRNA and reverse univer-sal qPCR reverse primer provided by the NCode VILOmiRNA cDNA synthesis kit (Invitrogen) The primers arelisted on Supplementary Table S1 The specificity of thePCR amplicon (size and product) and absence of primer-

dimer were verified by melt-curve analysis using BioRadCFX manager software (Biorad) PCR conditions were asfollows 1 cycle of 95C for 3min followed by 40 cycles of95C for 10 s and 60C for 60 s with a final melt curveanalysis step (heating the PCR mixture from 65 to 95C by05C every 5 s) Samples were normalized to the 6 SrRNA level for quantification of the mRNA transcriptand with the snU6 level for quantification of maturemiRNA Finally the relative levels of expression ofmiRNA and mRNA were determined using the 2Ct

method To quantify plasmid DNA content in tissuesabsolute quantitative genomic PCR was performed as pre-viously described (28) Briefly 50 ng of extracted genomicDNA samples were used in each quantitative PCRreaction performed in triplicate using CymR primersThe absolute value of RILES plasmids DNA amount intissues was determined using a standard curve performedwith 5 ml of several dilutions of known concentrations ofRILES plasmids in presence of CymR primers

Animal experiments

Animal housing and procedures were carried out accord-ing to the guidelines of the French Ministry of Agriculturefor experiments with laboratory animals (Law 87848 CPichon accreditation) Female 8-week-old outbred Swissmice (BALBc genetic background) and athymicnude mice were obtained from Harlam (France)Intramuscular injections of expression plasmids were per-formed as previously described 8 mg expression plasmid or2 mg inducible expression plasmid plus 6 mg pQE30plasmid formulated with the amphiphilic block copolymer704 (29) were administered into the tibialis anteriormuscles of mice Hydrodynamic injections were preparedin a saline physiological buffer corresponding to 10body volume of the mouse and were administered over a5-s period into the tail vein of mice (30) Atrophy wasinduced in female nude mice (Harlam) aged 8 weeks bysciatic nerve transaction as previously described (31) Theanimals were isofluorane-anesthetized and the lefthindlimb (at the level of the femur) of the mice wasexposed before making a small incision to isolate thesciatic nerve Then a segment of 5mm in length of thesciatic nerve was cut and carefully removed Muscle andskin incisions were subsequently closed using 4-0 sutures

Bioluminescence imaging

Bioluminescence imaging was performed using either theNightOWL I LB (Berthold Bad Wildbad Germany) orthe IVIS Lumina II (PerkinElmer) imaging scannercoupled to the Indigo Software (Berthold) or the LivingImage Software (PerkinElmer) respectively Briefly 2mgof in vivo luciferase substrate (beetle luciferin substratePromega) were injected intraperitoneally in each mouseFive minutes later the mice were isofluorane-anesthetizedand scanned The abdominal cavity and the lower legs ofmice were shaved once a week to allow accurate collectionof bioluminescence signals Light emissions werequantified from regions of interest (ROI) drawnmanually and quantified using the imaging softwareThe sensitivity of the imaging scanner was tested

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weekly with commercially available positive sources ofbioluminescence

Statistical analysis

Statistical analysis was performed using Prism (GraphPadsoftware) Dual comparisons were made by the two-tailedstudent t-test Plt 005 Plt 001 were considered assignificant

RESULTS

Molecular construct and design

We constructed a Cumate gene-switch expression systemby assembling into a single plasmid unit the expressioncassette encoding for the CymR repressor driven by theSV40 promoter and the inducible expression cassetteencoding for the luciferase gene reporter driven by theCMV5(Cuo) inducible promoter (Figure 1) This dual ex-pression system was called RNAi-Inducible LuciferaseExpression System and denoted RILES A multiplecloning site was then subcloned in the 30 location of theCymR repressor cDNA to insert on-demand a block offour perfect-match complementary sequences to RNAimolecules such as siRNA or miRNA The system isdesigned in such a way that it is the RNAi moleculethat induces the expression of the luciferase gene For

instance if a miRNA of interest is not expressed in thecell the CymR transcript is produced as well as the CymRrepressor protein Consequently the repressor proteinbinds to the Cuo operator and blocks transcription ofthe luciferase gene Under this configuration RILES isswitched-OFF and no bioluminescence activity isexpected In contrast if the miRNA of interest is ex-pressed in the cell it binds to the 30UTR region of theCymR transcript resulting in activation of the endogen-ous RNAi machinery Consequently no CymR repressorprotein is produced enabling the luciferase gene reporterto be transcribed Therefore under this configuration theRILES system is switched-ON and bioluminescenceactivity is detected

In vitro validation studies

We first performed a series of in vitro proof of principleexperiments to evaluate the specificity and the sensitivityof RILES in response to exogenous expressed RNAi mol-ecules such as siRNA molecules encoded by a shRNAplasmid and synthetic miRNAs Several RILES plasmidswere constructed (Supplementary Table 1) and denotedfor example pRILESsiRNA tGFPT or pRILES122Twhen the RNAi targeting cassette contained complemen-tary sequences to detect the siRNA tGFP or miRNA-122respectively These two RILES plasmids pRILESsiRNAtGFPT and pRILES122T were individually transfected

Figure 1 Schematic representation of the RILES method When present in cells target miRNA or siRNA binds to the four complementary-blocksequences located in the 30UTR of the CymR repressor transcript and activates the RNAi silencing complex (RISC) machinery The CymR mRNA isthen cleaved and degraded resulting in lack of repressor production The luciferase expression system is thus switched-ON generating a positivebioluminescence signal

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in HEK 293 cells in presence of increasing amounts of theshRNA plasmid (Figure 2A) or synthetic precursormiRNA-122 (Figure 2B) Forty-eight hours later theluciferase activity in cells was determined and expressedas relative fold of luciferase induction by normalizing thevalues to cells transfected with the pRILES plasmid aloneAs shown in Figure 2A and B increasing the amount ofshRNA tGFP plasmid (Figure 2A) or synthetic miRNA-122 (Figure 2B) in pRILES transfected cells also increasedthe luciferase fold induction values A maximum 9-fold(plusmn03 n=3 P 001) luciferase induction was found inresponse to 50 ng shRNA tGFP plasmid (Figure 2A) anda maximum of 26-fold (plusmn12 n=3 P 001) wasdetected in response to 40 nM of miRNA-122 (Figure2B) In the latter the luciferase fold induction wasfound well correlated (R2=09321) with theconcentration of miRNA ranging from 0 to 20 nMOverall our experiments indicated that RILES is able todetect 1 nM of synthetic precursor miRNA molecules intransfected cells this may represent the detection limit ofRILES It is worth noting that when the second gener-ation of synthetic miRNA molecules (miR Mimics) wasused the detection limit of RILES was as low as 03 nM

