MicroRNA signature in patients with hepatocellular carcinoma associated with type 2 diabetes

Table 1 Clinical and laboratory aspects of the recruited patients for Illumina sequencing in the profiling study

	Sex	Age in yr	AST in IU/L	ALT in IU/L	Total bilirubin in mg/dL	AFP in ng/mL	Albumin in g/dL	Hemoglobin in g/dL	BCLC score
HCC patients
1	Male	65	50.51	33.7	5.18	50.8	3.12	11.3	A
2	Male	55	38.9	44.29	1.92	17.8	3.03	12.2	A
3	Male	57	126.8	194.3	1.91	42.1	2.41	10.4	B
4	Female	59	31.42	26.56	1.38	25.82	2.63	9.4	B
5	Female	57	103.62	39.82	1.95	27.47	2.94	9.6	B
6	Male	57	39.5	43.19	1.81	15.8	2.96	10.2	A
7	Female	53	102.32	37.52	1.94	26.98	2.23	11.1	B
8	Female	60	28.62	21.77	1.2	2365	2.76	9.1	B
9	Male	57	137.9	204.3	1.71	41.7	2.43	10.0	B
10	Male	61	51.38	35.2	5.07	52.1	3.11	111.3	A
LC patients
1	Male	61	39.4	18.8	1.58	26	3.23	9.0
2	Female	59	36.8	59.7	1.51	43	3.45	10.6
3	Female	55	34.9	58.2	1.49	40.9	3.01	10.8
4	Female	52	50.1	52.3	1.58	32.9	2.54	9.2
5	Male	63	78.5	30.9	4.99	7.2	2.46	10.4
6	Female	57	51.6	55.5	1.69	33	2.25	10.5
7	Male	62	77.7	31.7	5.06	7.6	3.75	9.6
8	Male	59	95.4	74.3	1.22	3.6	2.78	10.3
9	Male	53	93.1	71.9	1.19	3.5	2.33	11.2
10	Male	60	38.9	18.2	1.53	25.9	2.54	10.9

AFP: Alpha-fetoprotein; ALT: Alanine amino transferase; AST: Aspartate amino transferase; BCLC: Barcelona clinic liver cancer; HCC: Hepatocellular carcinoma; LC: Liver cirrhosis.

Table 2 Clinical and laboratory aspects of the first confirmation analysis recruited patients

Parameter	LC, n = 50	HCC, n = 50	P value
Age in yr	55.63 ± 7.69	58.65 ± 8.97	NS
Male sex	66%	66%	NS
ALT in IU/L	76.98 ± 65.83	78.53 ± 43.19	NS
AST in IU/L	69.17 ± 63.36	62.78 ± 43.32	NS
Total bilirubin in mg/dL	2.53 ± 1.94	2.79 ± 1.78	NS
Albumin in g/dL	2.59 ± 1.47	2.45 ± 1.62	NS
ALP in IU/L	134.6 ± 83.5	103.18 ± 37.5	NS
Creatinine in mg/dL	0.98 ± 0.3	1.38 ± 0.87	NS
Hemoglobin in g/dL	11.42 ± 1.23	11.35 ± 1.68	NS
AFP in ng/mL	27.56 ± 41.23	165.49 ± 214.56	< 0.005
Platelet count as × 103/µL	96.7 ± 51.6	149.7 ± 129.3	< 0.005
Leukocyte count as × 103/µL	6.98 ± 2.98	7.61 ± 4.3	< 0.005
BCLC stage, n (%)
A		20 (40)
B		26 (52)
C		4 (8)

Data are presented as mean ± standard deviation. Clinical data was analyzed by analysis of variance analysis. Results were considered to be statistically significant if P < 0.05. AFP: Alpha-fetoprotein; ALP: Alkaline phosphatase; ALT: Alanine amino transferase; AST: Aspartate amino transferase; BCLC: Barcelona clinic liver cancer; HCC: Hepatocellular carcinoma; LC: Liver cirrhosis.

