Corresponding Author: Adquinta
Wulandini Putri
E-mail: [email protected]
INTRODUCTION
Diabetic Foot Ulcer (DFU) is a major problem for
people with diabetes. More than 15% of diabetic patients must treat DFU during
their lifetime (Jaroenarpornwatana et al., 2023). DFU severely impairs
patients' quality of life, causes long-term hospitalizations and causes more
than 70,000 lower limb amputations annually in the United States. The
prevalence of DFU in Indonesia is 8.7% (Yunir et
al., 2022). Diabetic Foot Ulcer (DFU) is
one of the most common complications of diabetes and chronic ulcers (Kurdi &
Priyanti, 2020). This disease causes foot
injuries and tissue damage in diabetic patients caused by abnormalities in the
nerves in the legs (Hidayat
& Nurhayati, 2014).
Studies have shown that
complicating factors in DFU wound healing include microbial infections,
epithelial cell damage, and decreased immunity (Maria Allen,
2022). Among all complications
caused by type II diabetes, DFU is the main reason for the hospitalization of
patients. Studies have shown that 25% of people with diabetes may develop foot
ulcers (Rodrigues et al., 2023). Current treatments developed
for Diabetic Foot Ulcer (DFU) include wound debridement, wound transport,
regulation of blood sugar levels, and disease management (VITA, 2022). Currently, there are many
technological developments for the treatment of Diabetic Foot Ulcer (DFU),
including stem cells, the use of hyperbaric O2, and the use of Becaplermin. New
technologies and drugs have been developed to treat DFU. However, the treatment
effect is considered sub-optimal due to its strong pathophysiological
processes. The annual cut-off
rate of patients with DFU is 5.1% (Theocharidis et al.,
2022). From the research of PN Theocharidis G, it is
possible to know the healing process of DFU by looking at gene expression.
Several other studies have examined differences in gene expression in the DFU
healing process using the Single-cell transcriptomic and Spatial
transcriptomics methods using the RNA sequencing method. Gene expression
analysis is an important technique used to compare differences between two or
more groups of genes. This technique involves using methods such as microarrays
or RNA sequencing (RNA-seq) which make it possible to examine gene activity
under various conditions or situations (PN Theocharidis G,
2020). In addition, more specific target gene expression
techniques, such as quantitative polymerase chain reaction (qPCR), are also
used to perform this analysis.
Based on the above background, this study aimed to
determine and analyze the expression of the diabetic foot ulcer (DFU) gene in
the homo sapiens species.
METHODS
This review is
based on research articles on the expression of the Diabetic Foot Ulcer gene in
the Homo sapiens species. The data source for this review was obtained from the
National Center for Biotechnology Information database; then, Geo Data Sets
were selected. Geo Data Sets is a database with curated gene expression
datasets and original series and platform records in the Gene Expression
Omnibus (GEO) repository. After that, enter search keywords to find relevant
research. Search keywords use a PICO strategy, such as: "Diabetic Foot
Ulcer". 127 DFU gene expression studies were obtained with Homo sapiens species,
2 DFU gene expression studies with Mus musculus species, and 1 DFU gene
expression study with Rattus norvegicus species. Articles will be analyzed and
selected with the inclusion and exclusion criteria provisions.
The inclusion
criteria are adjusted to the purpose of the review article, so the journal
inclusion criteria used are research data that produce primary data that
discusses the expression of the Diabetic Foot Ulcer gene, original articles,
research subjects are humans, selected type of sample type series, year of
publication of the journal at the period The last five years, namely 2018-2023,
full-text articles.
The exclusion
criteria that will be used to select articles are articles that do not
specifically discuss the expression of the Diabetic Foot Ulcer gene in the Homo
sapiens species, and the research subjects are Mus musculus or Rattus
norvegicus. Then, the literature review of journals, short reports, case
reports, and clinical trial reports.
Journals following
the criteria for keywords, titles, and abstracts are reviewed in full text to
find out the content and adjust it to the topic being studied. Journal search
is used for review by conducting searches and paying attention to inclusion and
exclusion criteria. The analysis carried out in this article review was carried
out descriptively.
RESULTS AND DISCUSSION
One hundred thirty titles were identified for the initial
review of search pages. The main search identified 130 journals, with 127 DFU
gene expression studies with Homo sapiens species, 2 DFU gene expression
studies with Mus musculus species, and 1 DFU gene expression study with Rattus
norvegicus species. After being selected, 120 articles did not meet the
inclusion criteria, and ten met the inclusion criteria. Articles that met the
inclusion criteria were then reviewed. Articles were studied based on the name
of the researcher, year, name of the gene that plays a role in healing DFU, the
methodology used, the results obtained and the outcome (Appendix).
