MicroRNA-31 Promotes Skin Wound Healing by Enhancing Keratinocyte Proliferation and Migration

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1 ORIGINL RTICLE McroRN- romotes Skn Wound Healng by Enhancng Keratnocyte rolferaton and Mgraton Dongqng L,XL, oxue Wang,, Floran Mesgen, ndor varcs, Enkö Sonkoly, Mona Ståhle and Nng Xu Landén Wound healng s a basc bologcal process restorng the ntegrty of the skn. The role of mcrorns durng ths process remans largely unexplored. By usng an n vvo human skn wound healng model, we show here that the expresson of mr- s gradually upregulated n wound edge keratnocytes n the nflammatory ( day after njury) through the prolferatve phase (7 days after njury) n comparson wth ntact skn. In human prmary keratnocytes, overexpresson of mr- promoted cell prolferaton and mgraton, whereas nhbton of mr- had the opposte effects. Moreover, we dentfed epthelal membrane proten (EM-) as a drect target of mr- n keratnocytes. The expresson of EM- n the skn was negatvely correlated wth the level of mr- durng wound healng. Slencng of EM- mmcked the effects of overexpresson of mr- on keratnocyte prolferaton and mgraton, ndcatng that EM- s a crtcal target medatng the functons of mr- n keratnocytes. Fnally, we demonstrated that transformng growth factor-β, whch s hghly expressed n skn wounds, upregulated mr- expresson n keratnocytes. Collectvely, we dentfy mr- as a key regulator for promotng keratnocyte prolferaton and mgraton durng wound healng. Journal of Investgatve Dermatology (5) 5, ; do:.8/jd.5.8; publshed onlne March 5 INTRODUCTION Cutaneous wound healng s a hghly orchestrated bologcal process, whch s essental to restore the ntegrty of the skn barrer. The classcal model of wound healng conssts of four sequental, yet overlappng phases: hemostass (up to several hours after njury), nflammaton ( days after njury), prolferaton ( days after njury), and tssue remodelng ( days- year after njury; Gosan and Detro, ). Reepthelalzaton s a crucal process, whch entals resurfacng of the wound wth new epthelum and requres proper mgraton and prolferaton of keratnocytes at the perphery of the wound (Santoro and Gaudno, 5). Defects of reepthelalzaton are assocated wth chronc non-healng wounds, whch affect about % of the populaton n developed countres and present a major and rsng health and economc burden to socety (Sen et al., 9). The Molecular Dermatology Research Group, Unt of Dermatology and Venereology, Centre for Molecular Medcne (CMM), Department of Medcne, Karolnska Insttutet, Stockholm, Sweden and Department of Dermatology, The Second fflated Hosptal of lan Medcal Unversty, lan, Chna Correspondence: Nng Xu Landén, Molecular Dermatology Research Group, Unt of Dermatology and Venereology, Centre for Molecular Medcne (CMM), L8:, Department of Medcne, Karolnska Insttutet, Stockholm SE-776, Sweden. E-mal: nng.xu@k.se bbrevatons: mrn, mcrorn; EM-, epthelal membrane proten ; pr-mr-, prmary mr- transcrpt; qrt-cr, real-tme quanttatve reverse transcrpton CR; srn, small nterferng RN; TGF-β, transformng growth factor-β Receved October ; revsed February 5; accepted February 5; accepted artcle prevew onlne 6 February 5; publshed onlne March 5 complex nature of wound healng process makes t challengng to develop effectve therapes. Current treatments for chronc ulcers focus manly on optmzaton of controllable healng factors e.g., nutrtonal support, nfecton clearance, and mechancal support. Few targeted approaches have been developed e.g., topcal applcaton of growth factor, such as platelet-derved growth factor (Sun et al., a). McroRNs (mrns) are ~ nt noncodng RNs, whch are beng recognzed as mportant gene regulators durng the last decade (He and Hannon, ). In humans, mrns are proposed to regulate ~ 6% of all proten-codng genes (Fredman et al., 9) and are thus nvolved n most bologcal processes nvestgated e.g., development, organogeness, apoptoss, and cell prolferaton (Ioro and Croce, ; Ha and Km, ). Deregulaton of mrn expresson has been mplcated n the pathogeness of many dseases, such as cancer, nfecton, and chronc nflammatory dseases (Bertero et al., ; Ioro and Croce, ; O'Connell et al., ). Increasng pre-clncal and clncal studes have demonstrated that modulaton of mrn expresson by admnstraton of specfc mrn mmcs or nhbtors may be benefcal for treatng dseases (Hydbrng and Badalan- Very, ; Nana-Snkam and Croce, ). Thus, mrns represent potentally mportant therapeutc targets. lthough n recent years ncreasng studes have revealed the mportant roles of mrns n skn bology and dseases (Schneder, ), the understandng of ther role n the wound healng process s stll n ts nfancy. MR- has been mplcated as an mportant regulator of keratnocyte 676 Journal of Investgatve Dermatology (5), Volume 5 5 The Socety for Investgatve Dermatology

