Downregulation of miRNAs during Delayed Wound Healing in Diabetes: Role of Dicer
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Delayed wound healing is a major complication associated with diabetes and is a result of a complex interplay among diverse deregulated cellular parameters. Although several genes and pathways have been identified to be mediating impaired wound closure, the role of microRNAs (miRNAs) in these events is not very well understood. Here, we identify an altered miRNA signature in the prolonged inflammatory phase in a wound during diabetes, with increased infiltration of inflammatory cells in the basal layer of the epidermis. Nineteen miRNAs were downregulated in diabetic rat wounds (as compared with normal rat wound, d 7 postwounding) together with inhibited levels of the central miRNA biosynthesis enzyme, Dicer, suggesting that in wounds of diabetic rats, the decreased levels of Dicer are presumably responsible for miRNA downregulation. Compared with unwounded skin, Dicer levels were significantly upregulated 12 d postwounding in normal rats, and this result was notably absent in diabetic rats that showed impaired wound closure. In a wound-healing specific quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) array, 10 genes were significantly altered in the diabetic rat wound and included growth factors and collagens. Network analyses demonstrated significant interactions and correlations between the miRNA predicted targets (regulators) and the 10 wound-healing specific genes, suggesting altered miRNAs might fine-tune the levels of these genes that determine wound closure. Dicer inhibition prevented HaCaT cell migration and affected wound closure. Altered levels of Dicer and miRNAs are critical during delayed wound closure and offer promising targets to address the issue of impaired wound healing.
Wound healing is a complex biological process involving an intricate crosstalk of cellular events including inflammation, reepithelialization and restoration of affected tissues (1,2). All these events are controlled by diverse growth factors, cytokines, matrix proteins and a variety of cell types. Homeostasis initiates the first response during wound healing, and this leads to movement of several cytokines, chemokines and growth factors (such as platelet-derived growth factor [Pdgf] and transforming growth factor β [Tgfβ]) toward the wound site. Invasion of neutrophils into the wound area primarily removes the resident microbes, and this step is followed by active movement of macrophages and monocytes that act to remove dead tissues and other debris materials (3,4). These phenomena involve the generation of reactive oxygen species and nitric oxide, and these inflammatory events are followed closely by reepithelialization that is achieved by migration and proliferation of keratinocytes and fibroblasts in and around the wound site. Also, epidermal cells undergo desmosomal dissolution and formation of peripheral cytoplasmic actin filaments. Subsequently, basement membrane proteins orderly reappear and epidermal cells tend to get attached to the basement membrane and dermis followed by formation of granulation tissue (1,5).
Any interference during these orchestrated events of wound healing leads to delayed wound healing, which is major cause of gangrene and limb amputations (6,7). Diabetes is a major cause of delayed wound healing, and various factors have been reported to be associated with this complication. Local ischemia due to vascular damage during diabetes significantly contributes to a delay in wound closure. Damage to the extracellular matrix (ECM), impaired granulocyte function and movement, altered neovascularization, increased proteinase activity and defective macrophage function all contribute to delayed wound healing during diabetes (8, 9, 10, 11). While a lot of studies report the involvement of several genes and pathways responsible for this complication, little is known of the role of microRNAs (miRNAs) in this phenomenon.
