Role of Decreased Expression of miR-155 and miR-146a in Peripheral Blood of Type 2 Diabetes Mellitus Patients with Diabetic Peripheral Neuropathy

Figures & data

Table 1 Comparisons of Clinical Parameters Among the NC, T2DM and DPN Groups [n/M(P25, P75)]

Variables	DPN Group (n=49)	T2DM Group (n=51)	NC Group (n=50)	P value
Sex (male/female)	26/23	28/23	26/24	0.957
Age (year)	56.18±9.96	53.8±10.90	52.36±8.85	0.159
Smoking history (%)	12 (24.5)	11 (21.6)	13 (26.0)	0.869
Drinking history (%)	13 (26.5)	15 (29.4)	14 (28.0)	0.950
Family history of diabetes (%)	15 (30.6)	16 (31.4)	11 (22.0)	0.510
BMI (kg/m^2)	24.14±3.13	24.65±3.16	23.47±2.47	0.130
Duration of diabetes (year)	10 (7, 15.5)	7 (3, 10)	–	0.002*
TBIL (umol/L)	12.83±4.53	14.13±4.35	14.68±3.94	0.091
AST (U/L)	14.5 (12.0, 18.0)	15.0 (12.0, 21.0)	16.35 (14.47, 19.42)	0.091
ALT (U/L)	17.0 (12.0, 23.5)	20.0 (15.0, 31.0)	16.9 (10.13, 29.7)	0.091
BUN (mmol/L)	6.41 (4.96, 8.55) ^■ ▲	5.12 (4.06, 6.30)	4.85 (4.30, 5.80)	<0.001*
Scr (umol/L)	66.0 (55.05, 83.0) ^■ ▲	56.0 (47.3, 63.0)	57.65(47.28, 67.03)	0.001*
FPG (mmol/L)	10.5 (9.1, 12.64) ^■ ▲	9.34 (6.8, 11.52) ^▲	5.21 (4.89, 5.36)	<0.001*
HbA1c (%)	10.0 (8.73, 11.85) ^■ ▲	9.1 (7.9, 10.0) ^▲	4.9 (4.6, 5.2)	<0.001*
TC (mmol/L)	4.41±0.78	4.37±0.94	4.47±0.79	0.831
TG (mmol/L)	1.36 (1.13, 1.97)	1.38 (0.96)	1.22 (1.00, 1.69)	0.214
VLDL (mmol/L)	0.50 (0.35, 0.67)	0.47 (0.32, 0.63)	0.38 (0.26, 0.71)	0.428
LDL-C (mmol/L)	2.74 (2.09, 3.15)	2.74 (2.05, 3.25)	-	0.754
HDL-C (mmol/L)	1.12 (0.84, 1.305)	1.02 (0.89, 1.17)	-	0.336
FCP (ng/mL)	0.96 (0.525, 1.655)	1.01 (0.66, 1.33)	-	0.934
SCV (m/s)	35.1 (33.25, 44.15) ^■	46.6 (42.4, 50.0)	-	<0.001*
MCV (m/s)	40.3 (36.65, 44.8) ^■	45.5 (40.8, 49.0)	-	<0.001*
MiR-155	3.58 (3.41, 3.78) ^■ ▲	3.77 (3.52, 3.89) ^▲	3.86 (3.68, 4.04)	<0.001*
MiR-146a	3.01 (2.6, 3.73) ^■ ▲	3.70 (3.26, 4.01) ^▲	4.21 (3.82, 4.63)	<0.001*

Note: Data are presented as mean ± standard deviation or numbers (%) or median with IQR; differences among the three groups were analysed using one-way analysis of variance or x² test, and least-significant difference (LSD) analysis was used for comparison between the two groups. *P < 0.05; ^■Compared to the T2DM group, P < 0.05; ^▲Compared to the control group, P < 0.05.

Abbreviations: T2DM, Type 2 Diabetes Mellitus; DPN, Diabetic Peripheral Neuropathy; BMI, Body Mass Index; TBIL, Total Bilirubin; ALT, Alanine Aminotransferase; AST, Aspartate Aminotransferase; BUN, Blood Urea Nitrogen; Scr, Serum Creatinine; FPG, Fasting Plasma Glucose; HbA1c, Glycated Haemoglobin; TC, Total Cholesterol; TG, Triglycerides; VLDL, Very Low-Density Lipoprotein Cholesterol; LDL-C, Low-Density Lipoprotein Cholesterol; HDL-C, High-Density Lipoprotein Cholesterol; FCP, Fasting C-peptide; SCV, Sensory Nerve Conduction Velocity; MCV, Motor Nerve Conduction Velocity; miR-155, microRNA-155; miR-146a, microRNA-146a.