2 DLet al. bology e.g., dfferentaton and har growth (Mardaryev et al., ; eng et al., ). Moreover, the upregulaton of mr- has been reported n several skn dseases characterzed by excessve keratnocyte prolferaton e.g., psorass, a chronc nflammatory skn dsease sharng many features wth wound healng (Xu et al., ; Morhenn et al., ) and cutaneous squamous cell carcnoma (Bruegger et al., ; Wang et al., ). We hypothesze that mr- may also play a role durng skn wound healng e.g., by regulatng keratnocyte prolferaton. Therefore, here we focused on mr- and studed ts expresson and functon n human skn wound healng. We show that mr- s gradually upregulated n wound edge keratnocytes n the nflammatory phase and prolferatve phase of wound healng and that t promotes cell prolferaton and mgraton by suppressng ts drect target gene, epthelal membrane proten (EM-). We dentfy transformng growth factor-β (TGF-β), whch s hghly expressed n the wound bed, as an nducer of mr- expresson. Taken together, our study suggests that mr- may be a potental therapeutc target for promotng skn wound healng. RESULTS MR- s upregulated n epdermal keratnocytes durng human skn wound healng To study the expresson of mrns durng human skn wound healng, we created surgcal wounds n the abdomnal skn of 7 healthy volunteers (Supplementary Table S onlne). Wound edge tssues were collected day (nflammatory phase) and 7 days (prolferatve phase) after njury (Supplementary Fgure S onlne). The hstologcal changes of the human skn wound durng healng process are shown n Supplementary Fgure S onlne. Heren, we focused on mr- and characterzed ts expresson pattern throughout the entre healng process. Real-tme quanttatve reverse transcrpton CR (qrt-cr) analyss revealed that the basal level of mr- n human skn s low but rapdly ncreasng.9-fold day after njury ( =.8; Fgure a). In the subsequent prolferatve phase (7 days post woundng), the expresson of mr- was contnuously upregulated 7.7-fold ( =.) compared wth the unwounded skn. Moreover, n the same wound bopses, we analyzed the expresson of prmary mr- transcrpts (prmr-), from whch mature mr- s processed (Fgure b). r-mr- was ncreased n the prolferatve phase (5.6-fold, =.7) compared wth the unwounded skn (Fgure b), postvely correlatng wth the level of mature mr- (R =.69, =.7; Fgure c). To dentfy the cell type(s) responsble for the dynamc changes of mr- expresson durng wound healng, we performed n stu hybrdzaton (Fgure d). In lne wth the qrt-cr results, the expresson of mr-was low n unwounded skn (day ), and ts sgnal was manly localzed n the basal layer of epdermal keratnocytes. The level of mr- was markedly upregulated n the prolferatve phase (day 7), spreadng to the suprabasal cell layers at the wound edge. Mnor amounts of mr- may also be present n dermal fbroblasts and nfltratng mmune cells. To clarfy the expresson pattern n detal, we separated the epderms and derms of the wound bopses from healthy donors (n = 5) by usng laser capture mcrodssecton and analyzed the expresson of mr- by qrt-cr (Fgure e and Supplementary Fgure S onlne). Our results show that mr- expresson s manly upregulated n the epderms compared wth the derms durng wound healng, whch s n lne wth the CR data of mr- expresson n whole skn bopses (Fgure a) and n stu hybrdzaton (Fgure d). Taken together, our results demonstrate that epdermal keratnocytes are the prmary cells of nterest for functonal studes of mr- durng skn wound healng. MR- promotes keratnocyte prolferaton Gven that mr- s hghly upregulated n keratnocytes at the wound edge n the prolferatve phase, we further studed the role of mr- n keratnocyte prolferaton. To ths end, we overexpressed or nhbted mr- n human prmary keratnocytes by transent transfecton of mr- specfc precursor RN (re-mr-) or