Increased Expression of miR-155 in Peripheral Blood and Wound Margin Tissue of Type 2 Diabetes Mellitus Patients Associated with Diabetic Foot Ulcer

Figures & data

Table 1 Comparisons of Clinical Parameters Among NC Group, T2DM Group, and DFU Group [n (%), (±S), M (P₂₅, P₇₅)]

Characteristics	NC (n=60)	T2DM (n=60)	DFU (n=112)	F/t/x^2 value	P value
Gender				1.183	0.562
Male	36 (60.0)	34 (56.7)	60 (53.6)
Female	24 (40.0)	26 (43.3)	52 (46.4)
Age (y)	54.7±10.1	55.1±11.9	54.5±10.6	1.327	0.315
Diabetes course (y)	–	0.3±0.2	11.3±5.2^d	8.946	<0.001
FPG (mmol/L)	4.8±0.6	9.9±2.5^b	11.2±3.8^bd	28.357	<0.001
HbA1c (%)	5.1±0.4	8.3±1.9^b	9.1±2.7^bc	26.123	<0.001
TG (mmol/L)	1.4±0.8	1.8±0.8^b	1.7±0.9^b	9.236	<0.001
TCH (mmol/L)	4.3±0.8	4.9±0.7	4.6±0.7	1.868	0.127
LDL-C (mmol/L)	2.4±0.3	2.8±0.5	2.6±0.5	1.981	0.092
HDL-C (mmol/L)	1.3±0.2	1.0±0.3^a	0.9±0.5^b	2.357	0.044
TcPO2 (mmHg)	76.4±7.2	70.5±8.9	48.5±10.2^bd	19.823	<0.001
ABI	1.12±0.16	1.05±0.21	0.84±0.32^bc	7.865	<0.001
CRP (mg/L)	7.2±1.1	9.2±1.2	48.2±19.3^bd	83.127	<0.001
ESR (mm/h)	10.2±2.1	12.7±3.1	48.2±19.5^bd	35.763	<0.001
ALB (g/L)	39.9±0.6	39.3±0.8	33.7±3.5^ac	2.964	0.023
WBC (×10^9)	4.2±0.8	4.7±0.9	11.2±4.2^bd	30.413	<0.001
Hb (g/L)	128.5±2.2	126.4±2.9	112.3±1.8^ac	2.745	0.031
P-miR-155	1.09 (0.61,1.92)	0.43 (0.19, 1.37)^b	4.38 (1.26,10.13)^bd	6.985	<0.001
T-miR-155	–	–	7.41 (2.47,13.86)	–	–

Notes: Data are presented mean±standard deviations or numbers (%) or median with IQR; differences among three groups analyzed using one-way analysis of variance or x² test, and least-significant difference (LSD) analysis was used for comparison between the two groups. Versus NC group, ^aP< 0.05, ^bP< 0.01; versus T2DM group, ^cP< 0.05, ^dP< 0.01.

Abbreviations: NC, normal control group; T2DM, type 2 diabetes group; DFU, diabetic foot ulcer group; FPG, fasting plasma glucose; HbA1c, glycosylated hemoglobin A1c; TG, triglycerides; TCH, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; TcPO2, transcutaneous oxygen partial pressure; ABI, ankle-brachial index; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; ALB, serum albumin; WBC, white blood cell count; Hb, hemoglobin; P-miR-155, miR-155 expression in peripheral blood; T-miR-155, miR-155 expression in the wound margin tissue; MiR, microRNA.

Table 2 Relationship Between miR-155 Expression Levels in Peripheral Blood and Clinical Characteristics of DFU [n (%)]

Characteristics	High Expression Group (n=72)	Low Expression Group (n=40)	x^2 value	P value
Age (y)			0.003	0.955
≥55	40 (55.6)	22 (55.0)
<55	32 (44.4)	18 (45.0)
Gender			0.287	0.592
Male	34 (47.2)	21 (52.5)
Female	38 (52.8)	19 (47.5)
Ulcer area (cm^2)			3.121	0.210
≤5	8 (11.1)	9 (22.5)
5–10	48 (66.7)	21 (52.5)
>10	16 (22.2)	10 (25.0)
Ulcer course (w)			6.844	0.033
≤6	17 (23.6)	19 (47.5)
6–10	44 (61.1)	16 (40.0)
>10	11 (15.3)	5 (12.5)
Wagner grade			9.151	0.010
II	4 (5.6)	10 (25.0)
III	56 (77.8)	26 (65.0)
IV	12 (16.7)	4 (10.0)
Amputation rate (%)			2.619	0.106
Amputated	16 (22.2)	4 (10.0)
Not amputated	56 (77.8)	36 (90.0)
Ulcer healing rate after 8 weeks (%)			4.352	0.037
Healed	32 (44.4)	26 (65.0)
Not healed	40 (55.6)	14 (35.0)

