A total of 39 patients with DFU and 22 trauma patients receiving debridement at Yijishan Hospital, Wannan Medical College (Wuhu, China) between January and February 2017 were enrolled. A total of 3 patients with DFU and 3 trauma patients were randomly divided into an ulcer group and a control group. The ulcer group contained one man and two women aged 50–69 years, and the control group contained one man and two women aged 48–65 years. There were no significant differences between the two groups. Patients with DM were excluded from the control group, which included patients whose wounds were caused by trauma, and for whom debridement indications were clear. Patients with a confirmed diagnosis of type 2 DM were included in the ulcer group. Their lower limb ulcers had persisted for longer than 1 month with no healing following medical treatment. Debridement indications were again clear for this group.

Complete skin tissues (2×2 cm) were collected from the center of the wound surfaces from patients in the two groups. Following washing, a number of tissue samples were fixed in 4% paraformaldehyde for 48 h at 25°C and embedded in paraffin. The remaining tissues were stored at −80°C. The present study was approved by the Ethics Committee of Yijishan Hospital, and all patients provided written informed consent.

Wound surface tissues were fixed with 4% paraformaldehyde, washed in purified water, dehydrated in ethanol, cleared by purified water, immersed in paraffin, embedded and sectioned at 5 µm. The sections were further dewaxed at 25°C, hydrated at 25°C and stained using hematoxylin and eosin (HE) for 30 sec at 25°C and Masson's trichome for 40 sec at 25°C (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) to observe histological changes, including alterations associated with blood capillaries, collagen and fibroblasts.

Skin tissues were frozen for 30 min at −20°C and cut into sections (8 µm). The sections were fixed in paraformaldehyde for 1 day at 25°C, washed a number of times with distilled water and subsequently dried at room temperature. Following this, the sections were treated with pure acetone at 4°C for 10 min and dried prior to further immunostaining. The sections were washed with PBS and 0.3% Triton X-100 for 30 min at 37°C, washed again with PBS and blocked using 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) for 30 min at 37°C. Primary antibodies against forkhead box P3 (1:100; Abcam, Cambridge, UK; cat. no. ab4728), cluster of differentiation (CD)3 (1:200; Abcam; cat. no. ab5690), CD4 (1:200; Abcam; cat. no. ab203034) or CD8 (1:150; Abcam; cat. no. ab4055) were added and incubated with the sections at 4°C overnight. Following washing with PBS, membranes were incubated at 37°C for 30 min with an appropriate fluorescent secondary antibody (1:500; Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. A-11034). Finally, sections were stained with DAPI for 5 min at 37°C, washed with PBS and observed via laser confocal scanning microscopy at ×100 magnification (Leica Microsystems GmbH, Wetzlar, Germany).

The total protein was extracted from the skin tissue using radioimmunoprecipitation assay buffer (Invitrogen; Thermo Fisher Scientific, Inc.), and its concentration was measured using a bicinchoninic acid assay kit (Invitrogen; Thermo Fisher Scientific, Inc.). The remaining protein was degenerated by boiling at 99°C for 10 min, and was subsequently stored at −20°C. Protein samples (20 µg) were run on 10% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes for 80 min. Membranes were blocked with 5% bovine serum albumin (Invitrogen; Thermo Fisher Scientific, Inc.) at 25°C for 1 h and incubated at 4°C overnight with primary antibodies (all purchased from Abcam) against the following proteins: Transforming growth factor-β (TGF-β; 1:5,000; cat. no. ab92486), interleukin (IL)-1β (1:1,000; cat. no. ab2105), IL-2 (1:1,000; cat. no. ab180780), IL-6 (1:500; cat. no. ab6672), IL-10 (1:1,000; cat. no. ab34843), IL-8 (1:1,000; cat. no. ab7747), interferon (IFN)-γ (1:400; cat. no. ab25101), tumor necrosis factor (TNF)-α (1:2,000; cat. no. ab6671) and GAPDH (1:5,000; cat. no. ab8245), β-actin (1:5,000; cat. no. ab8226). Following this, the membranes were washed three times with TBS containing 0.1% Tween-20 (TBST; 10 min/wash) and incubated with a secondary antibody (1:5,000; Cell Signaling Technology, Inc., Danvers, MA, USA) at room temperature for 1 h. Membranes were subsequently washed three times with TBST (10 min/wash) and developed using a gel imaging system (EMD Millipore, Billerica, MA, USA).

