MiR-29a Assists in Preventing the Activation of Human Stellate Cells and Promotes Recovery From Liver Fibrosis in Mice

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1 originl rtile The Amerin Soiety of Gene & Cell Therpy MiR-9 Assists in Preventing the Ativtion of Humn Stellte Cells nd Promotes Reovery From Liver Firosis in Mie Yoshinri Mtsumoto,,3, Sori Itmi, Mshiko Kurod 4, Ktsutoshi Yoshizto 5, Norifumi Kwd nd Yoshiki Murkmi Deprtment of Heptology, Grdute Shool of Mediine, Osk City University, Osk, Jpn; Deprtment of Medil Nutrition, Grdute Shool of Humn Life Siene, Osk City University, Osk, Jpn; 3 Current ddress: Deprtment of Nutrition Mngement, Osk University Medil Hospitl, Osk, Jpn; 4 Deprtment of Moleulr Pthology, Tokyo Medil University, Tokyo, Jpn; 5 Phoenixio Co. Ltd, Hiroshim, Jpn The mirorna-9 (mir-9) fmily is known to suppress the tivtion of hepti stellte ells (HSCs) nd reversily ontrol liver firosis; however, the mehnism of how mir-9 ontrols liver firosis remins lrgely unknown. This study ws onduted to lrify the mehnism of nti-firoti effet of mir-9 nd to explore if mir-9 is promising ndidte for nulei id mediine ginst liver firosis. Two liver firosis murine models (ron tetrhloride or thioetmide) were used. MiR-9 mixed with teloollgen ws systemilly dministered. Hepti firosis ws evluted y histologil nlysis nd the expression levels of firosisrelted genes. We oserved tht mir-9 tretment drmtilly elerted the reversion of liver firosis in vivo. Additionlly, mir-9 regulted the mrna expression of ollgen type I lph (COLA) nd pltelet-derived growth ftor C (PDGFC). We lso noted tht mir-9 signifintly suppressed COLA mrna expression nd ell viility nd signifintly inresed spse-9 tivity (P < 0.05) in LX- ells. Pretretment of mir-9 inhiited tivtion of LX- ell y trnsforming growth ftor et tretment. MiR-9 exhiited nti-firoti effet without ell toxiity in vivo nd diretly suppressed the expression of PDGF-relted genes s well s COLA nd indued poptosis of LX- ells. MiR-9 is promising nulei id inhiitor to trget liver firosis. Reeived Jnury 06; epted June 06; dvne online pulition August 06. doi:0.038/mt.06.7 INTRODUCTION Firosis nd tissue remodeling in the liver led to irrhosis. Liver firosis is hrterized y exessive prodution nd deposition of extrellulr mtrix (ECM) mterils, leding to the destrution of the norml hepti prenhym nd disruption of the liver struture., The development of hepti firosis is triggered y the tivtion nd prolifertion of resident hepti stellte ells (HSCs). 3 Ativtion of HSCs is indued y prrine peptide nd lipid meditors derived from dmged heptoytes, tivted Kupffer ells, nd endothelil ells. The utorine loop of trnsforming growth ftor β (TGFβ) lso ontriutes to liver firogenesis. 4 Clinilly, irrhosis is oserved in ptients with loholindued liver injury, hroni heptitis B nd C, nonloholi stetoheptitis, nd other heptitis. 5 In spite of the use, the risk of heptoellulr rinom inreses y the stge of liver firosis, therefore, ontrolling liver firosis is key to preventing heptorinogenesis. There is inresing evidene tht firosis is dynmi nd reversile proess nd removing the primry use is the most effetive pproh for firosis regression. In hroni heptitis C virus infetion, tretment with ntivirl drugs is usully reommended for heptitis C virus erdition. Other pprohes to improving liver firosis involve ontrolling firosis-relted moleules inlude: (i) reeptor lignds, for exmple, peroxisome prolifertor-tivted reeptor lph gonist or diponetin, (ii) inhiitors of TGFβ, nd (iii) induers of the poptosis of tivted HSCs or the mtrix degrdtion (reviewed in ref. 6). However, sine side effets, suh s liver toxiity, mke it diffiult to pply these methods linilly, it is ritil to develop diretting nd sfe gents to ontrol liver firosis. MiroRNAs (mirnas) re endogenous smll nonoding RNAs tht ontrol gene expression y degrding or suppressing the trnsltion of trget mrnas. 7 Aerrnt expression of mirna is ssoited with the progression of liver firosis. 8 0 For exmple, the expression of mir-3 nd the mir-9 fmily (mir-9, mir- 9, nd mir-9) is signifintly underexpressed in firoti livers, s demonstrted in humn liver irrhosis nd in liver injury models indued y ile dut ligtion nd ron tetrhloride (CCl 4 ) in mie. Downregultion of these mirnas n modify the tivity of HSCs. 3 Serum mir-9 is signifintly lower in ptients with dvned liver irrhosis thn in helthy ontrols or ptients with erly firosis. Further it hs een shown tht overexpression of mir-9 in murine HSC resulted in the downregultion of ollgen expression,4 through mehnism tht diretly trgets the mrna expression of ECM genes. These findings suggest tht mir-9 my e n importnt therpeuti trget in hroni liver disese. The first two uthors ontriuted eqully to this work. Correspondene: Yoshiki Murkmi, Deprtment of Heptology, Grdute Shool of Mediine, Osk City University, Osk , Jpn. E-mil: yoshimurk@med.osk-u..jp vol. 4 no. 0, ot. 06

