MicroRNA-129 and -335 Promote Diabetic Wound Healingby Inhibiting Sp1-Mediated MMP-9 ExpressionWei Wang,1 Chuan Yang,1,2 Xiao yi Wang,1 Li yan Zhou,1 Guo juan Lao,1,3 Dan Liu,1,2 Chuan Wang,1,2
Meng die Hu,1 Ting ting Zeng,1 Li Yan,1,2 and Meng Ren1,2
Diabetic wounds are recalcitrant to healing. However,the mechanism causing this dysfunction is not fully un-derstood. High expression of matrix metalloproteinase-9(MMP-9) is indicative of poor wound healing. In thisstudy, we show that specificity protein-1 (Sp1), a regula-tor of MMP-9, binds directly to its promoter and enhan-ces its expression. Additionally, we demonstrated thatSp1 is the direct target of twomicroRNAs (miRNAs), miR-129 and -335, which are significantly downregulated indiabetic skin tissues. In vitro experiments confirmed thatmiR-129 or -335 overexpression inhibits MMP-9 pro-moter activity and protein expression by targeting Sp1,whereas the inhibition of these miRNAs has the oppositeeffect. The beneficial role of miR-129 or miR-335 in di-abetic wound healing was confirmed by the topical ad-ministration of miRNA agomirs in diabetic animals. Thistreatment downregulated Sp1-mediated MMP-9 expres-sion, increased keratinocyte migration, and recoveredskin thickness and collagen content. The combinedtreatment withmiR-129 andmiR-335 induced a synergis-tic effect on Sp1 repression and MMP-9 downregulationboth in vitro and in vivo. This study demonstrates theregulatory mechanism of Sp1-mediated MMP-9 expres-sion in diabetic wound healing and highlights the poten-tial therapeutic benefits of miR-129 and -335 in delayedwound healing in diabetes.
Delayed wound healing is a common and serious compli-cation in patients with diabetes, affecting 15–20% of allpersons with diabetes (1–3). The reasons diabetic wounds
do not heal properly are unknown. However, an imbalancebetween the synthesis and degradation of the extracellularmatrix (ECM) leads to poor wound healing in patients withdiabetes (4).
Matrix metalloproteinases (MMPs) are a zinc-dependentendopeptidase family that degrades ECM componentsinvolved in tissue remodeling (5). The detrimental effectsof MMPs in diseased tissue are attributed to the rapidturnover of potential growth factors, receptors, and thenewly formed ECM, which are essential for wound healing(6). Elevated MMP-9 levels are present in various chronicnonhealing wounds, including diabetic foot ulcers (DFUs)(7–9). Our previous studies show that abnormally highMMP-9 expression contributes to delayed wound healingbecause of the imbalance between ECM synthesis anddegradation, and reducing MMP-9 expression promotesdiabetic wound healing (10,11).
MMP expression and activity are tightly regulated atmultiple levels (12), including gene transcription, post-transcriptional processing, and proenzyme activation(13,14). We recently demonstrated that methylation levelsat specific DNA loci in the MMP-9 promoter region areaffected by conditions resembling diabetes in the humankeratinocyte cell line HaCaT. Additionally, we investigatedthe MMP-9 promoter using bioinformatics analyses andidentified a specificity protein-1 (Sp1) binding site (15).However, Sp1 involvement in the regulation of MMP-9promoter activity is not well studied. Similarly, the regu-latory mechanisms of Sp1-mediated MMP-9 expressionare unknown.
1Department of Endocrinology, Sun Yat-Sen Memorial Hospital, Sun Yat-SenUniversity, Guangzhou, People’s Republic of China2China Diabetes-Related Chronic Wound Treatment Training Center, Guangzhou,People’s Republic of China3Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology,Texas A&M Health Science Center, Houston, TX
MicroRNAs (miRNAs) are highly conserved endogenoussmall noncoding RNA molecules involved in numerousbiological processes including diabetic wound healing.miRNAs regulate posttranscriptional gene expression bybinding to their target mRNAs, leading to mRNA degrada-tion or translation suppression (16–19). Although recentstudies revealed the important role ofmiRNAs in skin biologyand diseases, their role in diabetes wound healing is in itsinfancy.
We used serum miRNA expression profiling to identifynovelmiRNAs in diabetic wounds and found thatmicroRNA-129 (miR-129) and miR-335 were significantly downregu-lated in patients with diabetes compared with healthypatients. miR-129 and -335 play key roles in the regula-tion of cancer progression, chemoresistance, proliferation,and cell cycle control (20–23). However, their function indiabetic wound healing is unclear. Through bioinformaticsanalysis, we predicted that the transcription factor Sp1was a common target of miR-129 and -335. Therefore, weexplored the mechanism through which miR-129 and -335regulated Sp1-mediated MMP-9 expression and studiedtheir expression and function in the wound healing ofpatients with diabetes.