miRNAs are a class of endogenously expressed noncoding RNAs of ∼22 nucleotides in length that regulate gene expression primarily at the posttranscriptional level by translational repression or mRNA degradation/mRNA destabilization (12,13). They have been implicated in various physiological processes and pathological states, and altered levels of miRNAs have been reported in several diseases (14, 15, 16). In wound healing, too, few studies demonstrate the roles of miRNAs (17,18) and deregulated miRNA signatures are associated with impaired wound healing. miR-210 that is induced under hypoxic conditions targets E2f3 and inhibits keratinocyte proliferation during wound closure (19). Induction of miR-200b during wounding suppresses angiogenesis by targeting globin transcription factor 2 (Gata2) and vascular endothelial growth factor receptor 2 (Vefgfr2) (20). Several miRNAs including miR-130a and miR-21 are aberrantly expressed in venous ulcers, and they delay epithelization in an acute human skin wound model (21). miR-146a is downregulated in wounds of diabetic mice and correlates with the expression of inflammatory genes (22), thereby delaying the process of wound closure. In type 2 diabetic KKAy mice, 14 miRNAs were differentially regulated in the wounded skin, and miR-21 was identified to be critical in fibroblast migration (23). In chronic nonhealing diabetic ulcers, persistent elevated miR-198 expression impairs keratinocyte migration and reepithelialization, thereby delaying wound closure (24). Except for these sparse reports, not much has been reported on the roles and mechanisms of miRNAs in delayed wound healing during diabetes. Prolonged inflammation is one of the critical factors for delayed wound closure. A normal inflammatory phase during wound closure peaks at ∼1–4 d before moving into the next phase of granulation and proliferation. In a diabetic wound, the process many times is stuck in the inflammatory phase because of a variety of altered factors that prolong this phase and prevents the initiation of the next phase, thereby delaying the wound-healing process (25,26). Therefore, several studies are aimed at identifying processes that prolong the inflammatory phase.
In this study, we sought to interrogate miRNA signatures in the diabetic rat wound that is stuck in the inflammatory stage compared with the normal rat wound that shows a normal healing pattern. We report an altered miRNA signature in the wounded skin of diabetic rats during the prolonged inflammatory phase and present interacting networks of their predicted targets with specific wound-healing genes.
Materials and Methods
Animals and Induction of Diabetes
The study was approved by the Institutional Animal Ethical Committee. Male Sprague Dawley rats aged 10–11 wks (225–275 g) were procured from the National Institute of Nutrition (Hyderabad, India). Animals were allowed to acclimatize for 2 wks under a 12:12 light-dark cycle and were given food and water ad libitum throughout the experiment. Ten days before wounding, rats received intraperitoneal injection of streptozotocin (65 mg/kg body weight in citrate buffer 0.1 mol/L, pH 4.5.) to induce diabetes (n = 6 in each group). Control rats were injected with citrate buffer. On the third day after injection, blood glucose levels were measured by tail vein bleeding (Accu-Chek Glucometer). Rats with persistent blood glucose levels ≥250 mg/dL until wounding were confirmed diabetic and used for the experiment. Control rats had blood glucose levels in the range of 90–110 mg/dL. All animals were weighed at regular intervals and observed for side effects. While the control (normal) rats gained weight until the end of the experiment (245 ± 3 g [before] and 283 ± 4 g [end of the experiment]), there was a significant decrease in the body weights of streptozotocininduced diabetic rats (269 ± 3 g [before] and 153 ± 7 g [end of the experiment]). As compared with normal rats, the daily food and water intake increased in diabetic rats.
Creation of Wounds
Ten days after induction of diabetes, two full thickness excisional wounds with splints were created on the dorsal surface of the animals (n = 6 in each group). The rat’s dorsal surface hair was first trimmed and depilated 24 h before wounding. During the entire wounding procedure, rats were anesthetized with isofluorane (4% in 100% oxygen at a flow rate 1 L/min) and anesthesia was maintained using 1–3% isofluorane. Dorsal skin was wiped with an alcohol swab and 10% povidone-iodine solution was applied. Two symmetrical wounds were made on the dorsum of the rats on either side of the spine by using a sterile 5.0-mm diameter punch biopsy tool (Dermaindia). Silicone splints were placed around the wound area, and anchoring of the splint was done with four nylon sutures to ensure positioning. Immediately after splinting, wounds were dressed (Tegaderm, 3M). Rats were housed individually after the procedure to preserve skin and dressing integrity. On d 1, 7 and 12 postwounding (corresponding to normal phases of inflammation, granulation/proliferation and matrix remodeling, respectively), animals were euthanized by a single intraperitoneal injection of sodium thiopentone (Neon Laboratories Limited), and wounded tissue together with adjoining tissues of ∼0.5 mm were excised. Samples from normal and diabetic skin (unwounded [isolated on the same day as d-7-postwounding tissue] and wounded [d 1, 7 and 12 postwounding]) were washed in 1 × phosphate-buffered saline (PBS) and stored in −80°C for protein estimation or collected in RNA later solution (Ambion) for RNA isolation. Tissues in RNA later were kept at 4°C overnight and then frozen at −80°C until RNA isolation. Another set of wound tissue was collected from normal and diabetic rats (d 7 postwounding) and preserved in formaldehyde for hematoxylin-eosin (H&E) staining.
