mir-181a mediates tgf-β-induced hepatocyte emt and is dysregulated in cirrhosis and hepatocellular...

ORIG INAL ART ICLE

miR-181a mediates TGF-b-induced hepatocyte EMT and is dysregulated incirrhosis and hepatocellular cancerJennifer Brockhausen1, Szun S. Tay2, Candice A. Grzelak1, Patrick Bertolino1, David G. Bowen1, William M.d’Avigdor1, Narcy Teoh4, Sharon Pok4, Nick Shackel1, Jennifer R. Gamble3, Mathew Vadas3 andGeoff W. McCaughan1

1 Liver Injury and Cancer, Centenary Institute, Camperdown, NSW, Australia

2 Liver Immunology, Centenary Institute, Camperdown, NSW, Australia

3 Vascular Biology, Centenary Institute, Camperdown, NSW, Australia

4 Liver Research Group, ANU Medical School at The Canberra Hospital, Garran, ACT, Australia

Keywords

cirrhosis – EMT – hepatocellular carcinoma –

hepatocyte – microRNA – TGF-b

Abbreviations

CCL4, carbon tetrachloride; CDAA, choline

and amino acid-defined fed mice; DEN, N,

N-diethylnitrosamine; EMT, epithelial–

mesenchymal transition; GFP, green

fluorescent protein; HCC, hepatocellular

carcinoma; HCV, hepatitis C virus; miR,

microRNA; rAAV, recombinant adeno-

associated virus; SMA, smooth muscle actin;

TAA, thioacetamide; TGF-b, transforming

growth factor-b.

Correspondence

Geoff McCaughan, Head of Liver

Immunobiology Program, Centenary

Research Institute, Building 93, Royal Prince

Alfred Hospital, Camperdown, NSW 2050,

Australia.

Tel: +61 02 9565 6125

Fax: +61 02 9565 6101

e-mail: [email protected]

Received 15 October 2013

Accepted 23 February 2014

DOI:10.1111/liv.12517

AbstractBackground & Aims: Epithelial–mesenchymal transition (EMT) has beenimplicated in the processes of embryogenesis, tissue fibrosis and carcinogene-sis. Transforming growth factor-b (TGF-b) has been identified as a key driverof EMT and plays a key role in the pathogenesis of cirrhosis and hepatocellu-lar carcinoma (HCC). The aim was to identify microRNA (miR) expressionin TGF-b-induced hepatocyte EMT. Methods: We treated a human hepato-cyte cell line PH5CH8 with TGF-b to induce an EMT-like change in pheno-type and then identified dysregulated miRs using TaqMan Low DensityArrays. MiR expression was altered using miR-181a mimic and inhibitor inthe same system and gene changes were identified using TaqMan gene arrays.MiR-181a gene expression was measured in human and mouse cirrhotic orHCC liver tissue samples. Gene changes were identified in rAAV-miR-181a-expressing mouse livers using TaqMan gene arrays. Results: We identifiedmiR-181a as a miR that was significantly up-regulated in response to TGF-btreatment. Over-expression of a miR-181a mimic induced an in vitro EMT-like change with a phenotype similar to that seen with TGF-b treatmentalone and was reversed using a miR-181a inhibitor. MiR-181a was shown tobe up-regulated in experimental and human cirrhotic and HCC tissue.Mouse livers expressing rAAV-miR-181a showed genetic changes associatedwith TGF-b signalling and EMT. Conclusions: MiR-181a had a direct effectin inducing hepatocyte EMT and was able to replace TGF-b-induced effectsin vitro. MiR-181a was over-expressed in cirrhosis and HCC and is likely toplay a role in disease pathogenesis.

Epithelial–mesenchymal transition (EMT) is a class ofphenomena associated with three biological processes,which allow polarized epithelial cells to undergo multi-ple biochemical changes to adopt a mesenchymal phe-notype with enhanced invasive properties. Theseprocesses include type 1 EMT, which is associated withimplantation and embryogenesis, type 2 EMT with tis-sue fibrosis and remodelling and type 3 EMT with carci-nogenesis (1). The signalling pathways involved in EMThave cell-specific components, but transforming growthfactor-b (TGF-b) signalling is the commonest driver

and the miR-200, miR-205 series are the best-describedinhibitors of pathways that often involve the transcrip-tion factors Snail, Twist and Slug.

Hepatocyte EMT has been well recognized and novelinducers of EMT such as Maelstorm (acting through theC2H2 transcription factor Snail) have been linked tohepatocellular cancer (HCC) progression and prognosis.The role of EMT in fibrosis, often a clinical precursor tocancer, is less clear as activated stellate cells, rather thanthe hepatocyte, are involved in collagen deposition.However, cellular tracing studies have implied that

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hepatocytes may also contribute to fibrosis via an EMTprocess (2–5) and in particular, Snail has been shown asa key intermediate (4). Other studies have questionedwhether such cells actually contribute to fibrosis in vivoas they do not acquire mesenchymal marker expressionor exhibit morphological changes (6, 7). It has been sug-gested that stellate cell activation is carcinogenic by vir-tue of the cytokine secretion pattern and that the laydown of collagen is not essential (8).

Thus, there is considerable interest to understand themechanism of hepatocyte EMT as this appears to under-lie much of the invasive nature of HCC and may be acontributor to liver fibrosis. To examine this process, wehypothesized that miR-200, as seen in many epithelialtissues (9), is a master regulator of hepatocyte EMT. Thelink with miR-200 and EMT is driven by TGF-b, whichis well known to induce EMT in several epithelial tissues(10, 11). Therefore, we employed a screen that usedTGF-b to induce EMT in the non-transformed humanhepatocyte cell line, PH5CH8. To our surprise, whileTGF-b induced strong phenotypic markers of EMT,there was little decrease in expression of miR-200. How-ever, another microRNA, miR-181a was significantlyinduced early in the process of EMT. MiR-181a has beenshown to be induced by TGF-b in several systems, buthas now been linked to the EMT process in breast andovarian cancer (12–16). In this study, we show that inhepatocytes, miR-181a is able to reproduce the geneticand phenotypic effects of TGF-b. Its increased expres-sion is also closely linked in human specimens withcirrhosis and HCC and in animal thioacetamide (TAA),carbon tetrachloride (CCL4) models of liver fibrosis andthe N,N-diethylnitrosamine (DEN) model of HCCdevelopment. In keeping with hepatocyte EMT, thehepatocyte-specific over-expression of miR-181ainduced changes in genetic markers of EMT and fibrosis.