The process of converting gene
information into products that cells can recognise is called gene expression (Sandoval-Schaefer et al., 2023). The result
of this gene expression can be a protein and an RNA product such as tRNA or
snRNA (Ramirez-Acu�a et al., 2019). Gene expression measurement is important in drug discovery,
biomarker research, and gene pathway analysis. Gene expression analysis
involves genome expression techniques such as microarrays or RNA sequencing
(RNA-seq) and more specific target gene expression techniques such as qPCR (Kusnadi & Arumingtyas, 2020). Today, many publicly available gene expression data such as NCBI
GEO or ArrayExpress. This data set contains valuable information for
discovering and developing biomarkers and therapeutics.
Research on the expression of the
Diabetic Foot Ulcer gene in the Homo sapiens species aims to look at gene
expression that occurs in humans and to group genes that are influential in the
healing process of DFU. This systematic review identified ten gene codes
associated with DFU recovery. The following is a review of 10 articles that
meet the inclusion criteria:
GSE223964 Gene Code
The gene code was obtained from a previous study entitled "
Transcriptional heterogeneity in human diabetic foot wounds" (Santra et al., 2020). This study, using single-cell RNA sequencing in chronic foot
ulcers of non-diabetics (NDFUs) and diabetes (DFUs), in the DFUs group showed
transcriptional changes indicating reduced keratinocyte differentiation,
fibroblast function and alteration, and defects in macrophage metabolism and
ECM production compared to NDFUs. In addition, cellular interaction analysis
revealed major changes in several altered signal pathways in DFUs. These data
provide insight into the mechanism of wound healing in leg ulcers. They may
provide new therapies for the treatment of DFUs. This study used single-cell
RNA sequencing technology on cells taken from the foot wounds of a non-diabetic
individual at different times and from five diabetic patients. The cells were
isolated through mechanical and enzymatic breakdown, screening, and removal of
red blood cells, then retrieved via single-cell RNA sequencing technology using
the 10X Genomics platform (Sandoval-Schaefer et al., 2023).
GSE165816 Gene Code
Previous research with the article "Single-cell
transcriptomic landscape of diabetic foot ulcers" used single-cell samples
from skin cells of the feet and forearms and peripheral blood mononuclear cells
(PBMC), ten samples from non-diabetic subjects and 17 patients. Diabetes, 11
with DFU, and six without DFU. Fifty-four samples were analyzed using the
single-cell RNA sequencing technique and verified using the
Immunohistochemistry and Spatial Transcriptomics techniques. The study
demonstrated increased unique inflammatory fibroblast populations in DFU
patients with healing wounds (Theocharidis et al., 2022). Patients with DFU who recovered also showed increased
macrophages with M1 polarization. In contrast, DFU patients did not experience
an increase in M2 macrophages.
GSE166120 Gene Code
Previous research used ulcer tissue samples from DFU patients who
successfully healed and those who did not heal. Total sample 23. To understand
the molecular mechanisms and cell types involved in DFU healing using the
spatial transcriptome profiling method, with NanoString's GeoMx digital spatial
profiling platform on DFU tissue sections and comparing gene expression in the
same area within the same ulcer, as well as between patients 12 weeks after
surgery those who recovered from DFU (Talers, N=2) were compared to those who
did not (Non-Healers, N=2) (VA Theocharidis G, 2021).
GSE134431 Gene Code
Previous research used skin tissue samples from DFU patients, oral
wound tissue, and wound tissue, so 21 samples were obtained. The method used
was RNA-sequencing on tissue biopsies from patients with DFU and compared with
oral wounds and human skin to identify the mechanism. Molecular and
transcriptional networks that are disrupted in DFU. As a result of this study,
a unique inflammatory transcription exclusive to oral and skin wounds promotes
cell proliferation and survival of immune cells lacking in DFU. In addition,
identifying immune cell profiles shows the absence of macrophage and neutrophil
activation and proliferation in DFU. These results indicate that an impaired
immune response, including the activation, proliferation, and survival of
immune cells, contributes to the pathogenesis of DFU.