Notes: Data are presented numbers (%); differences between two groups analyzed using x² test. The cut-off point of miR-155 expression level for grouping was 4.38.

Table 3 Relationship Between miR-155 Expression Levels in Wound Margin Tissue and Clinical Characteristics of DFU [n (%)]

Characteristics	High Expression Group (n=80)	Low Expression Group (n=32)	x^2 value	P value
Age (y)			0.178	0.673
≥55	44 (55.0)	19 (59.4)
<55	36 (45.0)	13 (40.6)
Gender			0.289	0.591
Male	38 (47.5)	17 (53.1)
Female	42 (52.5)	15 (46.9)
Ulcer area (cm^2)			1.934	0.380
≤5	10 (12.5)	6 (18.8)
5–10	56 (70.0)	18 (56.2)
>10	14 (17.5)	8 (25.0)
Ulcer course (w)			6.786	0.034
≤6	14 (17.5)	16 (50.0)
6–10	50 (62.5)	8 (25.0)
>10	16 (20.0)	8 (25.0)
Wagner grade			8.756	0.013
II	5 (6.3)	7 (21.9)
III	61 (76.2)	16 (50.0)
IV	14 (17.5)	9 (28.1)
Amputation rate (%)			2.492	0.114
Amputated	21 (26.3)	4 (12.5)
Not amputated	59 (73.7)	28 (87.5)
Ulcer healing rate after 8 weeks (%)			4.444	0.035
Healed	30 (37.5)	19 (59.4)
Not healed	50 (62.5)	13 (40.6)

Notes: Data are presented numbers (%); differences between two groups analyzed using x² test. The cut-off point of miR-155 expression level for grouping was 7.41.

Figure 1 Curve of wound complete healing rate of DFU between miR-155 high expression group and low expression group by Kaplan-Meier survival curve analysis. (A) The cumulative rate of unhealed DFU in the high expression group of peripheral blood miR-155 was higher than that in the low expression group of peripheral blood miR-155 (log rank, P = 0.004). The estimated time median of wound complete healing in peripheral blood miR-155 high expression group and low expression group were 10.23 weeks and 9.51 weeks, respectively (P < 0.05). (B) The cumulative rate of unhealed DFU in the high expression group of wound margin tissue miR-155 was higher than that in the low expression group of wound margin tissue miR-155 (log rank, P < 0.001). The estimated time median of wound complete healing in wound margin tissue miR-155 high expression group and low expression group were 10.12 weeks and 8.67 weeks, respectively (P < 0.05).

Table 4 Correlations Between miR-155 Expression in Peripheral Blood and Other Clinical Parameters in NC Group, T2DM Group, and DFU Group (r)

Variables	NC (n=60)		T2DM (n=60)		DFU (n=112)
	r	P value	r	P value	r	P value
Age	0.041	0.825	0.052	0.812	0.048	0.785
Gender	0.102	0.346	0.039	0.857	0.049	0.722
Course of diabetes	–	–	0.101	0.426	0.142	0.302
Course of foot ulcer	–	–	–	–	0.295	0.041
Foot ulcer area	–	–	–	–	0.112	0.319
Wagner grade	–	–	–	–	0.309	0.025
FPG	−0.083	0.311	−0.298	0.039	0.181	0.098
HbA1c	−0.078	0.335	−0.287	0.046	0.173	0.102
TG	−0.015	0.888	−0.088	0.301	0.017	0.841
TCH	0.014	0.897	0.029	0.885	0.068	0.719
LDL-C	0.038	0.826	−0.011	0.903	0.055	0.768
HDL-C	0.067	0.553	0.046	0.652	−0.094	0.458
ALB	0.012	0.902	0.008	0.925	−0.019	0.826
TcPO2	−0.009	0.926	−0.068	0.474	−0.075	0.453
ABI	0.095	0.344	0.086	0.798	−0.103	0.382
CRP	0.085	0.501	0.123	0.295	0.315	0.021
ESR	−0.069	0.552	0.106	0.304	0.203	0.069
WBC	−0.011	0.828	0.178	0.224	0.296	0.042
Hb	0.093	0.415	−0.085	0.302	−0.081	0.336