Table 2 Comparison of Clinical Examinations, Biochemical Indicators and miRNA Among the Subgroups of DPN )/M(P25, P75)]

Variables	PDPN Subgroup (n=16)	Non-PDPN Subgroup (n=33)	NC Group (n=50)	T2DM Group (n=51)	P value
Age (year)	55.13±10.08	56.70±10.02	26/24	53.8±10.90	0.610
Duration of diabetes (year)	9.0 (6.25, 20.0)	10.0 (7.5, 15.0)	-	7 (3, 10)	0.949
BMI (kg/m^2)	23.88±2.93	24.27±3.26	23.47±2.47	24.65±3.16	0.692
TBIL (umol/L)	10.32±4.10	13.74±4.55	14.68±3.94	14.13±4.35	0.014*
AST (U/L)	14.00 (12.00, 16.65)	15.00 (11.50, 18.50)	16.35 (14.47, 19.42)	15.0 (12.0, 21.0)	0.353
ALT (U/L)	13.00 (11.30, 20.25)	18.00 (13.50, 24.50)	16.9 (10.13, 29.7)	20.0 (15.0, 31.0)	0.156
BUN (mmol/L)	6.25 (5.10, 9.05)	6.57 (4.94, 8.20)	4.85 (4.30, 5.80)	5.12 (4.06, 6.30)	0.693
Scr (umol/L)	62.65 (48.50, 74.25)	68.60 (59.40, 91.85)	57.65(47.28, 67.03)	56.0 (47.3, 63.0)	0.141
FPG (mmol/L)	9.20 (8.74, 12.63)	11.20 (9.53, 12.75)	5.21 (4.89, 5.36)	9.34 (6.8, 11.52)	0.082
HbA1c (%)	9.75 (8.70, 11.93)	10.00 (8.90, 11.80)	4.9 (4.6, 5.2)	9.1 (7.9, 10.0)	0.685
TC (mmol/L)	4.42±0.75	4.40±0.81	4.47±0.79	4.37±0.94	0.921
TG (mmol/L)	1.26 (0.96, 1.90)	1.43 (1.20, 2.06)	1.22 (1.00, 1.69)	1.38 (0.96)	0.197
VLDL (mmol/L)	0.45±0.21	0.56±0.22	0.38 (0.26, 0.71)	0.47 (0.32, 0.63)	0.125
LDL-C (mmol/L)	2.47 (1.94, 3.19)	2.85 (2.25, 3.13)	-	2.74 (2.05, 3.25)	0.502
HDL-C (mmol/L)	1.25 (0.77, 1.48)	1.08 (0.88, 1.27)	-	1.02 (0.89, 1.17)	0.430
FCP (ng/mL)	1.16 (0.48, 1.68)	0.94 (0.61, 1.65)	-	1.01 (0.66, 1.33)	0.848
SCV (m/s)	34.40 (32.25, 41.23)	38.70 (34.00, 44.30)	-	46.6 (42.4, 50.0)	0.236
MCV (m/s)	39.79±4.52	40.85±4.63	-	45.5 (40.8, 49.0)	0.451
MiR-155	3.49 (3.15, 3.68) ^▲	3.61 (3.50, 3.82) ^▲	3.86 (3.68, 4.04)	3.77 (3.52, 3.89)	<0.001
MiR-146a	3.49 (2.77, 3.90) ^▲	2.88 (2.50, 3.35) ^■▲	4.21 (3.82, 4.63)	3.70 (3.26, 4.01)	<0.001

Notes: Within the DPN group, patients were further divided into subgroups: those experiencing symptoms such as numbness, burning or stabbing pain in the extremities were classified into the painful neuropathy subgroup (n = 16), while those without such symptoms were categorised into the non-painful neuropathy subgroup (n = 33). Data are presented as mean ± standard deviation or numbers (%) or median with IQR; differences among the three groups were analysed using one-way analysis of variance or x² test, and least-significant difference (LSD) analysis was used for comparison between the two groups. *P < 0.05; ^■Compared to the T2DM group, P < 0.05; ^▲Compared to the control group, P < 0.05.