(data not shown) To assess the specificity of RILES theexperiments were conducted with a control mismatchshRNA plasmid (Figure 2A) or with irrelevant miRNAs(Figure 2CndashE) No significant luciferase induction wasdetected in these assays In contrast luciferase inductionwas detected only in cells transfected with pRILES122TpRILES133T and pRILES221T in presence of the cor-responding miRNA-122 miRNA-133 and miRNA-221(Figure 2CndashE) We also found that three different syn-thetic miRNAs investigated at the same concentrationwere all equally efficient in switching-ON the configur-ation of RILES and inducing the same level of luciferasegene expression (data not shown) We finally assessedwhether RILES could distinguish two closely relatedmiRNA sequences that differ by two nucleotides such astwo members of the miRNA-200 family Results indicatedthat the pRILES200cT did not distinguish these twomiRNA sequences (Supplementary Figure S1) Next wewanted to determine whether RILES could also monitorthe expression pattern of endogenous expressed miRNAsfrom established cell lines For this purpose we exploitedthe fact that HUH7 and HLE cell lines express oppositelevels of miRNA-122 (32) and miRNA-221 (33) These cell

Figure 2 Luciferase expression in HEK 293 cells transfected with several RILES plasmids Dosendashresponse study of luciferase expression in HEK 293cells transfected with (A) pRILESsiRNA tGFP T or (B) pRILES122T in presence of (A) increasing amounts of siRNA tGFP (pU6shRNA tGFP)and control siRNA (pU6shRNA Ctl) or (B) increasing concentrations of synthetic miRNA-122 Selective luciferase expression in HEK 293 cellstransfected either with (C) pRILES122T (D) pRILES133T or (E) pRILES221T in the presence of two concentrations of synthetic miRNA-122133 and 221 Forty-eight hours after transfection luciferase expression in cells was determined and expressed as fold induction relative to controlcells transfected with the plasmids alone and set to the arbitral value of 1 Data shown are the mean plusmnSD of one representative experimentperformed in triplicate and reproduced at least three times Statistics by the two-tailed t-test Plt 005 Plt 001 ns (no statistically significantdifference) compared to control cells

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lines were transfected individually with the pRILES122Tand pRILES221T and luciferase induction wasdetermined 3 days later by normalizing the luciferasevalues to those found in cells transfected with thecontrol untargeted miRNA RILES plasmid (pRILES)As shown in Supplementary Figure S2 the luciferase in-duction pattern detected in these cells was found to beremarkably similar to the miRNA expression patternmeasured by quantitative RT-PCR MiRNA-122 andmiRNA-221 were oppositely expressed in HUH7 andHLE cells while miRNA-133 was not significantlydetected Similar specificity of data was also found inC2C12 myoblast cells differentiated in myotubes in vitroto induce expression of the muscle-specific miRNA-133(34) Again data from quantitative RT-PCR and bio-luminescence analysis indicated a similar miRNA expres-sion pattern ie expression of miRNA-133 and almostundetectable expression of miRNA-122 and -221 indifferentiated C2C12 cells (Supplementary Figure S2)Remarkably RILES was also found to be functional inprimary hard-to-transfect cells The luciferase fold induc-tion in primary culture of human dermal fibroblastNHDF was similar to the endogenous expressionpattern of miRNA detected by quantitative PCR (datanot shown)

In vivo monitoring of miRNA-122 expression in the liver

We then examined whether similar data could be collectedthrough whole-body imaging of live-anesthetized animalsand in real time As miRNA-122 is exclusively expressedin the liver (32) and miRNA-133 is a muscle-tissuendashspecificmiRNA (34) we enquired whether RILES would have thepotential to discriminate the expression of these twomiRNAs in the liver of the mice To transfect the liverwe hydrodynamically injected (30) pRILES122T andpRILES133T and as control the untargeted miRNARILES plasmid (pRILES) Three days after administra-tion the mice were placed under a bioluminescencescanner to detect expression of the luciferase reportergene As shown in Figure 3A a strong bioluminescentsignal was detected in the abdominal area of thepRILES122T-treated group of mice compared with thebioluminescent signals collected in the pRILES133T- andthe pRILES-treated groups which both generated weaksignals The autopsy of one representative mouse pergroup of animals indicated that the liver was the sourceof emitted light (Figure 3B) Quantitative analysis showeda statistically significant 85-fold (plusmn15 n=3 P 001)increase in luciferase activity in the liver of mice adminis-tered with pRILES122T compared with the luciferaseactivity detected in the pRILES control group andnormalized to the arbitral value of 1 (plusmn05 n=3)(Figure 3C) No statistical difference (Pgt 005) wasfound between the pRILES133T and pRILES groupsindicating as expected that miRNA-133 was not ex-pressed in the liver To assess the specificity of our datawe compared the pattern of bioluminescence signalsdetected in mice with the expression pattern of miRNAdetected by quantitative RT-PCR Results indicated thatthese two methods generated similar data (Figure 3D

versus 3C) Quantitative RT-PCR demonstrated thatmiRNA-122 is expressed in the liver in contrast tomiRNA-133 which was almost undetectable in the liversamples To demonstrate that the bioluminescence signaldid not arise from an unspecific effect or from an uncon-trolled dose of administration of the RILES plasmids aquantitative genomic PCR was conducted to measure theabsolute amount of RILES plasmids present in the liverNo statistically significant difference (Pgt 005) was foundbetween the three groups of animals (Figure 3E)demonstrating that the bioluminescence signals aroserather from activation of the endogenous RNAi machin-ery To fully validate this point we conducted a relativequantitative RT-PCR analysis on the same tissue samplesto evaluate the expression level of the CymR repressortranscript The CymR transcript was found to be signifi-cantly 12-fold downregulated (plusmn18 n=6 P 001) inthe liver tissues of the pRILES122T-treated groupcompared with the pRILES control group (Figure 3F)A significant but less pronounced 2-fold (plusmn16 n=6Plt 005) downregulation of cymR transcript was foundin the liver tissues of the pRILES133T-treated groupwhereas this group did not generate significant biolumin-escence signals (Figure 3F versus 3C) These data indicatethat the expression of miRNA-133 in the liver is not suf-ficient to repress a sufficient amount of CymR transcriptto switch-ON the RILES in the liver of the mice