nhbtor (nt-mr-), respectvely. The effcency of overexpresson or nhbton of mr- was confrmed by qrt-cr (Supplementary Fgure S onlne). We performed the CyQUNT cell prolferaton assay for the keratnocytes wth modfed mr- expresson, whch showed that overexpresson of mr- ncreased, whereas nhbton of mr- decreased the number of vable cells n culture (Fgure a and Supplementary Fgure S5 onlne). We also observed the ncreased expresson of the prolferaton marker K-67 n keratnocytes wth overexpressed mr-, whereas K-67 was reduced upon mr- nhbton (Fgure b). These results were further confrmed by the 5- ethynyl- -deoxyurdne (EdU) ncorporaton assay. Inhbton of mr- decreased cell prolferaton, as evdenced by the reduced percentage of EdU-postve cells and the cells wthn S-phase (Fgure c). Furthermore, usng colony formaton assays, we found that mr- enhanced the long-term growth of keratnocytes (Fgures d and e). Collectvely, we demonstrate that mr- s a postve regulator of keratnocyte growth, reflected by both mmedate ncrease of cell cycle progresson and long-term promoton of self-renewal. MR- enhances keratnocyte motlty Mgraton of keratnocytes s one of the essental bologcal processes for epdermal repar (Haase et al., ); therefore, we amed to determne the role of mr- n keratnocyte mgraton. To that end, we performed scratch assays that showed that overexpresson of mr- sgnfcantly accelerated the healng rate of keratnocytes hours after scratchng (.8-fold, =.; Fgure a), whereas nhbton of endogenous mr-reduced the rate of keratnocyte mgraton (.9-fold, =. at 7 hours;.-fold, =. at hours; Fgure b and Supplementary Fgure S5 onlne). In lne wth ths, by usng the haptotactc transwell mgraton assay, we found that overexpresson of mr-strkngly ncreased (.6-fold, =.) the mgraton capacty of keratnocytes (Fgure c). Vce versa, nhbton of mr- resulted n a sgnfcant decrease (.-fold, =.) n cell 677

3 D L et al Tme after njury (days) Tme after njury (days) Log (mr-) y 7.8 Epderms Derms.6.. NS. y y y7 y y y7 Scramble ( ) mr- ( ) mr- ( ) mr- ( ) y R=.86 =.7.5. y mr- (RU)..5 Log (pr-mr-) r-mr- (RU) mr- (RU).8 Fgure. MR- s upregulated durng skn wound healng. The expressons of mr- (a) and of prmary mr- transcrpt (pr-mr-; b) were analyzed n wound bopses from healthy donors (n = 7) before or and 7 days after njury usng real-tme quanttatve reverse transcrpton CR (qrt-cr). (c) Correlaton of mr- wth pr-mr- expresson n human wound bopses and 7 days after njury, Spearman-correlaton on log-transformed values. (d) In stu hybrdzaton was performed on wound bopses from healthy donors (n = 7) usng mr--specfic probe or scrambled probe. Blue-green color ndcates mr- expresson. Black arrow demarcates wound edges on day, whereas red one ponts at newly formed epderms on day 7 post woundng. Scale bar = μm. (e) The Epderms and derms n wound bopses from healthy donors (n = 5) before or and 7 days after njury were separated by laser capture mcrodssecton. The expresson of mr- were analyzed usng qrt-cr. MR- and pr-mr- CR data are expressed n relatve unts (RU) compared wth RNU8 RN and 8S rbosomal RN, respectvely. Results for ndvdual donors and mean ± SD are shown. o.5 and o.; the Mann Whtney U-test. mgraton (Fgure d). Together, our data dentfy mr- as a postve regulator of keratnocyte motlty. EM- s targeted by mr- n keratnocytes MRNs exert bologcal functons through regulatng ther target genes. Thus, we amed to dentfy the target gene medatng the bologcal effects of mr- n keratnocytes. To predct the potental targets for mr-, we performed an ntegrated analyss usng three ndependent algorthms: TargetScan (Lews et al., 5), mranda (John et al., ), and mrwalk (Dweep et al., ). Hundred putatve mr- targets were commonly predcted by all three algorthms (Fgure a and Supplementary Table S onlne). To dentfy targets that are regulated by mr- n keratnocytes, we revsted the data from our prevous study (Xu et al., ), n whch transcrptome analyss was performed on keratnocytes after nhbton of mr- (GSE95). mong the targets commonly predcted by all three algorthms, four genes were 678 Journal of Investgatve Dermatology (5), Volume 5 sgnficantly upregulated (fold change., o.5) upon mr- nhbton (Supplementary Table S onlne). In ths study, we focused on one of them, EM-, as t had been prevously dentfied