H&E Staining of the Wounded Tissue
Formaldehyde-preserved wounded tissues from normal and diabetic rats (n = 6) were paraffin-embedded and cut at a thickness of 5 µm for H&E staining. Stained sections were then examined by light microscopy (20×, Eclipse 80i, Nikon), and images were acquired using the NIS-elements imaging software. Quantitative analysis of the inflammatory cells in normal and diabetic sections was done by dividing the stained sections into equal number of grids and counting the inflammatory cells in the same number of grids within the same region. Results are presented as number of inflammatory cells per mm2 of area.
RNA Isolation and miRNA Array
miRNA array was done in normal and diabetic rat skin and also in wounds of normal and diabetic rats (n = 6) (d 7 postwounding). Collection of unwounded skin tissues from normal and diabetic rats was done on the same day as that of wounds collected from normal and diabetic rats (d 7 postwounding). Total RNA (from normal skin, diabetic skin, normal skin wound and diabetic skin wound) was isolated by using the TRIzol reagent (Ambion, Life Technologies) according to the manufacturer’s protocol. The quality and yields of RNA were analyzed by agarose gel electrophoresis and quantified by using Nanodrop 2000 (Thermo Fisher Scientific). miRNA expression was evaluated with the TaqMan Array Rodent MicroRNA card A v2.0 (Applied Biosystems). RNA (500 ng) was reverse-transcribed with the Taqman MicroRNA Reverse Transcription Kit and Megaplex RT Primers (Rodent Megaplex RT Primers Pool A, Applied Biosystems). The cDNA was combined with a Universal PCR Master Mix, NoAmpErase UNG (Applied Biosystems) and loaded into the arrays. TaqMan real-time polymerase chain reaction (PCR) was performed in a 7900 HT Fast Real-Time PCR system (Applied Biosystems) according to the manufacturer’s protocol. Data were analyzed with the 2−ΔΔCt method, where the average Ct (ΔCt) is the normalized Ct values of the miRNA with that of U87 (reference control) and miRNAs altered in (a) unwounded diabetic rat skin compared with normal rat skin and (b) diabetic skin wound compared with normal skin wound in rats (d 7 postwounding), with a p value <0.05 (compared with the respective normal rats) considered as statistically significant.
Wound-Healing PCR Array
Gene expression profiling for genes specific to the wound-healing process was done using the Wound Healing RT2 Profiler PCR Array (SABiosciences). A total of 3 µg RNA (from normal rat skin, diabetic rat skin, normal rat skin wound or diabetic rat skin wound) of each normal and diabetic rat (n = 6) were reverse-transcribed, and the cDNA was subjected to PCR on a LightCycler 480 Real-Time PCR System (Roche) by using the Wound Healing RT2 Profiler PCR Array according to the manufacturer’s instructions. Expression data generated was background corrected and normalized to the built-in housekeeping genes’ panel by using the Web-based PCR array data analysis software (https://doi.org/pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php) available from SA Biosciences. Here, the relative amounts of transcripts in the diabetic skin compared with the normal rat skin or those in the diabetic rat skin wound compared with normal rat skin wound (d 7 postwounding) are calculated by the ΔΔCt method. Fold changes with a p value <0.05 were taken as statistically significant.