Table 3 Clinical and laboratory aspects of the second confirmation analysis recruited patients

Parameter	Healthy, n = 225	T2DM, n = 200	LC, n = 270	HCC, n = 200	P value
Age in yr	49.92 ± 7.14	53.68 ± 9.48	57.18 ± 7.30	56.23 ± 8.14	0.26
ALP in IU/L	64.2 ± 20.8	134.3 ± 36.9	103.18 ± 37.5	134.6 ± 83.5	< 0.0001
ALT in IU/L	30.2 ± 15.6	58 ± 18	31.8 ± 21.08	206.8 ± 133.31	0.001
AST in IU/L	28.6 ± 10.3	52.1 ± 18.0	58.18 ± 41.6	447.07 ± 249.90	< 0.0001
Bilirubin, total in mg/dL	0.6 ± 0.2	0.39 ± 0.19	4.64 ± 7.90	8.39 ± 8.83	< 0.0009
Bilirubin, direct in mg/dL	0.2 ± 0.1	0.86 ± 0.28	2.98 ± 6.5	6.36 ± 7.8	< 0.0001
Albumin in g/dL	4.3 ± 1.2	4.01 ± 0.57	2.53 ± 0.49	2.33 ± 0.39	< 0.0001
Hemoglobin in g/dL	11.9 ± 2.1	11.81 ± 4.2	9.20 ± 2.41	10.15 ± 1.98	< 0.0001
AFP in ng/mL	9.33 ± 1.82	3.72 ± 1.15	35.56 ± 41.23	267.8 ± 221.9	< 0.0001
Platelet count as × 103/µL	240 ± 97.6	320 ± 68.2	147.9 ± 129.6	97.8 ± 53.6	< 0.0001
leukocyte count as × 103/µL	6.8 ± 2.4	8.6 ± 2.42	6.17 ± 4.1	7.18 ± 3.18	< 0.0001
INR	1.25 ± 0.23	1.43 ± 0.51	1.81 ± 0.52	1.95 ± 0.63	< 0.0001
Creatinine in mg/dL	0.7 ± 0.2	0.98 ± 0.31	0.99 ± 0.25	1.41 ± 0.78	< 0.0001
GGT in U/L	12.0 ± 3.0	33.0 ± 12.8	41.54 ± 25.6	51.38 ± 32.86	< 0.0001
BUN in mg/dL	29.2 ± 3.4	70.0 ± 20.3	39.9 ± 28.6	71.54 ± 52.83	< 0.0001

Data are presented as mean ± standard deviation. Clinical data was analyzed by analysis of variance analysis. A result was considered to be statistically significant if P < 0.05. AFP: Alpha-fetoprotein; ALP: Alkaline phosphatase; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; BUN: Blood urea nitrogen; HCC: Hepatocellular carcinoma; INR: International normalized ratio; LC: Liver cirrhosis; T2DM: Type 2 diabetes patients.

Table 4 Imaging characteristics of hepatocellular carcinoma cases

Variable		n (%)
Size in cm, mean ± SD (range)	7.41 ± 3.137 (3.2-14)
Portal vein tumor thrombosis	Yes	35 (17.5)
	No	165 (82.5)
Number of focal lesions	Single	25 (12.5)
	Multiple	175 (87.5)
Site of focal lesions	Right lobe	112 (56.0)
	Left lobe	60 (30.0)
	Both lobes	28 (14.0)
BCLC stage	A	25 (12.5)
	B	81 (40.5)
	C	94 (47.0)

BCLC: Barcelona clinic liver cancer; HCC: Hepatocellular carcinoma; SD: Standard deviation.

Table 5 Illumina output sequencing data

Sample	LC	HCC
Total raw reads	29233450	29867922
Total raw bases	1461672500	1493396100
Total clean reads	17784320	21650404
Total clean bases	432618636	498024958
Clean reads rate, %	60.8	72.5
Low quality reads	10854	1494
Low quality rate, %	0.04	0.01
Without 3p adaptor reads	2626766	1093718
Without 3p adaptor rate, %	8.99	3.7
Without insert reads	984336	14858
Without insert rate, %	3.4	0.05
Poly A/T reads	1335340	6929464
Poly A/T rate, %	4.6	23.2
Ex-length reads	6491834	177984
Ex-length rate, %	22.2	0.6
Raw reads Q20, %	97.3	96.7
Raw reads Q30, %	94.5	95.5
Clean reads Q20, %	97.5	97.4
Clean reads Q30, %	94.6	96.4

HCC: Hepatocellular carcinoma; LC: Liver cirrhosis.

