Inhibition of Dexamethasone-induced Fatty Liver Development by Reducing mir-17-5p Levels

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1 originl rtile Inhiition of Dexmethsone-inue Ftty Liver Development y Reuing -5p Levels Willim W Du,, Fengqiong Liu 3, Sze Wn Shn,, Xini Ciny M,, Shn Gupt,, Tinru Jin 4, Dvi Spner, Sergey N Krylov 5, You Zhng 6, Wenhu Ling 3 n Burton B Yng, Sunnyrook Reserh Institute, Sunnyrook Helth Sienes Centre, Toronto, Ontrio, Cn; Deprtment of Lortory Meiine n Pthoiology, University of Toronto, Toronto, Ontrio, Cn; 3 Shool of Puli Helth, Sun Yt-sen University, Gungzhou, P.R. Chin; 4 Toronto Generl Reserh Institute, University Helth Network, Toronto, Ontrio, Cn; 5 Deprtment of Chemistry n Centre for Reserh on Biomoleulr Intertions, York University, Toronto, Ontrio, Cn; 6 Key L in Helthy Siene n Tehnology, Division of Life Siene, Grute Shool t Shenzhen, Tsinghu University, Shenzhen, P.R. Chin Stetosis is pivotl event in the initition n progression of nonloholi ftty liver isese (NAFLD) whih n e riven y peroxisome prolifertor-tivte reeptor-α (PPAR-α) ysregultion. Through exmining the effet of PPAR-α on ftty liver evelopment, we foun tht PPAR-α is trget of -5p. Trnsgeni mie expressing evelope ftty liver n proue higher levels of triglyerie n holesterol ut lower levels of PPAR-α. Etopi expression of enhne ellulr stetosis. Gin-of-funtion n lossof-funtion experiments onfirme PPAR-α s trget of -5p. On the other hn, PPAR-α oun to the promoter of n promote its expression. The fee-k loop etween -5p n PPAR-α plye key role in the inution of stetosis n ftty liver evelopment. Mie with high levels of -5p were sensitive to Dexmethsone-inue ftty liver formtion. Inhiition of -5p suppresse this proess n enhne PPAR-α expression in mie trete with Dexmethsone. Clofirte, Ciprofirte, n WY-4643: three gents use for tretment of metoli isorers, were foun to promote PPAR-α expression while eresing -5p levels n inhiiting stetosis. Our stuies show tht -5p inhiitor n gents use in metoli isorers my e pplie in omintion with Dexmethsone in the tretment of nti-inflmmtion, immunosuppression, n ner ptients. Reeive Novemer 4; epte 7 Mrh 5; vne online pulition 9 My 5. oi:.38/mt.5.64 INTRODUCTION Nonloholi ftty liver isese (NAFLD) represents spetrum of metoli synrome-ssoite liver isese progressing from simple stetosis, through nonloholi stetoheptitis (NASH) n firosis to irrhosis n heptoellulr rinom., While its risk ftors hve een lerly efine, the unerlying mehnisms of the isese progression remin poorly unerstoo. Although the generl pthogenesis inlues insulin resistne, oxitive stress, inflmmtion, heptoyte injury, ellulr poptosis, firosis, n rinogenesis, stetosis is the very initil n vitl step in the progress of NAFLD.,3 Gluoortiois re lss of steroi use extensively for ntiinflmmtion in ptients with llergies, sthm, utoimmune iseses, sepsis, n ners. They re lso use for ptients with metoli isorers. The sie-effets inlue hyperglyemi ue to inrese insulin resistne n impire gluose tolerne, leing to the evelopment of stetosis n ftty liver. 4 One of the most populr use rugs in this lss of sterois is Dexmethsone (), whih is 5 times more potent thn ortisol in its gluoortioi effet. Peroxisome prolifertor-tivte reeptor-α (PPAR-α) is nuler reeptor protein enoe y the PPARA gene. 5 It serves s trnsription ftor n mjor regultor of lipi metolism in the liver, whih is tivte uner nutrient-efiient onitions. 6 Inrese expression of PPAR-α promotes uptke, utiliztion, n tolism of ftty is. Upregultion of PPAR-α is epenent on the presene of ftty i synthse. 7 PPAR-α is lign-tivte trnsriptionl ftor tht n in to speifi PPAR-response elements of ertin genes with its heteroimeri prtner retinoi X for regultory purposes, 8 thus plying ruil role in intrellulr lipi metolism. Previous reports emonstrte tht PPAR-α exerte its role in hepti lipi metolism through tivting genes involve with ftty i β-oxition, 9 leing to upregultion of mlonyl-coa eroxylse to inrese trnslotion of ftty is into mitohonri for oxition,, n lowering the hepti sustrte for triglyerie synthesis y limiting its output from other orgns. Furthermore, PPAR-α reues e novo ftty i synthesis y loking enzymes like etyl-coa roxylse n ftty i synthse. 9 MiroRNAs (mirnas) re smll nonoing RNAs tht negtively regulte trget gene expression through prtil se piring with 3 untrnslte regions (UTRs).,3 With multiple n iverse trgets, mirnas ontrol ellulr proesses suh s prolifertion, 4,5 ivision, 6 ifferentition, 7 poptosis, 8 protein seretion, 9 n virl infetion. The speifi ontriution of The first two uthors ontriute eqully to this work. Corresponene: Burton B Yng, S, Sunnyrook Reserh Institute, Sunnyrook Helth Sienes Centre, 75 Byview Ave, Toronto M4N 3M5, Ontrio, Cn. E-mil: yng@sri.utoronto. Moleulr Therpy