GSE146028
Gene Code
In the previous study, seven types of immune cells were sampled,
consisting of 1 reference (CD14) and six differentiated cell types: M0, M1,
M2a, Mreg, Mreg_UKR, and PCMO. There are nine anonymized human donors, with at
least three for each cell type. The donor served as a biological replication
for statistical purposes of expression differences. The method used is the
characterization of regulatory macrophages that have clinical relevance, namely
Mreg and Mreg_UKR, programmable cells of monocyte origin (PCMO), as well as
comparison macrophages (M1, M2a, and M0) using flow-cytometry, RT-qPCR,
phagocytosis and secretome measurements, as well as RNA-Seq. The results
obtained in macrophage production can produce reproducible cell phenotypes. At
the same time, small changes introduced in a protocol can consistently affect
the phenotype of the final product. In addition, we have identified new
combinations of potential biomarkers specific to the process, which will
support further clinical product development and lead to a better understanding
of differentiation and activation of macrophage polarization (Gurvich et al., 2020).
GSE143735 Gene Code
Previous research was conducted using skin samples of DM
sufferers, with as many as 13 patients isolated on the volar part of the
forearm. A total of 5 patients recovered from DFU, four patients who did not
recover from DFU and four patients who did not have ulcers. The total sample
used was 14 samples. Multiplex array serum was used to detect systemic
cytokines, chemokines, and growth factors correlating with DFU healing. In
addition, forearm biopsies were used for histological analysis and
transcriptome bulks to ensure DFU healing results were reflected in
non-ulcerative skin sites. Analysis of RNA-seq bulks revealed extracellular
matrix (ECM) related genes that are increased in recoverable DFU types,
including MMP2 and the implication of IFNγ and IL13 as upstream regulators.
Based on analysis of transcriptome data with an error rate of discovery (FDR)
<0.05 and log2fold change (log2FC) >0.5, a total of 25 genes (3 up) were
differentially expressed when comparing Non-Healer and Healer DFU types, 916
(530 up-regulated) in Healers compared to DM, and 160 (89 up-regulated) in
Non-Healers compared to DM (PN Theocharidis G, 2020).
The results of this study show that the genes that increase in the
"Healer" group include molecules related to inflammation, such as
lymphoid chemokine ligand 19 (CCL19), complement component 6 (C6), lipoprotein
lipase (LPL), and beta-defensin 124 (DEFB124), as well as extracellular
matrix-related proteins such as pigment-epithelium derived factor (SERPINF1),
tenascin X (TNXB), biglycan (BGN), and matrix metalloproteinase-2 (MMP2).
Whereas in the "Non-Healer" group, the genes that experienced an increase
included members of the cytochrome P450 (CYP1A1), prostaglandin transporter
(SLCO2A1), and metabolic regulator G0/G1 switch gene 2 (G0S2).
GSE114248
Gene Code
Previous studies using wound biopsy samples were divided into two
groups: the Control Group, a group of patients who had wounds and were normal
on the seventh day from healthy donors, and the Test Group, a group of patients
with DFU (Diabetic Foot Ulcers). Circular RNA (circRNA) microarray analysis was
performed to examine the expression profile of circRNA in diabetic foot ulcers
(DFU) and human excision skin wounds seven days after injury. The result of this study is that there is
a regulation of circular RNA expression in diabetic chronic wounds. Increased
expression of hsa_circ_0084443 in diabetic chronic wounds. hsa_circ_0084443
localized in the cytoplasm of human epidermal keratinocytes. The
hsa_circ_0084443 expression modulation does not affect PRKDC expression.
hsa_circ_0084443 reduces keratinocyte motility and supports keratinocyte growth
(Wang et al., 2020).
GSE114236 Gene Code
Previous studies studied samples of human
primary epidermal keratinocytes transfected with 50 nM si-hsa_circ_0084443 or
si-Control for 24 hours (biological triples in each group). A total of 6
samples were used. Global transcriptome analysis was performed on keratinocytes
after decreasing hsa_circ_0084443 using the Affymetrix series. The results
showed that the expression level of hsa_circ_0084443 decreased during normal
skin wound healing. In contrast, a higher concentration level of
hsa_circ_0084443 was found in non-healing chronic diabetic foot ulcers compared
to normal wounds (Wang et al., 2020).