2 The Amerin Soiety of Gene & Cell Therpy MiR-9 Assists Reovery From Liver Firosis Reently, vrious forms of nulei id hve emerged s potentil tretments in the medil field. For instne, the use of nulei id ginst mir- for hroni heptitis C genotype infetion resulted in prolonged dose-dependent redutions in heptitis C virus levels without virl resistne. 5 Besides mir-, severl lterntive nulei id therpies re likely to e tested in future linil trils. 6 Bsed on these pplitions of mirnas, it is expeted tht mir-9 n ontrol liver firosis regrdless of the primry use. If liver firosis n e treted diretly, the development of irrhosis nd rinogenesis ould e repressed. Thus, the im of our study is to estlish methods of nulei id therpy for liver firosis using mir-9 nd to eluidte the mehnism of how mir- 9 represses liver firosis. RESULTS Liver firosis models Liver firosis ws indued in mie y dministering CCl 4 or thioetmide () for 5 weeks. Liver firosis level nd spontneous resolution fter esing firosis indution were exmined (Figure,g). CCl 4 nd livers demonstrted ridging firosis, ut this grdully reversed fter CCl 4 (Figure ) or (Figure h) tretment ws esed. During the 5-week dministrtion period, sirius red (SR)-positive re ws signifintly lrger in oth CCl 4 nd thn in oth groups of ontrol livers (orn oil or sline) (P < 0.05) nd time-dependently deresed in the - or -week oservtion groups (CCl 4 week, CCl 4 weeks; week, weeks oservtion) (P < 0.05) (Figure,i). Hydroxyproline level in the liver ws higher in CCl 4 nd models thn in the ontrol groups; in prtiulr, there ws signifint differene etween ontrol livers nd t 5 weeks (P < 0.05) (Figure d,j). To evlute the tivtion of HSC, the expression of four representtive genes ws exmined. The expression level of ollgen type I lph (COLA) in CCl 4 or ws signifintly higher thn in the ontrol nd - or -week oservtion groups (Figure e,k). The expression level of lph-smooth musle tin (α-sma) in CCl 4 ws signifintly higher thn the other groups. In CCl 4 nd, the expression level of mtrix metllopeptidse (MMP) ws signifintly higher thn the other groups (P < 0.05). In ddition, tissue inhiitor of metlloproteinse (TIMP) in ws signifintly higher thn in the others (P < 0.05). The expression level of mmu-mir-9 ws lower in CCl 4 - or -treted liver thn in the ontrols ut ws similr mong ll the groups (Figure f,l). Controlling liver firosis y dministering mir-9 Previous reports hve suggested tht mir-9 n ontrol HSC tivity. 9,0 The sequene etween mmu-mir-9 nd hs-mir- 9 ws onserved ross speies ( We evluted the ility of mir-9 to improve liver firosis using CCl 4 nd models estlished in Figure. MiRNA ws dministered for or weeks fter induing firosis for 5 weeks (Figure ). In CCl 4 model, histologil exmintion onfirmed tht mir-9 dministrtion for or weeks improved liver firosis (Figure,). Moreover, sed on the SR-positive re, we determined tht mir-9 elerted the resolution of liver firosis signifintly (P < 0.05) ompred to the negtive ontrol (NC) mirna groups (Figure d). Hydroxyproline level ws signifintly lower in -week/-week mir-9 groups thn in NC mirna groups (P < 0.05) (Figure e). The expression level of COLA mrna ws signifintly lower in the mir-9-treted groups thn in the CCl 4 -week oservtion groups (P < 0.05) (Figure f). In the model, histologil exmintion showed tht mir- 9 improved liver inflmmtion nd firosis (Figure 3,). The SR stining re in the groups dministered mir-9 hd signifintly deresed ompred to NC mirna or oth vehile groups (P < 0.05) (Figure 3). Hydroxyproline levels in mir-9-treted liver ws signifintly lower thn in NC mirna or oth vehile livers (P < 0.05) (Figure 3d). The expression level of COLA in the mir-9 group ws lower thn in the -week vehile group (Figure 3e). Elsti vn Gieson stining of liver smples showed tht elsti fiers deresed in the week/ weeks mir-9 group ompred with NC mirna group (Supplementry Figure S). Gene expression profile in mir-9-treted liver tissue