Journal Pre-proof

miR-23c regulates wound healing by targeting stromal cell-

derived factor-1α (SDF-1α/CXCL12) among patients with
diabetic foot ulcer

Karan Naresh Amin, Dhamodharan Umapathy, Arunkumar

Anandharaj, Jayasuriya Ravichandran, Changam Sheela
Sasikumar, Sathish Kumar Rajappan Chandra, Rajesh Kesavan,
Ramkumar Kunka Mohanram

PII: S0026-2862(19)30098-6
DOI: https://doi.org/10.1016/j.mvr.2019.103924
Reference: YMVRE 103924

To appear in: Microvascular Research

Received date: 21 April 2019

Revised date: 9 September 2019
Accepted date: 10 September 2019

Please cite this article as: K.N. Amin, D. Umapathy, A. Anandharaj, et al., miR-23c
regulates wound healing by targeting stromal cell-derived factor-1α (SDF-1α/CXCL12)
among patients with diabetic foot ulcer, Microvascular Research(2018), https://doi.org/
10.1016/j.mvr.2019.103924

This is a PDF file of an article that has undergone enhancements after acceptance, such
as the addition of a cover page and metadata, and formatting for readability, but it is
not yet the definitive version of record. This version will undergo additional copyediting,
typesetting and review before it is published in its final form, but we are providing this
version to give early visibility of the article. Please note that, during the production
process, errors may be discovered which could affect the content, and all legal disclaimers
that apply to the journal pertain.

© 2018 Published by Elsevier.

 Journal Pre-proof

miR-23c Regulates Wound Healing by Targeting Stromal Cell-Derived

Factor-1α (SDF-1α/CXCL12) Among Patients with Diabetic Foot Ulcer

Karan Naresh Amina, Dhamodharan Umapathya, Arunkumar Anandharajb, Jayasuriya

Ravichandrana, Changam Sheela Sasikumarc, Sathish Kumar Rajappan Chandrad, Rajesh
Kesavanc*and Ramkumar Kunka Mohanrama*

a. Life Science Division, SRM Research Institute, SRM Institute of Science & Technology,
Kattankulathur-603 203, Tamilnadu, India.
b. Indian Institute of Food Processing Technology, Pudukkottai Road, Thanjavur, 613005,
Tamilnadu, India.

of
c. Department of Podiatry, Hycare Super Speciality Hospital, MMDA Colony, Arumbakkam,
Chennai-600 106, Tamilnadu, India.
d. Interdisciplinary Institute of Indian System of Medicine (IIISM), SRM Institute of Science

ro
& Technology, Kattankulathur-603 203, Tamilnadu, India.
-p
Running Title: miR-23c Regulates Wound Healing via SDF-1α
re
lP
na

*Correspondence to:
Dr. K. M. Ramkumar
ur

SRM Research Institute, SRMIST, Kattankulathur-603 203, Tamil Nadu, India,

Tel.: +91-99407 37854, Fax: +91-442745 23437,
Jo

Mail: ramkumar.km@res.srmuniv.ac.in
and
Dr. Rajesh Kesavan
Hycare for Wounds (A Unit of NRA Advanced Wound Care Pvt Ltd.), Purasavakkam-600
084, Tamilnadu, India,
Tel: +91- 93607 78800

1
 Journal Pre-proof

Abstract

Diabetic Foot Ulcer (DFU) is most common in patients who have diabetic peripheral

neuropathy and angiopathy as well as a foot deformity. The delayed process of wound

healing in diabetic condition is mainly due to reduced expression of the growth factors,

persistent inflammatory response and endothelial dysfunction. Emerging evidence indicate

that miRNAs play a crucial role in regulating angiogenesis, collectively called as

“angiomiRs”. The present study aimed to screen the expressions of angiomiRs particularly

of
miR23 family and its association with the various angiogenic factors including SDF-1α in the

ro
tissue biopsies isolated from DFU patients. Among the 40 enrolled subjects for this study, 10

were subjected in each group as healthy controls, type 2 diabetic subjects (T2DM), T2DM
-p
subjects with uninfected DFU, and T2DM subjects with infected DFU. The miR23 family
re
such as hsa-miR-23a, hsa-miR-23b, hsa-miR-23c) and gene expression of angiogenic factors
lP

such as SDF-1α, HIF-1α, VEGF, eNOS were investigated in peripheral blood mononuclear

cells by qPCR. We found that the angiogenic factor SDF-1α was significantly decreased in
na

both the circulation and tissue biopsies of patients with T2DM and infected DFU. The SDF-

1α at the 3'-untranslated region pairs with target miRNAs namely hsa-miR-23a-3p, hsa-miR-
ur

23b-3p and hsa-miR-23c as established using miRNA target prediction algorithm. Further,
Jo

the tissue-specific expressions of miR-23a and miR-23b were found to be low whereas miR-

23c was increased in patients with infected DFU. Moreover, correlation analysis showed that

SDF-1α was found to have a significant inverse association with miR-23c. In conclusion,

miR-23c may function as a new regulator to inhibit angiogenesis by targeting SDF-1α.

