M OLECULAR AND C ELLULAR B IOLOGY,June2011,p.2513–2527Vol.31,No.12 0270-7306/11/$12.00doi:10.1128/MCB.01189-10
Copyright©2011,American Society for Microbiology.All Rights Reserved.
A KLF4–miRNA-206Autoregulatory Feedback Loop Can Promote or
Inhibit Protein Translation Depending upon Cell Contextᰔ
Chen-Chung Lin,1,2Ling-Zhi Liu,1†Joseph B.Addison,1,3William F.Wonderlin,1
Alexey V.Ivanov,1,3and J.Michael Ruppert1,2,3*
Department of Biochemistry,1Program in Cancer Cell Biology,2and The Mary Babb Randolph Cancer Center,3
West Virginia University,Morgantown,West Virginia26506
Received10October2010/Returned for modification21November2010/Accepted10April2011
Kru¨ppel-like factor4(KLF4),a transcription factor that regulates cell fate in a context-dependent fashi
on,
is normally induced upon growth arrest or differentiation.In many cancer cells there is dysregulation,with
increased expression in proliferating cells.To identify sequence elements that mediate KLF4suppression in
normal epithelial cells,we utilized a luciferase reporter and RK3E cells,which undergo a proliferation-
differentiation switch to form an epithelial sheet.A translational control element(TCE)within the KLF4
3-untranslated region interacted with microRNAs(miRs)206and344-1to promote or inhibit KLF4expres-
sion,respectively,in proliferating epithelial cells.Overall,the TCE suppressed expression in proliferating
primary human mammary epithelial cells,but this suppressive effect was attenuated in immortalized mam-
mary epithelial MCF10A cells,in which Dicer1and miR-206promoted KLF4expression and TCE reporter
activity.In contrast to MCF10A cells,in breast cancer cells the activity of miR-206was switched,and it
repressed KLF4expression and TCE reporter activity.As miR-206levels were KLF4dependent,the results
identify a KLF4–miR-206feedback pathway that oppositely affects protein translation in normal cells and
cancer cells.In addition,the results indicate that two distinct miRs can have opposite and competing effects
on translation in proliferating cells.
The zincfinger protein Kru¨ppel-like factor4(KLF4)regu-lates gene transcription and cell fate in a context-dependent fashion,promoting cell differentiation,tumor suppression, stem cell properties,and malignant transformation(2,21,40, 58).Although Klf4is dispensable for early development,anal-ysis of postnatal,Klf4-deficient mice revealed roles in forma-tion of the cutaneous water permeability barrier,
in formation of mucosecreting goblet cells in the gut or conjunctiva,and in late fetal or early postnatal cardiac development(23,24,30,42, 46,61).In addition to its developmental roles,KLF4regulates the phenotype of cancer cells and stem cells.While KLF4 appears to suppress tumor formation in tissues such as the gut (5,12,65),it can promote malignant properties in other tis-sues,such as the breast and skin(8,10,31,37,39,45,62). When expressed in adult somatic cells with other Yamanaka factors,KLF4can promote the formation of induced pluripo-tent stem(IPS)cells(38,47,48,58).
How KLF4mediates its pleiotropic effects is an area of current study.KLF4typically reduces cell proliferation rates, possibly through regulation of p21Waf1/Cip1or other factors(39, 64).Even though KLF4slows cell proliferation,human carci-nomas are often slow growing,and KLF4may promote malig-nant properties in this context through suppression of p53or by upregulation of Notch1and confer stem cell properties in embryonic stem(ES)cells through induction of factors such as Nanog(16,31,39,63).
A seminal observation by Yang and colleagues was the induction of endogenous Klf4transcripts and protein fol-lowing in vitro growth suppression(43,64).A variety of growth-suppressive signals lead to upregulation of KLF4, including contact inhibition,serum starvation,DNA dam-age,and differentiation signals,such as retinoids or cyclic AMP(3,43,54,59,64).These in vitro results suggest an inverse relat
ionship between KLF4levels and cell prolifer-ation rates and are supported by extensive analyses in vivo that revealed that KLF4mRNA and protein are selectively expressed in the postmitotic,differentiating cell layers of epithelia such as the skin,gut,and oral mucosa(10,11, 42,43).