To assess the transcript levels of Dicer in the normal and diabetic rat tissue (unwounded and wounded [d 1, 7 and 12 postwounding]), cDNA as prepared above was subjected to real-time PCR by using Dicer specific primers (forward 5′-TTCCA GAGTG CTTGA GGGAG-3′ and reverse 5′-CGCCT GAAGT TGAGC TCATC-3′). SYBR Green PCR Master Mix (Applied Biosystems) was used according to the manufacturer’s instructions (Applied Biosystems). Data were analyzed as described by Pfaffl (27), and results are expressed as the fold change (with respect to normal unwounded rat skin) in Dicer expression after normalization to 18S rRNA.
Rat skin (normal and diabetic, collected on the same day as wounds on d 7 postwounding) and skin wound tissues (d 1, 7 and 12 postwounding, n = 4) were lysed in RIPA lysis buffer containing protease inhibitors (Calbiochem). Protein samples (90 µg) were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting by using an anti-Dicer antibody (Santa Cruz Biotechnology). Immunoreactive bands were detected by using the ECIL Western Blotting Kit (Thermo Fisher Scientific). Hsc70 (Santa Cruz Biotechnology) was used as a loading control.
Generation of Networks
We sought to assess interactions between the altered wound-healing genes and the predicted targets to the differentially altered miRNAs. Potential regulators enriched for the altered wound-healing genes were extracted (p < 0.05) from ChEA within the ENRICHR tool (https://doi.org/amp.pharm.mssm.edu/Enrichr/). Targets to the altered miRNAs were identified by TargetScan (https://doi.org/www.targetscan.org/index.html). Of this list of potential miRNA-predicted targets, only those targets were prioritized that were also regulators to the altered wound-healing genes (from CHEA). Networks were generated with these regulators and the altered wound-healing genes using Cytoscape v3.0.2 (28) with nodes (as genes) and edges with a flat arbitrary numerical value of 1.0.
Transfection and Quantitative Reverse Transcriptase (RT)-PCR
HaCaT cells were plated in six-well plates and allowed to grow to 40% confluence in Dulbecco’s modified Eagle’s medium (DMEM) F12 media (Sigma) supplemented with 10% fetal bovine serum in an atmosphere of 5% CO2 at 37°C. They were transfected with either the scramble or Dicer siRNA (10–100 nmol/L; Dharmacon) by using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen). After 24 h, cells were collected and RNA was isolated as described above. A total of 2 µg RNA from each sample was reverse-transcribed, and the cDNA was subjected to real-time PCR as described above with human specific Dicer primers (sense CAGAC TGTCG TGCCG TATTG and antisense ATTTC CTGTG CAGCT CCTCT). Also, protein levels of Dicer were evaluated by Western blot analyses in the absence and presence of Dicer siRNA (10–100 nmol/L) as described above. Lamin (Santa Cruz Biotechnology) was taken as the loading control.
In scramble and Dicer siRNA (10–100 nmol/L) transfected HaCaT cells, a wound was made with a sterile 10-µL pipette tip, such that cells along the wound are scrapped off and a sharp visible line at the wound edge is left. Cells were washed with PBS to remove cell debris, and the culture medium was replaced with DMEM-F12 media. After 6, 12 and 24 h, cells were examined and photographed under a phase-contrast microscope (10×, Ti-S, NIKON). The time-dependent movement of cells into the wound area at 6, 12 and 24 h were monitored and quantified using the T-Scratch software (https://doi.org/cse-lab.ethz.ch/software).
For densitometric analyses of the Western blots, the Alpha DigiDoc 1201 software (Alpha Innotech Corporation) was used to evaluate protein expression.
All bars represent mean ± standard error of the mean (SEM) or standard deviation (SD), and data were analyzed using a Student t test. p Values <0.05 were considered statistically significant. Correlations between miRNA levels and decreased Dicer levels were evaluated using the online tool found at https://doi.org/www.alcula.com/calculators/statistics/correlation-coefficient, and p values were calculated (https://doi.org/www.danielsoper.com/statcalc3/calc.aspx?id = 44).