Keywords: AngiomiRs; Diabetic Foot Ulcer; HIF-1α; miRNA; miR-23c; SDF-1α/CXCL12;

VEGF

2
 Journal Pre-proof

1 INTRODUCTION:

Diabetic Foot Ulcer (DFU) is the most important complication of diabetes (Pemayun

et al., 2015), which affects up to 19-34 percent of T2DM patients in their lifetime and

contributes to both morbidity and mortality rate (Mayfield et al., 2004) with a substantial

physical, physiological and financial burden for the patients as well as to the community. It

has been well reported that a lower limb or part of a lower limb is lost in every 30 seconds

due to amputation somewhere in the world among patients with diabetes (Bakker et al.,

2005). A recent report from India had estimated that the care for patients with DFU is an

of
increasingly heavy economic burden and spent four times more than that of non-DFU

ro
patients with DM (Ghosh and Valia, 2017). The principal goal behind wound management is
-p
to achieve rapid wound healing at the site of infection, which is impaired in DFU. Despite an
re
increased insight into the pathologies underlying DFU, the effective treatment strategies are

still lacuna.
lP

Wound healing is a complex event that is conserved in all organ systems (Guo and
na

Dipietro, 2010). However, impaired wound healing is a hallmark of DFU (Jhamb et al.,

2016). Few studies highlighted that wound healing process can be stimulated by promoting
ur

angiogenesis through growth factors and evidenced by the safety and the clinical potential of
Jo

“therapeutic angiogenesis” that worn growth factors (Losordo and Dimmeler,

2004),(Altavilla et al., 2009) or cell-based strategies (Losordo and Dimmeler, 2004). Hence it

is of great importance to understand the pathophysiological mechanisms of angiogenic

response that occurs in DFU patients. An earlier studies from our laboratory demonstrated

single nucleotide polymorphisms of angiogenic cytokines/chemokine in the circulation of

DFU patients (Dhamodharan et al., 2015),(Pichu et al., 2015). Among the several angiogenic

proteins, stromal-cell-derived factor-1 (SDF-1α) has a crucial role in dysfunctional wound

healing of DFU (Ho et al., 2012),(Ho et al., 2010).

3
 Journal Pre-proof

SDF-1α is a peptide chemokine, referred as chemokine ligand 12 (CXCL12) which is

located on chromosome 10q 11.1 and detected in stem cells and stromal tissues of multiple

organs (Petit et al., 2007). Two different variants of SDF-1 have been reported that are SDF-

1α and SDF-1β (Shirozu et al., 1995) and the significance of these variants remains unknown.

Previous studies reported that HIF-1α is a direct regulator of SDF-1α and the regulatory axis

is important for the recruitment of monocyte/macrophage into the inflammatory sites

(Ceradini et al., 2004). In addition to this, a recent report from our laboratory have also

shown that SNPs among various cytokine/chemokine genes involved in the wound healing

of
process, SDF-1α showed a higher risk for emerging severe podal microbial infection,

ro
amputation and advanced grades of foot ulcer among diabetic patients (Viswanathan et al.,
-p
2018). The regulation of SDF-1α has been studied under invivo condition and reported that
re
impaired healing was associated with abnormal SDF-1α production, chronic inflammation

and decreased angiogenesis (Bermudez et al., 2011). However, the regulation of SDF-1α in
lP

the clinical settings of DFU are not yet studied.

na

miRNAs are small non-coding RNA’s that span between 18-24 nucleotides and

possess a pivotal role during post-transcriptional regulation of gene expression (Hashimoto

ur

and Tanaka, 2017). Numerous reports suggested the key involvement of miRNAs in various
Jo

disorders and diseases (Tufekci et al., 2014), (Giza et al., 2014). The differential expressions

of miRNAs in tissue biopsies of different organs have been well reported (Ludwig et al.,

2016). A set of miRNAs that are a key regulatory role in angiogenesis are termed as

“angiomiRs”. Pro-angiomiRs promote angiogenesis by regulating negative signals of the

angiogenic process, whereas anti-angiomiRs suppress angiogenesis by regulating positive

signals (Wang and Olson, 2009). Earlier studies have documented that few miRNAs regulate

angiogenesis process as demonstrated both cell lines and animal studies (Kuehbacher et al.,

2007), (Suarez et al., 2008), (Suarez et al., 2007), (Zhou et al., 2011). Although few reports

4
 Journal Pre-proof

documented the key regulatory role of miRNAs in both experimental and clinical diabetes (de

Candia et al., 2017), (Miao et al., 2018), (Vaishya et al., 2018), the role of angiomiRs in

regulating the angiogenic factors in DFU tissues biopsies have not been investigated. Based

on the In silico identification of conserved miRNAs which regulate SDF1 gene, we have

selected miR23 family due to its established role in angiogenesis as demonstrated using

preclinical studies (Zhou et al., 2011). However, the role of miR23 family members in the

progression of DFU patients is not explored. Thus, we made an attempt to study the

expression of the angiomiRs particularly miR23 family in the tissue biopsies of DFU patients

of
and its association with the angiogenic factors including SDF-1α.

2 Materials and Methods

ro
-p
2.1 Recruitment of Study Subjects
re
A case-control association study was conducted consisting of 40 subjects and grouped
lP

as Normal Glucose Tolerance (NGT) (n=10), T2DM patients free from other complications

(n=10), T2DM subjects with uninfected DFU (n=10); T2DM subjects with infected DFU
na

(n=10). Grading of the ulcer was in accordance with the Infectious Diseases Society of
ur

America (IDSA) and the International Working Group on the Diabetic Foot (IWGDF)

classification (Lipsky et al., 2004). Blood samples were obtained from all the subject groups
Jo

whereas wound tissue biopsies were isolated from patients with DFU (i.e. groups-III and IV).