as a drect target of mr- n esophageal squamous cell carcnoma (Zhang et al., ), contanng a conserved bndng ste for mr- n ts untranslated regon ( -UTR) of mrn (Fgure b; Lews et al., 5). Notably, EM- has been shown to regulate cancer cell prolferaton and mgraton n several prevous studes (Zhang et al., ; Sun et al., ; Sun et al., b; Sun et al., c; Sun et al., d). Frst, we examned whether EM- was drectly targeted by mr- n human prmary keratnocytes. We performed -UTR lucferase reporter assays wth reporter gene constructs contanng the full-length -UTR of EM- mrn n human prmary keratnocytes (Fgures b and c). The wldtype EM- -UTR lucferase actvty was decreased (.78fold, =.9) by mr- overexpresson, whereas mutaton

4 D L et al. 5,, Ctr R- Ctr R- RR-m re-m -m n-m e n r nt-mr-ctr Ctr Ctr - - R- -mr R- -mr m t e t r n n m re- 8 S nt-mr-ctr nt-mr- nt-mr- 6 Cells (%) EdU ntensty S 5, K-67 (RU) Fluorescence, G nt-mr-ctr re-mr- nt-mr- OD5 (% of control) ropdum odde re-mr-ctr S G/M EdU 8 6 Ctr R- Ctr R- mr R-m nt-m e t r n m re Fgure. MR- promotes keratnocyte prolferaton. (a) Keratnocytes were transfected wth nm pre-mr-/ant-mr- for 8 hours. The number of vable cells n culture was quantfied by the CyQUNT cell prolferaton assay. (b) The expresson of prolferaton marker K-67 was analyzed n the transfected keratnocytes usng real-tme quanttatve reverse transcrpton CR. ta are expressed n relatve unts (RU) compared wth 8 s rbosomal RN wth mean ± SD (c) Keratnocytes were transfected wth nm ant-mr- or ant-mr-control (Ctr) for 8 hours. Cell prolferaton and cell cycle progresson were measured by 5-ethynyl- -deoxyurdne (EdU) labelng and subsequent cell cycle analyss by flow cytometry. ercentage of cells n the G, S, and G/M phase of cell cycle and percentage of EdU+ cells are shown. (d) Colones formed by the transfected keratnocytes were staned wth crystal volet 8 days after transfecton. (e) Crystal volet was dssolved and absorbance value was measured at 5 nm. ta of one representatve experment out of four ndependent experments are shown wth mean ± SD o.5 and o.; Student s t-test. of the predcted target ste completely abolshed the effect of mr- on reporter gene expresson. These data demonstrate that mr- drectly regulates EM- expresson by bndng to the predcted target ste n ts -UTR. In lne wth ths, we found that overexpresson of mr- n keratnocytes sgnficantly decreased EM- expresson, whereas nhbton of mr- led to an ncreased expresson of EM-, both at mrn (Fgure d and Supplementary Fgure S6 onlne) and proten levels (Fgure e). Next, we analyzed the expresson of EM- n human n vvo wounds collected at dfferent stages of the healng process. EM- expresson level was rapdly ncreased.5-fold ( =.) day after njury n comparson wth the unwounded skn as measured by qrt-cr; however, ts expresson returned to basal levels 7 days after njury (Fgure f). Notably, there was a sgnficant negatve correlaton (R =.76, =.7) between the expresson of EM- and mature mr- n the human wound bopses and 7 days after njury (Fgure g). Usng mmunofluorescence stanng, we observed that EM- proten was expressed by all layers of epdermal keratnocytes n unwounded human skn, and ts sgnal was stronger n the granular layers (Fgure h). In lne wth the qrt-cr data, the expresson of EM- was hghly upregulated day after njury and dmnshed 7 days post woundng (Fgure h). Ths recprocal expresson pattern of mr- and EM- both n vtro and n vvo supports that mr- targets EM- n keratnocytes and the ncreased expresson of mr- may be crtcal n reducng the EM- level durng skn wound healng. Slencng of EM- promotes keratnocyte prolferaton and mgraton The functon of EM- n keratnocytes s largely unexplored. To determne whether the effects of mr- on keratnocyte prolferaton and mgraton could be medated through EM-, we slenced the expresson of EM- n keratnocytes by transfecton of two dfferent EM--specfic small nterferng RNs (srns).e., EM- s- and EM- s-. The slencng efficency of both srns was confirmed by qrtcr (Fgure 5a) and western analyss (Fgure 5b). Reduced expresson of EM- by srns led to a sgnficant ncrease n 679

