Role of Increased miR-222-3p Expression in Peripheral Blood and Wound Marginal Tissues of Type 2 Diabetes Mellitus Patients with Diabetic Foot Ulcer

Figures & data

Table 1 Comparisons of Clinical Parameters Among the Three Groups [n (%), (), M (P₂₅, P₇₅₎]

Variables	NC (n=70)	T2DM (n=70)	DFU (n=146)	F/t/x^2 value	P value
Gender				1.364	0.411
Male	40 (57.1)	39 (55.7)	78 (53.4)
Female	30 (42.9)	31 (44.3)	68 (46.6)
Age (y)	55.2±10.6	56.8±11.6	56.5±11.5	1.726	0.368
Diabetes duration (y)	–	0.4±0.2	12.3±6.5^d	9.831	<0.001
FPG (mmol/L)	4.9±0.5	9.2±3.1^b	12.7±3.9^bd	38.564	<0.001
HbA1c (%)	5.2±0.4	8.5±1.8^b	9.3±2.9^cd	33.712	<0.001
TG (mmol/L)	1.5±0.4	1.9±0.8^b	2.2±0.9^cd	8.975	<0.001
TCH (mmol/L)	4.6±0.7	5.0±0.9	4.8±0.7	1.792	0.221
LDL-C (mmol/L)	2.5±0.4	2.9±0.6	2.8±0.5	1.589	0.086
HDL-C (mmol/L)	1.5±0.3	1.1±0.4^a	0.9±0.5^bc	2.851	0.021
TcPO2 (mmHg)	77.1±7.8	72.1±9.1	51.7±11.3^bd	25.624	<0.001
ABI	1.13±0.12	1.07±0.18	0.85±0.21^bd	6.126	<0.001
CRP (mg/L)	5.9±0.8	8.2±1.3	49.3±18.7^bd	79.462	<0.001
ESR (mm/h)	10.5±2.4	12.2±2.6	47.8±19.1^bd	32.535	<0.001
WBC (×10^9)	4.6±0.5	4.7±0.8	11.5±3.9^bd	28.521	<0.001
ALB (g/L)	41.7±1.3	41.3±1.1	36.2.±3.2^bd	3.127	0.014
Hb (g/L)	129.1±1.5	128.5±1.7	115.3±3.8^bd	2.831	0.029
P-IL-6 (pg/mL)	12.8±3.9	19.5±7.5	38.6±10.4^bc	28.259	<0.001
P-IL-10 (ng/L)	4.5±0.9	4.1±1.2	3.6±1.4	1.803	0.218
T-VEGF	–	–	0.49 (0.15, 0.79)	–	–
T-CD31	–	–	0.64 (0.28, 1.01)	–	–
T-IL-6	–	–	2.92 (0.96, 4.11)	–	–
T-IL-10	–	–	0.52 (0.19, 0.96)	–	–
P-miR-222-3p	0.92 (0.61, 1.87)	1.98 (0.98, 3.62)^b	5.61 (1.98, 10.24)^bd	30.719	<0.001
T-miR-222-3p	–	–	7.93 (2.27, 12.86)	–	–

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, glycated hemoglobin A1c; TG, triacylglycerol; TCH, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; TcPO2, transcutaneous oxygen pressure; ABI, ankle brachial index; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; WBC, white blood cell; ALB, albumin; Hb, hemoglobin; P-IL-6, IL-6 level in peripheral blood; P-IL-10, IL-10 level in peripheral blood; T-VEGF, vascular endothelial growth factor mRNA expression in wound margin tissue; T-CD31, CD31 mRNA expression in wound margin tissue; T-IL-6, interleukin-6 mRNA expression in wound margin tissue; T-IL-10, interleukin-10 mRNA expression in wound margin tissue; MiR, MicroRNA.

Table 2 Relationship Between miR-222-3p Expression Levels in Peripheral Blood and Clinical Characteristics of DFU [n (%)]

Characteristics	High Expression Group (n=88)	Low Expression Group (n=58)	x^2 value	P value
Age (y)			0.458	0.501
≥55	48 (54.5)	28 (48.3)
<55	40 (45.5)	30 (51.7)
Gender			0.112	0.738
Male	48 (54.5)	30 (51.7)
Female	40 (45.5)	28 (48.3)
Ulcer area (cm^2)			2.139	0.342
≤5	7 (11.1)	8 (22.5)
5~10	59 (66.7)	40 (52.5)
>10	22 (22.2)	10 (25.0)
Ulcer duration (w)			6.483	0.039
≤6	12 (13.6)	18 (31.0)
6~10	61 (69.3)	32 (55.2)
>10	15 (17.1)	8 (13.8)
Wagner grade			7.523	0.023
II	7 (7.9)	14 (24.1)
III	68 (77.3)	36 (62.1)
IV	13 (14.8)	8 (13.8)
Severity of IDSA infection			6.155	0.046
Mild	8 (9.1)	13 (22.4)
Moderate	59 (65.9)	37 (63.8)
Severe	21 (25.0)	8 (13.8)
Amputation rate (%)			2.303	0.129
Amputated	23 (26.1)	9 (15.5)
Not amputated	65 (73.9)	49 (84.5)
Ulcer healing rate after 8 weeks (%)			4.684	0.030
Healed	37 (42.0)	35 (60.3)
Not healed	51 (58.0)	23 (39.7)

Notes: Data are presented numbers (%); differences between two groups analyzed using x² test. The cut-off point of miR-222-3p expression level for grouping was 5.61.