All tissue biopsies used in the present study were carefully isolated by a well-trained

podiatrist (Dr. Rajesh Kesavan) according to the defined criteria. The unrelated healthy

control individuals were without the history of diabetes mellitus, and had HbA1C less than

5.6%. The screening for healthy volunteers were done using oral glucose tolerance test

(OGTT) based on WHO criteria (Alberti and Zimmet, 1998) for the diagnosis of both NGT

and T2DM. All the study samples were collected from Hycare for Wounds (Chennai, India),

and the study protocol was approved by the institutional ethics committee with consent

5
 Journal Pre-proof

obtained from all study subjects (009/HYC/IEC/2017). The patients with pneumonia, type 1

diabetes, inflammatory bowel disease, sepsis and hematologic diseases were considered as

exclusion criteria. The sample collection from the study subjects was done during the part of

standard debridement, in an ambulatory ambiance setting with a well-trained footcare team

consisting of a diabetologist, podiatrist and vascular surgeon. A comprehensive quality

management system, with standard operating procedures were ensured that the samples are of

consistent quality and best fit for the intended analyses.

of
2.2 Tissue Biopsy collection procedure

ro
Prior to biopsy, 1% lidocaine was injected for local anesthesia. Full-thickness punch
-p
biopsy of 6 mm in diameter and 2-3 mm in depth were collected from the ulcer tissue

adjacent to intact peri-ulcer skin using a trephine before and after therapy. Samples were then
re
stored at -80°C for further studies.
lP

2.3Anthropometric and Biochemical parameters measurements

na

Anthropometric measurement were measured by use of standardized methods (Deepa

et al., 2003). Blood pressure was recorded with a mercury sphygmomanometer. All
ur

biochemical parameters were measured and recorded as described earlier (Umapathy et al.,
Jo

2018). Fasting Plasma Glucose (FPG) was measured by glucose oxidase-peroxidase method,

serum cholesterol by cholesterol oxidase-peroxidase-amidopyrine method, serum

triglycerides using glycerol phosphate oxidase-peroxidase-amidopyrine method, high-density

lipoprotein cholesterol (HDL-c) by the direct method (polyethylene glycol pretreated

enzymes) and creatinine with Jaffe's method was measured using a Randox Daytona fully

automated biochemistry analyzer (BioAgilytix, Durham, NC). The intra and inter assay

coefficients of variation for the biochemical assays ranged between 3.1% and 5.8%

6
 Journal Pre-proof

respectively. Low-density lipoprotein cholesterol (LDL-c) was calculated using the

Friedewald equation (Friedewald et al., 1972).

2.4 Isolation of Peripheral Blood Mononuclear Cells (PBMC) from blood using density

gradient centrifugation:

PBMCs were isolated using LymphoprepTM (Stemcell Technologies, Vancouver,

Canada) as described earlier (Riedhammer et al., 2016). Briefly, blood samples were

centrifuged for 20 min at 800g and plasma was carefully removed. Further, the pellet was

resuspended with phosphate-buffered saline (PBS) and then layered on top of Lymphoprep.

of
PBMCs layer was pipette out and washed with PBS and followed by centrifugation for 10

ro
min at 250g. Further, the pellets were resuspended in ammonium chloride potassium (ACK)
-p
lysis buffer followed by 10-15 mins of incubation and was finally washed with PBS.
re
2.5 Total RNA Extraction from both Skin Biopsies and PBMCs:

Total RNA from skin biopsies and PBMCs were extracted using RNeasy Mini Kit
lP

(Qiagen, CA, USA) following manufacturers protocol. Further, the RNA concentration was
na

measured using Agilent Bioanalyser (Agilent Technologies) and RNA Integrity value ≥8.0

were taken for the study. The cDNA synthesis was carried out using miScript® II RT Kit
ur

(Qiagen, CA, USA) and qPCR was performed using miScript® SYBR® Green PCR Kit
Jo

(Qiagen, CA, USA). The primer sequences used in this study are listed in Table 1. With

respect to other angiogenic cytokine/chemokine, SYBR® Premix Ex Taq™ II (Clontech,

Takara, Japan) were used for the expression level of GAPDH (Housekeeping gene) and

respective target genes such as (VEGF, HIF-1α, SDF-1α and eNOS). The expression of target

miRNAs and mRNA were determined using 2-ΔΔCt and normalized to U6snRNA and GAPDH

respectively.

2.6 Measurement of Nitrite levels

7
 Journal Pre-proof

Nitrite levels were measured in both plasma and tissue biopsy using a

spectrophotometric method (Griess reaction) (Bryan and Grisham, 2007).

2.7 miRNA target prediction by Bioinformatics tools

Of note, because of the thousands of potential interactions between a single miRNA

and target gene, bioinformatics prediction tools play a vital role in facilitating the task for

individuating and selecting putative target genes. The TargetScan software

(http://www.targetscan.org/), an online miRNA target prediction algorithm was employed to

of
predict the binding sequences (of SDF-1 UTR regions in both 3’ and 5’ ends. The online tool

ro
retrieved a list of miRNAs that bind in both the regions of SDF-1 to potentially silence the

expression. The list consists of miRNAs those bind to either conserved region or poorly
-p
conserved region of SDF-1 mRNA) of hsa-miR-23 family. In the present study, the miRNAs
re
23 family was chosen based on analyzed by TargetScan (v7.0). Table 1 shows the primer
lP

sequences and the amplicon sizes of the products.