RESEARCH DESIGN AND METHODS
Ethics StatementThe Institutional Review Board of the Sun Yat-Sen Me-morial Hospital of Sun Yat-Sen University (Guangdong,China) approved the study protocol, which was in accor-dance with the principles of the Helsinki Declaration II.Written informed consent was obtained from each partic-ipant before data collection. The Sun Yat-Sen UniversityInstitutional Animal Care and Use Committee approved allanimal studies.
MMP-9 Expression in Wound Fluids of Patients WithDiabetesDemographic information including age, sex, blood pres-sure, BMI, blood pressure (systolic and diastolic bloodpressure), fasting blood glucose, hemoglobin A1c (HbA1c),serum creatinine, triglycerides, total cholesterol, HDL cho-lesterol, LDL cholesterol, and blood cell count (red bloodcell, white blood cell, platelets, and hemoglobin) wererecorded for all patients with DFUs and control subjects(Supplementary Table 1). Wound fluids were collected fromthe ulcer site of 21 patients with DFUs as previously de-scribed (24). Samples were stored at 280°C for furtherquantification of MMP-9 expression. The MMP-9 concen-tration was measured using an MMP-9 activity assay kit(Abcam, Boston, MA). The ulcer grade was assessed usingthe Wagner classification (25).
Skin Tissue SpecimensDiabetic perilesional skin samples were obtained frompatients undergoing amputation surgery. Control (healthy)skin tissue samples were collected from the lower leg ofpatients without diabetes undergoing reconstruction due to
foot injury. The wound edge area was excised in the perile-sional area of the skin ulcer or the edge of the surgical in-cision during routine surgery. Samples were rapidly trimmedto small strips (size 1 3 1 3 1 mm), fixed in 4% para-formaldehyde at 48°C overnight, and embedded in paraffin.Each paraffin block was serially sectioned into 20 sections4 mm thick and used for immunohistochemistry (IHC).
Hematoxylin-Eosin Staining and Masson TrichromeStainingSkin tissues were stained with hematoxylin-eosin (HE).Epidermal and dermal thickness was measured on stainedslides using pictures taken at 310 magnification. Massontrichrome staining was performed using a staining kit(Shanghai Bogoo Biotechnology, Shanghai, China) accordingto the manufacturer’s instructions. For collagen quantifica-tion, Masson trichrome–stained skin areas were determinedusing ImageJ software (National Institutes of Health).
IHCParaffin-embedded sections of skin tissues were stainedusing the avidin–biotin complex method as previouslydescribed (26). Sections were incubated with polyclonalrabbit anti-Sp1 or anti–MMP-9 antibody at 4°C overnightfollowed by treatment with horseradish peroxidase–labeled anti-rabbit IgG. The streptavidin–biotin complexwas used to visualize the staining. For each section, fivehigh-power fields were observed, and 100 cells from eachfield were counted. Scoring was conducted according tothe immunoreactive score (IRS) standard as previouslyreported (26). The antibodies were purchased from Milli-pore (Temecula, CA).
Cell Culture and TreatmentPrimary human keratinocytes were obtained from thehospital during routine infant circumcision (26) and iden-tified as described previously (Supplementary Fig. 1).HaCaT cells were purchased from American Type CultureCollection (Manassas, VA) and cultured in keratinocyteserum-free medium (Gibco, Gaithersburg, MD). Afterreaching 80% confluence, cells were incubated overnight inkeratinocyte serum-free medium containing 0.5 mg/mLBSA (Sigma-Aldrich, St. Louis, MO) or advanced glycationend product (AGE)–BSA (Millipore). The supernatants ofHaCaT cells were collected to measure MMP-9 activityusing an MMP-9 activity assay kit (Abcam). HaCaT cellswere harvested for analysis of Sp1, MMP-9, and miRNAexpression. Where indicated, Sp1 small interfering RNA(siRNA)1–3 (GenePharma, Shanghai, China) was trans-fected into the cells, or the Sp1-specific inhibitor mithra-mycin A (100 nmol/L; Abcam) was added.
Quantitative Real-time PCR AnalysisRNA isolation and quantitative real-time PCR (qPCR)were performed as previously described (27). Briefly, totalRNA was extracted with TRIzol (Gibco) according to themanufacturer’s instructions. qPCR was performed usinga LightCycler 480 SYBR Green Master (Roche Diagnostics,
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Mannheim, Germany) in a LightCycler System. All datawere analyzed using the expression of GAPDH as an inter-nal RNA standard.
Western Blot AnalysisTo identify Sp1 and MMP-9 expression, lysates fromcultured keratinocytes, HaCaT cells, and skin tissues werecollected and analyzed. Nuclear proteins were separatedusing commercial NE-PER Nuclear and Cytoplasmic Extrac-tion Reagents (Thermo Fisher Scientific, Waltham, MA).Western blot analysis was performed as described previ-ously (28). For normalization, the same membrane wasimmunoblotted with anti–b-actin or anti-H3 antibody (CellSignaling Technology, Danvers, MA).