In vivo monitoring of myomiRNA expression in the tibialisanterior skeletal muscles

Next we wanted to evaluate the quantitative potential ofRILES We thus attempted to monitor the expressionpattern of miRNA-1 miRNA-133 and miRNA-206 inthe skeletal muscles of the anterior tibialis of the miceThese muscle-specific miRNAs and known to be differen-tially expressed in adult skeletal muscle tissue (34) Totransfect the skeletal muscles efficiently the RILESplasmids were formulated with the amphiphilic block co-polymer 704 (704) as described in (29) and then intramus-cularly administered in the two tibialis anterior muscles ofnaive mice Six days later (Figure 4A) strong biolumines-cence signals were detected in the lower legs of mice ad-ministered with pRILES bearing the myomiRNAtargeting cassette 1T -133T and -206T In contrast lowalmost undetectable bioluminescence signals were detectedin the pRILES and pRILES122T groups Quantificationof the bioluminescence signals indicated that the inductionindex of luciferase expression in the pRILES1T-pRILES133T- and pRILES206T-treated groups was re-spectively 37- (plusmn44 n=6 Plt 001) 33- (plusmn35 n=6Plt 001) and 17- (plusmn42 n=6 Plt 001) fold higher thanthe bioluminescence signal detected in the pRILES controlgroup and normalized to the arbitral value of 1 (plusmn04n=6) (Figure 4B) No statistical difference was foundbetween the pRILES122T and the pRILES group sug-gesting that in contrast to the liver miRNA-122 is notexpressed in the skeletal muscles We then compareddata generated using our method with those detectedusing quantitative RT-PCR Remarkably both methodsprovided similar data and conclusions (Figure 4B and C)

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MiRNA-122 was only faintly detected in the skeletalmuscle while miRNA-206 -133 and -1 showed anincreasing expression level (Figure 4C versus 4B) Theautopsy of one representative mouse per group indicatedthat all skeletal muscle tissues received the same amountof plasmids (Figure 4D) eliminating again the possibilitythat signals in mice could arise from a nonreproducibleadministration procedure of the plasmids in vivoImmunohistochemical analysis indicated that 80 of theskeletal fibers of the pRILES1T anterior tibialis tissueswere stained by the luciferase antibody with an expectedrestricted localization to the skeletal muscle cells which isin line with the unique cellular source of miRNA-1 expres-sion in the anterior tibialis (Supplementary Figure S3)whereas no significant staining was detected in the

pRILES and pRILES122T muscle tissues We alsoexamined the expression level of CymR repressor tran-script by quantitative RT-PCR We found a significantreduction of CymR transcript in all skeletal muscletissues investigated that was inversely correlated with thebioluminescence values (Figure 4E versus 4B) Indeed amean of 32-fold (plusmn25 n=3 Plt 001) of CymRdownregulation was found in the pRILES1T skeletalmuscle tissues which generated 37-fold (plusmn44) more bio-luminescence than pRILES control mice A mean of 28-fold (plusmn25 n=3 Plt 001) of CymR downregulation wasfound in the RILES133T skeletal muscle tissues whichgenerated 33-fold (plusmn35 n=6) more bioluminescencethan the pRILES control mice Remarkably an inter-mediate mean of 12-fold (plusmn19 n=3 Plt 005) of

Figure 3 Noninvasive bioluminescence imaging of the liver-specific miRNA-122 in mice Fifty micrograms of pRILES122T and pRILES133T werehydrodynamically injected in Swiss mice to transfect the liver Negative control included the pRILES not regulated by miRNA Bioluminescenceimaging was performed 3 days later and light emission quantified using ROIs covering (A) the whole abdominal cavity of the mice or (B) the liver ofone representative mouse per group (C) Quantitative bioluminescence values detected in mice described in A and expressed as luciferase inductionrelative to the control pRILES group of animals set arbitrarily to the value of 1 (D) Quantitative RT-PCR analysis of miRNA expression in the livertissues of another group of mice (E) Absolute quantification of plasmid content in the liver tissues of the mice described in B (F) Quantitative RT-PCR analysis of CymR expression in the liver tissues of the mice described in B Results are expressed as CymR fold change relative to controlpRILES-tissues set arbitrarily to the value of 1 Error bars in C meanplusmnSEM (n=6) of one representative experiment repeated two times Errorbars in D E F meanplusmnSD (n=3) of one representative experiment repeated at least three times Statistics by the two-tailed t-test Plt 005Plt 001 ns (no statistically significant difference) compared with the pRILES control group

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CymR downregulation was found in the pRILES206Tskeletal muscle tissues which generated an intermediate17-fold (plusmn42 n=6) bioluminescence signal in micecompared with pRILES control mice (Figure 4E versus4B) We also found a significant but lower 6-fold (plusmn13n=6 Plt 005) degradation of CymR transcript in thepRILES122T-treated group although miRNA-122 isweakly expressed in the skeletal muscles (Figure 4C) andunable to induce significant bioluminescence (Figure 4B)This indicated that the expression of miRNA-122 in theskeletal muscle is not sufficient to repress a sufficientamount of CymR transcript to switch-ON the RILESin vivo

Kinetics of miRNA expression in immunocompetent andimmunodeficient mice

As miRNA-133 is constitutively expressed in the adultstage of skeletal muscles (34) we conducted a biolumines-cence kinetic analysis of miRNA-133 expression in thetibialis anterior muscle of the mice pRILES133T withpRILES122T and pRILES as controls was formulatedwith the 704 copolymer and then intramuscularly injectedin the tibialis anterior muscle of mice The biolumines-cence signals emitted from the lower legs of animalswere collected at several time points and expressed asthe mean of bioluminescence as a function of time (day)