2 mirna n Ftty Liver selete mirnas in hepti isese evelopment n progression hs een esrie. It hs een suggeste tht mirna my e relte to hepti stetosis formtion in NAFLD through iretly trgeting ownstrem moleules. For exmple, mir-, one of the most unnt mirornas in the liver, regultes holesterol iosynthesis in norml heptoytes. mir- inhiition in norml mie resulte in reue plsm holesterol levels, inrese hepti ftty-i oxition, n erese in hepti ftty-i n holesterol synthesis rtes. Another mirna ppers to ply roles in regulting liver funtions. 3,4 In this stuy, we teste the effets n reltionship of, -5p, n PPAR-α in the evelopment of stetosis n ftty liver, using -overexpressing trnsgeni mouse moel. We foun tht enhne expression of PPAR-α n -5p. MiR-7-5p ws foun to repress PPAR-α expression, while PPAR-α oul in to the promoter of mir- 7 n inrese its trnsription, forming feek loop in the regultion of stetosis n ftty liver evelopment. We onlue tht n inhiitor of -5p oul e use to reue the ourrene of stetosis n ftty liver evelopment uring Dexmethsone tretment. Finlly, we foun tht rugs use for the tretment of metoli isorers (WY-4643, Clofirte, n Ciprofirte), oul erese -5p expression levels n the -inue stetosis n ftty liver evelopment. RESULTS inue expression of n PPAR-α is gluoortioi lss of steroi rugs with potent effets on nti-inflmmtion n immunosuppression n sie-effets tht inlue stetosis n ftty liver evelopment. 4 Sine PPAR-α regultes lipi metolism, 6 we hve, first of ll, teste whether or not ffete PPAR-α expression. HepG, SNU449, n mouse primry heptoytes were trete with, followe y rel-time polymerse hin retion (PCR) for quntittively ssessing PPARA mrna levels n western lot nlysis proe with n nti-ppar-α ntioy. exhiite ose-epenent effet of enhning PPARA mrna expression (Figure ). The effet of on stetosis ws onfirme in HepG ells y mirosopi exmintion n stetosis quntifition (Supplementry Figure S). Further nlysis inite tht ells trete with lso expresse inrese levels of -5p (Figure ). To nlyze whether or not plys role in inuing stetosis, we exmine HepG ells tht h een stly trnsfete with the expressing onstrut or ontrol GFP plsmi. Overexpression of ws onfirme y rel-time PCR (Supplementry Figure S). The ells were trete with olei i followe y Oil Re O stining to revel stetosis. Etopi expression of enhne stetosis (Figure ; Supplementry Figure S). We then vlite the effets of n seprtely. HepG ells were trnsfete with -5p inhiitor or ontrol oligonuleoties, followe y stetosis ssy. The experiment showe tht trnsfetion with -5p inhiitor suppresse ell stetosis ompre to the ontrol (Figure ), onfirming role of -5p on inuing stetosis. We exmine the omine effet of n -5p y treting HepG ells with -5p inhiitor n vrious onentrtions of, followe western lot nlysis. While ells trete with showe inrese in PPAR-α expression, tretment with -5p inhiitor promote PPAR-α levels (Figure e). To exmine the omine effets of n -5p inhiitor, we trete HepG ells with n with or without -5p inhiitor. Stetosis ssy showe tht h ose effet on promotion of stetosis n tretment with -5p inhiitor erese the -inue stetosis (Figure f). inue evelopment of ftty liver in trnsgeni mie We hve previously evelope trnsgeni mouse line overexpressing. 5 After onfirming inrese expression of (Supplementry Figure S), we exmine whether or not the expression of ffete liver funtion in oth trnsgeni (Tg) n wiltype (WT) mie n oserve extensive stetoti hnge in the Tg liver ut fewer in the WT liver (Figure, with lrger view provie, Supplementry Figure Se). Oil Re O stining of the liver tissues further onfirme lipi umultion in the Tg mie ut not in the WT mie (Figure ). We exmine totl of 4 trnsgeni livers n 3 wiltype livers n groupe them to severe (t lest seven lrge vuoles per exmintion fiel s shown in Figure, eh roun the size of heptoyte), meium (t lest three vuoles etete per exmintion fiel), n light (t lest one vuole per exmintion fiel) ftty liver. With this riterion, eh photo ws snne with Imge J softwre. Trnsgeni mie showe signifint severer ftty liver inienes s ompre with wiltype mie (Figure, P <.). Liver tissues from rnomly selete Tg mie n WT mie were sujet to nlysis of triglyerie ontent. The Tg mie livers exhiite signifintly higher levels of triglyerie thn the WT mouse livers (Figure, left). Mesurement of holesterol onentrtions in totl plsm inite tht the trnsgeni livers ontine higher levels of holesterol thn the wiltype (Figure, right). PPAR-α is iret trget of -5p Bioinformti nlysis inite tht potentilly trgete mny mrnas. We fouse on those tht re known to e importnt in lipi metolism n in the evelopment of ftty liver. After extensive nlysis with western lotting using skin n livers from the trnsgeni n wiltype mie n HepG ell lines stly trnsfete with n the ontrol vetor, we foun tht PPAR-α ws represse (Figure 3). The 3 UTR of PPAR-α ontins three potentil trget sites for -5p (Figure 3). It hs een reporte tht PPAR-α plys ruil role in intrellulr lipi metolism. 6 It regultes the expression of proteins involve in the trnsport n β-oxition of free ftty is, preominntly in the liver. 7 PPAR-α knokout mie file to show inrese trnsription of the enzymes involve in ftty i trnsport n β-oxition fter stimultion with firtes n evelope intrhepti umultion of lipi roplets fter weeks of regulr how iet. 8 To vlite trgeting of PPAR-α y -5p, we generte three luiferse