Flow Cytometric AnalysisHaCaT cells were incubated with annexin V–FITC inloading buffer with 10 mL propidium iodide, resuspendedin binding buffer, and detected using an FACS (BD Bio-sciences, Franklin Lakes, NJ) analyzer. Analysis was carriedout in triplicate for three separate experiments.
Chromatin Immunoprecipitation AssaysChromatin samples from HaCaT cells or primary kera-tinocytes were prepared and incubated with an anti-Sp1antibody or normal IgG control (Millipore). Pulled-downDNA fragments and input DNA were purified and usedas templates for qPCR analysis using primers designedto amplify the 2668- to 2538-bp region of the MMP-9promoter, which contains the putative Sp1 binding site.
Dual-Luciferase Reporter AssayHaCaT cells were cotransfected with the MMP-9 promoterluciferase reporter and the pRL-TK reporter plasmid(Promega, Madison, WI) in the presence of an empty vectoror a plasmid expressing Sp1 (Sangon Biotech Co., Shanghai,China). The luciferase activities were assessed using a Dual-Luciferase Reporter Assay System (Promega) according tothe manufacturer’s protocol. The relative luciferase activitywas normalized to the Renilla luciferase activity.
Argonaute-RNA ImmunoprecipitationAn anti-AGO2 antibody (Ab32381; Abcam) and the MagnaRIP RNA-Binding Protein Immunoprecipitation Kit (17-700; Millipore) were used to perform RNA immunopre-cipitation of AGO2-containing RNA-induced silencingcomplex according to the manufacturers’ instructions.
miRNA Microarray Analysis, Target Prediction,and ValidationMicroarray hybridization, data generation, and normali-zation were performed by KangChen Bio-tech (Shanghai,China) using standard protocols (28–30). The thresholdvalue used to screen differentially expressed miRNAs wasa fold change of$2.0 or#0.5, P, 0.01, and a normalizedsignal value indicating a relative abundance to the tran-script of $2.0. The Sp1 39 untranslated region (UTR)sequences containing putative wild-type (WT) and mu-tated (MUT) target sites for miR-129 or miR-335 werechemically synthesized and inserted into the pGL3-
luciferase reporter vector (Promega). Human embryonickidney 293T (HEK-293T) cells were cotransfected with thereporter vector containing either the WT (Sp1-WT) orMUT (Sp1-MUT) putative Sp1 binding site and miR-129or miR-335 mimics using the Lipofectamine 3000 trans-fection reagent (Invitrogen, Carlsbad, CA). After 48 h, celllysates were prepared and used for the detection of theluciferase activity measured with a Dual-Luciferase Re-porter Assay Kit (Promega).
miRNA Mimics and Inhibitor TransfectionmiR-129 or -335 mimics, inhibitors, and scrambled miRNA(negative control [NC]) were designed and purchased fromRiboBio (Guangzhou, China). HaCaT cells were cultureduntil 50–60% confluent and transiently transfected withmiR-129 or -335 mimics, inhibitors, or NC. (50 mmol/L)using Lipofectamine 3000 (Invitrogen), according to themanufacturer’s instructions. After 48 h, the cells wereharvested and used for further assays.
5-Ethynyl-29-Deoxyuridine DNA Proliferation Assay,CCK-8 Cell Proliferation Assay, and TranswellMigration AssayThe 5-ethynyl-29-deoxyuridine DNA proliferation assaywas performed according to the manufacturer’s instruc-tions (RiboBio). Images were obtained using an invertedfluorescence microscope. The percentage of 5-ethynyl-29-deoxyuridine–positive cells was calculated from fiverandom fields. Cell proliferation was determined usinga CCK-8 kit (CWBiotech, Beijing, China) according to themanufacturer’s instructions. The vertical migration abili-ties of HaCaT cells were assessed using a transwell migra-tion assay, as described previously (31). The stained cellsfrom five randomly chosen fields were counted undera light microscope.
In Situ Hybridization of miRNAThe hsa-miR-129 and hsa-miR-335 probes were labeledwith 59 digoxigenin. An enhanced sensitive in situ hybrid-ization detection kit I (POD; Boster Biological Technology,Wuhan, China) was used to perform in situ hybridiza-tion, according to the manufacturer’s protocol. All re-lated reagents were purchased from CWBiotech. For eachsection, five high-power fields were observed, and 100 cellsfrom each field were counted. The percentage of miRNA-positive cells was calculated.
In Vivo Wound ModelSprague-Dawley rats (200–250 g each) were obtainedfrom the Laboratory Animal Center of Sun Yat-Sen Univer-sity. A diabetic wound was made as previously described(11). miR-129 and/or -335 agomirs (500 mL of a 1-nmol/Lagomir solution in PBS) were injected intradermally intothe wound edges of the rats immediately and 4, 7, and10 days after wounding. A negative agomir injection groupwas used as the control. Wound images were immediatelyacquired after wounding (day 0) and on days 7, 10, and14. Wound closure was quantified as the percentage ofthe initial wound area size. Wound healing rate was
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calculated as the percentage of the original wound sizeusing the following formula: (initial area 2 final area)/initial area 3 100%. Wound samples were collected 14days after injury for histology, gene and protein expres-sion, and collagen deposition analyses.