Abbreviations: T2DM, Type 2 Diabetes Mellitus; PDPN subgroup, painful neuropathy subgroup; Non-PDPN subgroup, non-painful neuropathy subgroup; BMI, Body Mass Index; TBIL, Total Bilirubin; ALT, Alanine Aminotransferase; AST, Aspartate Aminotransferase; BUN, Blood Urea Nitrogen; Scr, Serum Creatinine; FPG, Fasting Plasma Glucose; HbA1c, Glycated Haemoglobin; TC, Total Cholesterol; TG, Triglycerides; VLDL, Very Low-Density Lipoprotein Cholesterol; LDL-C, Low-Density Lipoprotein Cholesterol; HDL-C, High-Density Lipoprotein Cholesterol; FCP, Fasting C-peptide; SCV, Sensory Nerve Conduction Velocity; MCV, Motor Nerve Conduction Velocity; miR-155, microRNA-155; miR-146a, microRNA-146a.

Table 3 Correlation Analysis of Peripheral Blood miR-155, miR-146a and Clinical Indexes in the DPN, T2DM and NC Groups

Variables	DPN Group				T2DM Group				NC Group
	miR-155		miR-146a		miR-155		miR-146a		miR-155		miR-146a
	rs	P	rs	P	rs	P	rs	P	rs	P	rs	P
Sex(male/female)	−0.176	0.225	−0.036	0.805	−0.181	0.204	−0.037	0.794	0.051	0.723	0.050	0.731
Smoking history (%)	−0.185	0.204	0.084	0.567	0.136	0.341	−0.288	0.040*	0.267	0.061	−0.092	0.526
Drinking history (%)	−0.165	0.257	−0.034	0.815	−0.021	0.884	0.164	0.250	0.114	0.432	0.117	0.419
BMI (kg/m^2)	−0.271	0.059	−0.212	0.144	0.063	0.660	0.085	0.552	0.155	0.283	−0.096	0.509
Age (year)	−0.012	0.933	0.112	0.442	−0.176	0.217	0.055	0.701	−0.117	0.417	0.227	0.113
Duration of diabetes (year)	0.140	0.336	−0.023	0.875	−0.175	0.219	0.278	0.049*	-	-	-	-
TBIL (umol/L)	−0.050	0.733	0.048	0.744	0.028	0.848	−0.070	0.627	0.157	0.277	0.149	0.303
AST (U/L)	0.154	0.290	−0.346	0.015*	0.136	0.343	−0.156	0.273	0.167	0.246	−0.050	0.730
ALT (U/L)	0.068	0.643	−0.402	0.004*	0.203	0.152	−0.168	0.238	−0.222	0.122	−0.303	0.033*
BUN (mmol/L)	−0.224	0.121	0.068	0.643	0.053	0.712	−0.242	0.087	−0.123	0.395	0.137	0.344
Scr (umol/L)	−0.034	0.814	−0.123	0.398	0.176	0.217	0.037	0.796	−0.305	0.031*	0.044	0.763
FPG (mmol/L)	0.362	0.011*	0.109	0.455	−0.007	0.960	0.016	0.913	0.019	0.896	−0.168	0.243
HbA1c (%)	0.087	0.554	−0.114	0.435	−0.076	0.595	−0.213	0.134	0.115	0.426	−0.041	0.775
TC (mmol/L)	0.239	0.098	0.098	0.501	−0.003	0.983	0.116	0.417	−0.014	0.923	−0.142	0.324
TG (mmol/L)	0.221	0.127	0.066	0.653	0.127	0.375	−0.090	0.531	0.038	0.792	−0.187	0.193
VLDL (mmol/L)	0.145	0.319	−0.034	0.818	−0.054	0.709	−0.085	0.555	−0.274	0.055	−0.239	0.095
FCP (ng/mL)	−0.058	0.691	−0.066	0.654	0.169	0.236	−0.078	0.588	-	-	-	-
SCV (m/s)	0.025	0.864	0.017	0.906	−0.120	0.403	0.133	0.350	-	-	-	-
MCV (m/s)	0.234	0.106	−0.090	0.537	−0.009	0.950	0.151	0.289	-	-	-	-
miR-155	-	-			-	-	0.005	0.975	-	-	−0.019	0.896
miR-146a	−0.185	0.204	-	-	0.005	0.975	-	-	−0.019	0.896	-	-

Note: *P < 0.05.

Abbreviations:T2DM, Type 2 Diabetes Mellitus; DPN, Diabetic Peripheral Neuropathy; BMI, Body Mass Index, TBIL, Total Bilirubin; ALT, Alanine Aminotransferase; AST, Aspartate Aminotransferase; BUN, Blood Urea Nitrogen; Scr, Serum Creatinine; FPG, Fasting Plasma Glucose; HbA_1c, Glycated Haemoglobin; TC, Total Cholesterol; TG, Triglycerides; VLDL, Very Low-Density Lipoprotein Cholesterol; LDL-C, Low-Density Lipoprotein Cholesterol; HDL-C, High-Density Lipoprotein Cholesterol; FCP, Fasting C-peptide; SCV, Sensory Nerve Conduction Velocity; MCV, Motor Nerve Conduction Velocity; miR-155, microRNA-155; miR-146a, microRNA-146a.