Mechanisms accounting for induction of KLF4upon growth arrest or differentiation potentially involve the gain of positive factors as well as the loss of suppressive influences on tran-scription,translation,or protein stability.In rapidly dividing colorectal cancer cells,ubiquitin-mediated proteolysis destabi-lizes KLF4,and protein stabilization therefore contributes to the induction of KLF4upon serum starvation(4).Since KLF4 can induce its own transcription,stabilization of the protein in growth-arrested cells can potentially lead to positive feedback (6,33).
Given its role as a stem cell factor that can promote malig-nant transformation,regulatory mechanisms that suppress KLF4in proliferating cells may be important to restrict cancer progression and/or the acquisition of stem cell phenotypes. Support for this notion includes the observation that KLF4is upregulated in the basal epithelial cells of dysplastic or malig-nant lesions in the skin and oropharynx(10,14,18)and of the
*Corresponding author.Mailing address:Department of Biochem-istry,P.O.Box9142,1Medical Center
Dr.,West Virginia University School of Medicine,Morgantown,WV26506.Phone:(304)293-5246. Fax:(304)293-4667.E-mail:mruppert@hsc.wvu.edu.
†Present address:Department of Pathology,Thomas Jefferson Uni-versity,Philadelphia,PA19107.
ᰔPublished ahead of print on25April2011.
2513
activity of KLF4as an oncogene when induced in the basal layer of mouse skin(10).
MicroRNAs(miRs),processed from pre-miR hairpin struc-tures by DICER1(DCR1),associate with Argonaute family members and other components to generate micro ribonucleo-proteins(miRNPs)that can suppress or promote protein trans-lation through regulatory elements within mRNAs(1,13,20, 27,36,44,51––53).In the current study we observed cell-type-specific effects of DCR1knockdown on cellular levels of KLF4. We identified a TCE coregulated by translation-stimulatory ,miR-206in human and rodent cells)and transla-tion-inhibitory ,miR-344in rodent cells).The TCE suppressed the activity of a luciferase reporter in proliferating epithelial cells,where endogenous KLF4was low,but pro-moted reporter activity in other contexts where the en
doge-nous protein was increased.These effects were attributed to induction of miR-206by KLF4,creating a positive feedback loop for translational control in primary human mammary epithelial cells(HMECs),MCF10A,and other epithelial cells. Unlike in normal epithelial cells,miR-206inhibited translation in malignant cells,indicating a switch from a positive to a negative regulator during tumor progression.The results iden-tify miR regulation of protein translation as a mechanism by which epithelial KLF4is suppressed in normal proliferating cells.Altered TCE activity contributed to the upregulation of KLF4in both immortalized epithelial cells and cancer cells. TCE regulation is intricate and involves competition between miRs that have opposing effects.Finally,the distinct effects of miR-206in normal cells and cancer cells may account for some of the distinct and context-dependent effects of KLF4on tu-mor progression(5,8,10,12,31,37,39,45,62,65).
MATERIALS AND METHODS
Cell lines and cell culture.Mammary epithelial MCF10A and breast cancer cell lines were obtained from ATCC.Primary HMECs(passage6)were pur-chased from Invitrogen.MCF10A and184A1cells were cultured as previously reported(7,8,34).HMECs were cultured in mammary epithelial basal medium (Lonza)supplemented with bovine pituitary extract,hydrocortisone,epidermal growth factor,insulin,gentamicin,amphotericin B,transferrin,and isopro-terenol.T47D and BT20cells were cult
ured in RPMI1640supplemented with penicillin,streptomycin,and10%fetal bovine serum.MCF7and BT474cells were cultured similarly except that insulin was added.MDA-MB-231and RK3E cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM)supple-mented with penicillin,streptomycin,and10%fetal bovine serum.4-Hydroxy-tamoxifen(4OHT)was dissolved in ethanol and,where indicated,was added to cultures at afinal concentration of0.3M.