Statistical AnalysesContinuous variables are presented as the mean6 SD, anddifferences among groups were tested using the Studentt test or one-way ANOVA. Statistical analysis was per-formed using SAS software, version 9.2 (SAS Institute Inc.,Cary, NC). All SPSSs were two-sided, and P , 0.05 wasconsidered statistically significant.
RESULTS
MMP-9 Expression in Diabetic WoundsMMP-9 levels in wound fluid were significantly higher inpatients with DFUs than in control subjects. Additionally,an increase in the Wagner grade of the lesion was asso-ciated with a dramatic increase in MMP-9 level (Fig. 1A).Similarly, improvement in DFUs was accompanied bya gradual decline of MMP-9 levels (Fig. 1B). MMP-9 ex-pression in skin tissues of patients with DFUs was in theepidermis (Fig. 1C). The MMP-9 staining intensity wassignificantly higher in the skin from patients with DFUscompared with control subjects. A significant increase inMMP-9 expression was also observed in skin tissues ofdiabetic rats compared with controls (Fig. 1C). Histologicalresults of perilesional skin biopsies from patients andanimal models showed that the epidermis and dermisfrom diabetic wounds were significantly thinner thanfrom control subjects (Fig. 1C andD). The collagen fibers ofthe diabetic skin were thin, degenerated, and fractured;additionally, dermal collagen was less dense and disor-dered (Fig. 1E).
Sp1 Is Necessary for MMP-9 Gene ExpressionSp1 was expressed predominantly in the cytoplasm ofkeratinocytes from normal skin tissues, whereas in di-abetic skin tissues, Sp1 was expressed in both the nucleusand cytoplasm of keratinocytes (Fig. 2A). We also observedsignificantly increased Sp1 immunostaining in the skinsamples from patients with diabetes and diabetic animalscompared with controls (Fig. 2B).
After 24-h exposure to 200 mg/L AGE-BSA, the Sp1mRNA expression in HaCaT cells and primary keratino-cytes was unchanged. However, MMP-9 mRNA expressionlevels increased significantly after AGE-BSA treatment(Fig. 2C). The Sp1 and MMP-9 protein expression in-creased significantly after 48 h of AGE-BSA treatmentcompared with the control group (Fig. 2D). Sp1 was ex-pressed predominantly in the cytoplasm of HaCaT cells,whereas AGE-BSA increased Sp1 activity and promoted itsnuclear translocation (Fig. 2E and F).
Sp1 Binds Directly to the MMP-9 PromoterBioinformatics analysis indicated a putative Sp1 bindingsite is located in a region between 2571 and 2540 bp on
the MMP-9 promoter (Fig. 3A). Therefore, we hypothe-sized Sp1 could act on the MMP-9 promoter and enhanceMMP-9 expression. Sp1 inhibition via RNA interference ortreatment with the specific Sp1 inhibitor mithramycin Asignificantly decreased MMP-9 expression (Fig. 3B) with-out influencing cell proliferation (Supplementary Fig. 2) orapoptosis (Fig. 3C).
To test whether Sp1 modulates MMP-9 promoter ac-tivity, we performed luciferase assays. Sp1 overexpressionsignificantly increased MMP-9 promoter activity (approx-imately eightfold) (Fig. 3D). The results from chromatinimmunoprecipitation (ChIP) and qPCR assays confirmedSp1 binds directly to the MMP-9 promoter and activatesits transcription in both HaCaT cells and primary kera-tinocytes (Fig. 3E). Moreover, the treatment with AGE-BSA induced a stronger binding of Sp1 to the MMP-9promoter than BSA treatment (Fig. 3F).
miR-129 and miR-335 Expression in Diabetic SkinTissues and CellsTo identify changes in miRNA expression in diabetic skinlesions, we conducted a comprehensive microarray analy-sis comparing miRNA expression profiles of serum samplesfrom five patients with diabetic wounds and five matchedcontrol subjects (Supplementary Table 2). Hierarchicalclustering identified 159 miRNAs significantly altered inthe serum of patients with diabetic wounds (Fig. 4A). Six ofthe 58 downregulated miRNAs (miR-32, -106a, -106b,-129, -155, and -335) were validated using qPCR inAGE-BSA–treated HaCaT cells (Fig. 4B). We focused onmiR-129 and -335 because they were significantly down-regulated in patients with diabetes, and their biologicalfunction in diabetic wound healing was unclear. A signif-icant downregulation of miR-129 and -335 expression wasconfirmed in the skin of patients and rats with diabeticwounds through qPCR (Fig. 4C) and in situ hybridiza-tion assays (Fig. 4D and E).