Delayed Wound Closure during Diabetes
miRNAs of the Skin Are Downregulated in a Diabetic Rat Wound
Having observed a decrease in miRNA and Dicer levels in the wounds of diabetic rat skin (compared with normal rat wounds on d 7 postwounding), we sought to evaluate the correlation coefficient between decreased miRNA levels and inhibited Dicer levels. Decreased miRNA levels showed positive correlation with decreased Dicer levels (r values ranging from 0.6 to 0.94 for all miRNAs), with several of them at a significant p value ≤0.05.
Wound-Healing Genes Are Altered in the Wounds of Diabetic Rats
In the diabetic rat wounded tissue on d 7 postwounding, 10 genes were significantly altered (p < 0.05) compared with normal rat wounded tissue (d 7 postwounding) (Figure 4B). In addition to the collagens that were altered in the diabetic rat skin, other members of the collagen family, namely, collagen type XIV α1 (Col14a1) and collagen type 3α1 (Col3a1) were significantly downregulated, suggesting additional decrease of collagen levels in wounds of diabetic rats. Wingless type mouse mammary tumor virus (MMTV) integration site family member 5A (Wnt5a), transforming growth factor β receptor 3 (Tgfβr3), insulin-like growth factor 1 (Igf1) and integrin α V (Itgav) were also markedly downregulated. Integrins and insulinlike growth factors are extremely critical during the wound-healing process, and their inhibition interferes with the normal process of wound healing and delays the same during diabetes (40). Three genes, namely Ccl7, Ctgf and Tgfβ1, were upregulated. Ccl7 is a chemokine specifically responsible for attracting monocytes and regulating macrophage function. Its upregulation in a diabetic wound presumably is an adaptive event to facilitate wound closure. Connective tissue growth factor (Ctgf) and Tgfβ1 are growth factors that promote migration, proliferation and angiogenesis; however, excessive and prolonged increases in these factors have been suggested to be antagonistic to the wound-healing process, and transgenic mice constitutively overexpressing Tgfβ1 exhibit significant delay in full thickness wound healing compared with nontransgenic mice (33).
In addition, we also evaluated the wound-healing genes’ signature during the healing process in normal rats (d 7 postwounding versus unwounded) to follow the pattern of genes’ behavior as the wounds heal during a normal healing process. As shown in Figure 4C, 40 genes were altered in the normal rat wound on d 7 postwounding compared with the unwounded normal rat skin. A total of 11 genes were downregulated, and 29 genes were upregulated. An identical analysis of the status of wound-healing genes in the diabetic rat wound (d 7 postwounding) versus the unwounded diabetic skin identified 34 genes to be differentially expressed compared with the unwounded diabetic rat skin (Figure 4D). Eight genes (namely, Rac1, Col4a1, Igf1, Plat, Mapk1, Ctnnb1, Itga4 and Rhoa) were significantly (p < 0.05) upregulated during the healing process in normal rats (Figure 4C) but were not altered in the diabetic rats (Figure 4D), suggesting that upregulation of these genes promotes and is critical during normal wound closure, and in the absence of such upregulation, as in diabetic rats, wound healing is severely impaired.
Interaction among the miRNA Predicted Targets and Altered Wound-Healing Genes
Inhibition of Dicer Prevents Keratinocyte Cell Migration
In this study, we demonstrate downregulation of miRNAs during delayed wound closure in diabetic rats. Delay in wound healing is a frequently encountered event during diabetes. Diverse physiological factors that contribute to wound-healing delay include but are not limited to impaired growth factor production, collagen accumulation, keratinocyte migration and proliferation and angiogenic response. A major determining factor for such a delay is a prolonged inflammatory phase that increases the time required for wound closure during diabetes (35). This step impedes the start of the next phase of wound healing, thereby causing a delay in granulation and proliferation. Several therapeutic approaches have been reported that address these impairments, but there has not been much success in preventing delayed wound closure during diabetes.
The emergence of miRNAs as critical regulators of cellular processes has added another regulatory element in the wound-healing process. miRNAs have been identified to be critical in wound healing (24,36, 37, 38). Our results demonstrate an inhibited miRNA signature in the wounds of diabetic rats accompanied by decreased Dicer levels. Interestingly, our results also show that inhibiting the levels of Dicer in HaCaT cells prevent cell migration and wound closure.