Plasmid construction.The pMIR-REPORT luciferase vector was purchased from Ambion/Invitrogen.pRL-TK was obtained from Promega(Madison,WI). KLF4(GenBank accession number AF105036)fragments were excised from pCTV3K-SCC7-1(10)and cloned into the3Ј-untranslated region(UTR)of pMIR-REPORT luciferase.For pMIR-REPORT-K1S(K1S),a446-bp SalI-SacI fragment was cloned into PmeI and SacI sites of the vector.SacI fragments of465 bp(K2S),495bp(K3S),and380bp(K4S)were cloned into the SacI sites of the vector.For K5S,a300-bp EcoRI fragment was cloned into the EcoRI site of the vector.For K6S,a558-bp EcoRI-SalI fragment was treated with Klenow poly-merase and doexynucleoside triphosphates(dNTPs)to generate blunt ends and cloned into the PmeI site of the vector.To insert a nearly full-length(FL)cDNA, a SalI-Asp718fragment that included the KLF4sequence in AF105036was treated with Klenow polymerase and dNTPs and then cloned into the PmeI site of the vector.
Subfragments of K5S were generated by PCR using K5S reporter as template. The PCR products were digested with HindIII and XhoI and cloned into the same sites of pMIR-REPORT luciferase.Primers were as follows(with the KLF4se-quence shown in uppercase letters):K5S-1781Sense,5Ј-gggagctcAATTCAGTATT TTTTACTTTTCAC-3Ј;Vector Antisense,5Ј-aggcgattaagttgggtaacgc-3Ј;K5S-1Antisense,5Ј-cccaagcttAAGATGACTCAGTTGGGAACTTG-3Ј;K5S-2Sense, 5Ј-gggagctcGTGAGTGGATAATCAGGAAAAATG-3Ј;K5S-2Antisense,5Ј-cccaa gcttCGGATTTAGAATTGGAATGATAG-3Ј;K5S-3Sense,5Ј-gggagctcACTTGA ATATTCCTGGACTTAC-3Ј;K5S-4Sense,5Ј-gggagctcCCAGCCAGAAAGCAC TACAATC-3Ј;K5S-4Antisense,5Ј-cccaagcttCTTTGATTTTTGTCTTTTGGATT C-3Ј;K5S-5Sense,5Ј-gggagctcAACAGATGGGGTCTGTGACTGG-3Ј;K5S-5Antisense,5Ј-cccaagcttCAACTTCCAGTCACCCCCTTGG-3Ј;K5S-6Antisense, 5Ј-cccaagcttCGGATTTAGAATTGGAATGATAG-3Ј;K5S-7Sense,5Ј-gggagctcG TGAGTGGATAATCAGGAAAAATG-3Ј.Cloned PCR products were confirmed to be wild type by sequence analysis.
To generate K5S reporters with mutant miR seed sites,mFold was used to identify seed sequence alterations that retained the secondary structure pre-dicted for the wild-type reporter(66).PCR-mediat
ed mutagenesis was per-formed,and constructs were confirmed by sequence analysis.To test for com-plementation of mutant reporters,the following small interfering RNAs (siRNAs)were purchased from Dharmacon(seed mutations are indicated in lowercase letters):MtmiR344B,5Ј-UGAaCgAGCCAAAGCCUGACUGU-3Ј; MtmiR206B,5Ј-UGGAuaGUAAGGAAGUGUGUGG-3Ј.
To generate retroviral vectors encoding HA-KLF4-FL and HA-KLF4-FL⌬K5S,a BspEI-SalI fragment containing the3Ј-UTR of KLF4was inserted into the BspEI and XhoI sites of pcDNA3.1-HA-KLF4(37)to generate pcDNA3.1-HA-KLF4-FL.pcDNA3.1-HA-KLF4-FL⌬K5S was generated by digestion with EcoRI followed by religation so as to delete the internal EcoRI fragment.BamHI-XbaI fragments were excised from these constructs and inserted into the same sites of the retroviral vector pB-puro.For rescue of KLF4knockdown cells,the retroviral vector pLJD-KLF4-ER T,conferring Geneticin resistance,was constructed by transferring the insert from pBpuro-KLF4-ER T(9).
KLF4knockdown(shKLF4)utilized pGIPZ lentiviral shRNAmir plasmid (Open Biosystems V2LHS_28277;mature sense,5Ј-GCCAGAAAGCACTACA ATC-3Ј;mature antisense,5Ј-GATTGTAGTGCTTTCTGGC-3Ј),which targets 3Ј-UTR sequence within the K5S fragment,and nonsilencing-GIPZ lentiviral shRNAmir control(shCtrl;RHS4346).psPAX2and pCMV-VSVg packaging plasmids were obtained from Addgene.