2.8 Statistical Analysis

na

Data are expressed as mean values for biochemical parameters and geometric mean

with 95% confidence interval values for cytokines. Student’s t-test was performed to compare
ur

groups for continuous variables, whereas χ2 test was used to compare proportions. Pearson’s
Jo

correlation analysis was carried out to determine the association of miRNAs with mRNA

expression and clinical parameters. p value less than 0.05 were considered statistically

significant.

8
 Journal Pre-proof

3 RESULTS:

3.1 Clinical characteristics of the study subjects

The levels of BMI, fasting glucose (FPG), systolic blood pressure (SBP), HbA1c,

postprandial glucose (PPG), serum triglycerides (TGL) and HOMA-IR were found to be

significantly high in T2DM as compared with controls, whereas HDL-c was significantly low

in T2DM subjects as compared with NGT (Table 2). However, other parameters such as age,

total serum cholesterol, diastolic blood pressure (DBP), urea, LDL-c, and creatinine didn’t

of
show any significant differences between T2DM and NGT subjects. Patients with subjects

ro
were further subdivided into uninfected and infected ulcers based on IDSA-IWGDF
-p
classification and the levels of FPG, DBP and TGL were significantly elevated in subjects

with infected when compared with uninfected DFU. With respect to other complications of
re
diabetes, 30.0% and 40.0% of uninfected and infected DFU patients had diabetic nephropathy
lP

(DN), respectively; 10.0% of both uninfected and infected DFU patients had diabetic

retinopathy (DR); 20.0%, and 10.0% of uninfected and infected DFU patients had both DN
na

and DR respectively; 20.0% and 10.0% of uninfected and infected DFU patients had
ur

myocardial ischemia respectively; 10.0% and 20.0% of uninfected and infected DFU patients
Jo

had coronary artery disease respectively; and 10.0% of both uninfected and infected DFU

patients had cardiovascular diseases. At the time of hospitalization, the location of foot

lesions was as follows: 1 patient of uninfected DFU had ulcer only in toes, 4 and 5 patients of

uninfected and infected DFU had ulcer localized in the plantar region respectively and 5

patients of both uninfected and infected DFU had an ulcer in the heel region.

3.2 Expression of the miR-23 family in different stages of DFU

The tissue-specific expression of miR-23a and miR-23b were found to be decreased in

patients with infected DFU [(P<0.013), (P<0.009) respectively] when compared with

9
 Journal Pre-proof

uninfected DFU (Fig. 1a and b). On the other hand, miR-23c expression (Fig. 1c) showed

1.5 fold increase in patients with infected DFU (P<0.005) and we have studied the expression

of their specific targets such as SDF-1α using qPCR.

3.3 Expression of SDF-1α in both circulation and tissue biopsies of DFU patients

Results of our study had shown that the circulatory SDF-1α showed a stepwise

significant decreased expression from control to infected DFU subjects, wherein T2DM,

uninfected and infected showed a decrease upto 0.6 fold (P<0.0009), 0.2 fold (P<0.003), 0.1

of
fold (P<0.003) respectively (Fig. 2a). Further, the tissue-specific expression of SDF-1α also

ro
showed a significant decrease upto 0.5 fold (P<0.005) in infected when compared to patients

with uninfected DFU (Fig. 2b).

-p
re
3.4 SDF-1α targeted miRNAs using TargetScan analysis
lP

S.Table 1 shows In silico identification of conserved miRNAs which regulates SDF-

1α gene. The findings of the present study showed that at the position starting from 2997 to
na

3004 of SDF-1α at the three prime untranslated regions (3'-UTR) pairs with only three target

miRNAs namely (hsa-miR-23a-3p, hsa-miR-23b-3p and hsa-miR-23c) with their CWCS and
ur

PCT scores as shown in Table 3. Based on the score for all targeted miRNAs, we have
Jo

chosen only the miRNAs 23 family which regulates angiogenesis (23a, 23b and 23c).

3.5 miR 23c regulates eNOS, HIF-1α and VEGF in DFU

As eNOS being one of the crucial factors towards the activation of SDF-1α during the

wound healing process, we attempted to study the expression of eNOS and nitric oxide (NO)

levels in the tissue biopsies of uninfected and infected DFU patients. Our results showed that

the tissue-specific expression of eNOS (P<0.009) (Fig. 3a) and the levels of NO (P<0.0005)

(Fig. 3b) were found to be significantly decreased in patients with infected DFU. The finding

of the present study showed that HIF-1α (P=0.0001) and VEGF (P=0.0001) were

10
 Journal Pre-proof

significantly decreased in the foot biopsy specimen of patients with infected DFU (Fig. 4a

&b).

3.6 Spearman’s correlation of tissue specific miR-23c levels with SDF-1α

Table 4 depicts the Spearman’s correlation of tissue specific miR-23c levels with