Materials and methods

Materials

Recombinant human TGF-b was purchased from R&DSystems (Minneapolis, MN, USA). Mouse type-1 colla-gen, FBS, hydrocortisone and linoleic acid were pur-chased from Sigma (St Louis, MO, USA). DMEM, P/S,ITS, LipofectamineTM 2000 was purchased from Invitro-gen (Carlsbad, CA, USA). PH5CH8 cells were a giftfrom Dr S Locarnini. Protease and phosphatase inhibi-tor cocktail tablets were purchased from Roche Diag-nostics (Branchburg, NJ, USA). Polyvinylidenedifluoride membrane, horseradish peroxidise-conju-gated antibodies, ECL-chemiluminescence and Hyper-film were from Amersham (Pittsburgh, PA, USA).TaqMan Array Human MicroRNA A+B Card Sets, Taq-Man Array Gene Signature Plates, High-Capacity cDNAReverse Transcription Kit, TaqMan MicroRNA ReverseTranscription Kit and TaqMan probes were fromApplied Biosystems (Foster City, CA, USA). RecoverAll

Total Nucleic Acid Isolation Kit was from Ambion(Carlsbad, CA, USA). mirCURY LNATM miR-181ainhibitor was from Exiqon, miRIDIAN miR-181a mimicfrom Dharmacon, pEZX-MR04 and pEZX-CR04 Omic-sLinkTM Expression plasmids, pEZX-AM02 and pEZXinhibitor control miArrestTM miRNA inhibitor Expres-sion clones from GeneCopoeiaTM (Rockville, MD, USA).Anti-E-Cadherin, anti- vimentin, anti-BIM, was pur-chased from Cell Signalling, anti-SMA from Abcam,anti-albumin from Sigma and anti-GAPDH from Invi-trogen. Alexa590 and Alexa488 secondary antibodieswere from Molecular Probes (Eugene, OR, USA).

Cell culture and treatments

The immortalized non-neoplastic human hepatocytePH5CH8 cell line was cultured in Dulbecco’s modifiedEagle’s medium:F12 (DMEM:12, Invitrogen) supple-mented with 1% fetal bovine serum (FBS), 1% Penicil-lin/streptomycin (P/S), 1% insulin, transferrin, selenium(ITS), 0.2 ng/ml epidermal growth factor (EGF), 50 lMhydrocortisone, 5 lg/ml linoleic acid, 0.1 ng/ml prolac-tin. The mouse hepatocyte AML12 cell line was culturedin DMEM:F12 medium supplemented with 10% FBS,1% ITS and 40 ng/ml dexamethasone. The humanhepatic stellate LX-2 cell line was cultured in DMEMsupplemented with 10% FBS and 1% P/S. Primaryhepatocytes were isolated as previously described (17).For TGF-b assays, cells were seeded on 0.1% collagen-coated plates at a density of 6 9 104 cells/ml and serumstarved overnight. TGF-b1 was added at 5 ng/ml for24 h to 7 days in serum-free media.

Immunofluorescence

PH5CH8 cells plated on 0.1% collagen-coated glass cov-erslips were fixed in ice-cold 1:1 MeOH:acetone andpermeabilized in 1% Triton X-100/PBS. Cells were incu-bated overnight at 4°C in 1:200 mouse anti-E-Cadherinor 1:500 rat anti-vimentin followed by 1:500 anti-rabbit-IgG Alexa594 or anti-rat-IgG Alexa480 conjugates for1 h at room temperature. For GFP visualization, mouseliver tissues were fixed in 4% paraformaldehyde for 4 h,15% sucrose/PBS for 4 h, 30% sucrose overnight at 4°Cand then embedded in OCT compound. Cells andcryosections were analysed by confocal laser scanningmicroscopy (Leica DM IRE2 inverted microscopeequipped with a Leica TCS SP2 system, Germany).

Immunoblotting

Whole cell extracts were rinsed in cold PBS and har-vested in 1% Triton X-100 extraction buffer (50 mMTris pH7.5, 150 mM NaCl, 1 mM EDTA, 1% TritonX-100 containing protease and phosphatase inhibitorcocktail tablets). Whole tissue lysates were homogenizedin complete RIPA buffer consisting of 50 mM Tris pH8, 150 mM NaCl, 20 mM NaF, 5 mM EDTA pH8, 10%

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miR-181a mediates TGF-b-induced hepatocyte EMT Brockhausen et al.

glycerol, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40 containing protease and phosphatise inhibitor cock-tail tablets. After centrifugation (13 000g for 15 min) toremove cell debris, the extract was boiled in loading buf-fer containing 1% SDS, 0.1M DTT. Extracted proteins(40 lg/lane by Bradford protein assay) were separatedby 10% or 15% sodium dodecylsulfate polyacrylamidegel electrophoresis (SDS-PAGE) and transferred topolyvinylidene difluoride membrane. Blots were incu-bated overnight at 4°C in 1:1000 anti-E-Cadherin,1:2000 anti-vimentin, 1:500 anti-SMA, 1:1000 anti-albu-min, 1:1000 anti-BIM, 1:5000 mouse anti-GAPDH, fol-lowed by incubation for 1 h at room temperature in1:1000–1:5000 horseradish peroxidise-conjugated anti-body. Bands were visualized by ECL-chemiluminescenceand Hyperfilm ECL. ImageJ 1.31v software (NIH; Beth-esda; http:rsb.info.nih.gov/ij/) was used for quantifica-tion of immunoblot band intensity and area, expressedas integrated pixel intensity values. Statistical analyseswere performed using Prism IV for Windows (Graph-Pad Software Inc., www.graphpad.com). One-way ANO-

VA was used for comparisons across three or moregroups, with Bonferroni Multiple comparison test. Sta-tistical significance required a P value ≤0.05.