miR-129 and -335 Regulate the TranscriptionFactor Sp1We then searched for possible miR-129 and -335 targetsusing three online bioinformatics algorithms: miRanda,TargetScan, and PicTar (Fig. 5A). The analysis indicatedmiR-129 and -335 had highly conserved binding sites inthe 39-UTR of Sp1 in several species. Argonaute-RNAimmunoprecipitation (AGO-RIP) results showed miR-129, miR-335, and Sp1 were enriched in the AGO-RIPfraction of HaCaT cells compared with the IgG control(Supplementary Fig. 3). To verify whether miR-129and miR-335 directly target the Sp1 39-UTR, we per-formed 39-UTR luciferase reporter assays. The luciferaseactivity of the Sp1 WT 39-UTR reporter decreasedtwofold after transfection with miR-129 or -335 mimicscompared with control. The mutation of the predictedtarget sites completely abolished the repressive effectof miR-129 or -335 mimics on the reporter gene expres-sion, demonstrating miR-129 and -335 directly targetSp1 (Fig. 5B).
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miR-129 and -335 Inhibit Sp1 and MMP-9 ExpressionBecause Sp1 is an important transcription factor for MMP-9, the downregulation of Sp1 by miR-129 and -335 mightlead to the downregulation of MMP-9 transcription. Toinvestigate this hypothesis, we evaluated Sp1 and MMP-9protein levels in HaCaT cells after treatment with miR-129or -335 mimics/inhibitors. miR-129 or -335 overexpression
in keratinocytes significantly decreased Sp1 expressionand MMP-9 activity, whereas opposite results wereobtained when inhibiting miR-129 or -335 (Fig. 5C, D,and F).
Interestingly, miR-129 and -335 mimics had a syner-gistic inhibitory effect on Sp1 and MMP-9 expression.Meanwhile, miR-129 and -335 inhibitors had synergistic
Figure 1—MMP-9 expression in wound fluid and skin wound tissues in diabetes. A: Relationship between MMP-9 levels in wound fluids ofdiabetic skin and the Wagner grade of the lesion. B: Representative images of the wounds and levels of MMP-9 for wound fluids at the firstwound dressing and at every visit thereafter until wound healing. C: IHC of MMP-9 in skin tissues of patients with diabetic wounds and anestablished diabetic animal model with skin lesions. The quantitative analysis of MMP-9 was performed according to the IRS. Bars representthe mean 6 SD. *P , 0.05. Original magnification 3400. D: Representative HE staining from diabetic skin tissues (DM) and control (CON).Epidermal and dermal thicknesseswere analyzed. Bars represent themean6SD. *P, 0.05. Original magnification3100. E: Masson stainingin skin tissues of patients with diabetic wounds and an established diabetic animal model with skin lesions. The number of areas stainingpositive was analyzed by ImageJ, and the average percentage was calculated. *P, 0.05. Original magnification3100. con., concentration.
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effects promoting Sp1 and MMP-9 expression (Fig. 5C,D, and F). After treatment with AGE-BSA, miR-129 or-335 ectopic expression also contributed to the down-regulation of Sp1 expression and MMP-9 activity (Fig. 5Eand F). Moreover, transfection of miR-129 or -335
mimics moderately induced the migratory capabilityof HaCaT cells, and their cotransfection significantlyinduced cell migration without affecting cell viability,proliferation (Supplementary Figs. 4–6), or apoptosis(Fig. 5G).
Figure 2—Expression and localization of Sp1 in AGE-BSA–treated skin cells and diabetic skin tissues. Histological analysis to detect Sp1expression in skin tissues from patients with diabetes (DM) (A) and a diabetic animal model (B). Normal skin tissues were used as control(CON). Original magnification 3400. Bars represent the mean 6 SD. *P , 0.05 vs. CON. HaCaT cells or primary human keratinocytes weretreated with 200mg/L AGE-BSA. ThemRNA (C) and protein expression (D) of Sp1 andMMP-9weremeasured using qPCR andWestern blot.b-Actin was used as an internal control. The histograms indicate the quantitative analysis of Sp1 and MMP-9 expression in humankeratinocytes and HaCaT cells. Each bar represents the mean6 SD. *P, 0.05. E: Localization and expression of Sp1 in HaCaT cells. HaCaTcells were treated with 200 mg/L AGE-BSA for 48 h. The localization and expression of Sp1 were assessed using immunofluorescence.Representative immunofluorescence images show the cellular localization of Sp1 (red) and nuclear staining with Cy3 (blue). Originalmagnification 3200. F: Cytoplasmic and nuclear proteins were collected and Sp1 expression was detected separately. The histogramsindicate the quantitative analysis of Sp1 expression in the nucleus of HaCaT cells. Bars represent the mean 6 SD. *P , 0.05.