Figure 4 Noninvasive bioluminescence imaging of the muscle-specific myomiRs-206 133 and 1 in mice Eight micrograms of pRILES122TpRILES133T pRILES1T and pRILES206T were formulated with the 704 amphiphilic block copolymer and intramuscularly injected in the leftand right tibialis anterior to transfect the skeletal muscles of Swiss mice Negative control included the pRILES not regulated by miRNABioluminescence imaging was performed 6 days later and light emission quantified using ROIs covering the lower legs of the mice (A)Representative bioluminescence images collected in the left lower legs of mice (B) Quantitative bioluminescence values detected in the mice describedin A and expressed as luciferase induction relative to the control pRILES group of animals set arbitrarily to the value of 1 (C) Quantitative RT-PCRanalysis of myomiR expression detected in the tibialis anterior muscles of another group of mice (D) Absolute quantification of plasmid content inthe skeletal muscle of the mice described in A (E) Quantitative RT-PCR analysis of CymR expression in the skeletal muscle of one representativescanned mouse described in A Results are expressed as CymR fold change relative to control pRILES-tissues set arbitrarily to the value of 1 Errorbars in B meanplusmnSEM (n=6) of one representative experiment repeated at least three times Error bars in C D E meanplusmnSD (n=3) of onerepresentative experiment repeated at least three times Statistics by the two-tailed t-test Plt 005 Plt 001 ns (no statistically significant differ-ence) compared with the pRILES control group

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As shown in Supplementary Figure S4 a peak of bio-luminescence was detected for each group of mice 3 daysafter the intramuscular administration Then a stable andconstant bioluminescence signal ranging from day 5 today 15 was observed irrespective of the group of miceAt this plateau a statistically significant difference(Plt 001) in luciferase expression was detected in thepRILES133T versus pRILES122T and pRILES groupsof mice However after day 15 bioluminescence signals inthe pRILES133T group rapidly decreased to finallybecome undetectable at day 20 To determine whetherthis loss of bioluminescence signal may result from anonspecific regulation of the inducible promoter we re-administered the same amount of RILES derivatedplasmids and collected the bioluminescence signals subse-quently The bioluminescence signals in mice were lowerthan those obtained after the first injection and were notstatistically different between the various groups of mice(Supplementary Figure S4) We conducted similar experi-ments in immunodeficient mice to determine whether theloss of luciferase expression was finally related to the de-velopment of an immune response directed against eitheror both the bacterial origin of the CymR repressor or theluciferase protein In these mice we found (Figure 5) thatthe bioluminescence signals detected at the plateau of ex-pression persisted over time and were stable for at least 34days (end point of our experiment) These data indicatethat using RILES the expression of miRNAs can bemonitored for a long period in immunodeficient mice

Dynamic monitoring of miRNA expression during atrophydevelopment in immunodeficient mice

To fully validate the potential value ofrsquo RILES andpropose a relevant application in the field of miRNAbiology we examined whether the system may besuitable to monitor the dynamic regulation of miRNAsunder pathophysiological conditions To assess thispoint we used a muscular atrophy model obtained bycomplete section of the sciatic nerve of the miceRecently miRNAs were found or alleged to be involvedin response to this injury (35) In a pilot experiment studywe found that among the three different myomiRsinvestigated by in vivo bioluminescence imagingmiRNA-206 was the most strongly regulated miRNA(data not shown) To first evaluate the regulation ofluciferase expression during development of this chronicdisease characterized by significant metabolic changes(36ndash38) we injected in the tibialis anterior of the micean expression plasmid containing the second expressioncassette of RILES plasmid (CMV5CuoF-luc pRILESFluc) for constitutive expression of the luciferase genereporter in the denervated skeletal muscle As expectedthe bioluminescence signals emitted from the control not-atrophied-leg injected with pRILESF-Luc were stablefor at least 35 days (Supplementary Figure S5A red lineand S5B control) whereas results obtained from thepRILESFluc-denervated skeletal muscle resulted in arapid loss (within 3 days) of the bioluminescence signal(Supplementary Figure S5A blue line and S5B section)From day 7 the signal remained almost stable for 35 days

This change in luciferase expression was paralleled by therate of muscle-weight loss (Supplementary Figure S5C)and may be a consequence of a specific transcriptionalprogram governing the expression of ubiquitin ligases asdescribed in (3637) On this basis we decided to deter-mine the kinetic of miRNA-206 expression during thesecond phase of this chronic muscular disease known asthe adaptive response phase (37) pRILES206T andpRILES were injected intramuscularly in the tibialisanterior muscle of mice and 3 days later the sciaticnerve of the left leg was surgically sectioned The micewere thereafter imaged twice a week for the first 2 weeksand then once a week for 3 weeks Bioluminescenceimaging of the atrophied legs of mice administered withthe control untargeted miRNA pRILES showed a lowand stable level of luciferase expression (mean of312 106 photonss plusmn463 103 n=5) during thewhole kinetic course of the study (Supplementary FigureS6A) Quantitative analysis of pooled data from fiveanimals administered with the pRILES206T showed amuch higher luciferase activity (mean of 426 107

photonss plusmn93 106 n=5) which decreased 2-fold im-mediately after nerve section probably in relation with theoverall loss of metabolic activity documented inSupplementary Figure S5 Then the luciferase activityreached a first plateau from days 3 to 7 and then increasedquickly and peaked at day 13 before finally dropping atday 22 to the value of day 6 until day 30 In sharpcontrast the luciferase activity in the nonatrophied legintramuscularly injected with the pRILES206T plasmidwas constant during the whole course of the kineticanalysis (data not shown) In a separate experimentmiRNA-206 expression was determined by quantitativeRT-PCR at days 6 (n=5) 12 (n=5) 19 (n=5) and 27(n=5) after muscle denervation Quantitative PCR datashowed a similar pattern of miRNA-206 expression asdetected by bioluminescence imaging (SupplementaryFigure S6A) characterized by an increase of miRNA-206expression detected from day 12 to day 19 and a decreaseby day 27 after denervation (Supplementary Figure S6B)Both bioluminescence and quantitative RT-PCR datashowed strong variability and the only statistical signifi-cance detected by quantitative RT-PCR data was foundby comparing day 6 with day 12 and day 1219 with day27 We then fully exploited the advantage of the noninva-sive nature of the RILES method to determine the sourceof variability in each individual mouse As shown inFigure 6 a strong heterogeneity in the level of miRNA-206 expression was observed between the mice althoughthe kinetic of miRNA-206 expression exhibited a bell-shaped aspect with a maximum bioluminescence valuebordered by two minimal values for all mice We foundthat expression of miRNA-206 peaked at three differenttime points either at day 13 (3 out of 5 mice) or day 10(1 out of 5 mice) or even later at day 18 (1 out of 5 mice)(Figure 6A) To quantify the fold induction change inmiRNA-206 expression we normalized the biolumines-cence values monitored for each mouse to the minimalvalue detected at day 6 after denervation and set to thearbitral value of 1 Data from quantitative PCR (supple-mentary Figure S6B) and in vivo bioluminescence