Gelatin Zymography

PH5CH8 cells were plated in 6-cm dishes and treatedwith 5 ng/ml TGF-b. Cell culture media were collectedand diluted in SDS-PAGE sample buffer. Samples andMMP-2/9 standard were loaded and separated onto a0.75-mm wide 8% SDS-PAGE gel (8% bis-acrylamide(v/v), 0.4M Tris pH 8.8, 0.01% APS, 0.01% SDS and0.01% TEMED with 1 mg/ml of MDPF-labelled gelatin)and topped with a 3% stacking gel (3% bis-acrylamide(v/v), 0.6M Tris pH6.8, 0.08% APS, 0.01% SDS, 0.01%TEMED). Gel was incubated at room temperature withdevelopment buffer 1 (50 mM Tris pH 7.5, 0.02%NaN3, 1 lM ZnCl2, 2.5% Triton X-100) and buffer 2(50 mM Tris pH7.5, 0.02% NaN3, 1 lM ZnCl2, 2.5%Triton X-100, 10 mM CaCl2) for 30 min each followedby buffer 3 (50 mM Tris pH7.5, 0.02% NaN3, 1 lMZnCl2, 10 mM CaCl2) at 37°C with rocking, overnight.Gels were visualized using long wave ultra violet lightbox (UVP, USA, www.uvp.com).

TaqMan MicroRNA Array Assays

Total RNAs were extracted from PH5CH8 cells andcDNAs were made using TaqMan MicroRNA ReverseTranscription kit using Megaplex primer human poolsets A v2.1 and B v3.0 according to the manufacturer’sprotocol. The mixture was applied to the eight individ-ual ports of TaqMan Array Human MicroRNA A+BCards Sets v2.0 and v3.0, which allow accurate quantifi-cation of 377 human microRNAs and three TaqManMicroRNA Assay endogenous controls. QPCR was per-formed using a 7900HT Fast Real-Time PCR system

(Applied Biosystems) using SDS software v2.4. Resultswere analysed using DataAssist Software v2.0 using thecomparative cycle threshold (2�DDCt) method to calcu-late fold changes.

TaqMan Gene expression array assays

Total RNAs were extracted from human PH5CH8 cellsor mouse liver tissue and reverse-transcribed using theHigh-Capacity cDNA Reverse Transcription Kit accord-ing to the manufacturer’s protocol. Customized humanand mouse TGF-b and EMT TaqMan Array Gene Signa-ture Plates for PCR amplification were designed usingthe assays validated by the manufacturer, which allowaccurate quantification of 92 genes and four endoge-nous controls. 50 ng cDNA was added per well of eacharray. QPCR was performed using a 7900HT Fast Real-Time PCR system using SDS software v2.4. Results wereanalysed using DataAssist Software v2.0 using the com-parative cycle threshold (2�DDCt) method to calculatefold changes.

RNA Extraction and real-time PCR analysis

Total RNA was extracted from cells and tissue using Tri-zol or RecoverAll Total Nucleic Acid Isolation Kit forparaffin-embedded tissue. Reverse transcription formRNAs was performed using High-Capacity cDNAReverse Transcription Kit or for microRNAs the Taq-Man MicroRNA Reverse Transcription Kit. The expres-sion of mRNA was measured using a TaqMan probe orTaqMan probe specific for miR-181a (AACAUUCAACGCUGUCGGUGAGU) normalized to U6 RNA. Theexpression of each gene and miR was defined from thethreshold cycle (Ct), and relative expression levels werecalculated using the 2�DDCt method.

miR mimics and inhibitors

PH5CH8 cells were transfected with miRIDIAN miR-181a mimic or mirCURY LNATM miR-181a inhibitorusing LipofectamineTM 2000 according to the manufac-turer’s protocol. After 24–48 h, cells were incubatedwith TGF-b as stated above.

In vivo tissues

Diethylnitrosamine (DEN)-induced HCC tissue was akind gift from Dr Narci Teoh. All human tissue wasapproved by the Sydney Local Health Network EthicsReview Committee, RPAH Hospital (Australia). Toinduce liver injury, 8-week-old C57BL/6 female micewere administered 300 mg/ml thioacetamide (TAA) adlibitum through the drinking water up to 24 weeks.Carbon tetrachloride (12% CCl4 in paraffin oil) wasinjected 5.36 ll/g twice weekly for 8 weeks. Tissuesfollowing rAAV injection were examined by SMA immu-nofluorescence, H&E and Sirius red staining. For GFP


Brockhausen et al. miR-181a mediates TGF-b-induced hepatocyte EMT

visualization, mouse liver tissues were fixed in 4% para-formaldehyde for 4 h, 15% sucrose/PBS for 4 h, 30%sucrose overnight at 4°C and then embedded in OCTcompound. Cryosections were stained 1:500 a-SMA for1 h and analysed by confocal laser scanning microscopy(Leica DM IRE2 inverted microscope equipped with aLeica TCS SP2 system, Germany). For H+E and Siriusred staining, tissues were collected in 10% bufferedformalin, embedded in paraffin and stained using stan-dard protocols. All animal experiments were approvedby the University of Sydney Animal Ethics Committee.

rAAV production and vector delivery

rAVV vectors were based on AAV type 2 pseudotypedwith AAV type 8 (rAAV2/8) to confer liver tropism. All

vectors were produced based on the pAM2AA backbonein which the human alpha-1-a-trypsin (hAAT) pro-moter coupled with two Apolipoprotein E (APoE) enh-ancers drives hepatocyte-specific transgene expression.MiR-181a-GFP and Scrambled-GFP were restrictiondigested from pEZX-MR04 and pEZX-CR04 Omic-sLinkTM Expression plasmids (GeneCopoeiaTM, Rockville,MD, USA) and cloned into XbaI/HindIII pAM2AAmulticloning site. Vectors were packaged by calciumphosphate triple transfection of human embryonickidney 293 cells with either miR-181a-GFP-pAM orScrambled-GFP-pAM with p5E18-VD2/8 and pXX6adenoviral helper plasmid. Cells were lysed by threefreeze/thaw cycles and 1011 viral genomes per mousewere delivered via intraperitoneal injection. All micewere purchased from the Animal Resource Centre