Transient-transfection,luciferase reporter,and translation efficiency assays. The data shown below in Fig.2to4were generated using“forward transfection,”indicating that cells werefirst plated at5ϫ104cells per well in12-well plates 24h prior to transfection.Forward transfections were performed when cells were at approximately30%confluence.Transfection mixtures included50ng of pMIR-Report vector,with or without insert,and20ng of pRL-TK.Trans IT-LT1 transfection reagent(3l;Mirus Bio LLC,Madison,WI)was used according to the manufacturer’s instructions,except that keratinocyte growth medium(Invi-trogen)was substituted for Opti-MEM.
For analysis of miR activities,subsequent experiments(summarized below in Fig.5to9)utilized“reverse transfection,”in which cells were added to wells that contained preformed lipid-nucleic acid complexes.Anti-miR inhibitors(AMs; single-stranded,chemically modified nucleic acids)or miR mimics(PMs;mature miR/miR*duplexes that are loaded onto RISC without additional processing) were obtained from Ambion/Invitrogen,including miR-344-1(IDAM10439, PM10439),miR-1(AM10617,PM10617),and miR-206(AM10409,PM10409), as well as AM negative control1(AM17010),and PM negative control1 (AM17110).Inhibitors and mimics were diluted to20M in water and used for cell transfections at afinal concentration of25nM in a0.50-ml total volume in 3.8-cm2wells(12-well plates).Briefly,lipid-RNA complexes were allowed to form by incubation for20mi
n in the wells by addition of100l Opti-MEM I medium(Invitrogen),2.0l of Lipofectamine RNAiMAX transfection reagent (Invitrogen),and nucleic acids.In a separate tube,lipid-DNA complexes were allowed to form for20min following addition of100l serum-free medium,3.0l Trans IT-LT1,and plasmid DNA(quantities were as described for forward transfections).Lipid-DNA complexes were then added to the wells containing lipid-RNA complexes.Following trypsinization and washing,cells were resus-pended in keratinocyte-SFM(Invitrogen;supplemented with epidermal growth factor and bovine pituitary extract)at3.3ϫ105cells/ml,and0.30ml was added to each well.Plates were incubated for2h at37°C in5%CO2,and then1.0ml of complete growth medium was added to each well.
Luciferase assays utilized the dual luciferase reporter assay system(DLR assay;Promega).Extracts were prepared in0.15ml of1ϫpassive lysis buffer (PLB),and luciferase activities were determined at24h posttransfection(for proliferating cells,at approximately50to60%confluence)or at72h posttrans-fection(postconfluence for24h[PC24h]).Briefly,20l of lysate was added to 50l of luciferase substrate,and luciferase activity was measured using a Mod-ulus microplate multimode reader(Promega).Experiments,performed in du-plicate,were repeated for a total of three or more independent experiments.
2514LIN ET AL.M OL.C ELL.B IOL.
To determine translation efficiency,culture wells were transfected in parallel to isolate either RNA or protein.To adjust for well-to-well variations in trans-fection efficiencies,thefirefly luciferase(FLuc)/Renilla luciferase(RLuc)ratio (RNA or Luc activity)wasfirst determined for each well,and the mean ratio was determined for a set of replicates.FLuc and RLuc RNA ratios were determined using the2Ϫ⌬CT method with glyceraldehyde dehydrogenase(GAPDH)as the control.Translation efficiency for each construct was calculated as follows: (FLuc/RLuc)RLU/(FLuc/RLuc)RNA.A detailed example calculation is available upon request from the authors.
Cell transfection and virus transduction.Human DCR1(NM_030621)knock-down experiments utilized ON-TARGET plus RNA interference(RNAi)du-plexes and,as a control,the siGENOME nontargeting siRNA2(Dharmacon D-001210-02).The sequence for catJ-003483-09(si-DCR1number09)is5Ј-UA AAGUAGCUGGAAUGAUGUU-3Ј(sense)and5Ј-P-CAUCAUUCCAGCU ACUUUAUU-3Ј(antisense);the sequence for catJ-003483-11(si-DCR1num-ber11)is5Ј-ACACAGCAGUUGUCUUAAAUU-3Ј(sense)and5Ј-P-UUUA AGACAACUGCUGUGUUU-3Ј(antisense).