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experiments (Figure 6A) indicated that at day 6 the ex-pression of miRNA-206 was indeed at its lowest valueNormalized data indicated that the luciferase inductionof miRNA-206 regulation ranged heterogeneously froma minimal value of 2-fold to a maximum value of 17-fold The amplitude of miRNA-206 regulation was alsofound to be dependent on the mouse extending from aminimum period of 4 days to a maximum period of 20days Similar trends of miRNA-206 expression were foundin other sets of experiments performed We invariablydetected a major peak of miRNA-206 expression at day13 (3 out of 5 mice) and two additional peaks detected atday 10 (1 out of 5 mice) and 16 (1 out of 5 mice) (data notshown) In addition the miRNA-206 induction level aswell as the amplitude of expression was variable betweenthe mice ranging from 2- to 12-fold induction and to 4ndash16days of time of regulation (data not shown) Altogetherthese data demonstrate the great potential of the RILESmethod to generate relevant information about miRNAregulation and emphasize the importance of the temporaldimension of miRNA analysis during the development ofbiological processes

DISCUSSION

Here we described a novel method RILES to monitor inreal time and at the whole-body-scale of live animals thedynamic expression pattern of miRNAs under bothphysiological and physiopathological conditions Using

RILES we established for the first time in mice the ex-pression kinetic of a miRNA in an animal disease modelduring a period of 30 days Our strategy is based on theuse of the cumate gene-switch system (24) in which theCuO an operator DNA-binding sequence of the CymRprotein repressor is positioned between the luciferasereporter gene and its promoter Therefore when themiRNA is functional the CymR repressor is no longerproduced and the luciferase reporter gene is then ex-pressed Thus a fundamental molecular mechanismintegrated in our system is that RNAi molecules(siRNA miRNA shRNA) have the capacity of specificinducers This is different from the traditional approachwhere the lsquoONOFFrsquo configuration of the expressionsystem is controlled by the addition of exogenousinducers such as cumate tetracycline or doxycycline forinstance (26) All of the regulatory elements for this systemto function were assembled in a single plasmid to easilytransfect mammalian cells for in vitro studies or for in vivobioluminescence studies We validated the method in vitrousing a panel of cell lines in vivo in normal organs(skeletal muscles and liver) and finally in a mousemodel of muscular atrophy We demonstrated inaddition that RILES is suited to the longitudinal studyof miRNA expression in both physiological and patho-physiological organs without repetitive administration ofRILES Using a model of muscular atrophy we were ableto determine the kinetic of miRNA-206 expression duringthe muscle regeneration phase of muscle atrophy (3637)

Figure 5 Kinetics of luciferase expression in the tibialis anterior muscle of immunodeficient mice Two micrograms of pRILES133T pRILES122Tand control pRILES combined with 6 mg of the pQE30 empty expression plasmid were formulated with the 704 amphiphilic block copolymer andintramuscularly injected in the left and right tibialis anterior to transfect the skeletal muscles of nude mice ROIs covering the lower legs of animalswere drawn and light emission was quantified over time from day 1 (before the intramuscular injection of RILES-derivates plasmids) to day 38 theend point of the assay (A) Representative bioluminescence images collected at day 12 from three out of five mice per group (B) Quantification ofbioluminescence signals emitted from mice and plotted as a function of time Error bars in B meanplusmnSEM (n=6) of one representative experimentrepeated two times Statistics by the two-tailed t-test Plt 005 Plt 001 compared with the pRILES control group

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The bioluminescence data indicated that the expression ofmiRNA-206 is individual-dependent finely regulated in atime-dependent manner and characterized by individualheterogeneity during development of the pathologyWhen compared with data generated from conventionalquantitative RT-PCR miRNA-206 expression was alsofound to be overexpressed but its expression remainedconstant for 7 days before returning to the basal levelThis discrepancy between the two approaches is explainedby the invasive nature of the quantitative RT-PCRmethod that generates a set of information from a hetero-geneous population collected at different time points Thisinvasive method is costly in terms of animal numbers andmore importantly lacks temporal resolution at the individ-ual level Hence conventional invasive methods under-value the crucial significance of the temporal regulationof miRNA expression in response to biological processes(6ndash10) In contrast because RILES is suited to a longitu-dinal study sharper insights into miRNA regulation canbe gathered providing a novel dimension of miRNA ex-pression analysis These information are obviouslyrelevant in the field of miRNA biology and more particu-larly in the field of muscle regeneration which is currently

the focus of intensive research (3839) In addition thesedata also have some therapeutic value They indicate thatunderestimating the expression kinetics of miRNA-206and the heterogeneity between individuals might reducethe benefit of a miRNA-based therapeutic approachaiming at restoring (replacement therapy) or reducing (an-tagonist therapy) the expression of miRNA-206 for thetreatment of muscular disease as proposed in (3940)Our RILES method has additional advantages over

current approaches devoted to the in vivo monitoring ofmiRNA expression First RILES is a positive-molecular-monitoring system that generates bioluminescence signalswhen the target miRNA is expressed in cells Thisapproach is more reliable than the negative-molecular-monitoring approach (1516) where expression ofmiRNA is indicated by a reduction in the bioluminescencesignal Second RILES is a sensitive method particularlywell adapted to in vivo functional studies in small animalsBioluminescence emission catalyzed by the enzymaticluciferase reaction is extremely efficient with a quantumyield of 88 with low or no background and remainsone of the most reliable reporter systems used to quantifygene expression in mammalian cells in vivo (41ndash43)