3 mirna n Ftty Liver 7 5 Optil ensity (54 nmol/l) e Reltive levels of PPAR-α mrna nmol/l 3 nmol/l HepG PPARα Dex β-tin Primry heptoyte HepG SNU449 HepG-GFP HepG-7.5 Olei i (mmol/l) 3 nmol/l 3 nmol/l 3 n 3 n 3 n.5 3 nmol/l nmol/l 3 nmol/l Ctrl Inhiitor Ctrl Inhiitor Ctrl Inhiitor Ctrl Inhiitor PPARα f Optil ensity (54 nmol/l) Optil ensity (54 nmol/l) Reltive levels of (,) HepG Primry heptoyte SNU449 nmol/l 3 nmol/l 3 nmol/l 3 nmol/l inhiitor.5 Olei i (mmol/l) +pcdna +mir7 inhiitor.5 β-tin.5.5 onentrtions (mmol/l) Figure Dexmethsone inues expression of n prolifertor-tivte reeptor-α (PPAR-α). () Primry heptoytes, HepG, n SNU449 ells were trete with Dexmethsone t the inite onentrtions for 8 hours, followe y nlysis of PPAR-α expression y rel-time polymerse hin retion using primers hu-ppr-sli n hu-ppr-84r (upper) n western lotting (lower). Up to 3 nmol/l, Dexmethsone enhne PPAR-α expression. () Anlyze for -5p levels showe tht Dexmethsone tretment inrese -5p expression. Error rs = SD, n = 3, P <.5, P <.. () HepG ells stly trnsfete with or GFP were sujet to stetosis y treting the ells with ifferent onentrtions of olei i followe y Oil Re O stining. The stine olor ws extrte for optil ensity mesurement. Expression of promote stetosis. n = 3, P <.5, P <.. () HepG ells trnsfete with inhiitor or the ontrol oligos were trete with olei i t the onentrtions inite followe y stetosis ssy. Introution of inhiitor erese ell stetosis. Asterisks inite signifint ifferenes. P <.; (n = 4). (e) Protein lystes prepre from -5p inhiitor- n ontrol oligos-trnsfete HepG ells, whih were ulture in vrious onentrtions of, were sujet to immunolotting proe with ntioies ginst PPAR-α. The sme memrne ws reproe for β-tin levels. Trnsfetion with -5p inhiitor enhne PPAR-α expression. (f) HepG ells were trete with exmethsone with or without -5p inhiitor followe y stetosis ssy. Tretment with -5p inhiitor signifintly erese stetosis. onstruts (Lu- PPAR-α, Lu- PPAR-α, n Lu- PPAR-α3, primer sequenes provie in Supplementry Figure S) hroring the three frgments with the ining sites of n three onstruts in whih the trget sites were mutte (Lu-PPAR-α mut, Lu-PPAR-α mut, n Lu-PPAR-α mut3) (Figure 3, etil of the sequenes re provie in Supplementry Figure S). HepG ells were otrnsfete with mimi n either one of the PPAR-α 3 UTR luiferse onstruts or the mutnt onstruts, followe y luiferse tivity nlysis. Signifint repression of luiferse tivity ws etete in the Lu-PPAR-α trnsfete ells (ompre with luiferse empty vetor or luiferse vetor with no ining site sequene, G3R, P <.), while trnsfetion with the mutnt onstruts reverse the effet (Figure 3). Moleulr Therpy 3

4 mirna n Ftty Liver wt39 wt75 wt466 wt39 tg974 tg97 tg89 tg88 tg57 tg4 tg88 5 µm 5 µm Numer of mie evelope ftty liver Severe Meium Light None Triglyerie ontent (mg/g of liver) Wt mie mie WT liver Tg liver WT plsm Tg plsm Cholesterol onentrtion (mg/l) Figure Expression of promote stetosis n ftty liver evelopment. () Histologil nlysis of liver setions from mir7-trnsgeni (tg974, tg97, tg89, tg57, tg4, n tg88) n wil type (wt39, wt75, n wt466) mie showing opious ftty liver prtiulrly in the trnsgeni livers. () Lipis in the liver setions were stine with Oil Re O. () Livers were hrveste from trnsgeni n wil-type mie, setione, n sujete to H&E stining for exmintion of ftty liver. We groupe them to severe (t lest seven lrge vuoles, eh roun the size of heptoyte, ourring in nery ells), meium (t lest three vuoles etete in nery ells), n light (t lest one vuole etete) ftty liver. Trnsgeni mie tene to hve fttier livers thn wiltype mie (χ test, P <.). () Left, Liver tissues were hrveste from trnsgeni n wil type mie n evlute for triglyerie ontent. Trnsgeni livers ontine higher levels of triglyerie, n = ; P <.. Right, Totl plsm levels were evlute for holesterol onentrtion. Trnsgeni livers ontine higher levels of holesterol, n = 5; P <.. To orroorte tht PPAR-α oul ontriute to stetosis of HepG ell, we generte three smll interfering RNAs (sirna) onstruts trgeting PPAR-α. PPAR-α silening ws onfirme y western lot in the sirna-trnsfete HepG ells (Supplementry Figure S3). Trnsfetion with PPAR-α sirna promote stetosis ompre with those trnsfete with ontrol oligonuleoties (Figure 3, left), whih ws olei i onentrtion epenent (Supplementry Figure S3). To test whether or not PPAR-α ws meiting the effet of in inuing stetosis, we re-expresse PPAR-α in the -trnsfete ell line (Supplementry Figure S3), followe y stetosis ssy. We etete signifint erese in stetosis ourrene in the ells fter retrnsfetion with the PPAR-α expression onstrut ompre with ontrols (Figure 3, right), whih ws olei i onentrtion epenent (Supplementry Figure S3). We lso performe omprehensive experiment to vlite the effet of PPAR-α, -5p, n PPAR-α sirna on stetosis. The experiment onfirme tht -5p promote stetosis, whih ws inhiite y PPAR-α (Figure 3e; Supplementry Figure S4). Consistent with these results, tretment with PPAR-α inhiitor inrese stetosis of HepG ells (Figure 4). This onfirme tht -enhne HepG stetosis ws meite y PPAR-α pthwy. PPAR-α enhne expression We then teste whether or not PPAR-α regulte expression. HepG ells were trnsiently trnsfete with PPAR-α 4

5 mirna n Ftty Liver Wt- Wt PPARα 5-86 PPARα 3 5 Skin β-tin PPARα PPARα WT PPrα β-tin PPARα β-tin Lu-PPARα Lu-PPARα-mut Lu-PPARα Lu-PPARα-mut Lu-PPARα3 Lu-PPARα3-mut.. Liver PPARα Cells.. PPARα Luiferse oing sequene XXX PPARα 3 UTR PPARα3.. Reltive luiferse units Optil ensity (54 nmol/l) Vetor Olei i (.5 mmol/l) NC sirna G3R PPARα sirna sirna3 PPARαmut Optil ensity (54 nmol/l) Olei i (.5 mmol/l) Vetor + PPAR-α G3R PPARα PPARαmut Figure 3 Trgeting prolifertor-tivte reeptor-α (PPAR-α) y. () Cell lyste prepre from trnsgeni n wiltype liver n skin, n - n ontrol vetor-trnsfete HepG ells ws nlyze on western lot for PPAR-α expression. PPAR-α ws represse y expression. Stining for β-tin from the sme memrne onfirme equl loing. () Upper, Computtionl nlysis inite tht mir- 7 potentilly trgete PPAR-α lote t nuleoties,,66,86,,56,53, 5,49 5,43. Lower, Three luiferse onstruts were generte, eh ontining frgment hroring the trget site of, prouing Lu-PPrα, Lu-PPrα, n Lu-PPrα3. Muttions were lso generte