To lrify the mehnism of the nti-firoti effet of mir-9, we performed omprehensive gene expression nlysis using mirorry. We hose genes with expression levels tht hd.5 times differene etween mir-9-treted group nd NC mirnatreted group. CCl 4 or livers tht underwent nd weeks of mir-9 tretment (- nd -week mir-9) hd 6 nd 5 upregulted genes nd 76 nd 8 downregulted genes, respetively, ompred to those treted with NC mirna (Figure 4 nd Supplementry Tles S nd S3). In CCl 4 nd groups tht reeived or weeks of mir-9 tretment, nine nd seven genes were ommonly upregulted nd downregulted, respetively (Figure 4 nd Supplementry Tle S4). Trget genes for mir-9 were predited from the 76 nd 8 downregulted genes using the online serh lgorithms of Trgetsn, mirtrse, nd mirorna.org. In ddition, genes tht were found in previous studies to e ssoited with firogenesis were seleted from the predited trget genes. Firinogen-like protein 7 nd pltelet-derived growth ftor C (PDGFC) 8,9 were hosen nd the expression level of these genes in -week mir-9 ws lower thn in NC mirna. Mitogen-tivted protein kinse kinse kinse kinse 4 (ref. 0) nd PDGFC were lso hosen nd genes with expression levels tht were lower in -week mir-9 livers thn in the NC mirna (Figure 4,). Applying these onstrints, we vlidted the expression of three genes y rel-time polymerse hin retion (PCR) nlysis (Figure 4d). In ddition, the expression of PDGFC ws ommonly lower in -week/-week mir-9 livers thn in the NC mirna groups. COLA, representtive ECM gene, expressed lower in mir-9-treted CCl 4 mouse thn in NC mirna-treted CCl 4 mouse. We lso identified COLA s mir-9 trget gene. MiRNA disposition kinetis of mir-9 We exmined mirna disposition kinetis of dministered mir- 9 in mie. Mie in whih liver firosis ws not indued were killed t 3, 6, 4, nd 48 hours fter eing dministered mir-9. Moleulr Therpy vol. 4 no. 0 ot

3 The Amerin Soiety of Gene & Cell Therpy MiR-9 Assists Reovery From Liver Firosis 0 week 5 weeks 6 weeks 7 weeks Control (orn oil) week oservtion weeks oservtion Control week oservtion weeks oservtion d e g f 0 week 5 weeks 6 weeks 7 weeks i Control (sline) week oservtion weeks oservtion h Control week oservtion weeks oservtion j k l Figure Preprtion of two hroni liver injury models. (,g) Tretment shedules for ron tetrhloride (CCl4) nd thioetmide (). (,h) Hemtoxylin nd eosin (upper rows) nd sirius red (SR) stining (lower rows) of liver. Sle r = 00 μm. (,i) SR stining positive re of liver. (d,j) Liver hydroxyproline levels. (e,k) The expression pttern of four firosis-relted genes (COLA, α-sma, MMP, nd TIMP) in liver. (f,l) The expression pttern of mir-9 in liver. P < 0.05 in Tukey HSD. Horizontl xis in ( f) nd (i l) shows whether CCl4 tretment ws done or not (CCl4) nd the timing of srifie (oservtion). In these mie, elevted mir-9 expression ws oserved in the erly phse fter mir-9 ws dministered (Figure 5). Liver mir-9 expression ws similr mong the CCl4 nd groups tht underwent -week nd -week mir-9 tretment (Figure 850 5). They were killed t 7 hours fter mir-9 ws dministered. In ddition, to onfirming tht mir-9 is ssoited with firosisregulted genes physiologilly nd funtionlly, we performed n rgonute-oimmunopreipittion (Ago-IP) nlysis to vol. 4 no. 0 ot. 06

4 The Amerin Soiety of Gene & Cell Therpy MiR-9 Assists Reovery From Liver Firosis 5 weeks 0 week 6 weeks 7 weeks mirna tretment for week mirna tretment for weeks week oservtion week vehile week NC mirna week mir-9 weeks oservtion weeks vehile weeks NC mirna weeks mir-9 f weeks mir-9 mir-9 mir-9 week Reltive expression week mir-9 e weeks week d weeks mir-9 mir-9 Figure Therpeuti effet of mir-9 in ron tetrhloride (CCl4)-treted mouse liver for nd weeks. () Summry of shedule for mirna tretment. (,) Hemtoxylin nd eosin (upper row) nd sirius red (SR) (lower row) stining in livers. Sle r = 00 μm. (d) SR stining positive re of livers. (e) Liver hydroxyproline levels. (f) The expression pttern of COLA in livers. P < 0.05 in Tukey HSD, P < 0.05 in student s t-test. oserve whether RNA-indued silening omplex (RISC) ould retin firosis-regulted genes when mir-9 ws overprodued in CCl4 -week NC mirna nd mir-9 liver (Figure 5). The mir-9 disposition kinetis in other orgns is shown in Supplementry Figure SA nd the expression of COLA ppers in Supplementry Figure SB. Moleulr Therpy vol. 4 no. 0 ot. 06 To determine if there ws ny toxiity ssoited with mir9 tretment, the rtion of ody weight (BW) (Supplementry Figure S3) nd the rtio of liver to BW (Supplementry Figure S4) were mesured. There ws not different mong eh mirnatreted groups. Alnine minotrnsferse nd sprtte minotrnsferse in serum inresed with CCl4 tretment, ut returned 85