Abbreviations: DFU, diabetic foot ulcer; IDSA, Infectious Disease Society of America.

Table 3 Relationship Between miR-222-3p Expression Levels in Wound Margin Tissue and Clinical Characteristics of DFU [n (%)]

Characteristics	High Expression Group (n=94)	Low Expression Group (n=52)	x^2 value	P value
Age (y)			0.137	0.712
≥55	50 (53.2)	26 (50.0)
<55	44 (46.8)	26 (50.0)
Gender			1.716	0.190
Male	54 (57.4)	24 (46.2)
Female	40 (42.6)	28 (53.8)
Ulcer area (cm^2)			1.606	0.448
≤5	8 (8.5)	7 (13.5)
5~10	63 (67.0)	36 (71.1)
>10	23 (24.5)	9 (15.4)
Ulcer duration (w)			7.505	0.023
≤6	13 (13.8)	17 (32.7)
6~10	64 (68.1)	29 (55.8)
>10	17 (18.1)	6 (11.5)
Wagner grade			7.468	0.024
II	8 (8.5)	13 (25.0)
III	71 (75.5)	33 (63.5)
IV	15 (16.0)	6 (11.5)
Severity of IDSA infection			8.213	0.016
Mild	8 (8.5)	13 (25.0)
Moderate	64 (68.1)	32 (61.5)
Severe	22 (23.4)	7 (13.5)
Amputation rate (%)			2.014	0.156
Amputated	24 (26.1)	8 (15.5)
Not amputated	70 (73.9)	44 (84.5)
Ulcer healing rate after 8 weeks (%)			4.828	0.028
Healed	40 (42.0)	32 (60.3)
Not healed	54 (58.0)	20 (39.7)

Notes: Data are presented numbers (%); differences between two groups analyzed using x² test. The cut-off point of miR-222-3p expression level for grouping was 7.93.

Abbreviations: DFU, diabetic foot ulcer; IDSA, Infectious Disease Society of America.

Figure 1 Kaplan-Meier survival curve analysis evaluated the complete wound healing rate of DFU between two expression groups. (A) Group with high miR-222-3p expression presented higher unhealed DFU cumulative rate relative to the low expression group in DFU patients’ peripheral blood (log rank, P = 0.011). The estimated median time of complete wound healing in groups with high and low miR-222-3p expressions were 9.64 weeks and 8.70 weeks, respectively, in DFU patients’ peripheral blood (P < 0.05). (B) Group with high miR-222-3p expression presented higher unhealed DFU cumulative rate relative to the low expression group in DFU patients’ wound margin tissue (log rank, P = 0.001). The estimated median time of complete wound healing in high and low expression groups were 9.76 weeks and 8.71 weeks, respectively, in DFU patients’ wound margin tissue (P < 0.05).

Table 4 Correlations Between miR-222-3p Expression in Peripheral Blood and Other Clinical Parameters in NC Group, T2DM Group, and DFU Group (r)

Variables	NC (n=70)		T2DM (n=70)		DFU (n=146)
	r	P value	r	P value	r	P value
Age (y)	0.059	0.684	0.046	0.767	0.051	0.713
Gender	0.087	0.572	0.052	0.703	0.098	0.426
Diabetes duration (y)	–	–	0.121	0.227	0.152	0.203
Foot ulcer duration (w)	–	–	–	–	0.298	0.031
Foot ulcer area (cm^2)	–	–	–	–	0.147	0.252
Wagner grade	–	–	–	–	0.326	0.021
Severity of IDSA infection	–	–	–	–	0.315	0.024
FPG (mmol/L)	0.101	0.326	0.286	0.035	0.273	0.048
HbA1c (%)	0.085	0.581	0.301	0.027	0.292	0.032
TG (mmol/L)	0.041	0.815	0.092	0.581	0.037	0.838
TCH (mmol/L)	0.022	0.894	0.028	0.865	0.043	0.813
LDL-C (mmol/L)	0.029	0.817	0.016	0.902	0.026	0.865
HDL-C (mmol/L)	−0.087	0.552	−0.291	0.032	−0.284	0.041
ALB (g/L)	0.015	0.903	0.068	0.625	−0.099	0.426
TcPO2 (mmHg)	−0.007	0.928	0.018	0.891	−0.298	0.029
ABI	0.025	0.872	0.073	0.658	−0.203	0.064
CRP (mg/L)	0.031	0.704	0.093	0.579	0.281	0.043
ESR (mm/h)	0.019	0.898	0.074	0.584	0.191	0.098
WBC (×10^9)	0.012	0.911	0.078	0.581	0.275	0.045
Hb (g/L)	0.083	0.593	0.024	0.882	−0.093	0.436
P-IL-6 (pg/mL)	0.091	0.583	0.176	0.118	0.319	0.021
P-IL-10 (ng/L)	−0.028	0.857	−0.102	0.397	−0.117	0.184
T-VEGF	–	–	–	–	−0.341	0.005
T-CD31	–	–	–	–	−0.291	0.033
T-IL-6	–	–	–	–	0.398	<0.001
T-IL-10	–	–	–	–	−0.195	0.072
T-miR-222-3p	–	–	–	–	0.435	<0.001