on the potentil trget sequene (re olor), resulting in two mutnt onstruts Lu-Pprα-mut, Lu-Pprα-mut, n Lu-Pprα3-mut. () HepG ells were otrnsfete with -5p mimi n luiferse reporter onstrut, whih ws engineere with one of the three PPAR-α 3 UTR frgments, PPARα (left), PPARα (mile), n PPARα3 (right), hroring -5p trget site or n pproprite mutnts (PPARαmut, PPARαmut, n PPARα3mut). An unrelte sequene ws use s ontrol (G3R) to ompre with the vetor, PPAR-α 3 UTR frgment, n PPAR-α mutnt group. Unpire Stuent s t-test for norml istriution ws performe. The ifferenes were onsiere sttistilly signifint t P <.5. Asterisks inite signifint ifferenes. P <.5, n = 6. The luiferse tivities erese when the ells were trnsfete the PPAR-α luiferse onstruts. Muttion of the ining site reverse -5p s effet. () Left, HepG ells trnsiently trnsfete with three PPAR-α sirnas or the ontrol oligo were sujet to stetosis ssy. Trnsfetion with PPAR-α sirnas enhne stetosis. P <.5, P <., n = 3. Right, HepG ells stly trnsfete with were trnsiently trnsfete with PPAR-α expression onstrut or ontrol vetor. Trnsfetion with PPAR-α reverse the effet of resulting in erese stetosis. P <., n = 3. (e) HepG ells trnsiently trnsfete with PPAR-α, -5p mimi or/n Ppr-α sirna, were sujet to stetosis y treting the ells with mmol/l olei i followe y Oil Re O stining. Expression of -5p or/n Ppr-α sirna promote stetosis, wheres expression of PPAR-α represse stetosis. Asterisks inite signifint ifferenes. P <.5, P <. (n = 3). e Density of oil stining (5 nmol/l) mmol/l olei i Vetor G3R PPARα3 PPARα3mut Vetor+oligo+oligo PPARα+oligo+oligo Vetor+-5p+oligo Vetor+oligo+PPARα sirna PPARα+-5p+oligo Vetor+-5p+PPARα sirna Moleulr Therpy 5

6 mirna n Ftty Liver Optil ensity (54 nm) NC 5 µmol/l µmol/l 5 µmol/l Reltive mirna levels.5.5 Pri- -5p -3p PPr-α inhiitor ppr-α ppr-α ppr-α Input Negtive Positive ppr-α trl ppr trl ppr trl ppr trl ppr 6 pgl3-mir7-promoter pgl3 Reltive signl to input (%) Ppr-α Reltive luiferse units (%) 8 4 Negtive Positive Ppr-α 5 5 5, Amount of ppr-α (ng) Figure 4 Expression of prolifertor-tivte reeptor-α (PPAR-α) enhne trnsription. () HepG ells were trete with PPAR-α inhiitor t the onentrtion inite followe y stetosis ssy. Tretment with PPAR-α ntgonist (GW647, Sigm) inrese stetosis. Asterisks inite signifint ifferenes. P <.; n = 4. () Reverse trnsription polymerse hin retion showing PPAR-α trnsfete HepG ells expresse high levels of pri- (left), -5p (mile), n -3p (right) ompre to ontrols. Asterisks inite signifint ifferenes. P <.5, P <.; n = 4. () Upper, Chromtins of ontrol- n PPAR-α-trnsfete HepG ells were isolte, igeste, n immunopreipitte with ntioies ginst PPAR-α, Histone H3 (positive ontrol), n rit IgG (negtive ontrol), followe y PCR with speifi primers (mir7- promot-for-pcr-f n mir7-promot-for-pcr-r) flnking frgment of DNA in the promoter. Lower, Grph showing tht PPAR-α-trnsfete ells h more PPAR-α ining to promoter. Asterisks inite signifint ifferenes. P <.; n = 4. () Luiferse ssys were performe in HepG ells otrnsfete with either PGL3 vetor, or PGL3--promoter, n vrious ose of PPAR-α. Inrese PPAR-α oses enhne luiferse tivities. Asterisks inite signifint ifferenes. P <.; n = 4. expression onstrut, followe y nlysis of expression. We foun tht the trnsfete ells expresse signifintly higher levels of Pri-, -5p, n -3p s ompre with the vetor ontrol (Figure 4). It ppere tht PPAR-α regulte trnsription. Chromtin from PPAR-α- n ontroltrnsfete HepG ells were isolte, igeste, n immunopreipitte with ntioies ginst PPAR-α, Histone H3 (serving s positive ontrol), n rit IgG (serving s negtive ontrol), followe y PCR with speifi primers flnking frgment of DNA in the promoter. It showe tht ntioy ginst PPAR-α, whih preipitte PPAR-α (Supplementry Figure S4), pulle own promoter (Figure 4). A luiferse onstrut ws generte y inserting the mir- 7 promoter into the pgl3-bsi Vetor (Promeg) s inite (Supplementry Figure S4), followe y trnsfetion of HepG ells with this promoter-ontining pgl3 onstrut or the ontrol pgl3 plsmi t vrious mounts. Insertion of promoter inrese luiferse tivities signifintly ompre with the ontrol (Figure 4). expression promote -inue ftty liver evelopment To test whether -5p oul moulte -inue ftty liver evelopment, we injete the trnsgeni mie with t onentrtion of 5 mg/kg everyy for 3 ys. The mie were kept for n itionl 3 ys. At this onentrtion, tretment enlrge the livers signifintly, ut i not show signifint effet on the wiltype group (Figure 5, upper). Also, the livers were yellow in olor, initing the presene of ftty liver (Figure 5, lower). However, the mouse livers showe erese weight ompre with wiltype group without tretment, lthough not rehing signifint level. The most likely resons were tht the mie use in this experiment were young (3 4 weeks ol), the livers of these young mie h not evelope ftty liver signifintly, n expression of retre some orgns, suh s liver growth uring evelopment. 5 Histologil nlysis lso exhiite lrge numer of lipi vuoles in the liver trete with, nother inition of ftty livers (Figure 5). The prffin setions were sujet 6