Table 6 MicroRNAs selected criteria for inclusion

MiRNA name	HCC	LC	Fold change	Log2 fold change	P value
hsa-miR-1	0.13	0.32	0.41	-1.29	0.168
hsa-miR-16	0.12	0.43	0.28	-1.84	0.001a
hsa-miR-21	4.21	3.82	1.10	0.14	0.801
hsa-miR-23b-3p	0.27	0.78	0.35	-1.51	0.001a
hsa-miR-26a	0.33	0.51	0.64	-0.64	0.009
hsa-miR-29a	0.54	0.42	1.29	0.37	0.001
hsa-miR-34-a	0.69	0.16	4.31	2.11	0.001a
hsa-miR-98	1.95	1.19	1.63	0.70	0.723
hsa-miR-122-5p	0.12	0.98	0.12	-3.06	0.001a
hsa-miR-125b-5p	0.01	0.05	0.20	-2.32	0.823
hsa-miR-127-3p	0.29	0.78	0.37	-1.43	0.059
hsa-miR-150-5p	2.16	0.78	2.78	1.48	0.071
hsa-miR-185-5p	0.21	0.88	0.24	-2.06	0.083
hsa-miR-198	0.09	0.29	0.31	-1.69	0.001a
hsa-miR-199a-3p	0.08	0.31	0.26	-1.94	0.001a
hsa-miR-221	9.40	3.31	2.84	1.51	0.001a
hsa-miR-224-5p	1.82	0.71	2.56	1.36	0.021
hsa-miR-494-3p	0.98	0.17	5.76	2.53	0.022
hsa-miR-877-5p	1.51	0.78	1.94	0.96	0.231
hsa-miR-4433b-3	1.52	0.45	3.38	1.76	0.072

Expression data is presented as average fold change = (2^-ΔΔCT) in the hepatocellular carcinoma group vs the liver cirrhosis group.

^aP < 0.005 with ± 1.5 log₂ fold expression change. HCC: Hepatocellular carcinoma; LC: Liver cirrhosis; miRNA: MicroRNA.

Table 7 Significant microRNAs expression as average fold change = (2^-ΔΔCT) in the liver cirrhosis group vs the hepatocellular carcinoma group

MiRNA name	HCC	LC	Fold change	Log2 fold change	P value
hsa-miR-23b-3p	2.44	0.78	0.32	-1.64	0.001a
hsa-miR-34a	1.88	0.16	0.09	-3.47	0.001a
hsa-miR-122-5p	0.34	0.98	0.12	2.87	0.001a
hsa-miR-198	0.88	0.29	0.33	-4.64	0.001a
hsa-miR-199a-3p	1.18	0.31	0.26	-1.94	0.001a
hsa-miR-221	1.13	3.31	2.92	1.55	0.001a

^aP < 0.005 with ± 1.5 log₂ fold expression change. HCC: Hepatocellular carcinoma; LC: Liver cirrhosis; miRNA: MicroRNA.

Table 8 Diagnostic ability for studied microRNAs to screening hepatocellular carcinoma among diabetics and controls