GSE132187 Gene Code
THP 1 cells under conditions of
hyperglycemia and normoglycemia. A total of 6 samples. THP-1 cells were
cultured under hyperglycemia or normoglycemia, and hypoxia and differentiated
into macrophages by PMA. LPS activates macrophages. The effects of hyperglycemia
and hypoxia on macrophage phenotypes were analyzed. For this purpose,
microarray analysis was performed to study the gene expression profile of
macrophages cultured in high glucose concentrations. The contribution of
hypoxia and hyperglycemia to chronic inflammation and potential synergistic
effects were evaluated in activated THP-1-derived macrophages. Long-term
effects after activation (17 h) were only observed in increased expression of
pro-inflammatory cytokines when hypoxia was combined with high glucose
concentrations. This study showed that hyperglycemia increased the expression
of pro-inflammatory cytokines such as TNF- α, IL-1,
IL-6, and chemokines and decreased the expression of two receptors involved in
phagocytosis (CD36 and class B collector type I receptors). Hyperglycemia and
hypoxia do not affect wound-healing molecules such as TGF - β1. Overall,
the study's results suggest that hyperglycemia acts synergistically with
hypoxia to maintain a state of chronic inflammation in macrophages (Morey et al., 2019).
GSE114908 Gene Code
Previous studies used samples of human epidermal
keratinocytes transfected with 20nM WAKMAR1 GapmeRs (GapmeR-WAKMAR1) and 20nM
oligo control (GapmeR-Ctrl) for 24 hours (biological triple in each group). In total, there are six samples. Global transcriptome analysis was
performed on keratinocytes after WAKMAR1 inhibition using the Affymetrix
sequence. Long noncoding RNAs (lncRNAs) are important regulators in cellular
physiology and pathology, making them promising therapeutic and diagnostic
entities. WAKMAR1 lncRNA was significantly decreased in wound edge
keratinocytes in venous ulcers and diabetic foot ulcers compared with normal
wounds. To study WAKMAR1-regulated genes, lncRNA transfection GapmeRs into
human primary epidermal keratinocytes to inhibit their expression. The results
of this study stated that WAKMAR1 is a lncRNA localized in the cell nucleus,
transcribed by RNAPII, and has polyadenylation. WAKMAR1 expression was decreased in wound edge
keratinocytes in chronic human wounds. WAKMAR1 expression is induced by TGF- β signalling in keratinocytes. WAKMAR1 regulates keratinocyte motility and
re-epithelialization processes in ex vivo human wounds. WAKMAR1 regulates a
network of genes that mediate pro-migrant functions in keratinocytes. WAKMAR1
activates E2F1 expression by inhibiting E2F1 promoter methylation (Li et al., 2019).
CONCLUSION
A total of 10 studies
of the gene code associated with Diabetic Foot Ulcer concluded as follows: 1)
Patients with DFUs (Diabetic Foot Ulcers) show transcriptional changes that
indicate a decrease in keratinocyte differentiation, changes in function and fibroblast
lines, as well as defects in macrophage metabolism, inflammation, and
production ECM compared to NDFUs (Non-Diabetic Foot Ulcers). 2) There are major
changes in several signalling pathways that change DFUs, as well as increased
populations of inflammatory fibroblasts and M1-polarizing macrophages in
recovered DFU patients. 3) There is a unique inflammatory transcription
exclusive to oral wounds and skin wounds in DFU that plays a role in promoting
cell proliferation and survival of deficient immune cells. 4) Identification of
the immune cell profile showed no activation and proliferation of macrophages
and neutrophils in DFU, indicating an impaired immune response. 5) The
macrophage production process can produce reproducible cell phenotypes. However,
small changes in the protocol can affect the phenotype of the final product. 6)
Certain genes related to inflammation and extracellular matrix were increased
in the "Healer" group, and certain genes related to metabolism were
increased in the "Non-Healer" group. 7) Hsa_circ_0084443 is a lncRNA
localized in the cytoplasm of human epidermal keratinocytes, and its expression
is related to the movement and growth of keratinocytes. The expression level of
hsa_circ_0084443 decreases during normal skin wound healing, whereas it
increases in diabetic chronic wounds. 8) Hyperglycemia increases the expression
of pro-inflammatory cytokines and reduces the expression of receptors involved
in phagocytosis. 10) Hyperglycemia and hypoxia do not affect wound healing molecules
such as TGF- β1.
WAKMAR1 is a lncRNA localized in the cell nucleus, induced by TGF- β
signals, and involved in regulating keratinocyte motility,
re-epithelialization of wounds, and expression of migration genes. WAKMAR1
activates E2F1 expression by inhibiting E2F1 promoter methylation.
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