7 mirna n Ftty Liver more pink roplets thn the wiltype livers (Figure 5, upper). Quntifition of the pink roplets inite signifint inrese in -trete livers (Figure 5, lower). In this experiment, pplition of inue ftty liver in oth trnsgeni n to immunohistohemil stining. A ler ownregultion of PPAR-α ws onfirme in the trnsgeni mie ompre with the wiltype (Figure 5). The frozen setions of livers were sujet to Oil Re O stining. The livers showe mny.8. (5 mg/kg) Weight of liver (g) Tretment with 5 mg/kg 5 mg WT Density of oil stining (54 nmol/l).3 (5 mg/kg) e 5 mg PGL3 Reltive luiferse tivity (%) P PM mg nmol/l 3 nmol/l nmol/l nmol/l µmol/l onentrtions Figure 5 Development of ftty liver in trnsgeni mie inue y exmethsone (). () Upper, Although the weight of liver erese in the -trnsgeni mie, tretment with (5 mg/kg ) signifintly inrese their liver weight ompre with wiltype. Asterisks inite signifint ifferenes, n = 5. Lower, Typil photo of livers from -trnsgeni n wil-type mie. () In HE stining, the trnsgeni mie exhiite lrge numer of lipi vuoles in the liver trete with. () Immunohistohemistry showing tht the trnsgeni mie expresse low levels of prolifertor-tivte reeptor-α (PPAR-α) in liver trete with. () Upper, in Oil Re O stining ssy, trnsgeni livers isplye more oil roplet stine y Oil Re O in the liver trete with. Lower, Quntifition nlysis inite tht h signifint effet on stetosis, n =. Sle rs for, 5 µm. (e) Luiferse ssys were performe in HepG ells trnsfete with PGL3 vetor, PGL3--promoter (-P), or PGL3--promoter-mutnt (-PM) in the presene of t vrious onentrtions. enhne luiferse tivities. Asterisks inite signifint ifferenes. P <.5, P <.; n = 4. Moleulr Therpy 7

8 mirna n Ftty Liver wiltype mie. However, the trnsgeni group evelope serious ftty liver. To test if oul enhne -5p tivity through upregultion, we onute luiferse ssy, y trnsfeting HepG ells with the luiferse plsmi pgl3, ontining the promoter or the mutnt promoter, in whih two PPAR-α ining sites were mutte (Supplementry Figure S4). Luiferse ssy showe tht enhne luiferse tivity when the onstrut ws inserte with the promoter (Figure 5e). Muttion of the PPAR-α ining sites signifintly erese luiferse tivity, ut oul not fully suppress the luiferse tivity. Perhps, PPAR-α oul in to other res of the promoter frgment resulting in erese luiferse tivities. Inhiition of -5p funtion olishe inue ftty liver evelopment To test the possiility of using -5p inhiitor s n gent to llevite -inue ftty liver evelopment, we trete mie with ifferent onentrtions of n foun tht t onentrtion of mg/kg oul inue formtion of ftty livers effetively. The trnsgeni n wiltype mie were trete with ( mg/kg) omine with -5p inhiitor. The experiment showe tht inlusion of -5p inhiitor erese the liver weight of oth types of mie (Figure 6, upper). In the trnsgeni mie, tretment with showe yellow livers, ut ition of -5p inhiitor olishe this effet (Figure 6, lower). We then exmine the effet of -5p inhiitor on PPAR-α expression y western lotting. Protein lystes were prepre from liver tissues n sujet to immunolotting proe with ntioies ginst PPAR-α. We onfirme tht oth trnsgeni n wiltype mie expresse high levels of PPAR-α fter tretment with n -5p inhiitor (Figure 6). Histologil nlysis of the livers inite tht vuoles in oth trnsgeni n wiltype livers signifintly erese when the mie were trete with -5p inhiitor (Figure 6). The liver setions were sujet to Oil Re O stining. Tretment with -5p inhiitor erese Oil Re O stining in oth the wiltype n trnsgeni mie signifintly (Figure 6). Immunohistohemil stining showe tht PPAR-α levels were lower in the liver thn in the wiltype liver ut higher in the liver trete with -5p inhiitor (Supplementry Figure S5). The sie-effet of -inue stetosis oul e inhiite y hemils tht erese -5p levels Our results showe tht PPAR-α n -5p re two essentil ftors tht ffete stetosis n ftty liver evelopment. We sought to fin hemil rugs tht moulte expression of PPAR-α n -5p n inhiit -inue stetosis. We selete Bezfirte, Fenofirte, Gemfirozil, Clofirte, Ciprofirte, n WY-4643 in the in vitro stuy. All of these hemils re potent PPAR-α gonists. While the first five regents re wiely use in lini, the lst one is still in prelinil tril. HepG ells were trete with these hemils, n sujet to rel-time PCR nlysis for expression of PPAR-α n -5p. We foun tht tretment with ll hemils/rugs promote PPAR-α expression (Figure 7). However, only Clofirte, Ciprofirte, n WY-4643 oul erese -5p levels (Figure 7). This effet ws more potent when omine with. Western lot nlysis proue onsistent results when the ells were trete with WY-4643, Clofirte, n Ciprofirte (Supplementry Figure S5). The tretments proue inonsisteny in protein n mrna levels oul e resulte from post-trnsriptionl regultion. Interestingly, only the three hemils/rugs tht erese -5p levels n enhne PPAR-α expression oul erese stetosis in the ells trete with (Figure 7). These results provie eviene initing the essentil roles of -5p in moulting -inue stetosis, n highlight the importne of seletion of suitle PPAR-α tivting regents to prevent stetosis in tretment. Those hemils with oth PPAR-α tivtion n -5p repression might e the proper hoie in lini. The effets of these ftors on stetosis n ftty liver evelopment, s well s their reltionship, re provie in Figure 7. Applition of enhne expression of oth n PPAR-α of liver ells, whih plye ifferent roles in the progression of stetosis n ftty liver evelopment. Current stuy inite tht expression of -5p promote stetosis progression n ftty liver evelopment, wheres tivtion of PPAR-α represse these proesses. Finl physiologil outome relies on the intertion etween -5p n PPAR-α. Sine -5p trgete PPAR-α n represse its expression, while PPAR-α promote trnsription, ny regents moulting the expression of one moleule woul ffet the other, n might hnge the liver ell phenotype. In our stuy, we pplie mir- 7-5p inhiitor n PPAR-α gonists to interrupt this feek regultion loop, n foun tht repression -5p expression or pplition of PPAR-α tivtors ws potentilly vlule strtegy to reverse -inue stetosis n ftty liver evelopment. DISCUSSION Stetosis is the erliest stge of NAFLD n is frequently oserve in metoli ysfuntions tht preee humn heptoellulr rinom. Hepti stetosis is often self-limite, ut it n lso progress to NASH. 9,3 Sine ell injury my our, when the pity of heptoytes to sfely store ft is overwhelme y ontinue uptke, lol synthesis, or impire egress of ftty is, these ftty is then eome toxi to the ell, using ell eth y the iret effets of lipi meitors on poptosis. 