Abbreviations: NC, normal control group; T2DM, type 2 diabetes group; DFU, diabetic foot ulcer group; IDSA, Infectious Disease Society of America; FPG, fasting plasma glucose; HbA1c, glycated hemoglobin A1c; TG, triacylglycerol; TCH, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; TcPO2, transcutaneous oxygen pressure; ABI, ankle brachial index; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; WBC, white blood cell; ALB, albumin; Hb, hemoglobin; P-IL-6, IL-6 level in peripheral blood; P-IL-10, IL-10 level in peripheral blood; T-VEGF, vascular endothelial growth factor mRNA expression in wound margin tissue; T-CD31, CD31 mRNA expression in wound margin tissue; T-IL-6, interleukin-6 mRNA expression in wound margin tissue; T-IL-10, interleukin-10 mRNA expression in wound margin tissue; MiR, MicroRNA.

Table 5 Correlations Between miR-222-3p Expression in Wound Margin Tissue and Other Clinical Parameters in the DFU Group (r)

Variables	DFU (n=146)
	r	P value
Age (y)	0.046	0.763
Gender	0.063	0.548
Diabetes duration (y)	0.179	0.117
Foot ulcer duration (w)	0.311	0.026
Foot ulcer area (cm^2)	0.189	0.102
Wagner grade	0.367	0.002
Severity of IDSA infection	0.338	0.015
FPG (mmol/L)	0.276	0.044
HbA1c (%)	0.297	0.035
TG (mmol/L)	0.028	0.872
TCH (mmol/L)	0.048	0.751
LDL-C (mmol/L)	0.034	0.886
HDL-C (mmol/L)	−0.295	0.036
ALB (g/L)	−0.106	0.314
TcPO2 (mmHg)	−0.283	0.039
ABI	−0.211	0.058
CRP (mg/L)	0.298	0.033
ESR (mm/h)	0.185	0.107
WBC (×10^9)	0.281	0.045
Hb (g/L)	−0.102	0.294
P-IL-6 (pg/mL)	0.342	0.008
P-IL-10 (ng/L)	−0.109	0.278
T-VEGF	−0.358	0.001
T-CD31	−0.306	0.028
T-IL-6	0.436	<0.001
T-IL-10	−0.172	0.126
P-miR-222-3p	0.435	<0.001

Abbreviations: DFU, diabetic foot ulcer group; IDSA, Infectious Disease Society of America; FPG, fasting plasma glucose; HbA1c, glycated hemoglobin A1c; TG, triacylglycerol; TCH, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; TcPO2, transcutaneous oxygen pressure; ABI, ankle brachial index; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; WBC, white blood cell; ALB, albumin; Hb, hemoglobin; P-IL-6, IL-6 level in peripheral blood; P-IL-10, IL-10 level in peripheral blood; T-VEGF, vascular endothelial growth factor mRNA expression in wound margin tissue; T-CD31, CD31 mRNA expression in wound margin tissue; T-IL-6, interleukin-6 mRNA expression in wound margin tissue; T-IL-10, interleukin-10 mRNA expression in wound margin tissue; MiR, MicroRNA.

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

Variables	β	SE	Wald	OR	95% CI	P value
Diabetes duration (y)	0.62	0.34	6.21	4.12	1.23~9.78	<0.001
HbA1c (%)	0.37	0.21	3.56	1.47	1.08~7.62	0.041
TcPO2 (kPa)	0.49	0.36	4.16	2.18	1.16~10.26	0.032
CRP (mg/L)	0.41	0.28	3.05	1.13	1.08~9.12	0.045
ALB (g/L)	0.35	0.17	2.98	1.12	1.13~8.75	0.048
P-miR-222-3p	0.75	0.48	5.83	3.85	1.18~12.37	0.008

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-IL-6, P-IL-10, miR-222-3p.

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-IL-6, IL-6 level in peripheral blood; P-IL-10, IL-10 level in peripheral blood; P-miR-222-3p, miR-222-3p expression in peripheral in blood; MiR, microRNA.

Figure 2 The biomarker potential of circulating miR-222-3p for DFU and control. ROC analysis assisted in evaluating the ability of circulating miR-222-3p to distinguish between two groups. The AUC of P-miR-222-3p specific to DFU diagnosis reached 0.803 (95% CI 0.713–0.884, P < 0.001), with the optimal cut-off point of 2.52, the sensitivity of 95.93%, and the specificity of 96.27%.

Figure 3 MiR-222-3p effectively served for DFU diagnosis and prognosis as a useful biomarker. ↑: increase; ↓: decrease.