Table 4 Correlation Analysis Between Peripheral Blood miR-155, miR-146a and Clinical Indexes in DPN Subgroups

	PDPN Subgroup				Non-PDPN Subgroup
	miR-155		miR-146a		miR-155		miR-146a
	rs	P	rs	P	rs	P	rs	P
Sex (male/female)	−0.161	0.552	−0.190	0.481	−0.066	0.714	−0.155	0.388
Smoking history (%)	−0.084	0.757	0.196	0.467	−0.111	0.537	−0.044	0.808
Drinking history (%)	−0.052	0.848	−0.052	0.848	−0.194	0.279	−0.062	0.730
BMI (kg/m^2)	−0.749	0.001*	0.254	0.343	−0.151	0.402	−0.368	0.035*
Age (year)	0.199	0.460	0.399	0.126	0.019	0.917	0.134	0.457
Duration of diabetes (year)	0.206	0.445	−0.155	0.566	0.147	0.413	−0.027	0.883
TBIL (umol/L)	−0.013	0.961	0.106	0.696	−0.227	0.204	0.214	0.232
AST (U/L)	0.239	0.373	0.103	0.704	0.061	0.737	−0.470	0.006*
ALT (U/L)	−0.173	0.521	0.004	0.987	0.020	0.913	−0.437	0.011*
BUN (mmol/L)	−0.174	0.520	0.341	0.196	−0.243	0.173	−0.099	0.585
Scr (umol/L)	−0.303	0.254	0.000	1.000	−0.068	0.705	−0.164	0.360
FPG (mmol/L)	−0.015	0.957	0.015	0.957	0.444	0.010*	0.376	0.031*
HbA1c (%)	0.027	0.922	−0.169	0.530	0.102	0.571	−0.038	0.832
TC (mmol/L)	0.241	0.368	0.059	0.829	0.293	0.098	0.079	0.661
TG (mmol/L)	−0.126	0.641	0.221	0.412	0.277	0.119	0.164	0.361
VLDL (mmol/L)	−0.179	0.506	0.197	0.464	0.147	0.415	0.048	0.790
FCP (ng/mL)	−0.182	0.499	0.269	0.313	−0.005	0.979	−0.189	0.292
SCV (m/s)	−0.093	0.733	0.155	0.568	0.043	0.811	−0.022	0.903
MCV (m/s)	0.363	0.168	−0.208	0.440	0.154	0.393	−0.049	0.787
miR-155	-	-	−0.318	0.231	-	-	0.101	0.577
miR-146a	−0.318	0.231	-	-	0.101	0.577	-	-

Note: *P < 0.05.

Abbreviations: T2DM, Type 2 Diabetes Mellitus; PDPN subgroup, painful neuropathy subgroup; Non-PDPN subgroup, non-painful neuropathy subgroup; BMI, Body Mass Index; TBIL, Total Bilirubin; ALT, Alanine Aminotransferase; AST, Aspartate Aminotransferase; BUN, Blood Urea Nitrogen; Scr, Serum Creatinine; FPG, Fasting Plasma Glucose; HbA_1c, Glycated Haemoglobin; TC, Total Cholesterol; TG, Triglycerides; VLDL, Very Low-Density Lipoprotein Cholesterol; LDL-C, Low-Density Lipoprotein Cholesterol; HDL-C, High-Density Lipoprotein Cholesterol; FCP, Fasting C-peptide; SCV, Sensory Nerve Conduction Velocity; MCV, Motor Nerve Conduction Velocity; miR-155, microRNA-155; miR-146a, microRNA-146a.