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Fig. 1. PH5CH8 cells undergo an EMT-like change in phenotype in response to TGF-b. (A) PH5CH8 cells were incubated 4 and 7 days with5 nM TGF-b and labelled with anti-vimentin (top panels) and E-Cadherin (bottom). Four days after treatment, cells became elongated andby day 7 displayed typical, TGF-b-induced properties including a mesenchymal phenotype and a decrease in cell contact. (B) Western blotanalysis was performed on cells seeded from the same cell population used in the previous slides. TGF-b-treated cells showed an increase invimentin protein levels after 4 and 7 days, whereas E-Cadherin protein levels were significantly reduced (one-way ANOVA, N = 3, **P < 0.01,***P < 0.001). The E-Cadherin-to-vimentin ratio (C) was reduced in TGF-b-treated cells (one-way ANOVA, N = 3, *P < 0.05). Sample blotsare shown on the right.



(Perth, WA, Australia) and experiments were approvedby the University of Sydney Animal Ethics Committee.

FLOW cytometry

Hepatocytes were perfused as stated above and dead cellswere excluded by 40-6-diamidino-2-phenylindole stain-ing. Flow cytometric acquisition was performed using aFACSCanto II flow cytometer (Becton Dickinson,Sydney, NSW, Australia) and analysis was performed usingFlowJo software (Tree star Inc., Ashland, OR, USA).

Results

TGF-b induces EMT in PH5CH8 hepatocytes

Treatment of PH5CH8 hepatocytes with 5 ng/ml TGF-binduced a fibroblast-like phenotype over a 7-day period(Fig. 1). Immunofluorescence analysis showed theinduction of vimentin and a reduction in E-Cadherinexpression (Fig. 1A), which was confirmed by Westernblot with a sequential increase in vimentin and decreasein E-Cadherin expression (Fig. 1B). The change in vi-mentin-to-E-Cadherin ratio (Fig. 1C) highlights theshift towards a mesenchymal phenotype. Western blotanalysis from cells treated 4 days with TGF-b alsorevealed an increase in smooth muscle actin (SMA)expression and zymography showed an increase inMMP2 expression (Fig. S1 A,B respectively).

mRNA profiling of TGF-b-treated PH5CH8 hepatocytes

To expand the molecular phenotype of the TGF-b-trea-ted PH5CH8 cells, mRNA profiling was undertakenusing EMT and TGF-b gene arrays. Cells were treatedfor 4 days with TGF-b; total RNA was extracted andanalysed using human TGF-b and custom human EMTTaqMan gene arrays (Table 1). Genes that were up- ordown-regulated in each array are listed in Table 1. TheEMT array showed significantly additional EMT-associ-ated genetic changes, notably the solid induction ofSnail and the metalloproteinases MMP2 and 9.

miR profiling of TGF-b-treated PH5CH8 hepatocytes

A key aim of these experiments was to identify mi-croRNAs that might regulate EMT. Total RNA wasextracted from TGF-b-treated PH5CH8 cells and anal-ysed using TaqMan array microRNA cards. Fourpotential miRNA candidates were revealed (Table 2).MicroRNAs over-expressed in this system includedmiR-181a and miR-483-5p (up-regulated 9.7-fold and2.8-fold, respectively, with TGF- b treatment) andthose down-regulated included miR-450-5p and miR-125a (down-regulated 0.4- and 0.7-fold respectively).Interestingly, no major changes in miR-200/205 werenoted. Based on these results, miR-181a was chosenfor further study.

miR-181a expression in TGF-b-treated PH5CH8hepatocytes

Up-regulation of miR-181a in TGF-b-treated PH5CH8cells was confirmed by real-time PCR showing a six- toeight-fold increase over 4–7 days of treatment (Fig. 2A).MiR-181a expression also increased 3.5-fold in AML12cells (a mouse hepatocyte cell line) treated 2 days with

Table 1. Genes that were up- or down-regulated after 4 days ofTGF-b treatment in PH5CH8 cells as seen by EMT or TGF-b TaqManarray analysis (n = 2)

EMT array TGF-b arrayGene Fold change Gene Fold change

UpARHGEF9 3.56 ACVR1 2.80CALD1 1.51 ACVR2A 1.71CAMK2N1 1.92 ACVR2B 1.26CDH2 1.89 FNTA 1.86CHD1L 5.37 GDF15 1.70COL3A1 2.41 HIPK2 1.61FN1 2.57 LTBP2 9.65ITGA5 1.53 LTBP4 2.14ITGAV 1.81 ROCK1 1.69ITGB1 1.65 SKP1 1.37JAG1 1.33 SMAD5 1.48MMP2 8.38 TGFA 3.48MMP9 3.72 TGFB1 4.25MSN 1.41 TNF 1.64NODAL 2.59 TGFBR1 1.64PDGFRB 4.72PLEK2 1.51SERPINE1 102.89SIP1 4.54SNAI1 5.57SNAI2 2.82SPARC 1.62TGFB1 4.75VIM 5.61WNT5B 4.35DownCDH1 0.68 BMP5 0.64CDKN1B 0.41 E2F5 0.44MST1R 0.86 EP300 0.62TFPI2 0.79 SMAD6 0.51TSPAN13 0.64 BMP5 0.64

Table 2. MicroRNAs that were significantly up- or down-regulatedafter 3 days of TGF-b treatment in PH5CH8 cells as seen by Taq-Man low-density array analysis (n = 3)

miR Fold change

UpmiR-181a 9.7miR-483-5p 2.81DownmiR-450b-5p 0.41miR-125a 0.72



TGF-b and 5-fold for primary mouse hepatocytes after2 days of TGF-b treatment (Fig. 2B, C). This up-regula-tion was not hepatocyte-specific as TGF-b-treated LX-2cells (a human stellate cell line) showed similar increasesin miR-181a (Fig. 2D).

miR-181a induces EMT

To explore the functional effects of miR-181a itself,we examined the effects of miR-181 over-expressionin the in vitro model of EMT. PH5CH8 cells trans-fected with miR-181a mimic showed up to 100-foldup-regulation. The transfected PH5CH8 hepatocyteswere then examined for phenotypic changes. Cellstransfected with miR-181a mimic alone (Fig. 3A, eand f) demonstrated a similar phenotypic change asthose treated with TGF-b by day 7 (c and d) com-pared to controls (a and b).