Dicer, a member of the RNase III superfamily of bidentate nucleases mediates the processing of pre-miRNAs into mature miRNAs in the cytoplasm. Across all homologs of Dicer in diverse species, the enzyme contains a putative helicase domain and a Piwi Argonaute and Zwille (PAZ) domain, tandem nuclease domains and a double-stranded ribonucleic acid (dsRNA) binding domain (39). In addition to varied roles of Dicer, it has also been identified in the epidermal layer of skin and in the hair follicle. Dicer knockout (KO) mice show follicular epithelial progenitors evaginating into the epidermal surface instead of into the dermis (40,41). Notch and sonic hedgehog and epithelial mesenchymal signaling are decreased by Dicer depletion, which results in deregulated skin physiology. These mice depict a marginal increase in apoptosis in the skin and start losing weight 1–2 d after birth (42,43). Oxidative insult can decrease Dicer levels as in observed in several pathological states (44,45). Although, in the context of endothelial cells, conditional Dicer inactivation reduces vascular endothelial growth factor (Vegf)-dependent angiogenesis and endothelial cell growth (46). These data suggest a critical role of Dicer in the skin. Our results demonstrate its involvement in keratinocyte migration, and decreased levels in a diabetic wound suggest that the levels of Dicer in the skin are presumably determining factors for impaired wound healing during diabetes. Interestingly, all the downregulated miRNAs in the diabetic rat wound (d 7 postwounding compared with normal rat wound) showed strong positive correlation with inhibited Dicer levels.
Our results show that compared with normal rat wounds (d 7 postwounding), several wound-healing specific genes are altered in the diabetic rat wound at this time point. Col14a1, Col3a1 and Col1a2 family of collagen proteins were downregulated. Collagen accumulation and wound remodeling typically is the last phase of the wound-healing process (47), and its inhibition, as seen in our study and also reported by others (34,48), is one of the main reason for the delay in wound closure. Ctgf and transforming growth factor β1 (Tgfβ1) were significantly upregulated. Both these factors are rapidly upregulated during injury and participate in initiating inflammatory events. However, in a diabetic wound, their chronic upregulation suggests the presence of excessive inflammation that is detrimental for wound repair. Ctgf, a cysteine-rich peptide of the CCN family (with CCN being an acronym for Connetctive Tissue Growth Factor, Cysteine-rich protein 61, Nephroblastoma Overexpressed gene), has been shown to be involved in fibrogenesis during tissue healing, and Ctgf-deficient mice demonstrate impaired extracellular matrix production (49,50). Its levels are regulated by Tgfβ, and both these transcripts are coordinately expressed during wound repair (51). K5.Tgfβ1wt mice (transgenic mice overexpressing the wild-type Tgfβ1 under the regulation of the K5 promoter) develop inflammatory skin disorders (52) and exhibit delayed wound healing (33). Wounds in these animals exhibit delayed reepithelialization and inflammatory cell infiltration compared with their nontransgenic counterparts. Persistent elevated levels of Tgfβ1 promote recruitment of macrophages into the wound area that produce excessive proteases and inhibit reepithelialization and wound closure. On the other hand, Tgfβ1 knockout mice display rapid epithelization and better wound-healing outcomes (53). While in a normal mouse, Tgfβ levels peak at d 3–4 postwounding and thereafter start decreasing, in a diabetic wound, Tgfβ levels start increasing from d 4, further increase on d 7 and remain elevated until d 13 (54). Overexpression of Tgfβ1 in keratinocytes and hair follicles has been shown to promote inflammation, hyperplasia and angiogenesis (55,56). These data suggest that elevated levels of growth factors are possibly responsible for prolonged inflammation and delayed wound closure during diabetes. Integrin α V was significantly downregulated, suggesting that this might be responsible for the inhibited integration of membrane components and, therefore, of wound closure. In addition to angiogenesis and vasculogenesis, integrins are critical in cell-cell adhesion and adhesion to the extracellular matrix and thereby might contribute to impaired wound healing. Ccl7 (a C-C motif chemokine 7) was increased by more than 2.5-fold, suggesting the presence of the inflammatory phase and the existence of inflammatory cells.