5 D L et al. re-mr- nt-mr-ctr h h 6 re-mr-ctr re-mr- Healng rate (%) h Healng rate (%) h h nt-mr-ctr nt-mr- Cell counts (% of control) 6 nt-mr- r t -C R e-m r - R m e- r 7h nt-mr-ctr h 7h re-mr-ctr re-mr- nt-mr- h Cell counts (% of control) re-mr-ctr 5 5 r Ct R- mr t n -m nt Fgure. MR- promotes keratnocyte mgraton. Scratch assays were performed to assess the mgraton rate of keratnocytes transfected wth nm pre-mr- (a) or ant-mr- (b) for 8 hours. hotographs were taken at ndcated tme ponts after scratch njury. The healng rates were quantfied by measurng the area of the njured regon. Representatve photographs of the transwell mgraton assay for the keratnocytes transfected wth nm pre-mr- (c) or ant-mr- (d) were taken under orgnal magnficaton. The number of keratnocytes passng through the membrane was counted. ta of one representatve experment out of four ndependent experments are shown wth mean ± SD o.5, o., and o.; Student s t-test. the level of prolferaton marker K-67 (Fgure 5c) and the colony formaton capacty of keratnocytes (Fgures 5d and e), ndcatng that EM- nhbts keratnocyte growth. Moreover, the results of scratch assay showed that slencng of EM- ncreased the mgratory capacty of keratnocytes (Fgures 5f and g). Taken together, slencng of EM- promotes prolferaton and mgraton of keratnocytes, whch phenocopes the effects of mr- overexpresson. These data ndcate that EM- s an mportant target medatng the bologcal functons of mr- n keratnocytes. MR- expresson s nduced by TGF-β n keratnocytes It has been shown that both TGF-β and TGF-β are crucal cytoknes n regulatng keratnocyte mgraton and prolferaton durng re-epthelalzaton, and ther levels are upregulated n skn wounds (Ramrez et al., ). We have prevously dentfied TGF-β as an nducer of mr- expresson n keratnocytes (Xu et al., ). Here we evaluated the effect of TGF-β on mr- expresson, 68 Journal of Investgatve Dermatology (5), Volume 5 showng that t sgnficantly ncreases the expresson of both mr- (.-fold, =.7; Fgure 6a and Supplementary Fgure S7 onlne) and ts prmary precursor pr-mr- (.fold, =.; Fgure 6b) n keratnocytes. Moreover, when we treated keratnocytes wth the TGF-β receptor nhbtor SB5, the nducble effect of TGF-β on mr- expresson was abolshed (Fgures 6c and d). ccordngly, we found that slencng of TGF-β expresson n keratnocytes wth srns decreased the expresson of both mr- and prmr- (Fgures 6e and f), ndcatng that the TGF-βmedated-regulaton of mr- expresson also nvolves autocrne-sgnalng pathways. In lne wth ths, we show that TGF-β sgnficantly nhbted EM- expresson n keratnocytes, whch may at least partally be medated by the ncreased expresson of mr- (Fgure 6g). Interestngly, we found that slencng of EM- expresson decreased mr- expresson n keratnocytes (Supplementary Fgure S8 onlne), suggestng that the hgh level of EM- durng the nflammatory phase may be another trgger factor of mr- expresson

6 D L et al. mranda 6-5 EM Mutaton Ctr R- Ctr - RR- mr -m re-m t-m ntn re 6 k EM- 8 β-ctn y..5 7 Tme after njury (days) Mut R=.7559 = y WT Log (EM-) re-mr- Vector EM- (RU) re-mr-ctr 5 re-mr-ctr mrwalk 8 5 nt-mr EM- (RU) 97 nt-mr-ctl Targetscan re-mr- 59 Lucferase actvty (% of control) 5 Has-mR Log (mr-). y 7 EM- DI Fgure. Epthelal membrane proten (EM-) s targeted by mr- n keratnocytes. (a) Venn dagram depctng the number of potental targets of mr- predcted by three bonformatcs methods. (b) Nucleotde resoluton of the predcted mr- bndng ste n UTR of EM- mrn. (c) Keratnocytes were transfected wth ng ml lucferase reporter plasmds contanng wld-type (WT) or mutant (Mut) EM- -UTR or empty vector (Vector) together wth nm pre-mr- or pre-mr-control (Ctr) and lucferase actvty was measured 8 hours later. (d e) Keratnocytes were transfected wth nm pre-mr-/ant-mr- for 8 hours and EM- expresson was analyzed by qrt-cr and western blottng. ta of one representatve experment out of four ndependent experments are shown wth mean ± SD o.5, o., and o.; Student s t-test. (f) EM- expresson was analyzed n wound bopses from healthy donors (n = 7) before or and 7 days after njury usng qrt-cr. ta are expressed n relatve unts (RU) compared wth 8 s rrn wth mean ± SD o.; the Mann Whtney U-test. (g) Correlaton of EM- mrn wth mr- expresson n human wound bopses and 7 days after njury, Spearman-correlaton on log-transformed values. (h) Immunofluorescence stanng of EM- (red) n human wound sectons (n = 7), whch were counterstaned usng DI (blue, nucleus). Scale bar = μm. -UTR, -untranslated