Anti-miR-181a prevents TGF-b-induced EMT

In parallel experiments, the miR-181a inhibitor was alsoused. RNA extracted from cells transfected with miR-181a inhibitor and treated with TGF-b showed a reduc-tion (close to baseline value of 1) in the TGF-b-inducedmiR-181a up-regulation normally seen with TGF-b(10-fold) or miR-181a mimic (100-fold; data notshown). Phenotypically, the inhibitor had a dramaticeffect (k and l) and prevented the TGF-b-inducedphenotypic change (l vs d).

The regulation of the EMT phenotype by miR-181mimic or inhibitor was confirmed by Western blots(Fig. 3B). After 7 days, E-Cadherin protein levelsdecreased in miR-181a and TGF-b-treated cells (Fig. 3Ca) with a concurrent decrease in vimentin protein(Fig. 3 Cb) and these changes were reversed using themiR-181a inhibitor. These experiments revealed thatmiR-181a could replace TGF-b as an inducer of in vitroEMT in this system and that in hepatocytes miR-181awas essential in TGF-b-mediated EMT.

mRNA profiling of PH5CH8 hepatocytes transfected withmiR-181a mimic/inhibitor and treated with TGF-b

We next analysed the mRNA changes induced by TGF-band the miR-181a. Total RNA was extracted fromPH5CH8 cells, which were (i) treated with TGF-b, (ii)transfected with miR-181a mimic or (iii) transfectedwith miR-181a inhibitor and treated with TGF-b andanalysed using EMT or TGF-b gene arrays. Table 3 indi-cates genes that were either up-regulated in cells treatedwith TGF-b or transfected with miR-181a mimic(groups i and ii) and concurrently down-regulated withmiR-181a inhibitor + TGF-b treatment or vice versa(up-regulated by a 1.5-fold increase or more/down-reg-ulated by 0.5-fold or less). Notable were the significantnumber of changes caused by the miR-181a mimic andthe reversal of these by the miR181a inhibitor. In partic-ular, Snail and MMP genes were fully reversed by themiR-181a inhibitor.

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Fig. 2. MiR-181a expression increases with TGF-b treatment. (A) PH5CH8 hepatocytes were treated for up to 7 days with 5 ng/ml TGF-b.RNA was extracted and QPCR was performed using miR-181a TaqMan probe. Seven-fold increases were seen by day 4 (one-way ANOVA,N = 9, **P < 0.01). (B) AML12 hepatocytes were treated for 2 days with 5 ng/ml TGF-b and RNA extracted. QPCR showed three-foldincreases in miR-181a levels after 24-h treatment (one-way ANOVA, N = 3, *P < 0.05). (C) Primary hepatocytes isolated from mouse liver weretreated for 2 days with TGF-b and showed an up-regulation in miR-181a levels. (D) LX-2 stellate cells were treated up to 2 days with TGF-band showed up to three-fold increases in miR-181a (one-way ANOVA, N = 3, ***P < 0.001).



BIM protein expression in PH5CH8 hepatocytestransfected with miR-181a mimic/inhibitor and treatedwith TGF-b

MiR-181a has been shown to have several interestingtargets including more than 100 C2H2 Zn finger tran-scription factors, the tumour suppressors RB1 andRBAK, and the pro-apoptotic protein BIM. BIM isknown to be expressed in hepatocytes and has been

shown to play a pro-apoptotic role in several signallingpathways (18, 19). Therefore, we investigated BIMexpression in our system. Cells treated with TGF-b,miR-181a mimic or TGF-b plus miR-181a mimic dis-played a marked reduction in BIM after 5 and 9 days oftreatment (Fig. 4A), and cells transfected with miR-181a inhibitor and treated with TGF-b showed nodecrease, indeed a slight up-regulation of BIM(Fig. 4A). As TGF-b is pro-apoptotic, the remaining

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Fig. 3. MiR-181a over-expression mimics TGF-b treatment. (A) PH5CH8 hepatocytes were transfected with miR-181a mimic (e–h) or miR-181a inhibitor (i–l) and treated from 4 to 7 days with 5 ng/ml TGF-b (c–d, g–h, k–l). At day 4, cells transfected with miR-181a mimic and/ortreated with TGF-b take on a mesenchymal appearance (c, e, g). By 7-day treatment, cells transfected with miR-181a mimic and/or treatedwith TGF-b acquire a fully mesenchymal phenotype (d, f, h). However, cells transfected with miR-181a inhibitor block the TGF-b-inducedtransformation (k, l). (B) Western blot analysis was performed on cells transfected with miR-181a mimic or inhibitor and/or treated with TGF-b and probed with anti-E-Cadherin and vimentin. (C) Lysates probed with anti-E-Cadherin and normalized to GAPDH (a) showed decreasesin E-Cadherin expression when transfected with miR-181a mimic or treated with TGF-b. Lysates probed with anti-vimentin (b) showeddecreases in vimentin expression when transfected with miR-181a mimic or treated with TGF-b. The E-Cadherin-to-vimentin ratio (D) wasreduced in cells transfected with miR-181a mimic or treated with TGF-b (one-way ANOVA, N = 3, *P < 0.05).



cells, which have evaded apoptosis, remained on the cul-ture dish and showed reduced levels of BIM (Fig. 4D),whereas the cells with increased BIM expression wouldhave undergone apoptosis and were washed away inthe cell media. Cells transfected with miR-181a mimicdisplayed a concurrent increase in vimentin and a

reduction in E-Cadherin protein (Fig. 4B, C). Samplesblots are illustrated in Figure 4D. However, BIM mRNAlevels remained unchanged following 7 days of TGF-btreatment (Fig. 4E), indicating that the effects of miR-181a on BIM are at the post-translational level.