5 The Amerin Soiety of Gene & Cell Therpy MiR-9 Assists Reovery From Liver Firosis week NC mirna week mir-9 weeks oservtion weeks vehile weeks NC mirna weeks mir-9 week weeks week weeks mir-9 mir-9 mir-9 d mir-9 week vehile week oservtion e week weeks mir-9 mir-9 Figure 3 Therpeuti effet of mir-9 in thioetmide ()-treted mouse liver for nd weeks. (,) Hemtoxylin nd eosin (upper row) nd sirius red (SR) (lower row) stining in livers. Sle r = 00 μm. () SR stining positive re of livers. (d) Liver hydroxyproline levels. (e) The expression pttern of COLA in livers. P < 0.05 in Tukey HSD, P < 0.05 in student s t-test. to norml when CCl4 tretment ws esed. MiR-9 injetion did not inrese these mrkers (Supplementry Figure S5). Funtionl nlysis of mir-9 in vitro To evlute the effet of mir-9 on HSC tivtion, we used n in vitro system. LX- is humn HSC ell line tht n e tivted y TGFβ (Figure 6). TGFβ tretment suppressed the expression level of mir-9 in LX- ells (Figure 6) nd inresed the expression levels of COLA, PDGFC, nd MMP (Figure 6). MiR-9 suppressed expression of these genes in 85 HSCs in the presene or sene of TGFβ (Figure 6). An immunolot showed tht COLA expression ws deresed y mir-9 tretment in presene of TGFβ (Figure 6d). XTT ssy reveled tht overexpression of mir-9 suppressed ell viility regrdless of TGFβ tretment (Figure 6e). In LX- ells with overexpressed mir-9, spse 9 tivity ws higher thn in NC mirna-trnsfeted LX- ells (Figure 6f). The effet of mir-9 in mouse-primry HSC ws lso exmined. In this ulture system, HSC ws tivted nd COLA vol. 4 no. 0 ot. 06

6 The Amerin Soiety of Gene & Cell Therpy MiR-9 Assists Reovery From Liver Firosis mir-9>nc mirna: week mir-9>nc mirna: weeks mir-9>nc mirna: nd weeks mir-9>nc mirna: week mir-9>nc mirna: weeks mir-9>nc mirna: nd weeks Miro rry d Figure 4 Mirorry nlysis of ron tetrhloride (CCl 4 ) nd thioetmide () liver firoti models nd mir-9 trget genes. () Venn digrm of the firosis-relted genes seletion riteri in six ptterns. Numer depited in ommon genes in two or four groups. Blk irle shows CCl 4 week, dotted irle shows week, old lk irle shows CCl 4 weeks, nd old dotted irle shows weeks. () MiR-9 trget site sequenes in 3ʹ UTR of PDGFC, FGL, MAP4K4, nd COLA. Numers left of 5ʹ-UTR sequenes show nuleotide from 5ʹ end. MAP4K4 nd COLA hve pleurl reognition site of mir-9. () The expression pttern of three trget genes from mirorry dt. (d) The expression pttern of three trget genes from rel-time PCR dt. PDGFC shows four smple s dt (CCl 4 week/ weeks nd week/ weeks). Fgl shows two smple s dt (CCl 4 / week). Mp4k4 shows two smple s dt (CCl 4 / weeks). P < 0.05 in student s t-test. expression inresed over time. MiR-9 ws trnsfeted t 4 hours fter isoltion (dy ). The ells were hrvested t dy 3 nd 4 (Supplementry Figure S6A). The expression of COLA in mir-9-treted HSC ws inhiited ompred to NC mirnatreted HSC (Supplementry Figure S6B). Pretretment of mir-9 n inhiit HSC tivtion MiR-9 ws trnsfeted with LX- ells nd the ulture medium ws hnged 6 hours lter to remove ny remining mir-9; TGFβ ws then dded t onentrtion of.5 ng/ml. The ells were hrvested t 4 nd 48 hours fter trnsfetion (Figure 7). Moleulr Therpy vol. 4 no. 0 ot

7 MiR-9 Assists Reovery From Liver Firosis The Amerin Soiety of Gene & Cell Therpy Reltive expression Reltive expression 0 0 mmu-mir hours 6 hours 4 hours 48 hours CCI 4 week weeks Reltive expression mmu-mir-9 Reltive expression 0 Figure 5 Phrmodynmis of mir-9 in mie. () The expression ptterns of mir-9 in liver 3, 6, 4, nd 48 hours fter injeting mir-9 without tretment with ny firoti induing regents. () The expression pttern of mir-9 in week/ weeks therpeuti ron tetrhloride (CCl 4 ) models. () The expression pttern of mir-9 in week/ weeks therpeuti thioetmide () models. (d) The expression pttern of mir-9 in o-immunopreipitted mirna with Ago in -week therpeuti CCl 4 model liver. White rs show NC mirna-treted liver nd lk rs show mir-9-treted liver. P < in student s t-test. week weeks Reltive expression 0 NC mirna mir-9 NC mirna mmu-mir-9 mir-9 The expression of COLA mrna ws higher in LX- ells treted with TGFβ thn those without TGFβ tretment. However, pretretment with mir-9 signifintly