regon; DI, ',6-damdno--phenylndole; qrt-cr, real-tme quanttatve reverse transcrpton CR; rrn, rbosomal RN. n addton to TGF-β. Collectvely, our results suggest that the ncreased levels of TGF-β n wounds contrbute to the upregulaton of mr- n the keratnocytes. DISCUSSION In ths study, we characterze the mr- expresson durng human n vvo wound healng process. We show that mr- s manly expressed n epdermal keratnocytes, n accordance wth our prevous observaton (Xu et al., ). The expresson of mr- s low n the ntact skn; however, upon woundng, ts level gradually ncreases from the nflammatory phase through the prolferatve phase. In comparson wth the expresson pattern of ts prmary transcrpt, pr-mr-, whch s only upregulated n the prolferatve phase, we conclude that mr- may be regulated durng wound healng at dfferent levels: the rased amounts of mature mr- n the nflammatory phase s most lkely due to the more efficent processng of exstng pr-mr-, whereas n the subsequent prolferatve phase the further upregulaton of mr- expresson also requres ncreased transcrpton of the mr- gene. The next queston was whether ths characterstc expresson pattern of mr- correlates wth ts bologcal functon durng skn wound healng. By usng multple functonal assays n human prmary keratnocytes wth modfied mr- expresson, we demonstrate that mr- promotes both the prolferaton and mgraton of keratnocytes, whch are crucal 68

7 DLet al. EM- (RU) EM- re-mr-ctr re-mr- EM- s- EM- s- K-67 (RU) 6 EM- s- EM- s- EM- s- EM- s- OD5 (% of control) 9 8 β-ctn EM- s-. EM- s EM- s- EM- s- h 8 h Healng rate (%) EM- s- EM- s EM- s- EM- s- Fgure 5. Slencng of epthelal membrane proten (EM-) expresson ncreases keratnocyte prolferaton and mgraton. Keratnocytes were transfected wth nm EM- small-nterferng RNs (EM- s- or EM- s-) or srn-negatve control () for 8 hours. The slencng effcency was analyzed by measurng EM- mrn wth qrt-cr (a) and western blottng (b). (c) The expresson of prolferaton marker K-67 was analyzed n these transfected cells. ta are expressed n relatve unts (RU) compared wth 8 s rrn wth mean ± SD colones formed by the transfected keratnocytes were photographed (d) and quantfed (e). Scratch assays were performed to assess the mgraton rate of keratnocytes transfected wth EM- srns. hotographs were taken at ndcated tme ponts after scratch njury (f), and the healng rates were quantfed (g). ta of one representatve experment out of four ndependent experments are shown wth mean ± SD o.5, o., and o.; Student s t-test.qrt-cr, real-tme quanttatve reverse transcrpton CR; rrn, rbosomal RN. cellular events durng the prolferatve phase of wound healng and essental for re-epthelalzaton. Mgraton of keratnocytes at the wound edge s stmulated by lack of contact nhbton and many factors actve durng wound healng e.g., epdermal growth factor, keratnocyte growth factor, and TGF-βs (Renke and Sorg, ). Our study adds mr- to a growng number of mrns mplcated n the regulaton of keratnocyte mgraton e.g., mr- (hmed et al., ; Yang et al., ), mr- (Vtcche et al., ), mr-8-p (Bertero et al., ), and mr- (melo et al., ). Keratnocytes n the wound margns wth mtotc ablty, whch may nclude epdermal stem cells, transently amplfyng cells and early dfferentated cells (L et al., ), start to prolferate toward the end of nflammatory phase and provde more cells for mgraton to close the wound (Morasso and Tomc-Canc, 5). Thus, the hgh expresson of mr- n keratnocytes n the prolferatve phase may contrbute to the re-epthelalzaton process. In addton to ts effects on keratnocyte growth and motlty durng wound healng, mr- s nvolved n several other physcal processes of the skn. Durng the har cycle, mr- s upregulated n the anagen phase and nhbton of mr- accelerates anagen development (Mardaryev et al., ). lso, mr- regulates epdermal keratnocyte dfferentaton by enhancng Notch sgnalng (eng et al., ). Moreover, the upregulaton of mr- has been reported n several skn dseases, such as psorass, whch shares many features wth wound healng (Morhenn et al., ). MR- s overexpressed n epdermal keratnocytes of psorass patents, promotng cytokne and chemokne producton by keratnocytes, and contrbutes to skn nflammaton (Xu et al., ). Recently, the ncreased expresson of mr- was found n cutaneous squamous cell carcnoma, and t functons as an