Figure 6 Real time monitoring of miRNA-206 regulation during development of skeletal muscle atrophy Two micrograms of pRILES206Tcombined with 6 mg of the pQE30 empty expression plasmid were formulated with the amphiphilic block polymer 704 and intramuscularlyinjected in the tibialis anterior to transfect the skeletal muscles of nude mice Three days later (day 0) bioluminescence activity in the lower legsof mice was measured and the left sciatic nerves were cut surgically to induce denervation and atrophy Mice (n=5) were thereafter scanned twice aweek for the first 3 weeks and then once a week till day 35 (end point of our experiment) (A) Kinetic of miRNA-206 expression detected in eachindividual mouse by bioluminescence imaging Results are expressed as relative fold of luciferase induction by normalizing the bioluminescence valuesto the minimal value found before the bioluminescence peak for each single mouse (B) Representative bioluminescence images collected from onerepresentative mouse from the pRILES group and two representative mice from the pRILES206T group at days 7 (d7) 13 (d13) 22 (d22) and 30(d30) The number of each mouse for identification during the longitudinal study is given

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However environmental factors such as oxygen ATPtemperature and pH change have been shown to impacton the catalytic activity of the luciferase enzyme (44) Inthis case the luciferase gene in RILES can be substitutedby other optical reporter genes as the Tomato (45) orisotopic reporter genes such as the sodiumiodideSymporter (NIS) for instance (46) Third RILES doesnot require a complicated procedure to generate differentexpression plasmids to monitor miRNAs We designed aneasily interchangeable miRNA T cassette that can bestraightforwardly manipulated and used in any laboratoryskilled in standard molecular biology techniques andequipped with standard in vitro and in vivo biolumines-cence equipment This contrasts with the multistep pro-cedure and handling required to synthesize and labelmolecular fluorescent probes (47) that also are wellknown to have a low signal-to-background ratio whenused in vivo (42) Again when considering fluorescent mo-lecular beacons as a positive miRNA monitoring system(1718) careful design of the probes needs to be performedfirst in cellulo before being validated in vitro and this pro-cedure has to be repeated for each miRNA of interest (48)However the use of an expression plasmid to monitor theexpression of miRNA also has some limitations First thelong-term expression of a miRNA targeting sequence(miRNA T) in transfected cells might compete with theendogenous mRNA targets of the miRNA of interest Asa result RILES might interfere with the biological processstudied Other studies have addressed such issues (49ndash51)and demonstrated that a saturable effect of the miRNA Tcassette in cells is found only when the expression of trans-genes bearing the miRNA targeting sequence is driven bya strong promoter We anticipated this point and used theweak SV40 promoter to drive expression of the engineeredCymR transcript In addition we did not find any statis-tical difference in terms of miRNA-206 expressionbetween RILES-transfected tibialis muscle and not-trans-fected control muscle (data not shown) This indicates thatthe fraction of miRNA-206 bound to the CymR repressortranscript is not compensated by overexpression ofmiRNA-206 and strongly suggests that RILES is minim-ally interfering in cells Second when a long term moni-toring study of miRNA expression has to be performedRILES has a restricted application to immunodeficientmice Although we did not demonstrate the presence ofan immunological response developed against the pro-karyotic origin of the CymR repressor molecule it islikely that CymR is immunogenic in immunocompetentmice as already demonstrated with other prokaryotic-in-ducible expression systems (5253) The use of transgenicanimals bearing RILES in their genomes might overcomethis limitation and will provide in the meantime a reliabletool to monitor the expression of miRNAs during embry-onic development Finally as for most of the inducibleexpression systems described promoter leakiness in theabsence of inducer is a drawback (26) We did find somebasal expression of the luciferase gene in the absence ofRNAi molecules but the leakiness was low and not a limi-tation for our study as evidenced by our results It isworth noting that in HEK 293 cells the time frame forpRILES to be accurately switched-OFF and consequently

for the luciferase basal expression level to be at its minimalvalue is rapid ie 12 h after transfection (SupplementaryFigure S6) The observation that at this same time pointthe pRILES122T is adequately switched-ON in the samecells transfected with synthetic miRNA-122 indicates thatthe delay for the RNAi machinery to suppress expressionof CymR protein is also remarkably fast (SupplementaryFigure S7) This supports the notion that using RILES itmight be possible to monitor changes in miRNA expres-sion for periods at least equal to or above 12 h

Overall our data provide further compelling evidenceof the great potential of the endogenous RNAi machineryto control the expression of a transgene in a sequence-specific manner Indeed miRNA-target sequencessubcloned in the 30UTR region of a transgene cassettehave been shown to be a reliable tool to restrict transgeneexpression in specific cell types and lineages or to differ-entiate states (54ndash56) In all the systems currently used theendogenous RNAi machinery is used to repress expressionof the transgene (eg reporter gene therapeutic gene viralgene) (56) The success of this approach was proved by theexperimental evidence that de-targeting a transgene ex-pression in hematopoietic cells using target sequences ofhematopoietic lineage-specific miRNA-1423 enabledstable and long-lasting transgene expression in micewithout inducing an immune response overcoming oneof the most current issues in gene therapy (57) WithRILES the expression of the transgene is not suppressedby the endogenous RNAi machinery but rather inducedTherefore RILES can be used as a novel method toprogram the expression of transgenes in any targetedcells as long as a specific miRNA is expressed in thesecells We provided evidence that this programmingapproach is as robust specific and tight as the de-targetingapproach The transgene expression eg the luciferasereporter gene was found to be tightly controlled bytissue-specific miRNAs such as miRNA-122 in the liverand myomiRs-206 -1 and -133 in the skeletal muscles Wedemonstrated that the amplitude of luciferase expressionwas correlated with the amount of miRNA expressed ex-ogenously and endogenously and specific to the targetmiRNA sequence We also demonstrated that expressionof the luciferase protein was spatially restricted to skeletalmuscle cells when the RILES plasmid was customized tobe responsive to the muscle-specific miRNA-1 We finallyprovide the molecular evidence that RILES is tightlycontrolled by the endogenous RNAi machinery as expres-sion of the luciferase reporter gene was found to be in-versely correlated with the rate of repressor CymRtranscript degradation detected by quantitative PCR Itis worth noting that another advantage of an RNAi-indu-cible expression system such as RILES is that such systemswill overcome the low in vivo pharmacokinetics of exogen-ous inducers in small animals and in humans (2658) thathave compromised the stringent modulation of transgeneexpression in gene therapy (26) However the immuno-genicity of the prokaryotic form of the repressor is cur-rently an important issue that requires furtheroptimization to be applicable in immunocompetent indi-viduals Nevertheless we envision that in addition to theapplication of RILES in the field of miRNA biology