Lentiviral particles were packaged in HEK293T cells following calcium phos-phate-mediated cotransf
ection of shRNA vector,psPAX2,and pCMV-VSVg. Retroviral transduction of pBpuro constructs into RK3E cells was performed using supernatants of BOSC23cells as described previously(10).Retroviral transduction of human cells utilized the supernatant of AM12amphotropic packaging cells(a gift from L.T.Chow,University of Alabama at Birmingham). Following incubation with lentiviral or retroviral particles,cells were selected in puromycin(0.5g/ml)or Geneticin(200g/ml).
Cell growth rate and cell cycle analysis.Cells were maintained at subconflu-ence.Following trypsinization,1ϫ103cells were transferred into96-well plates and cultured for the indicated intervals.Cell number was determined using the CellTiter-Glo luminescent cell viability assay(Promega).Fluorescence-activated cell sorting analysis of cell cycle distribution was performed following staining of cells with propidium iodide using the Cycle Test Plus DNA reagent kit(BD Biosciences).
Metabolic labeling,immunoprecipitation,and protein half-life determination. To assess the rate of KLF4protein synthesis,2.2ϫ106cells were seeded in a 10-cm dish.Where miRs were analyzed the dishes contained preformed lipid-RNA complexes.After culture for48h the cells were washed twice in phosphate-buffered saline(PBS)and then cultured for an additional2h in l-methionine/l-cysteine(Met/
Cys)-free DMEM supplemented with10%FBS,20 mM HEPES-NaOH(pH7.2)and l-Gln.35S[Met/Cys](Perkin Elmer)was added directly to the culture medium at200Ci/ml.Labeled cells were rinsed twice in cold PBS and then scraped in200l of boiling denaturation buffer(BDB;0.5% SDS,50mM Tris-HCl[pH8.0],10mM dithiothreitol).Extracts were passed through a26-gauge needle10times to reduce the viscosity,heated to100°C for 5min,and then diluted with4volumes of radioimmunoprecipitation assay (RIPA)buffer without SDS(150mM NaCl,50mM Tris-HCl[pH7.5],5.0mM EDTA,1.0mM NaF,1%sodium deoxycholate,1%NP-40,and0.25mM phen-ylmethylsulfonylfluoride).Incorporated counts per minute were determined by trichloroacetic acid precipitation,and6.0ϫ107cpm were subjected to immu-noprecipitation(IP)using monoclonal anti-hemagglutinin(HA)–agarose(clone HA-7;Sigma-Aldrich).IPs were washed in RIPA containing0.1%SDS,eluted in SDS sample buffer,and separated by SDS-PAGE(10%).The gel was processed for autoradiography using EN3HANCE as recommended by the supplier (PerkinElmer),and the dried gel was exposed tofilm atϪ80°C.The yields were analyzed by digitized phosphorimaging(Typhoon imager;GE Healthcare). The HA-KLF4protein half-life was estimated by immunoblot analysis of cycloheximide(CHX)-treated cells using anti-HA antibody.HA-KLF4protein levels were normalized to a stable protein,-actin,and protein half-life was determined from thefirst-order rate constant during thefirst1h of CHX treatment.The estimated half-life was further confirmed by comparison of the IP yield of radiolabeled
protein when cells were labeled ,to50% of steady state),to approximately the steady state).
In vitro Transwell migration and invasion assays.For migration assays1.0ϫ104cells were plated in the top chamber(24-well insert;pore size,8.0m;BD Biosciences).For invasion assays,1.0ϫ104cells were plated in the top chamber over a Matrigel-coatedfilter(24-well insert;pore size,8.0m).In both assays, cells were plated in growth medium containing0.5%FBS,and growth medium containing10%FBS was used as a chemoattractant in the lower chamber.After 24h,cells that did not migrate or invade through the pores were removed by scraping,and cells on the lower surface of the membrane were stained using the Diff-Quik stain set(Siemens)and counted.
Immunoblot analysis.Cells were washed twice in PBS and then incubated for 30min in ice-cold lysis buffer:150mM sodium chloride,1.0%(wt/vol)sodium deoxycholate,1.0%(vol/vol)Triton X-100,5.0mM EDTA,50mM Tris-HCl(pH 7.5),0.25mM phenylmethylsulfonylfluoride,1.0mM benzamidine,1.0mM pepstatin,1.0g/ml leupeptin,1.0g/ml aprotinin,0.4mM sodium orthovana-date,and1.0mM sodiumfluoride.Extracts were centrifuged at15,000ϫg for15 min at4°C.Protein concentrations were quantified by using the Bradford assay (Bio-Rad).