Abbreviations: NC, normal control group; T2DM, type 2 diabetes group; DFU, diabetic foot ulcer group; FPG, fasting plasma glucose; HbA1c, glycosylated hemoglobin A1c; TG, triglycerides; TCH, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; ALB, serum albumin; TcPO2, transcutaneous oxygen partial pressure; ABI, ankle-brachial index; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; WBC, white blood cell count; Hb, hemoglobin; MiR, microRNA.

Table 5 Correlation Between miR-155 Expression in Wound Margin Tissue and Other Clinical Parameters in the DFU Group (r)

Variables	DFU (n=112)
	r	P value
Age	0.031	0.802
Gender	0.113	0.305
Course of diabetes	0.138	0.279
Course of foot ulcer	0.304	0.012
Foot ulcer area	0.179	0.114
Wagner grade	0.357	0.009
FPG	0.193	0.067
HbA1c	0.184	0.095
TG	0.011	0.893
TCH	0.052	0.746
LDL-C	0.101	0.493
HDL-C	−0.048	0.682
ALB	−0.081	0.795
TcPO2	−0.108	0.327
ABI	−0.115	0.286
CRP	0.298	0.025
ESR	0.201	0.064
WBC	0.275	0.043
Hb	−0.107	0.291
P-miR-155	0.418	< 0.001

Abbreviations: DFU, diabetic foot ulcer group; FPG, fasting plasma glucose; HbA1c, glycosylated hemoglobin A1c; TG, triglycerides; TCH, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; ALB, serum albumin; TcPO2, transcutaneous oxygen partial pressure; ABI, ankle-brachial index; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; WBC, white blood cell count; Hb, hemoglobin; P-miR-155, miR-155 expression in peripheral blood; MiR, microRNA.

Table 6 The Multivariate Stepwise Logistic Regression Analysis of Risk Factors for Diabetic Foot Ulcer

Variable	β	SE	Wald	OR	95% CI	P value
Course of diabetes (y)	0.73	0.52	5.51	4.69	1.98~6.22	<0.001
HbA1c (%)	0.57	0.33	3.86	1.83	1.17~7.51	0.031
TcPO2 (mmHg)	0.39	0.25	3.01	1.14	1.09~8.17	0.038
CRP (mg/L)	0.42	0.29	2.96	1.12	1.06~6.75	0.046
P-miR-155	0.81	0.43	7.12	3.98	1.25~8.62	0.003

Notes: Multivariate unconditional logistic regression analysis adjusted for sex, age, the course of diabetes, FPG, HbA1c, TG, LDL-C, HDL-C, Hb, ALB, TcPO2, ABI, CRP, WBC, ESR, P-miR-155.

Abbreviations: FPG, fasting plasma glucose; HbA1c, glycosylated hemoglobin A1c; TG, triglycerides; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; Hb, hemoglobin; ALB, serum albumin; TcPO2, transcutaneous oxygen partial pressure; ABI, ankle-brachial index; CRP, C-reactive protein; WBC, white blood cell count; ESR, erythrocyte sedimentation rate; P-miR-155, miR-155 expression in peripheral in blood; MiR, microRNA.

Figure 2 The biomarker potential of circulating miR-155 for DFU and control. ROC analysis was used to evaluate the ability of circulating miR-155 to distinguish between two groups. MiR-155 distinguished DFU patients from controls with area under curve (AUC) of 0.794 (95% CI, 0.726–0.863, P < 0.001), the best cut-off point of miR-155 was 1.01, the sensitivity was 96.82%, and the specificity was 95.93%. DFU: diabetic foot ulcer.

Figure 3 MiR-155 is a potentially valuable biomarker for diagnosis and prognosis of DFU. ↑: increase; ↓: decrease.