oncomr by ncreasng the motlty and colony-formng ablty of cancer cells (Bruegger et al., ; Wang et al., ). Taken together, mr- s a multfunctonal mrn playng mportant roles n both physologcal and pathologcal condtons of epdermal keratnocytes. Identfcaton of the drect target of mr- s crtcal for understandng ts mechansm of acton n keratnocytes. In ths study, we demonstrate that mr- targets EM- n keratnocytes, n accordance wth a prevous report n esophageal squamous cell carcnoma (Zhang et al., ). In lne wth ths, we observed a recprocal expresson pattern between mr- and EM- n the epdermal keratnocytes at human wound edge durng healng process, ndcatng that mr- may control the level of EM- n vvo. However, n the nflammatory phase, the expresson of mr- s only ncreased slghtly, whch cannot explan the surge n EM- expresson at ths tme pont. resumably EM- expresson may be trggered by other unknown mechansms. revously, EM- has manly been descrbed as a tumor suppressor e.g., t nhbts tumor cell growth and motlty n gastrc carcnoma (Sun et al., b), colorectal carcnoma 68 Journal of Investgatve Dermatology (5), Volume 5

8 DLet al. mr- (RU) Control TGF-β r-mr- (RU) Control TGF-β mr- (RU) TGF-β SB r-mr- (RU) 5 TGF-β SB mr- (RU) r-mr- (RU) EM- (RU) TGF-β/ TGF-βR SMD mr- SMD EM- & other targets Mgraton rolferaton TGF-β s TGF-β s Control TGF-β pr-mr- SMD mr- gene Re-epthelazaton Keratnocyte Fgure 6. Transformng growth factor-β (TGF-β) nduces mr- expresson n keratnocytes. The expressons of mr- (a) and of pr-mr- (b) were analyzed n keratnocytes treated wth ng ml TGF-β for 8h by qrt-cr. TGF-β receptor nhbtor, SB5, was appled 5 mn before addng TGF-β. MR- (c) and pr-mr- expressons (d) were analyzed 8 hours later usng qrt-cr. Keratnocytes were transfected wth TGF-β srn for 8 hours and the expressons of mr- (e) and of pr-mr- (f) were measured by qrt-cr. (g) EM- expresson was analyzed n keratnocytes treated wth ng ml TGF-β by qrt-cr. mrn and mrn CR data are expressed n relatve unts (RU) compared wth RNU8 RN and 8S rrn, respectvely. ta of one representatve experment out of four ndependent experments are shown wth mean ± SD o.5, o., and o.; Student s t-test. (h) Schematc summary of the regulaton and functon of mr- durng skn wound healng. EM-, epthelal membrane proten ; mrn, mcrorn; qrt- CR, real-tme quanttatve reverse transcrpton CR; rrn, rbosomal RN; srn, small nterferng RN. (Sun et al., c), breast carcnoma (Sun et al., d), nasopharyngeal cancer (Sun et al., ), and esophageal squamous cell carcnoma (Zhang et al., ). Here we found that EM- also suppressed the growth and mgraton of normal epdermal keratnocytes. Durng the transton from the nflammatory to the prolferatve phase, the expresson of mr- s hghly upregulated, whch results n the reduced expresson of ts target, EM, and thus assurng proper reepthelalzaton durng skn wound healng. Therefore, n ths study, we dentfed a mechansm medatng the bologcal functons of mr- n skn wound repar.e., mr- unblocks the EM--dependent represson of keratnocyte prolferaton and mgraton. nother mportant queston s why mr- s upregulated n the wound edge keratnocytes durng the healng process. revously, we performed a systematc screen usng dfferent cytoknes, growth factors, and factors modulatng cell dfferentaton and dentfed TGF-β as a potent regulator of mr- expresson n keratnocytes (Xu et al., ). Here we expanded the prevous fndngs and show that TGF-β can also nduce the expresson of both mature mr- and prmary transcrpts of mr-. Bndng of TGF-βs to ther receptors leads to the actvaton of downstream Smad protens, whch nduce the expresson of TGF-β target genes (Ramrez et al., ), e.g., mr-, as shown n our study. It has been known that, upon njury, both TGF-β and TGF-β are hghly upregulated n the skn wounds, promotng keratnocyte mgraton and re-epthelalzaton (Ramrez et al., ), and these effects may be at least partally medated through nducton of mr- expresson. Moreover, we found that slencng of EM- expresson decreased the level of mr- n keratnocytes, suggestng that the hgh level of EM- durng the nflammatory phase may be another trgger factor of mr- expresson n addton to TGF-β. The EM--nduced-mR--expresson may functon as a negatve feedback to turn-off the adverse effects of EM- durng the later phase of wound healng. Taken together, our studes propose a model, n whch mr- s nduced by TGF-β and TGF-β n wound edge keratnocytes. Overexpresson of mr- ncreases the prolferaton and mgraton of keratnocytes at least partally by targetng EM- (Fgure 6h). These fndngs suggest that ncreasng the level of mr- n skn wounds may be a strategy to mprove healng. Thus, mr- may be a promsng canddate for further nvestgatons of mrn-based therapeutcs. MTERILS ND METHODS RN extracton, qrt-cr, n stu hybrdzaton, cell culture methods, cell growth, and motlty analyss are descrbed n the Supplementary Materals and Methods onlne. 68