There were at least three replicates in the control test. Therefore, three tissue specimens were randomly selected from each group to conduct the lncRNA gene chip assay. The other tissue specimens from the patients were used for further test and verify. RNA was extracted from three randomly selected skin tissue samples obtained from the control and ulcer groups using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Quantified RNA was transcribed into fluorescent-labeled complementary (c)RNA using the Quick Amp Labeling kit (Agilent Technologies, Inc., Santa Clara, CA, USA) at 65°C. Following this, cRNAs were hybridized onto the Arraystar Human LncRNA Microarray v4.0 (Arraystar Inc., Rockville, MD, USA) and the fluorescence intensity was determined using an Agilent G2565BA microarray scanner (Agilent Technologies, Inc.). Images were input into Agilent Feature Extraction software version 10.5.1, and the data underwent quantile normalization and further analysis using Agilent Gene Spring software GX 12.0 (both Agilent Technologies, Inc.). A total of 20 lncRNAs and co-expressing mRNAs exhibiting maximum levels of upregulation and downregulation were screened for and verified via reverse transcription quantitative polymerase chain reaction (RT-qPCR), using GAPDH as an internal reference.

Total RNA was extracted from control and ulcer tissues using TRIzol^®, according to the manufacturer's instructions. lncRNA and mRNA expression levels were determined using the Ribo^™ SYBR Green mRNA/lncRNA RT-qPCR Starter kit (Guangzhou RiboBio Co., Ltd., Guangzhou, China), using GAPDH as an internal reference. Realtime PCR system (2XMaster Mix: 5 µl, 10 µM PCR-specific primer F: 0.5 µl, 10 µM PCR-specific primer R: 0.5 µl water added for total volume: 8 µl) was used to prepare all the DNA samples for PCR reaction. The conditions for RT-qPCR were: 95°C for 10 min; 40 cycles, then 95°C for 10 sec and 60°C for 60 sec. Dissociation curves revealed no nonspecific amplification. The primers used for the RT-qPCR assay are listed in Tables I and II. The results were analyzed using the 2^−∆∆Cq method (15), and all experiments were repeated in triplicate.

An lncRNA antisense probe was synthesized by Novatech Enterprise Co., Ltd. (Wuxi, China). Skin tissues were embedded in paraffin, cut into sections (4 µm), dewaxed and sealed with sealing solution for 15 min. Pre-hybridization solution (100–150 µl, Novatech Enterprise Co., Ltd. Wuxi, China) was added to the sections, which were incubated at 37°C for 1 h. The lncRNA antisense probe was diluted in pre-hybridization solution, degenerated at 65°C for 5 min and rapidly cooled in iced water for 10 min. Following this, the pre-hybridization solution was discarded from the sections, which were uniformly covered with 100–150 µl degenerated probe and incubated at 4°C overnight. The sections were subsequently washed, sealed for 30 min with sealing solution at 37°C and then stained with DAPI for 1–5 min at room temperature. Following this, the sections were washed with running water, air-dried, sealed with neutral gum and observed under a fluorescence microscope (Leica Microsystems GmbH) at ×100 magnification. Peptidyl-prolyl cis-trans isomerase B (Aviva Systems Biology, San Diego, CA, USA) was used as the internal control.

Full-length candidate lncRNAs were established via 5′ and 3′ rapid amplification of cDNA ends (RACE) using a FirstChoice^® RLM-RACE kit (Invitrogen; Thermo Fisher Scientific, Inc.). Candidate lncRNA non-coding verification was performed by cloning the full-length ENST00000411554 sequence into pcDNA3.1 vectors (Guangzhou RiboBio Co., Ltd.). A total of three Myc-tagged proteins encoding three different sequences were inserted into the C-terminus of the lnc-testicular cell adhesion molecule 1, pseudogene. In addition, the full-length ENST00000411554 sequence was also cloned into pFlag-CMV-2 vectors (Guangzhou RiboBio Co., Ltd.) containing Flag-tagged protein at the N-terminus using three different cloning techniques (16). The plasmids were mixed with Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and transferred into 293T cells (1×10⁶/vector, Invitrogen; Thermo Fisher Scientific, Inc.). pcDNA3.1 vectors containing a Myc-tagged green fluorescent protein (GFP) or pFlag-CMV-2 containing a Flag-linked GFP were used as positive controls. An empty plasmid was used as the negative control. The total RNA was isolated and subjected to RT-qPCR to determine the vector-mediated gene transduction 48 h post-transfection. After a 72-h time interval, the protein was extracted and Myc-tagged or Flag-tagged proteins were detected via western blotting as described above.