suppressed COLA gene expression espeilly in the TGFβ-treted groups. Moreover, on dy, in LX- ells trnsfeted with mir-9, COLA gene expression did not differ etween TGFβ-treted ells nd nontreted ells (Figure 7). The expression level of PDGFC in LX- ells with overexpressed mir-9 ws lower thn in NC mirnatreted ells on dy in the TGFβ nontreted group (Figure 7). In TGFβ-treted LX- ells, Western lot nlyses showed tht the expression level of COLA ws lower in mir-9 overexpressed ells thn in NC mirna-treted ells in presene of TGFβ (Figure 7). DISCUSSION In this study, we demonstrted two wys of ontrolling liver firosis with mir-9: one is through prevention nd the other is y repir. Controlling liver firosis through prevention In the ourse of preventing liver firosis, ECM prodution must e repressed y ontrolling mrna expression of ECM, inhiiting inflmmtion, nd y induing poptosis or quiesene of ECM-produing ells. MiR-9 n diretly ontrol COLA s trget gene nd repress the prodution of ECM (Figure 8). Dy Dy 0 Dy Dy Spred mirna trnsfetion Hrvest TGFβ d e f COLA βtin Figure 6 In vitro nlysis of mir-9 funtion. () Time ourse of experiment for LX- ells. () The expression level of mir-9 in LX- treted with TGFβ. () The expression level of firosis-relted genes in LX-. (d) Western lotting of COLA nd β-tin in LX- ells. (e) Cell viility test in LX- mesured y XTT ssy. (f) Apoptosis test in LX- verified y Cspse 9 tivity. (,e,f) White rs show NC mirna trnsfeted LX- nd lk rs show mir-9 trnsfeted LX-. P < 0.05 in student s t-test vol. 4 no. 0 ot. 06

8 The Amerin Soiety of Gene & Cell Therpy MiR-9 Assists Reovery From Liver Firosis Dy Dy 0 6 hours Dy Dy Spred mirna trnsfetion Hrvest Hrvest TGFβ Reltive expression COLA PDGFC MMP TGFβ() TGFβ() TGFβ() TGFβ() TGFβ() TGFβ() TGFβ() TGFβ() TGFβ() TGFβ() TGFβ() TGFβ() Dy Dy Dy Dy Dy Dy COLA βtin TGFβ() NC mirna mir-9 NC mirna TGFβ() mir-9 Figure 7 Inhiitory effet of TGFβ tivtion in LX- ells. () Time ourse of experiment. MiR-9 ws trnsfeted to LX- ells; the medium ws hnged fter 6 hours nd TGFβ ws dded. The ells were isolted 4 nd 48 hours fter trnsfetion. () The expression of firosis-relted genes in LX- ells. White rs show NC mirna trnsfeted LX- nd lk rs show mir-9 trnsfeted LX-. () Western lotting of COLA nd β-tin in LX- ells on dys. P < 0.05 in Tukey HSD, P < 0.05 in student s t-test. MiR-9 tretment downregulted the COLA expression in tivted HSC (Figure 6,d; Supplementry Figure S6), nd pretretment with mir-9 inhiited the upregultion of COLA resulting from TGFβ tretment (Figure 7,). The expression of COLA nd α-sma mrna inresed fter CCl 4 or tretment nd deresed fter these regents were esed. COLA mrna in model nd α-sma mrna in oth models hd lredy delined to nonstimulted stte y week of the spontneous reovery period (Figure e,k), nd no mir-9 effet ws oserved. However, the inhiition of COLA mrna expression in mir-9-treted mie ws oserved in CCl 4 model (Figure f). In regrds to the trget genes for mir-9 introdued in this study, ell viility ws downregulted (Figure 6e) nd spse9 tivity ws upregulted (Figure 6f) in mir-9-treted LX- ells. The PDGF pthwy is well known to e ssoited with firosis nd ell prolifertion, 9 nd mir-9 diretly reognized PDGFC (Figure 4). PDGF hs lso een implited in rs homolog fmily memer A mediting tin reorgniztion, hyperplsi of stress fier, nd ell dhesion., In ddition, PDGF elerted ell prolifertion vi mitogen-tivted protein kinse 8. 3 Previous reserh hs shown onnetion etween v-rf-leukemi virl onogene nd serum response ftor in the PDGF pthwy, nd these genes regulte ell prolifertion. 4,5 Mirorry nlysis showed tht the expression level of these PDGF-relted genes were lower in mir-9 liver thn in NC mirna liver (Figure 8). Thus, we elieve mir-9 my diretly regulte PDGFC expression s trget genes, nd then suppress ell migrtion nd ell prolifertion. Prior studies hve shown tht derese in firinogen-like protein expression in T ells inhiited HSC tivtion. 6 Our findings onur s mirorry onfirmed tht mir-9 tretment deresed firinogen-like protein (Figure 4 d nd Figure 8). Additionl reports hve suggested tht mitogen-tivted protein kinse kinse kinse kinse 4 plys role in heptoellulr rinom prolifertion nd epithelil mesenhyml trnsition nd metstsis. 