SDF-1α and other biochemical parameters. Correlation analysis showed that SDF-1α was

found to have a significant inverse association [(r= -0.56; p=0.028)], whereas eNOS exhibited

a positive association with miR-23c [(r= 0.79; p=0.006)]. Correlation of miR-23c with

of
various biochemical parameters for DFU showed, HbA1c [(r= 0.65; p=0.021)], serum

ro
triglycerides [(r= 0.79; p=0.004)] and HDL-c [(r= 0.72; p=0.01)] to have a significant

positive association.
-p
re
lP
na
ur
Jo

11
 Journal Pre-proof

4 DISCUSSION

DFUs are one of the leading causes of gangrene and amputation in 24% of people

with diabetes. Experimental evidence demonstrated that diminished cell and growth factor

response, reduced angiogenesis and decreased peripheral blood flow are contributing to the

delay in the process of wound healing among subjects with diabetes. The wound healing

arises as a cellular response to injury and comprises activation of platelets, fibroblasts,

keratinocytes, endothelial cells and macrophages. . The growth factors, chemokines &

of
cytokines released by these cell types are essential to manage and maintain healing process;

ro
however it is inadequate in diabetes. During wound healing, chemokines are critical for white

blood cell recruitment to the site of injured tissues (Brem and Tomic-Canic, 2007).
-p
Particularly, SDF-1α is a key chemokine involved in angiogenesis and helps in recruiting
re
endothelial progenitor cells (EPCs) to the site of infection (Ho et al., 2012). SDF-1α
lP

signaling is also involved in tissue homeostasis by coordinating immune cell migration (Yu et

al., 2016), neo-vascularization (Petit et al., 2007) and stem cell proliferation (Kortesidis et al.,
na

2005). Further Karin et al., reported that multiple faces for SDF-1α involved in regulating the
ur

immune system during various disease and disorders. It has been well documented that under

autoimmune disease, SDF-1α shifts its potential from pro-inflammatory function (during non-
Jo

inflammatory state) to anti-inflammatory role during site specific inflammation (Meiron et

al., 2008), (Karin, 2010).

Gallagher et al., reported that hyperoxia improves the mobilization of EPCs from the

bone marrow into the circulation in diabetic animals. Also, local injection of the chemokine

SDF-1α recruits these EPCs to the infected site thereby leads to enhanced wound healing.

Thus, SDF-1α act as a possible target for the wound healing process in diabetes (Gallagher et

al., 2007). It has been well documented that, miRNAs play a crucial role in various diseases

and disorders (Tufekci et al., 2014), (Giza et al., 2014). There are more than 1000 miRNAs

12
 Journal Pre-proof

reported to be under the influence of miRNAs. They act by targeting the 3’ or 5’untranslated

region (UTR) of genes with the significance of suppressing the synthesis of respective

protein. Along this line, differential expression of miRNAs in human tissue biopsies of

different organs have been reported recently (Ludwig et al., 2016).

Pillai et al., demonstrated the regulation of SDF-1α by miRNAs in human bone

marrow stromal cells (Pillai et al., 2010). Recently Oikawa et al., have demonstrated the role

of endothelial miRNA-23 clusters in angiogenesis using vascular endothelial cell (EC)-

of
specific miR-23 cluster double-knockout (DKO) mice (Oikawa et al., 2018). Nevertheless,

few data are available on the association of SDF-1α and miRNAs, especially in subjects with

ro
DFU. This study examined the angiomiRs regulating SDF-1α using bioinformatics tool and
-p
to investigate their expression in combination with the target gene (SDF-1α) in the tissue
re
biopsies of DFU patients. The findings of our study showed that SDF-1α at the 3'-UTR pairs
lP

with only three target miRNAs namely (hsa-miR-23a-3p, hsa-miR-23b-3p and hsa-miR-23c).

The expression of miR-23a and miR-23b were significantly downregulated in foot ulcer
na

biopsy of patients with infected DFU Whereas the tissue specific expression of miR-23c was
ur

significantly increased in patients with infected DFU. Also, the tissue specific expression of

SDF-1α was significantly decreased in patients with infected DFU.

Jo

Earlier studies from our laboratory have reported that single nucleotide

polymorphisms of angiogenic cytokines/chemokine genes in the circulation of DFU patients

(Dhamodharan et al., 2015). In the current study, SDF-1α expression was found to be

decreased among tissue biopsy of infected DFU patients. This result of the current study was

coincided with a previous report by Gallager et al., on diabetic wounds in mice, where

reduced expression of SDF-1α was observed in isolated epithelial and myofibroblasts cells

from the wound tissue. This is the first report highlighting the expression of SDF-1α in the

wound biopsy of DFU patients. The expression of SDF-1α is predominantly localized to

13
 Journal Pre-proof

EPCs, however during diabetic wound condition; the EPCs undergo reduced proliferation

correlated with decreased expression of SDF-1α (Gallagher et al., 2007).

In contrast, an earlier report from Iranian population had shown an elevated level of

circulatory SDF-1α in type 2 diabetic patients (Derakhshan et al., 2012). Further the current

study was conducted in the tissue biopsy isolated from DFU subjects whereas the above study

had performed circulatory protein levels. Therefore, it seems that decreased SDF-1α

expression in the tissues of DFU patients could be under the influence of epigenetic agents

of
such as miRNAs which may play vital role in the expression of various chemokines. Pillai et

al., demonstrated the regulation of SDF-1α by miR-886-3p using SDF-1α expressing and

ro
non-expressing cell lines (Pillai et al., 2010). Similarly Arabanian et al., showed another
-p
approach of conducting a library screening for SDF-1α modulating miRNAs with a
re
regulatory potential and the results of his study showed only three miRNAs (miR-23a, miR-
lP

222 and miR-130) are functionally related in human bone marrow stem cells (Arabanian et

al., 2014). In the current study, we made prediction on the biological targets of miRNAs by
na

finding out for the presence of conserved 8mer, 7mer, and 6mer sites that match the seed
ur

region of each miRNA using bioinformatics tools. Based on the CWCS score for all targeted

miRNAs, we found that miRNAs 23 clusters (23a, 23b and 23c) were found to be strongly
Jo

paired with SDF-1α. The existing study showed decreased expression of miR-23a and miR-