During the healing process in normal rats (normal rat skin wound [d 7 postwounding] compared with unwounded normal rat skin), eight genes (Rac1, Col14a1, Igf1, Plat, Mapk1, Ctnnb1, Itga4 and Rhoa) were upregulated and showed no significant alteration during the delayed healing process in diabetic rat wounds (diabetic rat skin wound [d 7] compared with unwounded diabetic rat skin), suggesting that these genes are possibly critical in promoting normal wound closure. By not being altered during delayed wound closure in diabetic rats, they are presumably preventing the timely closure of wounds. Igf1 is induced in wounds, both in animals (57,58) and humans (59,60), but is missing in chronic wounds (61). Proliferation and migration of keratinocytes and fibroblasts is promoted by Igf1 in vitro (62,63) and, interestingly, its levels are reduced in cells isolated from diabetic foot ulcers (64) and in diabetic mouse wounds (65). Igf1 treatment or overexpression has been shown to promote wound healing in diabetic animals (66,67). Plat (tissue-type plasminogen activator) was also significantly upregulated during healing in normal rats, but was notably missing during impaired healing in diabetic rats. It is a secreted serine protease that converts the proenzyme plasminogen to plasmin and plays a critical role in cell migration and tissue remodeling. Keratinocytes use plasmin activated by plasminogen activators for paving their way through the matrix during wound healing (68). Plasminogen-deficient mice depict a delay in wound healing, as evidenced by decreased migration of keratinocytes from the wound edges and their decreased ability to proteolytically dissect their way through the matrix (69,70). Another upregulated gene, Ctnnbl, also known as β-catenin, belongs to the adherens junction proteins that mediate adhesion between cells and is important in cell communication, growth, embryogenesis and wound healing (71). Its levels are elevated in mesenchymal cells during the proliferative phase of wound repair (72), and it is thought that Ctnnbl regulates the growth of dermal fibroblasts (73,74). Macrophage-specific deletion of β-catenin in mice shows defects in migration and adhesion to fibroblasts and consequently inefficient skin wound healing. Macrophages overexpressing β-catenin rescued impaired wound-healing in irradiated mice (75). Rac1, another protein upregulated in normal rats during wound healing, participates in the coordinated and orchestrated process of wound healing. Along with RhoA, another member of the small GTP-binding family of Rho-GTPases, Rac1 is required for the reorganization of the cytoskeleton for efficient wound closure (76).
Also, several regulators of the altered wound healing genes were identified to be predicted targets of the altered miRNAs. This result suggests that miRNAs that are deregulated in the wound during diabetes might be indirectly responsible for the differential status of wound-healing genes. These interactions are an area worthy of investigation.
Our results provide evidence for an altered miRNA and wound-healing genes’ status that is associated with the prolonged inflammatory phase in the wounds of diabetic rats. The downregulated miRNA signature is presumably due to decreased levels of Dicer and, to the best of our knowledge, this is a novel finding of an inhibited status of Dicer in a wound during diabetes and of its critical involvement in delaying wound closure. A recent report demonstrates compromised wound closure in keratinocyte-specific Dicerablated mice, suggesting the significance of Dicer in keratinocytes during restoration of skin function postwounding. Absence of Dicer in these animals and the consequent downregulation of miRNAs were responsible for desilencing p21Waf1/Cip1 expression, and this disrupted the skin barrier function. The authors suggest that p21Waf1/Cip1 might be a potential drug target to improve barrier function of the skin during Dicer loss (44). Identification of decreased Dicer levels during impaired wound healing in diabetes in our study, therefore, offers a valuable tool to be addressed such that the delay in the wound-healing process during diabetes might be targeted.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
The authors thank the Council of Scientific and Industrial Research (CSIR), New Delhi, India, for financial help (BSC0302). S Bhattacharya received a fellowship from CSIR.
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