6,7 We therefore elieve tht mir-9 my inhiit ell tivtion nd prolifertion y trgeting these genes (Figure 4 d nd Figure 8). In our mirorry nlysis, we oserved genes relted to liver firosis progression tht were downregulted in mir- 9-treted liver, however they were not trget genes for mir- 9 (Figure 8). Interleukin et regultes inflmmtion signl pthwy 8 nd elertes the expression level of MMP nd TIMP. 9 It ws reported tht N-my downstrem regulted regultes ngiogenesis in heptoellulr rinom, 30 nd B-ell leukemi/lymphom protein promotes poptosis. MiR-9 indiretly regulted these firosis-relted genes resulting in the suppression of liver firosis in this study. This hs led us to onlude tht mir-9 n prevent nd improve liver firosis diretly or synergistilly. Controlling liver firosis through repir In helthy liver, ECM is regulted y severl gene expressions, for exmple, MMP nd the TIMP fmily. In dmged liver, interleukin et n promote the expression of MMPs. In ddition, the expression of TIMPs is lso upregulted to inhiit MMP tivity nd led to ECM umultion 9 (Figure e,k nd Figure 8). It ws reported tht MMP expression inresed nd remined elevted during ECM umultion, however the upregulted TIMP expression deresed fter esing firosis indution in CCl 4 model mie. 9 The delined TIMP expression promoted MMP tivtion nd then led firolysis to the spontneous reovery proess. In oth CCl 4 nd liver firosis models, hydroxyproline nd SR stining re delined more rpidly in the mir-9treted group thn in the NC mirna-treted group (Figure e nd Figure 3 d). To lrify how mir-9 ontriute to the liver firolysis proess, we used quntittive PCR to ompre the Moleulr Therpy vol. 4 no. 0 ot

9 MiR-9 Assists Reovery From Liver Firosis The Amerin Soiety of Gene & Cell Therpy mir-9 Cell migrtion Cell prolifertion mir Inflmmtion Angiogenesis Apoptosis ECM resolution Figure 8 Shem of mir-9 for liver firosis. The summry of hypothetil effet of mir-9 on liver firosis in this study. The grphs show mirorry dt. White rs show NC mirna-treted liver nd lk rs show mir-9-treted liver. () MiR-9 n ontrol COLA, PDGFC, FGL, nd MAP4K4 s trget genes. The expression of four PDGFC pthwy genes, RhoA, MAPK8, RAF, nd SRF, were downregulted in the mir-9-treted group. () IL-B, NDRG, nd BCL were indiretly ontrolled y mir-9 nd expression pttern of these genes ws oserved in mirorry. expression rtio of MMP nd TIMP etween mir-9- nd NC mirna-treted mie liver were nlyzed (Supplementry Figure S7). CCl 4 or tretment upregulted MMP/TIMP expression rtio, however it eme downregulted during the spontneous reovery proess. In the group dministered mir-9, the rtio of MMP/TIMP remined higher thn in the NC mirna group, nd this my hve led to the more rpid progression of firolysis. MiR-9 tretment improved liver firosis through repir proess nd ould ontrol firolysis-relted genes indiretly euse MMP nd TIMP were not trget gene for mir-9. In the spontneous reovery period, liver firosis in model improved more rpidly thn in CCl 4 model. The effets of mir- 9 in oth models were not ompletely replited euse of the differene of their spontneous reovery time. However, the potentil of mir-9 to improve liver firosis ws ommon in oth models. This suggests tht liver firosis resulting from different uses my e ured using only one mirna. Phrmodynmis of mir-9 Ateloollgen, pepsin-treted type I ollgen lking telopeptides t the N nd C terminls tht onfer ntigeniity, hs een developed s highly ioomptile mteril. 3,3 Surfe hrge is importnt for ellulr uptke of nnoprtiles, nd positively hrged nnoprtiles show higher ell uptke thn negtively hrged prtiles. 33 We onfirmed the presene of phrmodynmis in mir-9 in mouse liver model using teloollgen. We surmised tht mir-9 rehed the liver in the erly phse of tretment (Figure 5). Elevted mir-9 expression ws not oserved in firoti mie (Figure 5) tht were killed 7 hours fter the finl injetion of mir-9. It would e fir to ssume therefore tht lthough mir-9 rehed the liver in the very erly phse vol. 4 no. 0 ot. 06