23b whereas upregulated expression of miR-23c in tissue biopsies isolated from the patients

with infected DFU. Earlier study by Shen et al., described that the level of miR-23a was

found to be down-regulated after the differentiation of 3T3-L1 cells, an in vitro model system

of adipocytes (Shen et al., 2016). Whereas Huang et al., showed that miR-23a was found to

be over-expressed at the early stage of differentiation 3T3-L1 adipocytes followed by a rapid

down-regulation during its maturation process (Huang et al., 2016). Although the role of

miRNAs has been reported in the regulation in various cell lines, may still require cross talk

14
 Journal Pre-proof

with other cell types found in the wound. To the best of our knowledge, this is the first report

on the tissue specific regulation of miR-23c on SDF-1α levels among subjects with infected

DFU. However, the implications of miR-23c up-regulation in diabetic wound require further

investigation.

Author contributions

DU, RKM and RK designed the experiment. KNA and DU drafted the manuscript.

KNA, DU, AA, CSS, and SKRC performed the experiment. AA, RK and RKM analyzed and

of
interpreted the data. DU, AA, CSS, SKRC and RKM contributed towards the discussion and

ro
reviewed the manuscript.

Acknowledgements
-p
re
The authors gratefully acknowledge the facilities provided by “SRM-DBT Partnership

Platform for Contemporary Research Services and Skill Development in Advanced Life
lP

Sciences Technologies” (Order No. BT/PR12987/INF/22/205/2015). The authors

na

acknowledge Mr. Vimal Kumar Ramachandran (Study co-coordinator, Hycare Super

Speciality Hospital), Ms. Teena Rajan (Research Scholar, SRM Research Institute, SRMIST),
ur

Ms. Trishla Adhikari and Ms. Awantika Rai (B.Tech-Biotechnology graduates, SRMIST) for
Jo

their kind help.

15
 Journal Pre-proof

References

Alberti, K. G., Zimmet, P. Z., 1998. Definition, diagnosis and classification of diabetes
mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus
provisional report of a WHO consultation. Diabet Med. 15, 539-53.
Altavilla, D., et al., 2009. Polydeoxyribonucleotide (PDRN): a safe approach to induce
therapeutic angiogenesis in peripheral artery occlusive disease and in diabetic foot
ulcers. Cardiovasc Hematol Agents Med Chem. 7, 313-21.
Arabanian, L. S., et al., 2014. MicroRNA-23a mediates post-transcriptional regulation of
CXCL12 in bone marrow stromal cells. Haematologica. 99, 997-1005.
Bakker, K., et al., 2005. 2005: The International Diabetes Federation focuses on the diabetic
foot. Curr Diab Rep. 5, 436-40.
Bermudez, D. M., et al., 2011. Inhibition of stromal cell-derived factor-1alpha further impairs
diabetic wound healing. J Vasc Surg. 53, 774-84.

of
Brem, H., Tomic-Canic, M., 2007. Cellular and molecular basis of wound healing in diabetes.
J Clin Invest. 117, 1219-22.

ro
Bryan, N. S., Grisham, M. B., 2007. Methods to detect nitric oxide and its metabolites in
biological samples. Free Radic Biol Med. 43, 645-57.
Ceradini, D. J., et al., 2004. Progenitor cell trafficking is regulated by hypoxic gradients
-p
through HIF-1 induction of SDF-1. Nat Med. 10, 858-64.
de Candia, P., et al., 2017. A unique plasma microRNA profile defines type 2 diabetes
re
progression. PLoS One. 12, e0188980.
Deepa, M., et al., 2003. The Chennai Urban Rural Epidemiology Study (CURES)--study
design and methodology (urban component) (CURES-I). J Assoc Physicians India.
lP

51, 863-70.
Derakhshan, R., et al., 2012. Increased circulating levels of SDF-1 (CXCL12) in type 2
diabetic patients are correlated to disease state but are unrelated to polymorphism of
na

the SDF-1beta gene in the Iranian population. Inflammation. 35, 900-4.

Dhamodharan, U., et al., 2015. Genetic association of IL-6, TNF-alpha and SDF-1
polymorphisms with serum cytokine levels in diabetic foot ulcer. Gene. 565, 62-7.
ur

Friedewald, W. T., et al., 1972. Estimation of the concentration of low-density lipoprotein

cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 18,
499-502.
Jo

Gallagher, K. A., et al., 2007. Diabetic impairments in NO-mediated endothelial progenitor

cell mobilization and homing are reversed by hyperoxia and SDF-1 alpha. J Clin
Invest. 117, 1249-59.
Ghosh, P., Valia, R., 2017. Burden of Diabetic Foot Ulcers in India: Evidence Landscape
from Published Literature. Value in Health. 20, A485.
Giza, D. E., et al., 2014. Key principles of miRNA involvement in human diseases.
Discoveries (Craiova). 2, e34.
Guo, S., Dipietro, L. A., 2010. Factors affecting wound healing. J Dent Res. 89, 219-29.
Hashimoto, N., Tanaka, T., 2017. Role of miRNAs in the pathogenesis and susceptibility of
diabetes mellitus. J Hum Genet. 62, 141-150.
Ho, T. K., et al., 2012. Stromal-Cell-Derived Factor-1 (SDF-1)/CXCL12 as Potential Target
of Therapeutic Angiogenesis in Critical Leg Ischaemia. Cardiol Res Pract. 2012,
143209.
Ho, T. K., et al., 2010. Angiogenic effects of stromal cell-derived factor-1 (SDF-1/CXCL12)
variants in vitro and the in vivo expressions of CXCL12 variants and CXCR4 in
human critical leg ischemia. J Vasc Surg. 51, 689-99.