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RILES may serve as a platform for the development of anovel generation of controllable gene expression systemswith potential research and therapeutic applications (59)In line with this perspective a recent work by Naldini andhis collaborators (60) demonstrated that lentiviral vectorsencoding for the tTR-KRAB or the tTR repressor proteinplaced under the control of hematopoietic lineage-specificmiRNAs were found to be a reliable method to positivelyidentify and select in vitro a subset of hematopoietic stemand progenitor cell populations When transplanted intomice these selected cell populations were able to repopu-late their respective spleen and bone marrow nichesRemarkably the expression of the reporter gene withinthe niche was found to be consistent with the pattern ofactivity of the miRNAs expressed by the different types ofimplanted stem cells These results are of importance asthey suggest that it might be possible to transfer the ex-pression of a therapeutic transgene to a subset of well-defined differentiated cell populations originated fromtransplanted stem cells in vivo This work provides experi-mental evidence that programming the expression of atherapeutic transgene in targeted cells might be possibleaccording to the differential expression pattern ofmiRNA

In summary we have provided here a complete proof ofprinciple study and demonstrated that the use of an indu-cible expression system placed under the control of theendogenous RNAi machinery is a robust and reliablemethod to induce the expression of a transgene in targetcells We have demonstrated that when a sensitive imaginggene reporter is used as transgene this system offers thepossibility to study the dynamic regulation of miRNA inphysiological and pathophysiological contexts BecauseRILES offers a temporal analysis of the expression ofmiRNAs at the individual level this method enables tocollect more relevant biological information than does aconventional quantitative RT-PCR approach ThereforeRILES has applications in the field of miRNA biologyand may also represent a novel method to program theexpression of therapeutic transgenes (cDNA shRNAmiRNA mimic antagomiRNA RNAi sponge etc) inspecific target cells with possible applications in the fieldof gene and cell therapy

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online

ACKNOWLEDGEMENTS

We thank S Lerondel A Lepape M Le Mee S Retifand J Sobilo (TAAMCIPA CNRS UPS 44 Orleans) fortheir technical assistance and for access to the LuminaBioluminescence scanner We also thank Pascal Pineau(Institut Pasteur Paris) for providing the HLE cell lineand Athanassia Sotiropoulos (Institut Cochin Paris) forexpertise and technical support in initiating the surgicalprocedure to induce muscular atrophy

FUNDING

La Ligue Contre le Cancer du Loiret (to PB) Vaincre laMucoviscidose ANR and Plan Cancer 2009ndash2013 (toGV) La Region Centre PhD funding (to SE)Funding for open access charge La Ligue Contre leCancer du Loiret Region centre

Conflict of interest statement None declared

REFERENCES

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9 BrenneckeJ HipfnerDR StarkA RussellRB andCohenSM (2003) bantam encodes a developmentally regulatedmicroRNA that controls cell proliferation and regulates theproapoptotic gene hid in Drosophila Cell 113 25ndash36

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11 ShahabSW MatyuninaLV MezencevR WalkerLDBowenNJ BenignoBB and McDonaldJF (2011) Evidence forthe complexity of microRNA-mediated regulation in ovariancancer a systems approach PLoS One 6 e22508

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15 KoHY HwangDW LeeDS and KimS (2009) A reportergene imaging system for monitoring microRNA biogenesis NatProtoc 4 1663ndash1669

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18 LuJ and TsourkasA (2009) Imaging individual microRNAs insingle mammalian cells in situ Nucleic Acids Res 37 e100

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20 ZhengG CochellaL LiuJ HobertO and LiW (2011)Temporal and spatial regulation of microRNA activity withphotoactivatable cantimirs ACS Chem Biol 6 1332ndash1338

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21 KimE YangJ ParkJ KimS KimNH YookJI SuhJSHaamS and HuhYM (2012) Consecutive targetable smartnanoprobe for molecular recognition of cytoplasmic microRNAin metastatic breast cancer ACS Nano 6 8525ndash8535

22 LiuW OwenDP FisherKD SeymourLW andStevensonM (2009) Establishment of a positive-readout reportersystem for siRNAs J RNAi Gene Silencing 5 331ndash338

23 YaoQ ZhangA MaH LinS WangX SunJ and ChenZ(2012) Novel molecular beacons to monitor microRNAs in non-small-cell lung cancer Mol Cell Probes 26 182ndash187

24 MullickA XuY WarrenR KoutroumanisM GuilbaultCBroussauS MalenfantF BourgetL LamoureuxL LoRet al (2006) The cumate gene-switch a system for regulatedexpression in mammalian cells BMC Biotechnol 6 43

25 RossiFM and BlauHM (1998) Recent advances in induciblegene expression systems Curr Opin Biotechnol 9 451ndash456

26 StiegerK BelbellaaB Le GuinerC MoullierP and RollingF(2009) In vivo gene regulation using tetracycline-regulatablesystems Adv Drug Deliv Rev 61 527ndash541

27 BillietL GomezJP BerchelM JaffresPA Le GallTMontierT BertrandE CheradameH GueganP MevelMet al (2012) Gene transfer by chemical vectors and endocytosisroutes of polyplexes lipoplexes and lipopolyplexes in a myoblastcell line Biomaterials 33 2980ndash2990

28 ChisholmEJ VassauxG Martin-DuqueP ChevreRLambertO PitardB MerronA WeeksM BurnetJ PeerlinckIet al (2009) Cancer-specific transgene expression mediated bysystemic injection of nanoparticles Cancer Res 69 2655ndash2662

29 ChevreR Le BihanO BeilvertF ChatinB BarteauBMevelM LambertO and PitardB (2011) Amphiphilic blockcopolymers enhance the cellular uptake of DNA moleculesthrough a facilitated plasma membrane transport Nucleic AcidsRes 39 1610ndash1622