3 Alterntively, liertion of oxiize lipis n their peroxition prouts (hemotti lehyes n orgni is) my e instrumentl in reruiting n perpetuting the inflmmtory response tht hrterizes NASH. 9 Stetosis oul therefore provie the fountion for ftty liver n NASH, whih n le to hepti firosis or irrhosis, n my progress to heptoellulr rinom. 3 We foun tht etopi expression of -5p oul inue stetosis n ftty liver evelopment y repressing PPAR-α expression. In our stuy, etopi expression of mture mirna-7-5p signifintly reue expression of PPAR-α through trgeting PPAR-α 3 UTR (Figure 3). It is ntiipte tht isruption of PPAR-α signling n promote lipi uptke n/or inhiit lipolysis s eviene y inrese lipi umultion. Sine PPAR-α elongs to the nuler hormone reeptor superfmily, it hs the potentil to regulte gene trnsription (inluing mirnas). In our stuy, we unover the reltionships of -5p,, n PPAR-α in ell stetosis n evelopment of ftty liver. 8

9 .8 Weight of liver (g) mirna n Ftty Liver Dex Trnsgeni mie.6 5 Inhiit 5 Inhiit PPARα β-tin DMSO + trl oligo mg/kg + mg + trl oligo mg/kg + inhiit + mg + mir7 inhiit + mg + trl oligo + mg + mir7 inhiit + mg + trl oligo + mg + mir7 inhiit Density of oil stining (54 nmol/l).4 Trnsgeni mie.3.. mg/kg mg/kg + inhiit Figure 6-5p inhiitor enhne prolifertor-tivte reeptor-α (PPAR-α) expression n llevite exmethsone ()-inue ftty liver. () Upper, Trete with higher onentrtion of ( mg/kg), the -trnsgeni n wil-type mie h similr liver weight. However, tretment with ontrol oligo (trl oligo) with rnom sequene or -5p inhiitor erese liver weight of oth the wiltype n -trnsgeni mie. Asterisks inite signifint ifferenes. P <.; n = 5. Lower, Typil photo of trnsgeni livers trete with or without n mir7-5p inhiitor. () Protein lystes prepre from liver tissues trete with ifferent onentrtion of n -5p inhiitor were sujet to immunolotting proe with ntioies to PPAR-α n β-tin. Immunolot showe tht Both trnsgeni n wiltype mie expresse high levels of PPAR-α fter tretment with n -5p inhiitor. () The trnsgeni n wiltype livers trete with or without n -5p inhiitor were sujet to HE stining. Vuoles in oth trnsgeni n wiltype livers signifintly erese when the mie were trete with -5p inhiitor. () Left, The liver setions were lso sujet to Oil Re O stining. Tretment with -5p inhiitor erese Oil Re O stining. Right, Quntifition nlysis inite tht treting the mie with -5p inhiitor signifintly erese oil umultion in the livers. Asterisks inite signifint ifferenes. P <.; n =. Sle rs for,, 5 µm. In the first prt of our work (Figures 4), while we provie eviene to unerstn the reltionship, we foun fee-k loop regultion etween -5p n PPAR-α. Computtionl nlysis showe tht PPAR-α is potentil trget of -5p: there re three potentil ining sites in PPAR-α 3 UTR for mir7-5p (Figure 3). Etopi expression of represse PPAR-α protein levels in the -trnsfete ells, n in the skin n liver of trnsgeni mie. Luiferse ssy further onfirme tht -5p oul repress luiferse tivities when Moleulr Therpy the onstruts hrore one of the three ining sites. On the other hn, PPAR-α oul iretly in to the promoter of the 9 luster (Figure 4). In the luiferse ssy, we showe tht this ining signifintly enhne promoter tivities. This fee-k loop regultion suggests tht, while oth -5p n PPAR-α plye importnt roles in the progression of stetosis n ftty liver evelopment, the fee-k loop my filitte lne for the regultion. Inee, oul upregulte PPAR-α expression, ut the overll outome of is to prevent 9

10 mirna n Ftty Liver Reltive PPAR- mrna levels Bezfirte Fenofirte 3 Gemfirozil 4 WY Clofirte 6 Ciprofirte Reltive -5p levels Bezfirte Fenofirte Gemfirozil WY-4643 Clofirte Ciprofirte Non Non- Density of oil stining (54 nmol/l) Bezfirte Fenofirte 3 Gemfirozil 4 WY Clofirte 6 Ciprofirte e Stetosis 4 3-e 5 PPARα Non- Ftty liver Figure 7 Seleting hemils tht n erese the sie-effet of exmethsone () in inuing stetosis. () Bezfirte, Fenofirte, Gemfirozil, Clofirte, Ciprofirte, n WY-4643 were ll issolve in Dimethyl sulfoxie (DMSO). Totl RNAs were prepre from HepG ells trete with 5 µmol/l Bezfirte, Fenofirte, Gemfirozil, Clofirte, Ciprofirte, or µmol/l WY-4643 for 48 hours, followe y rel-time polymerse hin retion nlysis. Cells trete with ll rugs/hemils proue signifintly higher levels of prolifertor-tivte reeptor-α (PPAR-α) trnsription fter ulture with or without. () The RNAs were lso sujet to ssy for -5p levels. Without tretment, Fenofirte n Gemfirozil inrese, while Clofirte erese -5p levels. In the presene of, Bezfirte, Fenofirte, n Gemfirozil inrese, while WY-4643, Clofirte, n Ciprofirte erese -5p levels. () In ell stetosis ssy y treting the ells with.5 mmol/l Olei i for 4 hours, ll hemils erese stetosis without the tretment of. However, in the presene of, Fenofirte, n