Following electrophoresis,proteins were transferred to nitrocellulose.Anti-bodies to DCR1(H-212;Santa Cruz Biotechnology),HA(12CA5;Roche)and cyclin D1(H-295;Santa Cruz Biotechnology)were used at0.40g/ml.Antibod-ies to KLF4(H-180;Santa Cruz Biotechnology)and p21(C-19;Santa Cruz Biotechnology)were used at0.25g/ml.p27antibody(C-19;Santa Cruz anti-body)was used at0.10g/ml.Antibody to-actin(C-4;Santa Cruz Biotechnol-ogy)or-tubulin(clone2-28-33;Santa Cruz Biotechnology)was used at0.20g/ml.Bound antibodies were detected using the Pierce enhanced chemilumi-nescence Western blotting substrate(Thermo Scientific).Scanned images were quantitated using ImageJ software,with normalization to the loading control. Reverse transcription and real-time PCR detection of miRNAs.To profile miR levels in proliferating versus confluent RK3E cells,total RNA was extracted using the mir Vana miRNA isolation kit(Ambion/Invitrogen)according to the manufacturer’s instructions.A total of381individual miRs were analyzed in triplicate using TaqMan rodent microRNA array cards as recommended by the manufacturer(Applied Biosystems).Briefly,miRNA was converted to cDNA by using a pool of stem-loop hairpin reverse transcription(RT)primers,and this product was delivered to single wells preloaded with miR-specific primers and probes.Real-time PCR was performed on a7900HT ABI Prism sequence de-tector system.miRNA fold changes were determined using the⌬⌬C T method with18S rRNA as the internal control(32).
Individual miRs were analyzed by stem-loop reverse transcription followed by quantitative,real-time PCR(SL-RTQ)using TaqMan microRNA assays(Ap-plied Biosystems)according to the manufacturer’s instructions;the individual miRs were miR-206(4373092),miR-1(4395333),miR-344-1(4373340),and U6 snRNA(4395470).For PCRs we utilized an Mx3005P real-time PCR system (Stratagene).miRNA fold changes were determined using the⌬⌬C T method with U6snRNA as the internal control.Three independent experiments were performed,with analysis of each RNA sample in duplicate fashion for each experiment.
Quantitative,real-time RT-PCR(qRT-PCR)analysis of mRNA.Total RNA was isolated using an RNeasy minikit(Qiagen)and reverse transcribed using SuperScript II reverse transcriptase(Invitrogen).PCR primers and TaqMan probes were from Assays-on-Demand(Applied Biosystems):human and rat KLF4(ID Hs00358836_m1and Rn00821506_g1)or human and rat GAPDH (Hs99999905_m1and Rn99999916_s1).Firefly and Renilla luciferase transcripts were analyzed using SYBR GreenER qPCR SuperMix(Invitrogen),with nor-malization to GAPDH and the following primers:Firefly-Luc-forward,5Ј-G CCCGCGAACGACATTTA-3Ј;Firefly-Luc-Reverse,5Ј-TTTGCAACCCCT TTTTGGAA-3Ј;Renilla-Luc-forward,5Ј-GCAGCATATCTTGAACCATTC AAA-3Ј;Renilla-Luc-Reverse,5Ј-CATCACTTGCACGTAGATAAGCATT ATA-3Ј;GAPDH-forward,5Ј-TCAC
CACCATGGAGAAGGC-3Ј;GAPDH-Reverse,5Ј-GCTAAGCAGTTGGTGGTGCA-3Ј.These GAPDH primers recognize mouse,rat,and human forms.
Statistical analyses.Data were analyzed using the unpaired t test or else a one-way analysis of variance(ANOVA)with Tukey’s multiple comparison test, both using GraphPad Prism5(GraphPad Software,San Diego,CA).Differ-ences were considered significant when the two-tailed analysis yielded a P value ofϽ0.05.