30 BonamassaB HaiL and LiuD (2011) Hydrodynamic genedelivery and its applications in pharmaceutical research PharmRes 28 694ndash701

31 JungnickelJ HaaseK KonitzerJ TimmerM and GrotheC(2006) Faster nerve regeneration after sciatic nerve injury in miceover-expressing basic fibroblast growth factor J Neurobiol 66940ndash948

32 ChangJ NicolasE MarksD SanderC LerroABuendiaMA XuC MasonWS MoloshokT BortR et al(2004) miR-122 a mammalian liver-specific microRNA is processedfrom hcr mRNA and may downregulate the high affinity cationicamino acid transporter CAT-1 RNA Biol 1 106ndash113

33 PineauP VoliniaS McJunkinK MarchioA BattistonCTerrisB MazzaferroV LoweSW CroceCM and DejeanA(2010) miR-221 overexpression contributes to liver tumorigenesisPNAS 107 264ndash269

34 ChenJF MandelEM ThomsonJM WuQ CallisTEHammondSM ConlonFL and WangDZ (2006) The role ofmicroRNA-1 and microRNA-133 in skeletal muscle proliferationand differentiation Nat Genet 38 228ndash233

35 BiggarKK and StoreyKB (2011) The emerging roles ofmicroRNAs in the molecular responses of metabolic ratedepression J Mol Cell Biol 3 167ndash175

36 GoldbergAL (1969) Protein turnover in skeletal muscle IIEffects of denervation and cortisone on protein catabolism inskeletal muscle J Biol Chem 244 3223ndash3229

37 SacheckJM HyattJPK RaffaelloA JagoeRT RoyRREdgertonVR LeckerSH and GoldbergAL (2007) Rapiddisuse and denervation atrophy involve transcriptional changessimilar to those of muscle wasting during systemic diseasesFASEB J 21 140ndash155

38 GullerI and RussellAP (2010) MicroRNAs in skeletal muscletheir role and regulation in development disease and functionJ Physiol 588 4075ndash4087

39 WilliamsAH ValdezG MoresiV QiX McAnallyJElliottJL Bassel-DubyR SanesJR and OlsonEN (2009)MicroRNA-206 delays ALS progression and promotesregeneration of neuromuscular synapses in mice Science 3261549ndash1554

40 LiuN WilliamsAH MaxeinerJM BezprozvannayaSSheltonJM RichardsonJA Bassel-DubyR and OlsonEN

(2012) microRNA-206 promotes skeletal muscle regeneration anddelays progression of Duchenne muscular dystrophy in miceJ Clin Invest 122 2054ndash2065

41 PrescherJA and ContagCH (2010) Guided by the lightvisualizing biomolecular processes in living animals withbioluminescence Curr Opin Chem Biol 14 80ndash89

42 XieBW MolIM KeereweerS van BeekER QueISnoeksTJA ChanA KaijzelEL and LowikCWGM (2012)Dual-wavelength imaging of tumor progression by activatable andtargeting near-infrared fluorescent probes in a bioluminescentbreast cancer model PLoS One 7 e31875

43 KangJH and ChungJK (2008) Molecular-genetic imaging basedon reporter gene expression J Nucleic Med 49 164Sndash179S

44 ThorneN IngleseJ and AuldDS (2010) Illuminating insightsinto firefly luciferase and other bioluminescent reporters used inchemical biology Chem Biol 17 646ndash65759

45 WinnardPT KluthJB and RamanV (2006) NoninvasiveOptical Tracking of Red Fluorescent Protein-Expressing CancerCells in a Model of Metastatic Breast Cancer Neoplasia 8 796ndash806

46 BarilP Martin-DuqueP and VassauxG (2010) Visualization ofgene expression in the live subject using the NaI symporter as areporter gene applications in biotherapy Br J Pharmacol 159761ndash771

47 JohnssonN and JohnssonK (2007) Chemical tools forbiomolecular imaging ACS Chem Biol 2 31ndash38

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49 PapapetrouEP KovalovskyD BeloeilL SantrsquoangeloD andSadelainM (2009) Harnessing endogenous miR-181a to segregatetransgenic antigen receptor expression in developing versus post-thymic T cells in murine hematopoietic chimeras J Clin Invest119 157ndash168

50 BrownBD CantoreA AnnoniA SergiLS LombardoADella ValleP DrsquoAngeloA and NaldiniL (2007) A microRNA-regulated lentiviral vector mediates stable correction ofhemophilia B mice Blood 110 4144ndash4152

51 GentnerB SchiraG GiustacchiniA AmendolaM BrownBDPonzoniM and NaldiniL (2009) Stable knockdown of microRNAin vivo by lentiviral vectors Nat Methods 6 63ndash66

52 RichardP PollardH LanctinC Bello-RoufaıM DesigauxLEscandeD and PitardB (2005) Inducible production oferythropoietin using intramuscular injection of block copolymerDNA formulation J Gene Med 7 80ndash86

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54 BrownBD VenneriMA ZingaleA Sergi SergiL andNaldiniL (2006) Endogenous microRNA regulation suppressestransgene expression in hematopoietic lineages and enables stablegene transfer Nat Med 12 585ndash591

55 SachdevaR JonssonME NelanderJ KirkebyA GuibentifCGentnerB NaldiniL BjorklundA ParmarM andJakobssonJ (2010) Tracking differentiating neural progenitors inpluripotent cultures using microRNA-regulated lentiviral vectorsProc Natl Acad Sci USA 107 11602ndash11607

56 BrownBD and NaldiniL (2009) Exploiting and antagonizingmicroRNA regulation for therapeutic and experimentalapplications Nat Rev Genet 10 578ndash585

57 AnnoniA BrownBD CantoreA SergiLS NaldiniL andRoncaroloMG (2009) In vivo delivery of a microRNA-regulatedtransgene induces antigen-specific regulatory T cells and promotesimmunologic tolerance Blood 114 5152ndash5161

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59 RuderWC LuT and CollinsJJ (2011) Synthetic biologymoving into the clinic Science 333 1248ndash1252

60 AmendolaM GiustacchiniA GentnerB and NaldiniL (2013)A double-switch vector system positively regulates transgeneexpression by endogenous microRNA expression (miR-ONVector) Mol Ther 21 934ndash946

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