Circulating microRNAs in patients with non-alcoholic fatty liver disease

Table 1 Clinical and demographic characteristics of non-alcoholic fatty liver disease patients and controls

Variable	NAFLD (n = 20)	Control (n = 20)	P
Age (yr)	42.75 ± 8.17	44.50 ± 9.31	0.761
Sex (male/female)	9 (45.0)/11(55.0)	9 (45.0)/11 (55.0)	0.761
BMI (kg/m2)	31.86 ± 4.76	27.26 ± 3.07	0.006
Waist circumference (cm)	103.25 ± 13.65	90.75 ± 11.02	0.014
Systolic blood pressure (mmHg)	120.25 ± 8.66	120.25 ± 7.69	0.999
Diastolic blood pressure (mmHg)	76.50 ± 6.09	77.50 ± 8.96	0.761
Fasting glucose (mg/dL)	97.85 ± 13.88	86.80 ± 11.92	0.034
Triglycerides (mg/dL)	151.10 ± 65.06	167.70 ± 51.46	0.645
HDL-C (mg/dL)	43.00 (36.50-48.50)	45.00 (38.45-49.95)	0.761
LDL-C (mg/dL)	125.50 (100.00-139.00)	108.00 (89.25-121.25)	0.511
Insulin (mIU/mL)	14.81 (11.88-22.75)	11.76 (8.61-15.70)	0.229
HOMA-IR	4.05 (2.71-5.51)	2.21 (1.77-3.12)	0.034
Total biluribin (mg/dL)	0.65 (0.50-0.80)	0.60 (0.55-0.80)	0.761
Direct biluribin (mg/dL)	0.20 (0.12-0.25)	0.20 (0.10-0.20)	0.761
AST (IU/L)	45.00 (34.50-59.00)	21.00 (18.00-27.00)	< 0.001
ALT (IU/L)	68.50 (52.50-85.50)	18.50 (14.00-29.00)	< 0.001
AP (IU/L)	76.00 (55.50-96.00)	69.00 (57.00-78.50)	0.761
GGT (IU/L)	48.00 (33.00-73.00)	20.00 (14.50-33.00)	< 0.001
Creatinine (mg/dL)	0.87 ± 0.15	0.80 ± 0.18	0.401
WBC (μL)	6709.50 ± 1709.47	6618.00 ± 1425.37	0.892
Trombocyte (103/μL)	252.10 ± 64.85	275.90 ± 69.82	0.511
Hct	43.37 ± 3.53	44.25 ± 4.19	0.761

Values are expressed as n (%), mean ± SD or median (25^th-75^th percentiles). P values were adjusted with FDR to control multiple testing problem. NAFLD: Non-alcoholic fatty liver disease; BMI: Body mass index; HDL-C: High density lipoprotein cholesterol; LDL-C: Low density lipoprotein cholesterol; HOMA-IR: Homeostasis model assessment insulin resistance index; AST: Aspartate aminotransferase ; ALT: Alanine aminotransferase; AP: Alkaline phosphatase; GGT: Gamma glutamyl transferase; WBC: White blood cell.

Table 2 Log₂ (Fold change) ratios of differentially expressed miRNAs which were statistically significant by t test (P < 0.05) and further confirmed by SAM test (q < 5%)

miRNAs	log2 (Fold change)		q value (%)
	Mean	SEM
Over-expressed
-	-	-	-
Under-expressed
miR-197	-1.61	0.54	0
miR-146b	-1.52	0.55	0
miR-181d	-2.49	0.50	0
miR-99a	-1.92	0.53	0

A q value less than 5% by SAM corresponds to a P value less than 0.05 by t test. SEM: Standard error of mean.

Table 3 Point-biserial correlation coefficients between NAS-scale parameters and miRNAs in non-alcoholic fatty liver disease patients

	miR-197	miR-146b	miR-10b	miR-181d	miR-34a	miR-122	miR-99a	miR-29a
Steatosis	0.229	0.191	0.179	0.179	0.245	0.067	0.311	0.256
Inflammation	-0.4571	-0.396	-0.4921	-0.402	-0.302	-0.23	-0.431	-0.315
Ballooning	0.154	0.114	0.175	0.254	0.165	-0.037	-0.029	0.096
Fibrosis	-0.112	-0.171	-0.213	-0.098	0.033	-0.137	-0.197	-0.103
NAS	0.051	0.015	0.035	0.069	0.124	-0.027	0.074	0.041

¹Correlation is significant at the 0.05 level (2-tailed).

Table 4 Correlation analysis between laboratory parameters and miRNAs in non-alcoholic fatty liver disease patients

	miR-197	miR-146b	miR-10b	miR-181d	miR-34a	miR-122	miR-99a	miR-29a
AST	-0.257	-0.331	-0.225	-0.252	-0.084	-0.225	-0.138	-0.322
ALT	-0.131	-0.147	-0.080	-0.215	0.122	0.103	0.077	-0.143
GGT	-0.327	-0.225	-0.303	-0.4641	-0.29	-0.231	-0.4791	-0.221
Triglyceride	0.008	-0.039	0.009	-0.035	-0.075	0.087	-0.239	-0.058
HDL-C	0.027	0.051	-0.031	0.042	0.052	0.111	0.16	0.034
LDL-C	0.078	0.042	0.067	0.015	0.139	0.102	0.213	0.155
HOMA-IR	-0.055	0.117	0.011	-0.015	0.195	0.205	0.221	0.177

¹Correlation is significant at the 0.05 level (2-tailed). ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; HOMA-IR: Homeostasis model assessment insulin resistance index; GGT: Gamma glutamyl transferase; HDL-C: High density lipoprotein cholesterol; LDL-C: Low density lipoprotein cholesterol.

Table 5 Statistical diagnostic measures for miR-197, miR-146b, miR-181d and miR-99a in identifying non-alcoholic fatty liver disease

miRNA	Cut-off value	SEN (95%CI)	SPE (95%CI)	PPV (95%CI)	NPV (95%CI)
miR-197	≤ -1.0144	60.0 (36.1-80.9)	95.0 (75.1-99.9)	92.3 (64.0-99.8)	70.4 (49.8-86.2)
miR-146b	≤ -0.7689	55.0 (31.6-76.9)	100.0 (83.0-100.0)	100.0 (71.3-100.0)	69.0 (49.2-84.7)
miR-181d	≤ -0.2861	70.0 (45.7-88.0)	85.0 (62.1-96.6)	82.4 (56.6-96.0)	73.9 (51.6-89.7)
miR-99a	≤ -0.1562	65.0 (40.8-84.5)	95.0 (75.1-99.2)	92.9 (66.1-98.8)	73.1 (52.2-88.4)

SEN: Sensitivity; SPE: Specifity; PPV: Positive predictive value; NPV: Negative predictive value.