RESULTS
Identification of posttranscriptional control elements in the KLF4transcript.A signature feature of KLF4is its induction upon growth arrest or differentiation(43,64).To determine whether sequence elements within the KLF4mRNA can func-tion in posttranscriptional regulation,we utilized subconfluent and postconfluent cultures of adenovirus E1A-immortalized rat kidney cells(RK3E)(41).These diploid epithelial cells proliferate rapidly at subconfluence,with a doubling time of approximately12h.Under these conditions the levels of Klf4 mRNA(Fig.1A)and protein(Fig.1B,lane1)were low.At confluence the cells form an epithelial sheet of1to3cell layers that persists intact for several weeks,with apical tight junc-tions,adherens junctions,and frequent desmosomes(10).Typ-
V OL.31,2011miR-206REGULATION OF KLF4IN EPITHELIAL CELLS2515
ical of epithelial cells in vivo ,these cells show peripheral stain-ing of E-cadherin and -catenin,with very low expression of mesenchymal markers,such as vimentin (10,29).Similar to differentiated epithelial cells in vivo ,postconfluent RK3E had increased amounts of Klf4mRNA and protein (Fig.1A and B,lanes 2to 4)and increased amounts of the cyclin-dependent kinase inhibitors p21Waf1/Cip1and p27Kip1(Fig.1B).Cell cycle analysis revealed a decrease in S phase from 36.0%in prolif-erating cells to 20.8%in postconfluent cultures (Fig.1C).These studies identified a simple in vitro model of the prolif-eration/differentiation switch that epithelial cells undergo in vivo .
To screen for elements that could confer differential expres-
sion in proliferating versus postconfluent cells,we inserted the KLF4cDNA (i.e.,KLF4-FL,including the protein-coding re-gion and 3Ј-UTR)into the 3Ј-UTR of a FLuc expression vector (Fig.2A).KLF4-FL or the empty vector was cotransfected with an RLuc expression vector into RK3E cells.Following normalization to RLuc,the effect of KLF4-FL sequence on FLuc activity was determined by comparison to the vector control (Fig.2B).In proliferating cells,normalized FLuc ac-tivity derived from FLuc-KLF4-FL was about 60%lower than vector.In postconfluent cells there was likewise a suppressi
on of FLuc derived from KLF4-FL ,but the effect was attenuated compared to proliferating cells (Fig.2B,PC24h).
To distinguish effects on mRNA from effects on translation efficiency,we analyzed FLuc and RLuc mRNA levels by qRT-PCR (Fig.2C).Relative translation efficiency (RTE)was cal-culated using Luc activity and RNA levels (Fig.2D).As shown
FIG.1.KLF4and KLF4-regulated cell cycle inhibitors are induced during formation of a confluent epithelial sheet in vitro .(A)Real-time qRT-PCR analysis of endogenous Klf4.Total RNA was isolated from subconfluent (proliferating)or postconfluent RK3E epithelial cells (three independent experiments;bars show standard errors).(B)Klf4and the cell cycle regulatory proteins cyclin D1,p21Waf1/Cip1,and p27Kip1were analyzed by immunoblotting.-Tubulin served as a load-ing control.(C)Cell cycle distribution of RK3E cells (three indepen-dent experiments performed in duplicate;bars show standard
errors).
FIG.2.The KLF4transcript can suppress translation of a linked reporter prior to the epithelial proliferation-differentiation switch.(A)Full-length human KLF4cDNA (KLF4-FL)was inserted in the sen
se orientation into the 3Ј-UTR of FLuc.(B)Following transfection of KLF4-FL or vector in parallel,the normalized FLuc activity ratio was determined (i.e.,FLuc KLF4-FL /FLuc vector ).Proliferating,RK3E cells were harvested at 50%confluence;PC24h,RK3E cells were harvested 24h after reaching confluence.(C)Wells transfected in parallel (see panel B)were used for preparation of RNA.Luc mRNAs were analyzed using qRT-PCR with normalization to the Gapdh gene.(D,left)Calculation of relative translation efficiency (RTE)of con-struct X relative to construct V.(Right)The RTE of KLF4-FL was calculated from the data shown in panels B and C (three independent experiments;bars show standard errors).
2516LIN ET AL.M OL .C ELL .B IOL .
in Fig.2C,insertion of theϳ2.6-kb KLF4-FL sequence did not decrease the abundance of FLuc transcripts relative to vector in proliferating cells.Furthermore,in postconfluent cells there was only a modest suppression of27%.Rather,reduced FLuc activity in proliferating cells(Fig.2B)was entirely attributed to inefficient translation(Fig.2D).In these experiments the RTE was reduced by72%(PϽ0.001).After adjusting for mRNA abundance,the RTE in postconfluent cells was similar for the vector and KLF4-FL(Fig.2D,PC24h).These results sug-gested that KLF4transcripts are inefficiently translated into protein when cells are proliferating,a condition where only low levels of endogenous K
lf4are observed(Fig.1B).These map-ping studies identified translational control as a mechanism leading to differential expression of KLF4in proliferating and differentiating epithelial cells.