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ChIP experiments were performed to investigate Sp1binding to the MMP-9 promoter in the presence of miRNAinhibitors. We found that miR-129 or -335 knockdownresulted in increased binding of Sp1 to the MMP-9 pro-moter, and miR-129 and -335 inhibitors had a synergeticeffect on Sp1 binding activity (Fig. 5H).
miR-129 and -335 Promote Diabetic Wound ClosureIn VivoWe injected miR-129 or -335 agomir or control oligosintradermally into the wound edges of diabetic rats im-mediately and 4, 7, and 10 days after the skin was in-jured (Fig. 6A). At 14 days, the wound healing rate was
significantly higher in the miR-129 (79.5 6 2.1%) or -335(82.4 6 4.5%) agomir group than in the controls (69.1 62.2%; P , 0.05). The combination miR-129 and -335agomir group (mix agomirs group) showed an 84.4%wound contraction (Fig. 6B and C).
Consistently, we observed decreased expression of Sp1and MMP-9 in the skin of the rats injected with the agomirin comparison with controls (Fig. 6D and E). The combinedinjection of miR-129 and -335 showed synergistic inhib-itory effects on Sp1 and MMP-9 expression in vivo.
Histological results showed that the epidermis anddermis of the control rats were significantly thinnerthan those of the rats injected with the miRNA agomirs
Figure 3—Sp1 binds directly to the MMP-9 promoter. A: Schematic representation of the putative Sp1 binding site on the promoter of MMP-9. B: Western blot analysis indicating the expression of Sp1 and MMP-9 in HaCaT cells treated with 50 nmol/L Sp1 siRNA (siRNA-1, -2, and-3) or 100 nmol/L of the Sp1 inhibitor mithramycin A (100 nmol/L). Scramble siRNA was used as NC. b-Actin was used as an internal control.Data are themean of three independent experiments performed in triplicate. Bars represent themean6 SD. *P, 0.05 vs. NC.C: Quantitativeresults of flow cytometric analysis of HaCaT cells transfected with Sp1 siRNA-1 (si-SP1-1) or the specific inhibitor mithramycin A (threeindependent experiments). D: Luciferase reporter assay on HaCaT cells cotransfected with Firefly luciferase constructs containing theMMP-9 promoter (2668 to2538 bp) and an Sp1 overexpression plasmid. *P, 0.05 vs. empty vector.E: ChIP assayswere performed to confirm thebinding of Sp1 to theMMP-9 promoter in HaCaT cells or primary keratinocytes using an anti-Sp1 antibody. Isotype IgGswere used as an NC.The histogram shows the average of three independent ChIP assays. *P, 0.05. F: HaCaT cells were treatedwith 200mg/L AGE-BSA for 48 h.ChIP assays were performed to compare the binding of Sp1 to the MMP-9 promoter with or without AGE-BSA treatment. The histogramshows the results of three independent experiments. *P , 0.05 vs. control group (BSA treatment). NF-KB, nuclear factor-kB.
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(Fig. 6F). A clear and distinct collagen layering was found inthe miRNA agomir-treated group, whereas in the controlgroup, collagen bundles were loosely packed (Fig. 6F).
These results indicate that miR-129 and -335 promotewound healing through the downregulation of Sp1-mediatedMMP-9 expression in vivo.
DISCUSSION
We report the novel finding that, in diabetes, the expres-sion of the transcription factor Sp1 is increased, and itsnuclear translocation is activated; Sp1 binds directly to theMMP-9 promoter, increasing MMP-9 expression in kera-tinocytes, which is associated with poor wound healing.miR-129 and -335 were markedly downregulated in theskin of patients with DFUs, as well as in animal modelsof diabetic wounds. These two miRNAs act cooperativelyto decrease MMP-9 levels by directly targeting the Sp1 39-UTR. In vivo upregulation of miR-129 and -335 promotedwound closure through the suppression of Sp1-mediatedMMP-9 expression in a diabetic wound model. Thus,targeting miR-129 and -335 may be a valid approach topromote wound healing in patients with diabetes.
In the pathogenesis of diabetic wounds, MMP-9 isactivated in skin cells, which impairs the balance ofECM synthesis and degradation, leading to unhealedwounds (7,8,32,33). In this study, we explored the asso-ciation between MMP-9 levels in wound fluid and theseverity of DFUs. The gradual decline of MMP-9 levelsduring the dynamic observation of the wound healingprocess further verified the essential role of MMP-9 indiabetic wound healing. In support of this, we and others(10,11) have shown that MMP-9 inhibition promoteswound healing. We then provided mechanistic detailsshowing that MMP-9 is activated in the skin of patientswith diabetes. MMP gene expression is regulated at thetranscriptional level, and the MMP-9 promoter contains cis-acting regulatory elements that bind several transcriptionfactors (34–36). In a previous study analyzing the tran-scriptional activity of a truncated MMP-9 promoter, wedemonstrated that Sp1 is the most important transcriptionfactor regulating MMP-9 expression (15). Sp1 is associatedwith the development and progression of various chroniccomplications in diabetes (37,38). However, the clinicalsignificance and biological role of Sp1 in diabetic skin tissues
Figure 4—miR-129 and -335 are significantly downregulated in diabetic wounds. A: miRNA array analysis showed the downregulatedmiRNAs in skin tissues from patients with DFUs and control subjects (CON; n = 5/group) (greater than twofold; P , 0.05). B: DifferentiallydownregulatedmiRNAswere validated by qPCR in AGE-BSA–treatedHaCaT cells. Bars represent themean6SD. *P, 0.05 vs. control (BSAgroup).C: qPCR analysis of miR-129 and -335 levels in normal skin tissues (CON) and skin tissues fromdiabetic wounds (DM). Bars representthe mean6 SD. *P, 0.05 vs. CON. D: Localization of miR-129 and -335 as detected by in situ hybridization in skin tissues from normal skinand diabetic wounds. Original magnification3400. E: The histogram indicates the percentage of miR-129 and -335–positive cells. *P, 0.05vs. CON.