All experiments were repeated at least three times. All statistical data are expressed as the mean ± standard deviation. Statistical analysis was performed using the SPSS 18.0 software package (SPSS, Inc., Chicago, IL, USA). Comparisons of the measurement data between two groups were performed using a Student's t-test. Pearson's test was used for correlation analysis. P<0.05 was considered to indicate a statistically significant difference.

HE staining of the control wound surface revealed a clear tissue structure with numerous neutrophil granulocytes and fibroblast infiltration, while Masson staining revealed thin collagenous fibers in a loose arrangement. HE staining of the ulcer group indicated a disordered tissue structure, the formation of granulated tissue, increased numbers of neutrophil granulocytes and fibroblast infiltration. Masson staining of the ulcer group revealed atrophic and decreased numbers of collagenous fibers in a disordered arrangement (Fig. 1).

Immunofluorescence staining revealed increased expression of CD3 and CD8 in the wound surface of tissues obtained from the ulcer group compared with the control group; however, there was no marked difference in the levels of CD4 expression between the two groups (Fig. 2). These results indicated a decreased CD4:CD8 ratio in DFU tissues, which suggested that cellular immune dysfunction was present in patients with type 2 DM and DFUs.

RT-qPCR and western blotting demonstrated that IL-1β, IL-2, IL-10, IFN-γ and TNF-α expression levels were significantly upregulated in skin tissues obtained from the ulcer group compared with the control group; however, no significant differences in the expression levels of TGF-β, IL-6 and IL-8 were revealed between the two groups (Fig. 3). These results suggested that IL-1β, IL-2, IL-10, IFN-γ and TNF-α may be associated with the onset of DFUs.

To investigate the involvement of lncRNA in DFUs, lncRNA/mRNA gene chip technology was used to detect lncRNA and mRNA expression in the wound surface tissues obtained from the ulcer and control groups. The expression levels of 2,142 lncRNAs were revealed to be upregulated, while 1,332 lncRNAs were downregulated with a differential multiple of >2. A total of 20 lncRNAs exhibiting significant differential expression between the two groups were selected for subsequent RT-qPCR analysis. The results demonstrated that five lncRNAs were upregulated, while eight lncRNAs were downregulated, which was consistent with the results of the chip screening analyses (Fig. 4).

Following the analysis of the aforementioned 13 lncRNAs and target genes, it was revealed that lncRNA-ENST00000411554 (location, chromosome 22: 22,390,743–22,394,463; transcript length, 779 bp) was located in a key upstream regulatory region of target gene, MAPK1. Of the numerous signaling pathways involved in the regulation of DFU, the MAPK signal transduction pathway is one of the most important (17). Therefore, to investigate whether there was a negative correlation between lncRNA-ENST00000411554 and MAPK1 expression in control and DFU tissues, an additional 19 control and 36 ulcer wound surface tissue samples were investigated via lncRNA FISH and RT-qPCR. The results revealed that lncRNA-ENST00000411554 expression was significantly downregulated and MAPK1 expression was significantly upregulated in DFU tissues compared with the control tissues, and thus exhibited a negative correlation (Fig. 5). lncRNA FISH also revealed differential expression of lncRNA-ENST00000411554 in control and DFU tissues (Fig. 6).

To verify the non-coding characteristics of lncRNA-ENST00000411554, lncRNA-ENST00000411554 was cloned into pcDNA3.1 and pFlag-CMV-2 vectors and subsequently tagged with Myc or Flag, respectively. Following this, these vectors were transfected into 293T cells and investigated via western blotting. The results did not reveal any detection of Myc- or Flag-tagged proteins via western blotting, which suggested that lncRNA-ENST00000411554 does not have any protein-coding abilities (Fig. 7).