In vivo regulation of miR-181a

TGF-b is a well-characterized cytokine known to be up-regulated within the liver in almost all forms of chronicliver disease (20). If miR-181a is indeed a mediator ofTGF-b, then diseased tissues should display appropriatechanges in its level. We therefore looked to quantifymiR-181a within experimentally induced chronic liverdisease models and in human liver tissues. MiR-181awas shown to be up-regulated two- to threefold in aTAA model of cirrhosis after 24 weeks of treatment(Fig. 5A) and four-fold in a CCL4 model of liver fibrosisafter 6 weeks of treatment (Fig. 5B). Mice treated withthe hepatotoxin DEN showed an up-regulation of miR-181a at 9 and 12 months in the HCCs and surroundingtissues as did mice with metastatic HCCs at 12 months(Fig. 5C). Human alcoholic cirrhotic livers showed up-regulation of miR-181a (Fig. 5D), and further analysisof human liver samples showed an increase in miR-181aexpression in HCV (hepatitis C virus)-induced cirrhotictissue with or without a HCC present compared withdonor tissue (Fig. 5E). Interestingly, up-regulation inHCV cirrhotic tissue where there was a HCC presentwas less than in HCV cirrhotic patients alone. There wasan increase in miR-181a in HCC tissue compared withdonor liver tissues (Fig. 5F, bars 1 and 2). However,miR-181a levels were reduced in the HCC nodules com-pared with the surrounding cirrhotic tissue from whichthe HCC was extracted (Fig. 5F, bars 2 and 3).

In vivo rAAV microRNA expression

To determine whether miR-181a could induce EMT-likecharacteristics in vivo, we used a recombinant adeno-associated virus (rAAV) system, which is hepatocytespecific. The miR-181a-GFP-rAAV and the controlGFP-rAAV were equally effective in transducing HEK-293 cells as judged by Flow cytometry (Fig. 6C). Liverstransduced with rAAV showed high expression levels ofmiR-181a-GFP and scrambled-GFP after 1 and 8 weeksof expression (Fig. 6A). miR-181a expression wasincreased four- to eight-fold as measured using real-time PCR in hepatocytes from GFP-positive livers(Fig. 6 Ba) and from total GFP-positive liver samples(Fig. 6 Bb). Analysis of the genes up- or down-regulatedin these hepatocytes as a result of the miR-181a over-expression (Table 4) indicates that miR-181a alone caninduce EMT-associated gene expression as BMP1, thecollagens, MMP2/MMP9 and Snail were all up-regu-lated. However, no change in actual cellular phenotype(hepatocyte size, shape or morphology) on H & E stain-ing was seen (data not shown). There was also no

Table 3. TGF-b and EMT genes that were up- or down-regulatedwith miR-181a mimic or inhibitor in vitro or in vivo*

TGF-b Mimic lnh + TGF-b

EMT arrayARHGEF9 up up downCALD1 up up downCAMK2N1 up up downCDH2 up up downCHD1L up up downFN1 up up downITGA5 up up downITGAV up up downITGB1 up up downMMP2 up up downMMP3 up up downMMP9 up up downMSN up up downPDGFRB up up downPLEK2 up up downSERPINE1 up up downSIP1 up up downSNAI1 up up downSNAI2 up up downTGFB1 up up downVIM up up downWNT5B up up downCDH1 down down upCDKN1B down down upMST1R down down upNUDT13 down down upTFPI2 down down upTGFB2 down down upTGFB3 down down upTSPAN13 down down upTGF-b arrayBMPER up up downFNTA up up downLTBP4 up up downSKP1 up up downTGFB1 up up downTGFBRl up up downTGFBRAPl up up downTNF up up downBMP5 down down upE2F5 down down up

*Results reflect increases greater than 1.5-fold or decrease greater than

0.5-fold. Genes that were up- or down-regulated with TGF-B, miR-

181a mimic or miR-181a inhibitor + TGF-B. PH5CH8 cells were treated

for 4 days with TGF-B, miR-181a mimic or miR-181a inhibitor + TGF-B;

RNAs were analysed using customized human and mouse EMT or TGF-

B TaqMan Array Gene Signature Plates. EMT or TGF-B-related genes

that were up-regulated with TGF-B or 181a mimic treatment and

down-regulated with miR-181a inhibitor + TGF-B are shown.



change in fibrosis markers (Sirius Red staining, vimen-tin and a-SMA immunofluorescence). As GFP-rAAVinjection showed no expression in other tissues and wasconfined to hepatocytes, other tissues were not exam-ined for altered microscopic appearance.

Discussion

The key findings in this study are firstly that miR-181acan induce an EMT-like response in hepatocytes and

can mimic the effects of TGF-b and secondly over-expression of miR-181 in vivo can induce many EMT-associated genes (Fig. 3 and Table 3). Thus, we wouldsuggest that miR-181a is of major importance in liverdisease. In support of this, we further show that miR-181a is dysregulated in chronic liver disease and HCC(Fig. 5).

In our initial investigations to determine miRNAsthat are critical to EMT in the liver, we were surprisedby the lack of regulation of the miR-200 series, as this

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Fig. 4. PH5CH8 cells transfected with miR-181a mimic or inhibitor and treated with TGF-b showed changes in BIM protein. (A) Cells treatedwith TGF-b, transfected with miR-181a mimic or both showed decreases in BIM protein at 5 and 9 days with corresponding increases invimentin (B) or decreases in (C) E-Cadherin protein (one-way ANOVA, N = 3, *P < 0.05). Samples blots are shown in (D). BIM mRNA levelswere unchanged (E).



has been linked to TGF-b-induced EMT in other sys-tems (Table 2). Indeed, downstream targets of the miR-200 series, Twist, Slug and ZEB were also not changedin the hepatocytes confirming the lack of involvementof this pathway in hepatocyte EMT (Table 3). As ourresults here show that miR-181a is important in hepato-cyte EMT, it suggests a tissue specific use of miRNAs forthis phenotypic change.