Table 5 Logistic Regression Analysis of Risk Factors of Clinical Outcome

Clinical outcome	Independent variable	β	OR	95% CI	P
DPN	Duration of diabetes (year)	0.147	1.158	1.040~1.291	0.008*
	Age (year)	−0.021	0.979	0.923~1.040	0.495
	BMI	0.013	1.013	0.815~1.261	0.905
	ALT	−0.036	0.964	0.884~1.052	0.412
	AST	0.004	1.004	0.829~1.216	0.964
	FPG	0.317	1.374	1.059~1.782	0.017*
	HbA1c	0.240	1.271	0.878~1.840	0.204
	BUN	0.197	1.217	0.826~1.794	0.320
	Scr	0.047	1.048	1.006~1.091	0.023*
	miR-155	−1.976	0.139	0.032~0.592	0.008*
	miR-146a	−1.316	0.268	0.133~0.539	<0.001*
PDPN	Duration of diabetes (year)	0.101	1.106	0.967~1.265	0.140
	Age (year)	−0.056	0.946	0.865~1.035	0.225
	BMI	−0.083	0.921	0.716~1.184	0.519
	ALT	−0.005	0.995	0.899~1.102	0.928
	AST	−0.137	0.872	0.673~1.130	0.301
	FPG	−0.333	0.717	0.500~1.029	0.071
	HbA1c	0.279	1.332	0.857~2.038	0.206
	BUN	0.443	1.557	1.003~2.419	0.049*
	Scr	−0.025	0.975	0.948~1.003	0.077
	miR-155	−2.117	0.120	0.012~1.241	0.075
	miR-146a	0.530	1.699	0.677~4.264	0.259

Notes: *P < 0.05. Multivariate unconditional logistic regression analysis adjusted for the course of diabetes, age, BMI, ALT, AST, FPG, HbA1c, BUN, Scr, miR-155, and miR-146a showed that increased diabetes duration, higher FPG, and elevated Scr levels were independent risk factors for DPN, while higher levels of miR-155 and miR-146a were potential protective factors against DPN. Multivariate unconditional logistic regression analysis adjusted for the same factors revealed that higher BUN levels were an independent risk factor for the occurrence of PDPN, whereas miR-155 and miR-146a, among other variables, did not show significant statistical relevance.

Abbreviations: DPN, Diabetic Peripheral Neuropathy; PDPN, painful neuropathy; BMI, Body Mass Index; ALT, Alanine Aminotransferase; AST, Aspartate Aminotransferase; FPG, Fasting Plasma Glucose; HbA1c, Glycated Haemoglobin; BUN, Blood Urea Nitrogen; Scr, Serum Creatinine; miR-155, microRNA-155; miR-146a, microRNA-146a.

Table 6 Predictive Value of ROC Curve of miR-155, miR-146a, Course of the Disease and All the Indexes in the Peripheral Blood

Index	AUC	95% CI	Optimal Critical Value	Sensitivity (%)	Specificity (%)	P value
miR-155	0.641	0.532~0.750	3.835	0.918	0.373	0.015*
miR-146a	0.722	0.621~0.824	3.185	0.571	0.843	<0.001*
Duration of diabetes (year)	0.681	0.578~0.785	6.5	0.796	0.49	0.002*
miR-155 and miR-146a	0.775	0.685~0.866	-	0.837	0.608	<0.001*
miR-155 and disease duration	0.720	0.621~0.819	-	0.918	0.431	<0.001*
miR-146a and disease duration	0.788	0.695~0.881	-	0.694	0.863	<0.001*
miR-155, miR-146a and disease duration	0.818	0.733~0.903	-	0.796	0.784	<0.001*

Note: *P < 0.05.

Figure 1 The biomarker potential of circulating miR-155 and miR-146a for distinguishing between DPN and T2DM was assessed through ROC curve analysis. Individually, circulating miR-155 showed a sensitivity of 91.8%, specificity of 37.3%, and AUC of 0.641(95% CI 0.532–0.750, P<0.05), while miR-146a had a sensitivity of 57.1%, specificity of 84.3%, and AUC of 0.722(95% CI 0.621–0.824, P<0.001).

Figure 2 The Diagnostic Value of Combining Multiple Indicators for DPN;The AUC for the combined diagnostic use of miR-155 and miR-146a specific to DPN diagnosis reached 0.775 (95% CI 0.685–0.866, P < 0.001), with a sensitivity of 83.7% and a specificity of 60.8%. The AUC of disease duration specific to DPN diagnosis reached 0.681(95% CI 0.578–0.785, P= 0.002), with a sensitivity of 79.6% and a specificity of 49%.The AUC for the combined use of miR-155 with disease duration, miR-146a with disease duration, and all three factors combined were 0.720(95% CI 0.621–0.819, P<0.001), 0.788(95% CI 0.695–0.881, P<0.001), and 0.818(95% CI 0.733–0.903, P<0.001), respectively.

Figure 3 miR-155 and miR-146a effectively served as useful biomarkers for the diagnosis and prognosis of DPN.↑: increase; ↓: decrease.

Abbreviations: DPN, Diabetic Peripheral Neuropathy; SCV, Sensory Nerve Conduction Velocity; MCV, Motor Nerve Conduction Velocity; miR-155, microRNA-155; miR-146a, microRNA-146a.

Data Sharing Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.