Gemfirozil inrese, while WY-4643, Clofirte, n Ciprofirte erese stetosis. () Digrm showing the effets of gluoortioi, mir- 7, n PPAR-α on stetosis n evelopment of ftty liver. Results from the figures re inite y figure numers. inhiition of stetosis n ftty liver evelopment, rther thn iretly inuing these proesses. PPAR-α ppers to e ul-role regultor in the formtion of stetosis n ftty liver. Inhiition of PPAR-α tivity promote stetosis. In ition, the levels of PPAR-α were muh lower in the ftty livers thn in the norml livers. Although these re orreltionl results, they support the notion tht PPAR-α n inhiit ftty liver evelopment. PPAR-α oul in to promoter n promote -5p expression. This seeme to proue n opposite funtion in the evelopment of stetosis n ftty liver. However, the upregulte -5p oul turn k to inhiit PPAR-α expression, forming the fee-k loop. This fee-k loop woul llow the system to hieve lne when trete with or other gents involve in the system. Nevertheless, repression of PPAR-α levels ppers to e n essentil step in the inution of stetosis n ftty liver evelopment regulte y -5p. It shoul e note tht there re mny potentil trgets of -5p. Other trgets (for exmple, ATP-ining ssette trnsporter ABCA) oul lso ontriute to the mir- 7-5p-inue stetosis n ftty liver evelopment. Further stuies re neee to provie omprehensive piture for unerstning -5p inution of stetosis n ftty liver evelopment. Our experiments show tht -5p is key plyer in meiting s effet on inuing stetosis n ftty liver formtion

11 mirna n Ftty Liver (Figure 7). Even without tretment, upregultion of mir- 7-5p oul inue stetosis in ells n ftty liver formtion in mie. High levels of -5p expression (in the -trnsgeni mie) inrese the sensitivity of mie to -inue ftty liver evelopment ompre to wiltype mie, whih showe little hnge when trete with low onentrtion of. Thus, we sought to evelop n pproh to inhiit stetosis n ftty liver formtion in the seon prt of our stuies. We foun tht the inhiitor of -5p oul suffiiently inhiit stetosis n ftty liver evelopment. While high onentrtion of oul inue evelopment of ftty liver, injetion of -5p inhiitor oul ompletely inhiit ftty liver evelopment. These results suggest tht -5p inhiitor n esensitize livers to tretment. It my thus e supplementry gent for those who re eing trete with n oul potentilly evelop ftty liver s result of the sie-effet of. We lso sreene numer of hemils/rugs tht re known to ply roles in treting hyperholesterolemi. It ws expete tht mny of these hemils/rugs might inrese PPAR-α expression. However, we looke for hemils/rugs tht oul erese mir- 7-5p levels. Clofirte, n Ciprofirte, n WY-4643 oul ll erese -5p expression n inhiit stetosis when eing omine with. In prtiulr, Clofirte showe the strongest effet on reuing -5p expression of stetosis levels. Clofirte, n orgni ompoun, is lipi-lowering gent n is use for ontrolling high holesterol in the loo. However, this rug ws isontinue in ue to verse ffets. Ciprofirte is firte, n WY-4643 is PPAR-α gonist, oth of whih re use for metoli isorers. Sine oth Ciprofirte n WY-4643 oul promote PPAR-α expression n inhiit the proess of stetosis, they re the iel gents to e omine with in the tretment of nti-inflmmtion n immunosuppression, s well s in some ses for ner ptients. We ntiipte these finings will stimulte further reserh into hemil gents tht reue n gluoortioi-inue sie effets. MATERIALS AND METHODS Genertion of onstruts n genotyping of trnsgeni mie. A DNA sequene ontining two humn pre- units ws inserte into mmmlin expression vetor pegfp-n in the restrition enzyme sites BglII n HinIII. Trnsgeni mie expressing were generte s esrie previously. 5 A luiferse reporter vetor (pmir-report; Amion) ws use to generte the luiferse onstruts. Three frgments of the 3 UTR of mouse PPAR-α were lone y RT-PCR using three pirs of primers (mus-ppr-mir7-si n mus-ppr-mir7-mlui for frgment-; mus-ppr-mir7-si n mus-ppr-mir7-mlui for frgment-; mus-ppr-mir7-si n mus-ppr-mir7-mlui for frgment-3). Three muttion primers (mus-ppr-mir7-mlui-mut, mus-pprmir7-mlui-mut, n mus-ppr-mir7-mlui-mut) were use to generte muttions in the -5p trget sites. PCR prouts were igeste with SI n MluI n the frgments were inserte into SIn MluI opene pmir-report luiferse vetor. The promoter of ws lone using two primers mir7promotsi n mir7promothiniii. The first potentil PPAR- ining site ws mutte with two primers Dex-prom-mut-BglII-F n Dex-prom-mut-BglII-R, while the seon potentil ining site ws mutte with two primers De-prom-mut-XhoI-R n Depromoter-XhoI-F. Rel-time PCR. The experiment ws performe s previously esrie. 33,34 In rief, totl RNA ws extrte from ~ 6 ells or ~5 mg tissues, n sujet to DNA synthesis using μg RNA. Suessive PCR ws performe y QuntiMir-RT Kit using μl DNA s templte (Qigen, Vleni, CA, misript Reverse Trnsription Kit, t#86; misript Primer Assy, t#84; misriptsybr GreenPCR Kit, t#873). Western lot. Cell lystes were otine from the ultures or extrte from frozen tissues n sujete to SDS-PAGE eletrophoresis n western lot nlysis s esrie. 