16
 Journal Pre-proof

Huang, Y., et al., 2016. Effects of MicroRNA-23a on Differentiation and Gene Expression
Profiles in 3T3-L1 Adipocytes. Genes (Basel). 7.
Jhamb, S., et al., 2016. Genetic and molecular basis of diabetic foot ulcers: Clinical review. J
Tissue Viability. 25, 229-236.
Karin, N., 2010. The multiple faces of CXCL12 (SDF-1alpha) in the regulation of immunity
during health and disease. J Leukoc Biol. 88, 463-73.
Kortesidis, A., et al., 2005. Stromal-derived factor-1 promotes the growth, survival, and
development of human bone marrow stromal stem cells. Blood. 105, 3793-801.
Kuehbacher, A., et al., 2007. Role of Dicer and Drosha for endothelial microRNA expression
and angiogenesis. Circ Res. 101, 59-68.
Lipsky, B. A., et al., 2004. A report from the international consensus on diagnosing and
treating the infected diabetic foot. Diabetes Metab Res Rev. 20 Suppl 1, S68-77.
Losordo, D. W., Dimmeler, S., 2004. Therapeutic angiogenesis and vasculogenesis for
ischemic disease. Part I: angiogenic cytokines. Circulation. 109, 2487-91.
Ludwig, N., et al., 2016. Distribution of miRNA expression across human tissues. Nucleic

of
Acids Res. 44, 3865-77.
Mayfield, J. A., et al., 2004. The epidemiology of lower-extremity disease in veterans with

ro
diabetes. Diabetes Care. 27 Suppl 2, B39-44.
Meiron, M., et al., 2008. CXCL12 (SDF-1alpha) suppresses ongoing experimental
autoimmune encephalomyelitis by selecting antigen-specific regulatory T cells. J Exp
-p
Med. 205, 2643-55.
Miao, C., et al., 2018. MicroRNAs in the pathogenesis of type 2 diabetes: new research
re
progress and future direction. Can J Physiol Pharmacol. 96, 103-112.
Oikawa, S., et al., 2018. Role of endothelial microRNA-23 clusters in angiogenesis in vivo.
Am J Physiol Heart Circ Physiol. 315, H838-H846.
lP

Pemayun, T. G., et al., 2015. Risk factors for lower extremity amputation in patients with
diabetic foot ulcers: a hospital-based case-control study. Diabet Foot Ankle. 6, 29629.
Petit, I., et al., 2007. The SDF-1-CXCR4 signaling pathway: a molecular hub modulating
na

neo-angiogenesis. Trends Immunol. 28, 299-307.

Pichu, S., et al., 2015. Impact of the hypoxia inducible factor-1alpha (HIF-1alpha) pro582ser
polymorphism and its gene expression on diabetic foot ulcers. Diabetes Res Clin
ur

Pract. 109, 533-40.

Pillai, M. M., et al., 2010. MiR-886-3p down regulates CXCL12 (SDF1) expression in
human marrow stromal cells. PLoS One. 5, e14304.
Jo

Riedhammer, C., et al., 2016. Peripheral Blood Mononuclear Cells: Isolation, Freezing,
Thawing, and Culture. Methods Mol Biol. 1304, 53-61.
Shen, L., et al., 2016. MicroRNA-23a regulates 3T3-L1 adipocyte differentiation. Gene. 575,
761-4.
Shirozu, M., et al., 1995. Structure and chromosomal localization of the human stromal cell-
derived factor 1 (SDF1) gene. Genomics. 28, 495-500.
Suarez, Y., et al., 2007. Dicer dependent microRNAs regulate gene expression and functions
in human endothelial cells. Circ Res. 100, 1164-73.
Suarez, Y., et al., 2008. Dicer-dependent endothelial microRNAs are necessary for postnatal
angiogenesis. Proc Natl Acad Sci U S A. 105, 14082-7.
Tufekci, K. U., et al., 2014. The role of microRNAs in human diseases. Methods Mol Biol.
1107, 33-50.
Umapathy, D., et al., 2018. Potential of circulatory procalcitonin as a biomarker reflecting
inflammation among South Indian diabetic foot ulcers. J Vasc Surg. 67, 1283-1291
e2.

17
 Journal Pre-proof

Vaishya, S., et al., 2018. MicroRNA, Proteins, and Metabolites as Novel Biomarkers for
Prediabetes, Diabetes, and Related Complications. Front Endocrinol (Lausanne). 9,
180.
Viswanathan, V., et al., 2018. Single nucleotide polymorphisms in cytokine/chemokine genes
are associated with severe infection, ulcer grade and amputation in diabetic foot ulcer.
Int J Biol Macromol. 118, 1995-2000.
Wang, S., Olson, E. N., 2009. AngiomiRs--key regulators of angiogenesis. Curr Opin Genet
Dev. 19, 205-11.
Yu, Y., et al., 2016. Stromal cell-derived factor-1-directed bone marrow mesenchymal stem
cell migration in response to inflammatory and/or hypoxic stimuli. Cell Adh Migr. 10,
342-59.
Zhou, Q., et al., 2011. Regulation of angiogenesis and choroidal neovascularization by
members of microRNA-23~27~24 clusters. Proc Natl Acad Sci U S A. 108, 8287-92.