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are unknown. The present results show Sp1 is upregulatedin diabetic skin tissues and AGE-BSA increased Sp1 activityand promoted its nuclear translocation in keratinocytes.At the same time, ectopic Sp1 expression increased
MMP-9 levels in HaCaT cells. Using both inhibitorsand siRNAs, Sp1 was a potent mediator of MMP-9upregulation in response to AGE-BSA. This finding issimilar to the recently reported Sp1 role in MMP-9
Figure 5—Sp1 is targeted by miR-129 and -335 in keratinocytes. A: Predicted miR-129 and -335 target sequences in Sp1 39-UTR in human,rat, and mouse species. B: The WT Sp1 39-UTR or the MUT 39-UTR was inserted into the Dual-Luciferase Reporter plasmid. HEK-293T cellswere cotransfected with miRNA mimics and luciferase reporters harboring WT or MUT Sp1 39-UTR fragments. Dual-luciferase assays wereconducted to compare the relative luciferase activities (Firefly/Renilla) among different groups. *P, 0.05 vs. NC. O.D, optical density. HaCaTcells were transfected with miR-129 or -335mimics (C) or inhibitors (D). The expression level of Sp1 andMMP-9 was detected usingWesternblot analysis. Bars represent the mean6 SD. *P, 0.05. E: Expression of Sp1 and MMP-9 in AGE-BSA–treated HaCaT cells transfected withmiR-129 or -335 mimics detected usingWestern blot analysis. Bars represent the mean6 SD. *P, 0.05. F: MMP-9 activity wasmeasured inthe supernatants of HaCaT cells. Bars represent the mean 6 SD. *P , 0.05. G: HaCaT cells were transfected with miR-129 and/or -335mimics and treated with AGE-BSA. Flow cytometry analysis was used to determine the rate of apoptosis. Data are reported as the mean6SD. H: HaCaT cells were transfected with miR-129 and/or -335 mimics. ChIP assays were performed to compare the binding of Sp1 to theMMP-9 promoter. The histogram indicates the results of three independent experiments. *P , 0.05 vs. NC. **P , 0.05 vs. single inhibitor(miR-129 inhibitor or miR-335 inhibitor) group.
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expression induction by reactive oxygen species in macro-phages of pulmonary fibrosis (39). Our results clearlydemonstrate that diabetes-induced Sp1 expression isclosely related to MMP-9 activation. Further, ChIP anddual-luciferase assays verified that the binding of Sp1 tothe MMP-9 promoter was functional: Sp1 could activate
MMP-9 transcription via directly binding to the MMP-9promoter region. Although previous studies suggestedSp1 might be involved in the regulation of MMP tran-scription (40,41), this is the first study reporting the directbinding of Sp1 to the MMP-9 promoter in diabetic skincells.
Figure 6—In vivo delivery of miR-129 and -335 accelerates wound healing in diabetes. A: miR-129 and/or -335 agomirs were injectedintradermally into the wound edges in rats (n = 10 for each group) immediately and on 4, 7, and 10 days after wounding. Skin biopsies at thewound site were collected on day 14 after injury. B: Representative wound healing images following surgery and 7, 10, and 14 days aftertreatment with miR-129 agomir, miR-335 agomir, or agomir mix. Wound sizes were calculated using ImageJ. C: Wound closures werequantified and are presented as the percentage of the initial wound area. Values are the mean6 SD for each group. *P, 0.05 vs. NC. D: Theexpression of Sp1 and MMP-9 was detected in the wounded skin 14 days after injury using Western blot assays. The histogram representsthe results of three independent experiments. *P, 0.05. E: IHC of Sp1 and MMP-9 in skin tissues from the agomir-injected and NC groups.The quantitative analysis of Sp1 and MMP-9 was performed according to the IRS. *P, 0.05. Original magnification3400. F: RepresentativeHE and Masson staining of skin tissues from the agomir-injected and NC groups. Epidermal and dermal thicknesses were analyzed. Thequantitative analysis of Masson staining positive areas was performed using ImageJ, and the average percentage of stained areas wascalculated. *P , 0.05. Original magnification 3100.