Our data on the TGF-b and miR-181a-induced EMTchanges show both induced and expected morphologi-cal changes, MMP2 increases, a corresponding increasein SMA, vimentin and a decrease in E-Cadherin proteinby 7 days (Figs 1, 3 and S1). The similarity of TGF-band miR-181a was also confirmed by examining changesin mRNA expression using EMT and TGF-b gene arraysat day 4 (Table 1). A very similar set of genes showed

up-regulation, which included LTBP2, TGF-b1, TGF-a,ARHGEF9, CHD1L, MMP2, MMP9, PDGFRB,SERPINE, SIP1, SNAI1, VIM, WNT5B and highlydown-regulated genes included SMAD6 and CDKN1B.Interestingly, miR-181a over-expression also duplicatedsome of the more immediate downstream targets ofTGF-b and induced TGFb itself suggesting a feedbackloop commonly seen with miRs (Table 3). The impor-tance of miR-181a in the TGF-b pathway of EMTinduction was demonstrated by the miR-181a inhibitoras this was able to suppress at both the morphologicaland molecular levels.

MiR-181a has been described as having a propensityfor targeting C2H2-type transcription factors, andindeed, we find that Snail, a factor known to mediateEMT, is regulated. Another reported target of miR-181a,

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Fig. 5. MiR-181a is expressed in liver injury models. QPCR showed that miR-181a expression is increased in mice after a (A) 24-week treat-ment with TAA (N = 3), (B) 6 weeks with CCl4 (N = 5) and (C) 9 months with DEN (N = 4). MiR-181a expression is increased in 9-month-old mice with developed HCCs and the surrounding tissue (C) and 12-month-old mice with developed and metastatic HCCs. (D) Humanalcoholic cirrhosis (N = 5) showed increases in miR-181a (one-way ANOVA, **P < 0.01). MiR-181a expression is increased in (E) human HCV-induced HCC liver tissue (N = 6) and HCV-infected liver tissue (one-way ANOVA, *P < 0.05, ***P < 0.001). RNAs extracted from paraffin-embedded human HCC liver tissue (F) and human cirrhotic liver tissue (F) showed higher increases in miR-181a in cirrhotic tissue (N = 10,one-way ANOVA, *P < 0.05).



recently seen to be involved in breast cancer metasta-sis, is the anti-apoptotic protein BIM. BIM is indeedregulated by miR-181a in hepatocytes and furtherinhibition of miR-181a reversed BIM down-regulation(Fig. 4). This is represented by the population ofremaining cells treated with miR-181a mimic andTGF-b, which have evaded apoptosis and subse-quently showed reduced levels of BIM expressioncompared with miR-181a inhibitor-treated cells(Fig. 4D). Previously, chronic TGF-B-b stimulation

via an autocrine loop was shown to decrease BIM lev-els and protect BIM levels in breast cancer cells andto protect cells from apoptosis (21). BIM function ispositively and negatively regulated by transcription,phosphorylation and degradation (22); we wouldtherefore expect the total protein detected in ourWestern Blots to include phosphorylated and non-phosphorylated BIM. However, BIM mRNA levelsremained unchanged suggesting a post-transcriptionalmechanism, which is typical of the actions of manymiRs (19).

The role of EMT in carcinogenesis is well docu-mented, and in the liver, the importance of EMT in can-cer progression was recently underlined by the discoveryof the role of Maelstorm, a gene over-expressed in can-cer and driving the expression of the transcription factorSnail. The origins of HCC also include the activation ofTGF-b signalling (23), which has been identified as amaster regulator or EMT (24). We observed increasedlevel of miR-181a in experimental and human HCCcompared with normal liver (Fig. 5). However, inhuman disease, it should be noted that levels in HCCtissues were lower than in the surrounding cirrhoticliver. This is consistent with the findings of Gramantieriet al., 2007 (25) in which genome-wide miRNA micro-arrays identified miR-181a/c as down-regulated in HCCvs the cirrhotic liver. Intriguingly, the levels of miR-181a in cirrhotic liver that contained at a distant a HCChad lower levels than cirrhotic livers that had no HCC.

We believe that the overall experimental data in thisstudy are novel and integrate well into the known litera-ture regarding the miR-181series. MiR-181a can func-tion as both a tumour suppressor and an oncogenedepending on the targeted gene. MiR-181a levels wereshown to be down-regulated in primary squamous lungcell carcinoma (26), oral squamous cell carcinoma (27),non-small-cell lung cancer (28), glioblastoma tumours(29) and up-regulated in breast cancer (14), MCF-7breast cancer cells (30), EpCam/AFP positive HCC (31,32) and gastric cancer (33). TGF-b has also been shownto up-regulate miR-181a in breast cancer cells (16), in aHepG2 HCC cell line (13, 16), and miR-181 wasrequired for activin/TGF-b-mediated cell migration andinvasion in human breast cancer (34). Mir-181a was up-regulated at the early stages of hepatocarcinogenesis incholine and amino acid-defined fed mice [CDAA; (13)]and is associated with poor prognosis of colorectal can-cer (35). Furthermore, miR-181a has recently beenshown to be a TGF-b-regulated ‘metastamir’ in breastcancer by promoting EMT (14). Inactivation of miR-181a elevated BIM protein levels and miR-181a expres-sion was linked to decreased overall survival in humanbreast cancer patients. The family member miR-181b,which shares an identical seed sequence to miR-181a,was also found to be up-regulated in a feeding CDAAdiet mouse model, which induces HCC (15). MiR-181bincreased the expression of MMP2/9, promotedgrowth and invasion of HCC cells and up-regulation

Table 4. Mice expressing rAAV miR-181a for 4 weeks showed up-or down-regulation of TGF-P or EMT genes*