of
ro
-p
re
lP
na
ur
Jo

18
 Journal Pre-proof

Table 1. Primer sequences and the amplicon sizes used in the study

Amplicon size
S. No Gene symbol Primer sequences
(bp)

1 hsa-U6 CGCAAGGATGACACGCAAATTC 22
2 hsa-miR-23a-3p ATCACATTGCCAGGGATTTCC 21
3 hsa-miR-23b-3p ATCACATTGCCAGGGATTACC 21
4 hsa-miR-23c ATCACATTGCCAGTGATTACCC 22
F: AAG AAG GTG GTG AAG CAG GC
5 GAPDH 114
R: GTC AAA GGT GGA GGA GTG GG
F: CGC ACT TTC ACT CTC CGT CA
6 SDF-1α 116
R: AGC ACG ACC ACG ACC TTG

of
F: AGC ATC CCT ACT CCC ACC AG 105
7 eNOS
R: CCC ACC TCC CAG TTC TTC AC

ro
F: GGC AAC CTT GGA TTG GAT GG
8 HIF-1α 185
R: TCT CCG TCC CTC AAC CTC TC
F: CTA CCT CCA CCA TGC CAA GT
9 VEGF-A
-p
R: GCA GTA GCT GCG CTG ATA GA
109
re
lP
na
ur
Jo

19
 Journal Pre-proof

Table 2. Clinical and biochemical characteristics of the study subjects

NGT (n=40) KDMa (n=40) Uninfected (n=40) Infectedb(n=40)

Clinical Parameters (n=40)
Age (years) 49.95±9.21 52.4±8.88 55.9±8.7 57.8±10.38

BMI (Kg/m2) 23.62±1.85 27.52±3.42** 26.47 ± 3.92 27.94±6.95

SBP (mm Hg) 116.4±9.53 134.0±19.55** 135.0±10.8 120.0±19.44

DBP (mm Hg) 81.82±8.93 90.0±19.44 76.0±10.75 94.0±12.65**

Fasting plasma glucose
95.09±8.45 158.7±42.12*** 162.4±25.05 208.0±39.61**
(mg/dL)

of
Postprandial plasma glucose
109.9±15.74 227.1±44.59*** 167.6±16.3 185.8±20.04
(mg/dL)
8.91±1.10***

ro
Glycated hemoglobin (%) 5.32±0.37 8.77±1.87 9.25±2.27
Total serum cholesterol
165.1±26.13 176.6±36.73 146.0±35.2 148.2±37.21
(mg/dL)
-p
Serum triglycerides (mg/dL) 103.5±33.86 152.3±22.79*** 129.4±48 151.2±39.38**
re
HDL- cholesterol (mg/dL) 46.09±5.48 34.8±4.84** 35.38±6.20 34.4±8.92
lP

LDL- cholesterol (mg/dL) 84.18±8.98 97.5±30.69 82.25±24.12 89.9±32.83

Urea (mg/dL) 28.14±8.69 23.88±9.37 25.8±10.2 26.7±7.91

na

Creatinine (mg/dL) 0.95±0.11 0.87±0.24 1.07±0.25 1.25±0.86

HOMA-IR 1.69±0.90 4.53±3.73*** 7.50±5.29 8.245±4.45

ur

All data are reported as mean ± SD for continuous variables. BMI- Body Mass Index; SBP- Systolic
Blood Pressure; DBP- Diastolic Blood Pressure; HDL- High Density Lipoprotein; LDL- Low Density
Jo

Lipoprotein.
a
Indicates comparison was made between KDM and NGT subjects.
b
Indicates comparison was made between Uninfected and Infected DFU subjects.
*
P< 0.05.
**
P< 0.01.
***
P< 0.001.

Table 3: miRNA Prediction Score

Position on Targeted miRNA’s Context++ PCT

SDF-1α 3’UTR score

20
 Journal Pre-proof

2997-3004 hsa-miR-23a-3p -0.35 0.38

2997-3004 hsa-miR-23b-3p -0.35 0.38

2997-3004 hsa-miR-23c -0.32 0.38

of
ro
-p
re
lP
na
ur

Table 4: Spearman’s Correlation analysis of miR23c with SDF-1α and other

Jo

biochemical variables

miR23c
Parameters
r P

SDF-1α -0.565 0.028

eNOS 0.798 0.006

Age 0.313 0.379

FBG 0.298 0.347

PPG 0.203 0.527

HBA1C 0.658 0.021

TRI 0.791 0.004

21
 Journal Pre-proof

HDL 0.726 0.018

LDL -0.068 0.852

of
ro
-p
re
lP
na
ur
Jo

22
 Journal Pre-proof

Highlights
 miRNAs play a crucial role in regulating angiogenesis, collectively called as
“angiomiRs”
 The angiogenic factor SDF-1α was significantly decreased in both the circulation and
tissue biopsies of patients with T2DM and infected DFU
 The tissue-specific expressions of miR-23a and miR-23b were found to be low
whereas miR-23c was increased in patients with infected DFU
 Correlation analysis showed that SDF-1α was found to have a significant inverse
association with miR-23c
 miR-23c may function as a new regulator to inhibit angiogenesis by targeting SDF-1α

of
ro
-p
re
lP
na
ur
Jo

23
Figure 1
Figure 2
Figure 3
Figure 4