Fragment K5S represents a3-TCE that suppresses trans-lation in proliferating cells.We tested subfragments of the KLF4cDNA for regulation of translation in proliferating cells (Fig.3A).K5S,a3Ј-UTR-derived fragment of306nucleotides (nt),was sufficient to suppress Luc activity by58%in prolifer-ating cells(Fig.3B)and suppressed RTE by42%(Fig.3C).As KLF4-FL had a larger effect on RTE(Fig.2D),other regula-tory elements may remain to be identified,or else the TCE may be context sensitive.As observed for KLF4-FL,the effect of K5S on RTE was specific,because there was no suppression in postconfluent cells(Fig.3C).
A100-nt subregion of the TCE contains translation regula-tory activity.To aid in the identification of regulatory miRs,we tested100-to200-nt subfragments of K5S for differential re-porter activity(Fig.4A).Of these,only K5S-5was differentially active(Fig.4B).For this construct,the RTE was0.56Ϯ0.04 (proliferating cells)and0.84Ϯ0.08(PC24h).While several other fragments inhibited translation,these failed to show dif-ferential activity and were not further investigated.These stud-ies supported context dependence,as several larger fragments that included the K5S-5region showed either no differential activity(K5S-7)or else an attenuated difference(K5S-6). Therefore,in addition to t
he nucleotide sequence,the second-ary structure appears likely to be important for activity.Indeed the predicted structure of K5S indicates a highly structured stem with a more open region in the vicinity of K5S-5(mfold data not shown).
Based upon target site prediction,at least eight different miRs,including miR-1,-26a,-26b,-145,-206,-344,-465,and -613have the potential to regulate through K5S-5(Table1).In different contexts,KLF4translation was previously found to be suppressed by miR-10b,miR-200c,miR-145,and miR-1(50, 55––57).Profiling indicated that three of these known inhibi-
tory miRs(miR-200c,-145,and-10b)are expressed at in-creased levels in PC24h RK3E cells compared with prolifer-ating cells(Table1),even though translation of reporters is upregulated(Fig.2D and3C)and endogenous Klf4is induced (Fig.1A and B).miR-1was unchanged(Table1).Unlike these miRs,miR-344-1(miR-344)was suppressed by67-fold in PC24 h cells,potentially contributing to the induction of Klf4.
We therefore used gain-and loss-of-function methods to investigate the functions of miR-344and two miRs with over-lapping,evolutionarily conserved binding sites,miR-1and miR-206(Fig.4C and D).Unlike other vertebrates,mice have an insertion of4bases in the center of the miR-344seed(Fig.4
C)(20).Interestingly,rather than disrupting the Klf4–miR-344base pairing,this GGAT insertion duplicates the adjacent seed sequence and recreates an miR-344seed with extensive3Јpairing for both miR-344and miR-1/206(Fig.4E and data not shown).As vertebrates therefore appear to strictly conserve the binding site for miR-344,the site for miR-1/206,and the 6-nt spacing between the two seeds(Fig.4C and E),we con-structed K5S FLuc reporters with mutations at the seed re-gions(Fig.4F).
miR-344and miR-206mediate proliferation-dependent translation of the TCE.To confirm the results obtained by miR profiling(Table1),we used gene-specific SL-RTQ assays to FIG.3.A cis-acting TCE derived from the KLF43Ј-UTR inhibits translation of a linked reporter in proliferating epithelial cells.
(A)Fragments of KLF4were inserted into the3Ј-UTR region of FLuc.
(B)Following transfection of RK3E cells,FLuc activity relative to the vector was determined using the DLR assay as shown in Fig.2B(five independent experiments;bars show standard errors).(C)RTE of the TCE construct(K5S,corresponding to bases1781to2086of GenBank accession number AF105036)in proliferating and PC24h cells(three independent experiments;bars show standard errors).
V OL.31,2011miR-206REGULATION OF KLF4IN EPITHELIAL CELLS2517