Assay Up Down

TGF-b arrayBmpl upBmp7 downCaldl upCdknlb downChdll upColla2 upCol Sal upCol5a2 upEpcam upFnl upGngll upIgfbp4 uplllrn upMmp2 upMmp9 upNotchl upOcln downRacl upSnail upWnt5a upWnt5b upEMT arrayAcvrl upAcvr2a upBmp7 downBmper upBmpr2 downChrd downCull upE2f5 downEp300 upFmod downFnta downGdf2 upHipk2 upLtbp3 upRbll downSmadl downSmad2 downSmad9 upSmurfl downTabl upTsc22dl down

*Results reflect increases greater than 1.5-fold or decrease greater than

0.5-fold.



of miR-181b at the early stages of feeding-promotedhepatocarcinogenesis. It is clear that miR-181 familymembers are mediators in TGF-b-induced signallingpathways and in various types of cancer. In hepatocytes,the role of miR-181 thus appears, both from our experi-ments and from the literature, as having a potentialoncogenic role.

In addition to its role in carcinogenesis, TGF-b alsoplays a central role in regulating the progression fromthe early stages of fibrosis to cirrhosis. TGF-b is themain cytokine in the pathogenesis of liver fibrosis,which induces a wound-healing response (20). It drivesproduction of extracellular matrix proteins from hepaticstellate cells and liver myofibroblasts via endocrine andautocrine mechanisms. To establish an associationbetween miR-181a expressions in fibrotic liver disease,we examined hepatic expression levels in a series of dif-ferent mouse liver models and in human cirrhosis. Inmurine models of fibrosis (TAA and CCL4), there was

an increase in miR-181a expression levels (Fig. 5). Inhuman tissues, miR-181a was up-regulated in humanalcoholic cirrhosis and HCV-induced cirrhotic tissuesamples. These results suggest that miR-181a is morehighly up-regulated prior to the development of a carci-noma, during the chronic TGF-b response. This mayreflect TGF-b effects on two separate cell populations,the myofibroblast and the hepatocyte, whereas in HCC,the effects may reflect changes within the hepatocytepopulation alone.

To test whether manipulation of hepatocyte miR-181a in vivo altered cellular phenotype or liver geneexpression, we used a rAAV approach (Fig. 6). Recom-binant AAV approaches are commonly being used totransduce mouse liver as a means of gene expression(36). A hepatocyte-specific rAAV2/8 over-expressingmiR-181a-GFP or scrambled control was used as anin vivo gene delivery system. After 4 weeks of miR-181a-GFP expression, TGF-b and EMT genes were

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Fig. 6. Mice livers are transducable with rAAV expressing miR181a-GFP. (A) Mice were injected with 1 9 1011 copies of hepatocyte-specificrAAV-expressing miR-181a GFP or Scrambled-GFP. Mice culled after 1-week expression (top panel) revealed GFP-positive livers. Mice culledafter 8-week expression (bottom panel) were cryosectioned and revealed positive GFP-positive liver sections. (B) Hepatocytes were perfusedfrom 1-week and 4-week livers from mice injected with PBS control or miR-181a-GFP-rAAV (a). RNA was extracted and QPCR showedincreases in miR-181a after 1- and 4-week post-injection. Total liver was isolated from mice injected for 1 and 4 weeks with PBS control ormiR-181a-GFP-rAAV (b). RNA was extracted and QPCR showed increases in miR-181a after 1- and 4-week post-injection (one-way ANOVA,N = 3, *P < 0.05, **P < 0.01, ***P < 0.001). (C) Hepatocytes were perfused from 1-week and 4-week livers from mice injected with PBScontrol, miR-181a-GFP-rAAV or GFP-rAAV-positive control. Cells underwent FLOW cytometric analysis; GFP-rAAV-positive control hepato-cytes were near 100% GFP positive; miR-181a GFP hepatocytes were 92% positive.



altered in total liver (Table 4). However, there were novisible differences in cell phenotype or increased fibrosisbetween miR-181a expressing mice and controls (datanot shown). We should note that this may not be incon-sistent with our in vitro results where distinct andmarked phenotypic changes were seen. In our in vitroexperiments, the PH5CH8 cells have low levels of TGF-b gene expression at baseline (data not shown). Theselevels were enhanced by TGF-b itself and the miR-181amimic (Tables 1 and 3). Thus, the phenotypic changesmay require a TGF-b-activated pathway separate fromthe miR-181a pathway, although miR-181a is critical asthe inhibitor blocked the phenotypic changes. Thus, inthe absence of significant levels of TGF-b in normalliver, the addition of miR-181a results in a subset ofgene expression changes consistent with EMT, but withno associated phenotypic changes. The use of the rAAVexpression system in liver damage models, where highlevels of TGF-b would be present, has, however, proveddifficult. In unpublished experiments, we found thatrAAV expression was quickly lost following treatmentswith TAA and CCL4, likely as a result of the high hepa-tocyte turnover. Thus, miR-181a knockout mice (hepa-tocyte and non-hepatocyte specific) will be required totest whether in vivo manipulation of miR-181a coulddirectly alter the development of liver injury and HCC.

In conclusion, our data indicate that miR-181a hasa direct effect on inducing hepatocyte EMT and canreplace TGF-b-induced effects in vitro. Hepaticexpression in vivo is associated with increased liverinjury and occurrence of HCC. A functional in vivorole was associated with over-expression of EMT-asso-ciated gene expression in the short term, but furtherclarification is required to define major in vivo pheno-type changes.

Acknowledgement

Origins of PH5CH8: The authors acknowledge ProfessorN. Kato (37).

Conflict of interest: The authors do not have any dis-closures to report.

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Supporting information

Additional Supporting Information may be found in theonline version of this article:

Fig. S1. (A) PH5CH8 cells express albumin and showincreased expression of SMA after 48-h TGF-b treat-ment as shown by Western blot. (B) Gelatin zymogra-phy showed increased MMP2 proteolytic activity in theculture media of TGF-b-treated cells up to 3 days oftreatment.



mir-181a mediates tgf-β-induced hepatocyte emt and is dysregulated in cirrhosis and hepatocellular...

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