10 The Amerin Soiety of Gene & Cell Therpy MiR-9 Assists Reovery From Liver Firosis of tretment it did not sty for long time. In ddition, Ago-IP nlysis showed tht mir-9 expression ws onentrted in RISC in CCl 4 -week mir-9-treted liver (Figure 5). Administering mir-9 hs effets on nti-firosis in CCl 4 nd hroni liver injury models. Generlly, mirna hs lower gene-silening ility thn sirna nd mirna expression vetor; however, mirna ontrols more genes thn oth. To linilly pply nulei id-sed mediine, mirna expression y virl vetor is highly effiient gene trnsporter; however, the drwk is it results in high ntigeniity. In our study, hemtoxylin nd eosin stining showed tht dministering mir-9 did not result in ell toxiity or inflmmtion (Figure, nd Figure 3,), ody nd liver weight (Supplementry Figures S3 nd S4), nd serum lnine minotrnsferse nd sprtte minotrnsferse (Supplementry Figure S5). We hve presented method for reversing firosis without heptotoxiity nd hve shed light on how mir-9 ontrols liver firosis. The results presented here n mke n impt on the linil pplition nd nulei id drug tehnology. Additionl reserh tht lrifies the nti-firoti effet of mir-9 would e instrumentl not only for understnding liver firosis ut lso other firoti diseses. MATERIALS AND METHODS Mouse model of liver firosis. C57BL/6J 6-week-old mle mie (Jpn SLC, Shizuok, Jpn) were limted for week, nd lighting onditions were ontrolled in hours light nd hours drk yle. The niml protool ws pproved y the Institutionl Animl Cre nd Use Committee of the Osk City University (Permission Numer: 3043). Two hroni liver injury mouse models were reted to indue firosis y using: (i) CCl 4 nd (ii). CCl 4 ws mixed with orn oil nd intrperitonelly injeted t dose of 0.5 µl/g BW every 3 dys over 5 weeks for totl of injetions (Figure ). 34 ws resolved in sline nd given to mie t eslted doses from 00 µg/g BW (st dministrtion), 00 µg/g BW (from nd to 5th dministrtion), 300 µg/g BW (from 6th to 9th dministrtion), to 400 µg/g BW (from 0th to th dministrtion) every 3 dys (Figure g). 35 Control mie for CCl 4 nd were dministered orn oil only or sline only, respetively, nd killed t week 5. Development of liver firosis nd its spontneous resolution were onfirmed in oth CCl 4 nd models. Mie were oserved for or weeks without ny tretment fter esing 5 weeks of intoxition. Eh mouse model ws divided into three groups depending on the oservtion period: 0 week (5 week CCl 4 or ), week (5 week CCl 4 or week oservtion), or weeks (5 week CCl 4 or weeks oservtion) (Figure,g). At lest three mie were prepred for eh group. Administrtion of mir-9 in vivo. To evlute the effet of mir-9 on the resolution of liver firosis, mie with developed liver firosis y CCl 4 or were dministered mir-9 for or weeks immeditely fter the firosis indution period. Fifty mirogrms of doule-strnd mture mmu-mir-9 (mir-9) or nonspeifi oligonuleotide (negtive ontrol for mir-9: NC mirna) (Bon, Fukuok, Jpn) ws mixed with 00 µl of teloollgen (Atelo gene; KOKEN, Tokyo, Jpn) nd injeted vi the til vein every 3 dys. 36 MiRNA ws dministrted two times in mie treted for week nd four times in mie treted for weeks. Thus, mie for eh firoti model were now spred over eight groups: oserved without ny tretment ( week oservtion/ weeks oservtion), dministrted only teloollgen, vehile for nuleotides ( week vehile/ weeks vehile), dministrted NC mirna ( week NC mirna/ weeks NC mirna), or dministrted mir-9 ( week mir-9/ weeks mir- 9) (Figure ). Histologil nlysis. Livers were fixed with 0% formlin nd two seril prffin setions were mde t 5 μm. One setion ws used for hemtoxylin nd eosin. The other setion ws stined in pirosirius red (Sigm-Aldrih, St. Louis, MO) solution nd ounter stined with Fst Green (Sigm- Aldrih). 37 The SR-positive re ws quntified y MICROANALYZER (Nihon Poldigitl, Tokyo, Jpn). To vlidte the positive re of SR, five imges ontining t lest one portl vein were hosen. Hydroxyproline ssy. Liver hydroxyproline ws mesured using Hydroxyproline Assy kit (BioVision, Milpits, CA) following the mnufturer s instrution. The detiled proedures re s previously desried. 37 Cell ulture nd mirna trnsfetion. Humn HSC ell line LX-, donted y Sott L. Friedmn (Division of Liver Diseses, Mount Sini Hospitl), ws ultured with Duleo s modified medium (WAKO, Osk, Jpn) nd supplemented with 0% fetl ovine serum, 00 U/ml peniillin, 00 μg/ml streptomyin (Thermo Fisher Sientifi, Wlthm, MA), nd nonessentil mino id (Thermo Fisher Sientifi). 0 5 of LX- ells were seeded to six-well plte (Thermo Fisher Sientifi). 