35,36 Oil Re O stining. Frozen liver setions were fixe in % formlin n riefly wshe with wter for minutes. The setions were rinse with 6% isopropnol n stine with freshly prepre Oil Re O working solution for 5 minutes. Setions were rinse with 6% isopropnol, lightly stine with luminium hemtoxylin n mounte in glyerine jelly. Inution of stetosis in HepG ell y olei i tretment. HepG ells were ulture in 96-well tissue ulture pltes to su-onfluene. The meium ws hnge into serum-free Duleo s moifie Egle s meium, followe y treting the ells with µl olei i (OA) solution ontining ifferent onentrtions (.5 mmol/l) of OA overnight. The meium ws remove n the ells were trete with µl fixtive solution (% formlin) t room temperture for minutes, followe y mirosopi exmintion. For quntifition, stetosis ws inue y OA n stine with Oil Re O. After wshing n ompletely rying, μl of % isopropnol ws e to eh well, followe y inuting t room temperture for minutes to relese Oil Re O from stetosis stining. The extrtion solution ws trnsferre to nother 96-well plte whih ws sujete to OD mesurement t wvelength of 54 nm using miroplte reer (Bio-Tek Instrument, Winooski, VT). Liver triglyerie ontent mesurement. Liver smples were weighte ( 3 mg) n 3 µl of ethnoli KOH ( prts EtOH mixe with prt 3% KOH) were e to eh smple, followe y inution t 55 C overnight. Dilute EtOH (H O:EtOH=:) ws e into eh tue to ring the volume to ml. The tues were entrifuge for 5 minutes n the superntnt ws trnsferre into new tues. The ilute EtOH ws e gin into eh tue to ring the volume to. ml. After vortex, µl of the mixture ws trnsferre into new Eppenorf tues in triplites, followe y ition of 5 µl M MgCl n inution on ie for minutes. The tues were entrifuge for 5 minutes n the superntnt ws trnsferre into new tues. Triglyerie regent (Sigm, St. Louis, MO, tlog # F648) ws reonstitute oring to mnufturer s instrutions for etermintion of glyerol ontent. One milliliter of reonstitute regent ws pipette into eh uvette. Liver lystes, stnrs, n ontrol lnks were e into seprte uvettes, n inute t 37 C for 5 minutes. Asorne vlues were mesure t 54 nm. Luiferse ssy. The ssy ws performe s previously esrie. 35 Chromotin immunopreipittion (ChIP) ssy. ChIP ws performe using Simple ChIP hromtin IP kit (Cell Signling) oring to the mnufturer s instrutions. Briefly, ells were trete with formlehye solution. The hromtin ws isolte, igeste, n immunopreipitte with ntioy ginst PPAR-α. The pture hromtin ws elute, unross-linke, n the DNA ws reovere. The ChIP isolte DNA ws sujet to PCR using speifi primers (mir7-promot-for-pcr-f n mir7-promot-for-pcr-r) flnking piee of DNA frgment of the promoter region. inution of mouse ftty liver. The trnsgeni mie use in this experiment were etween 4 5 weeks of ge. They were mintine uner -hour light/-hour rk photoperio t room temperture of 3 C n reltive humiity of 5%. All niml experiments Moleulr Therpy

12 mirna n Ftty Liver were pprove y the Animl Cre Committee of Sunnyrook Reserh Institute, Ontrio, Cn. soium phosphte (Omeg, Montrel, Cn) ilute in.9% NCl ws ministere i.p. to wiltype or trnsgeni mie t ose of 5 mg/kg or mg/kg oy weight every y for 3 ys. Eh group h 5 mie. The mie were srifie on y 6. In the group trete with -5p inhiitor (GenePhrm, Shnghi), -5p inhiitor (-5p ntisense mimi, 6 µg) ws injete i.p. with the inhiitor- PEG-Au NP omplexes hours efore injetion, n repete 48 hours fter initil injetion. Synthesis of the eliver omplexes (inhiitor-peg-au NP) ws performe s previously esrie. 37 Briefly, nmol thiol moifie mir- 7-5p inhiitor ws issolve in 8 µl of RNse-free wter. The mpeg- SH (PG-TH-k, Nnos) ws mixe with -5p inhiitor (: molr rtio). Then -nm gol nnoprtiles (AuNP, Cytoignostis) were mixe with -5p inhiitor-peg t weight rtio of : for onjugtion. The mixture ws gently shken t 6 C for 3 minutes n trnsferre into syringe. Mie were srifie on y 6 for liver hrvest. The left loe of the liver ws kept frozen for immunolotting or PCR, while itionl prts were fixe in % formlin. After 4 hours, the right loe of liver ws proesse to otin frozen setions, followe y Oil Re O stining n ounterstining with Myer s Hemtoxylin. To quntify Oil Re O stining, µl of isopropnol ws e to the setions n inute for minutes with gently shking. The superntnt ws trnsferre to 96-well pltes. Asorne vlue ws mesure t 54 nm with isopropnol s lnk. A frgment of liver ws proesse to prffin eme n setioning. The setions were sujete to either H&E stining or immunohistohemil stining with ntioies ginst PPAR-α (Snt Cruz, Dlls, TX, Ct# s-9). Sttistil nlysis. All experiments were performe in triplite or higher. Dt were expresse s men ± SE. Unpire Stuent s t-test n χ test for norml istriution ws performe. The ifferenes were onsiere sttistilly signifint t p <.5. SUPPLEMENTARY MATERIAL Figure S. Expression of. Figure S. Sequenes of primers n frgments of PPAR-α 3 UTR. Figure S3. Effets of silening n overexpression of PPAR-α. Figure S4. Trgeting nlysis of PPAR-α y -5p. Figure S5. Effets of PPAR-α inhiitors. ACKNOWLEDGMENTS This work ws supporte y Chin-Cn Joint Helth Reserh Grnt (from the Ntionl Nturl Siene Fountion of Chin, No. 3949, to Y.Z. n Cnin Institutes of Helth Reserh, CCI 5, B.B.Y.), grnt from Cnin Institutes of Helth Reserh (CHRP ) n Nturl Sienes n Engineering Reserh Counil of Cn (CPG 34747) to S.N.K n B.B.Y, n Disovery Grnt from the Nturl Sienes n Engineering Reserh Counil of Cn (NSERC; 7937-) to B.B.Y., who is the reipient of Creer Investigtor Awr (CI 748) from the Hert n Stroke Fountion of Ontrio. W.W.D. is supporte y fellowship from Cnin Brest Cner Fountion of Ontrio. F.L. is supporte y Sholrship from Chin Sholrship Counil. Referenes. Anstee, QM (). Animl moels in nonloholi stetoheptitis reserh: utility n linil trnsltion. Liver Int 3: Ytsuji, S, Hshimoto, E, Tori, M, Tnii, M, Tokushige, K n Shirtori, K (9). 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