1636 miRNAs and Wound Healing Diabetes Volume 67, August 2018
Next, we focused on Sp1 posttranslational regulation.miRNAs play pivotal roles in different phases of woundhealing (42). We analyzed the miRNA signature in theserum of patients with DFUs and identified 159 differen-tially expressed miRNAs. Consistent with previous stud-ies, the wound-healing miR-32, -106a, -106b, and -155(5,43–45) were altered in our comparative miRNA array.Among all of the downregulated miRNAs, miR-129 and-335 showed the most significant changes. miR-129 and-335 play key roles in tumorigenesis (20–23), whereastheir roles in wound healing remain unknown. In thisstudy, in diabetic conditions induced by AGE-BSA treat-ment, miR-129 and -335 expression significantly de-creased in HaCaT cells and primary cultured keratinocytes.
The main function of miRNAs is to regulate posttran-scriptional gene expression by binding to target mRNAsleading to their degradation, causing translation suppres-sion or gene activation. First, the inverse correlationbetween miR-129 and miR-335 with Sp1 in skin samplesof patients with diabetic wounds was demonstrated,suggesting that miR-129 and -335 may regulate Sp1 andare therefore clinically relevant. Then, Sp1 was identifiedas a common target of miR-129 and -335 through AGO-RIP experiments and 39-UTR luciferase reporter assays.Interestingly, miR-129 and -335 showed combinatorialeffects on Sp1 repression in keratinocytes. The synergeticeffect cannot be attributed to the dose of miRNAs used inthe experiments because the same total amount of miR-NAs was used each time. Recent studies focusing on thecombinatorial effect of miRNAs establish that a singlemRNA molecule can be targeted by multiple miRNAs(46,47). For example, miR-30d, miR-181a, and miR-199a-5p act cooperatively to decrease the levels of 78-kDaglucose-regulated protein in cancer (48). The results pre-sented in this study suggest that the combined action ofmultiple miRNAs might be important to achieve efficientSp1 downregulation.
The modulation of miRNA expression through the ad-ministration of specific miRNA mimics or inhibitors mighthave therapeutic potential for nonhealing wounds (49,50).In the current study, subcutaneous injection of miR-129 or-335 agomir accelerated diabetic wound healing, improvedthe skin thickness in a diabetic wound animal modelthrough decreased MMP-9 expression, increased collagencontent, and enhanced migration of keratinocytes. Asmentioned above, in vitro experiments showed that theoverexpression or inhibition of miR-129 or -335 resultedin the downregulation or upregulation of Sp1 expression,respectively, and the concomitant change of MMP-9 proteinlevels in keratinocytes. Therefore, we hypothesized thatthe mechanism through which miR-129 and -335 promotewound healing in diabetic skin tissues might involve thesuppression of Sp1-regulated MMP-9 expression.
The combined local application of miR-129 and -335in vivo synergistically downregulated MMP-9 expressionthrough targeting Sp1 and induced the recovery of skinthickness and collagen content. However, miR-129 and
-335 did not act synergistically in wound contraction com-pared with the single miRNA injection. The combination ofthe two miRNA mimics may induce the overinhibition ofMMP-9 expression. Despite excessive MMP activity delay-ing wound healing, their overdownregulation might inhibitECM degradation and subsequent keratinocyte migration.This indicates that MMP-9 expression must be tightlycontrolled during wound healing.
In summary, our findings demonstrate an importantrole for Sp1-mediated MMP-9 expression in diabeticwound healing. miR-129 and -335 inhibit MMP-9 expres-sion by targeting Sp1. Upregulation of miR-129 or -335favors wound healing through MMP-9 downregulation.Collectively, these results establish the molecular mech-anism by which miR-129 and -335 modulate the matrix-remodeling enzyme MMP-9 via Sp1 and suggest thatthis mechanism could be exploited to accelerate woundhealing in vivo. These findings provide insight into theregulation of MMP-9 expression in diabetic wound healingby miRNAs and provide new therapeutic targets and strat-egies to ameliorate complications from diabetic wounds.
Funding. This work was supported by grants from the National Natural ScienceFoundation of China (81770827, 81471034, 81370910, and 81670764), the 863Program for Young Scientists (S2015AA020927), Science and Technology Plan-ning Project of Guangdong Province (2016B020238001), and the Special Fund forScience and Technology Development of Guangdong Province (2016A01010301).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. W.W. and C.Y. researched the literature, designedthe methods and experiments, analyzed the data, performed the statisticalanalysis, interpreted the results, and wrote and approved the manuscript. X.y.W.performed the collection of clinical data and the measurement of MMP-9 inwound fluid. L.y.Z. and G.j.L. worked together on the collection of associated dataand their interpretation, performed the molecular biological experiments, andcollaborated with all of the other authors. L.y.Z. and T.t.Z. performed the animalexperiments. D.L., C.W., and M.d.H. worked together on the isolation, culture, andassociated assessment of keratinocytes; approved the manuscript; and collab-orated with all of the other authors. L.Y. and M.R. also designed the experiments,discussed analyses and their interpretation, and helped to edit, revise, present,and approve the manuscript. L.Y. and M.R. are the guarantors of this work and, assuch, had full access to all of the data in the study and take responsibility for theintegrity of the data and the accuracy of the data analysis.
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