38 Forty miromolr of mir-9 or NC mirna ws trnsfeted to ells y lipofetmine RNAiMAX regent (Thermo Fisher Sientifi). In se of TGFβ tretment (Figure 5), ells were seeded t -dy supplemented with.5 ng/ml TGFβ (Sigm-Aldrih). In the next dy (dy 0), mirnas were trnsfeted for 48 hours nd ells were hrvested t dy. In the study tht nlyzing the effet of mir-9 to prevent HSCs tivtion (Figure 6), the ells were seeded t -dy. At dy 0, mirnas were trnsfeted to ells nd.5 ng/ml TGFβ ws dded to medium 6 hours fter trnsfetion. The ells were hrvested t dy or dy, respetively. RNA extrtion nd mrna nd mirna expression nlysis. Totl RNA ws extrted y mirnaesy mini kit (QIAGEN, Venlo, Netherlnds) ording to the mnufturer s instrution. Complementry DNA ws synthesized using the Trnsriptor First Strnd DNA Synthesis Kit (Rohe, Bsel, Switzerlnd). For rel-time PCR nlysis, FstStrt Universl SYBR Green Mster mix (Rohe) ws used. The sequenes of primers re listed in Supplementry Tle S. The retion ws performed in triplite. TqMn mirna ssy (Thermo Fisher Sientifi) ws used to quntify the reltive expression level of hs-mir-9 (ssy ID 00447) nd U8 (ssy ID 0004) s n internl ontrol. Complementry DNA ws synthesized using the TqMn mirna RT Kit (Thermo Fisher Sientifi). For rel-time PCR nlysis, Fst Strt Universl proe mster (Rohe) ws used. StepOnePlus Rel-Time PCR System (Thermo Fisher Sientifi) ws used to detet mirna nd mrna. The retion ws performed in triplite. Cell viility nlysis. 0 4 LX- ells were seeded to 96-well plte. Cell Prolifertion Kit II (XTT) (Rohe) ws used ording to the mnufturer s instrution. Briefly, 48 hours fter mirna trnsfetion, 50 μl of XTT regent ws dded to ells nd inuted for 6 hours t 37 C. The sorne t 450 nm ws mesured. Cspse 9 tivity ssy. 0 4 LX- ells were seeded to 96-well plte. Cspse-Glo 9 Assy kit (Promeg, Mdison, WI) ws used ording to the mnufturer s instrution. Immunopreipittion of Ago. CCl 4 -treted -week mir-9 nd NC mirna mie livers were used. MiroRNA Isoltion Kit, Mouse Ago (WAKO) ws used ording to the mnufture s instrution. The expression of mir-9 onjugted with Ago ws onfirmed y rel-time PCR. Moleulr Therpy vol. 4 no. 0 ot

11 MiR-9 Assists Reovery From Liver Firosis The Amerin Soiety of Gene & Cell Therpy Mirorry nlysis. Complementry RNA from 00 ng totl RNA ws leled with ynine-3 using the Low Input Quik Amp Leling Kit, oneolor (Agilent Tehnologies, Snt Clr, CA) ording to the mnufturer s protool. Frgmented RNA smples were hyridized to SurePrint G3 Mouse Gene Expression 8 60 K Mirorry Kit (G485A) (Agilent). After hyridiztion, pltes were snned (Snner G539A) nd nlyzed y Feture Extrtion (v0.0..). Dt were nlyzed with GeneSpring GX (v3.0.0) nd registered in NCBI Gene Expression Omnius repository (GenBnk ession no. GSE6743). Immunolot. Whole ell lyste ws homogenized in 50 mmol/l Tris-HCl (ph 8.0), 50 mmol/l NCl, 0.% sodium dodeyl sulfte, 0.5% deoxyholi id, nd % NP-40. The quntified superntnts (0 µg/well) were oiled in equl volumes of 0% glyerol ontining 5 mmol/l Tris-HCl (ph 6.8), 4% sodium dodeyl sulfte, 0% -merptoethnol, nd 0.0% romophenol lue. PAGEL (5 0%) (ATTO, Tokyo, Jpn) ws used for sodium dodeyl sulfte polyrylmide gel eletrophoresis nd trnsferred to polyvinylidene difluoride memrnes (Millipore, Billeri, MA) y sumrine type lotting. Memrnes were inuted with 5% skim milk/tris-uffered sline with Tween 0 for loking. Primry ntiodies ginst COLA (s-8784; Snt Cruz, Dlls, TX) or β-tin (s-69879; Snt Cruz), nd horserdish peroxidse-onjugted got nti-rit or nti-mouse seondry ntiodies (Dko, Glostrup, Denmrk) were used. Immunoretivity ws visulized y hemiluminesene (GE Helthre, Bukinghmshire, Englnd). Sttistil nlysis. For sttistil nlysis, nlysis of vrine nd Tukey honestly signifint differene (HSD) post ho test were used for multiple group dt, nd student s t-test ws used for independent two group dt (SPSS; IBM, Chigo, IL). P vlues <0.05 were onsidered sttistilly signifint. SUPPLEMENTARY MATERIAL Figure S. EVG stining of mie liver. Figure S. The expression of mir-9 nd Col in tissues fter mir-9 dministrtion. Figure S3. The hnges of ody weight in weeks treted mie. Figure S4. The rte of liver weight/ody weight. Figure S5. ALT nd AST in serum. Figure S6. The effet of mir-9 on COLA expression in primry mie HSC. Figure S7. The lne of MMP nd TIMP expression y rel-time PCR nlysis in time ourse. Tle S. Primer list for rel-time PCR. Tle S. Genes with hnging expression level in liver treted with mir-9 for week. Tle S3. Genes with hnging expression level in liver treted y mir-9 for weeks. Tle S4. Genes with hnging expression level in liver treted with mir-9 for nd weeks. ACKNOWLEDGMENTS Study onept nd design: Y.M. nd Y.M. Aquisition of dt: Y.M. nd S.I. Anlysis nd interprettion of dt: Y.M., S.I., nd Y.M. Drfting the mnusript: Y.M., S.I., nd Y.M. Critil revision of the mnusript for importnt intelletul ontent: M.K., K.Y., N.K., nd Y.M. Sttistil nlysis: Y.M. All uthors red nd pproved the finl mnusript. 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