9 DLet al. Human wound bopses Healthy volunteers (n = 7) were enrolled at Karolnska Unversty Hosptal Dermatology Clnc, Stockholm, Sweden (Supplementary Table S onlne). Full-thckness skn wounds were made wth a -mm bopsy punch n the abdomnal regon of the healthy volunteers. On day and day 7 after njury, the wound edge area was excsed wth a 6-mm bopsy punch and snap frozen (Supplementary Fgure S onlne). The clncal materal was obtaned after wrtten nformed consent, and the study was approved by the Stockholm Regonal Ethcs Commttee and conducted accordng to the Declaraton of Helsnk rncples. Laser capture mcrodssecton Frozen wound bopses were embedded n Tssue-Tek (Thermo Scentfc, Waltham, M), and 8-μm tssue sectons were cut and staned wth hematoxyln. Laser capture mcrodssecton was performed wth Leca LMD7 (Leca, Bernred, Germany). RN from mcrodssected tssue was purfed usng the mrneasy Mn Kt (Qagen, Hlde, Germany). lasmds mutageness and Lucferase reporter assays Lucferase reporter plasmds contanng -UTR of the EM- gene and empty lucferase vector were obtaned from ctve Motf (Carlsbad, C). The mutaton was generated at the predcted target ste of mr- usng the QuckChange XL ste-drected mutageness kt (Stratagene, Lo Jolla, C) accordng to the manufacturer s nstructons. The sequences of CR prmers for mutaton are as follows: forward 5 -gagaaagctaataaagaaaagtacggatgttgtctcagattctccct ggggt- ; reverse 5 -accccagggagaatctgagacaacatccgtacttttctttattagctt tctc-. The mutaton was verfed by sequencng (Eurofns Genomcs, Ebersberg, Germany). lasmds ( ng ml ) were cotransfected wth pre-mr- or pre-mr-control (Ctr; nm) nto human prmary keratnocytes usng Fugene HD transfecton reagent (romega, Madson, WI). Forty-eght hours later, Lucferase actvty was analyzed usng a LghtSwtch Lucferase ssay Kt (ctve Motf) accordng to the manufacturer s nstructons. Western blottng EM- proten expresson was detected wth mouse ant-human EM- antbody (:,; bcam, Cambrdge, UK) and then vsualzed usng horseradsh peroxdse (HR)-conjugated antmouse antbody (:,; ko Cytomaton, Glostrup, Denmark). s loadng control, the blots were re-probed usng HR coupled anthuman actn antbody (:,) (Sgma-ldrch, St Lous, MO). Immunofluorescence stanng Frozen human wound sectons (5 μm) were rnsed wth phosphatebuffered salne, followed by ncubaton wth.% H O for mnute. fter blockng wth % bovne serum albumn, the sldes were ncubated wth rabbt ant-human EM- antbody (:, bcam) at C overnght. Matched IgG sotype controls were ncluded for each stanng. rmary antbodes were detected wth lexa Fluor 59-conjugated goat ant-rabbt IgG (H+L; Lfe Technologes, Carlsbad, C) and vsualzed wth a fluorescence mcroscope. Statstcal analyss ll data are presented as mean ± SD. Mann Whtney U-test or Student s t-test was used to determne the sgnfcance between two groups. Correlaton of the expresson of dfferent genes was made usng earson s correlaton test on log-transformed data. -values o.5 were consdered to be statstcally sgnfcant. CONFLICT OF INTEREST The authors state no conflct of nterest. CKNOWLEDGMENTS We thank all healthy donors partcpatng n ths study. We thank nna-lena Kastman and Helena Grehsel for excellent techncal support. Ths work was supported by the Swedsh Research Councl (Vetenskapsrådet, project number: K-85X-5--), La Roche-osay Foundaton, Hedlunds Foundaton, Welander and Fnsens Foundaton, Sgurt and Elsa Goljes Memoral Foundaton, and Karolnska Insttutet. SULEMENTRY MTERIL Supplementary materal s lnked to the onlne verson of the paper at REFERENCES hmed MI, Mardaryev N, Lews CJ et al. () McroRN- s an mportant downstream component of BM sgnallng n epdermal keratnocytes. 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