Serum miRNA	AUC value	Sensitivity	Specificity	PPV	NPV	Accuracy
miR-16
HCC against ctrl	0.897	81.0%	92.0%	90.6%	86.2%	90.1%
HCC against LC-T2D	0.864	84.9%	87.5%	83.0%	97.5%	88.7%
miR-122-5p
HCC against ctrl	0.793	79.8%	76.2%	80.0%	81.0%	81.3%
HCC against LC-T2D	0.802	83.2%	86.1%	69.0%	86.0%	83.2%
miR-198
HCC against ctrl	0.798	81.4%	79.4%	77.1%	82.0%	80.3%
HCC against LC-T2D	0.791	80.2%	85.2%	54.0%	86.0%	80.0%
miR-199a-3p
HCC against ctrl	0.901	99.7%	97.7%	86.7%	100%	96.7%
HCC against LC-T2D	0.843	82.6%	85.3%	75.7%	96.6%	83.6%
miR-221
HCC against ctrl	0.782	79.4%	80.4%	87.1%	93.7%	81.2%
HCC against LC-T2D	0.780	78.6%	81.8%	34.9%	91.0%	60.8%
miR-23b-3p
HCC against ctrl	0.792	81.0%	83.2%	76.1%	82.0%	79.8%
HCC against LC-T2D	0.689	72.5%	69.5%	30.6%	73.4%	72.7%
miR-34a
HCC against ctrl	0.761	96.4%	59.5%	83.4%	90.7%	75.4%
HCC against LC-T2D	0.789	96.4%	69.3%	46.0%	93.2%	78.2%

AUC: Area under the curve; ctrl: Control; HCC: Hepatocellular carcinoma; LC-T2D: Liver cirrhosis in type 2 diabetic; miRNA: MicroRNA; NPV: Negative predictive value; PPV: Positive predictive value.

Table 9 Significant relations among candidate serum microRNAs in hepatocellular carcinoma patients

MiRNA name	miR-16	miR-122-5p	miR-198	miR-199a-3p	miR-221	miR-23b-3p	miR-34a
miR-16		NS	r = 0.587	NS	NS	r = 0.682	r = 0.350
			P = 0.001			P ≤ 0.001	P = 0.003
miR-122-5p	NS		NS	r = 0.519	r = 0.372	r = 0.377	r = 0.421
				P = 0.001	P = 0.003	P ≤ 0.001	P = 0.003
miR-198	r = 0.587	NS		NS	r = 0.365	NS	r = 0.450
	P = 0.001				P = 0.004		P ≤ 0.001
miR-199a-3p	NS	r = 0.519	NS		r = 0.381	r = 0.519	NS
		P = 0.001			P = 0.002	P = 0.001
miR-221	NS	r = 0.372	r = 0.365	r = 0.381		r = 0.415	r = 0.392
		P = 0.003	P = 0.004	P = 0.002		P = 0.008	P ≤ 0.001
miR-23b-3p	r = 0.682	r = 0.377	NS	r = 0.519	r = 0.415		r = 0.429
	P ≤ 0.001	P ≤ 0.001		P = 0.001	P = 0.008		P = 0.005
miR-34a	r = 0.350	r = 0.421	r = 0.450	NS	r = 0.392	r = 0.429
	P = 0.003	P = 0.003	P ≤ 0.001		P ≤ 0.001	P = 0.005

HCC: Hepatocellular carcinoma; miRNA: MicroRNA; NS: Nonsignificant correlation.

Table 10 Levels of serum microRNA s in correlation to type 2 diabetes mellitus-related parameters

		hsa-miR-16	hsa-miR122-5p	hsa-miR-198	hsa-miR199a-3p	hsa-miR221	hsa-miR23b-3p	hsa-miR34a
HbA1c, %	r2	0.312	0.027	0.017	0.087	0.156	0.384	0.498
	P	< 0.001a	0.758	0.369	0.08	0.003a	< 0.001a	< 0.001a
GLU in mg/dL	r2	0.269	0.353	0.099	0.17	0.109	0.409	0.087
	P	0.001a	< 0.001a	0.026	0.003a	0.016	< 0.001a	0.08
Insulin in mU/L	r2	0.091	0.412	0.041	0.47	0.031	0.497	0.063
	P	0.003a	< 0.001a	0.209	< 0.001a	0.003a	< 0.00a	0.003a
HOMA-IR	r2	0.096	0.419	0.013	0.098	0.49	0.492	0.027
	P	0.03	< 0.001a	0.396	0.001a	< 0.001a	< 0.001a	0.001a
ISI	r2	0.095	0.421	0.102	0.019	0.499	0.545	0.0693
	P	0.0074a	< 0.001a	0.023	0.004a	< 0.001a	< 0.001a	0.005a
HIRI	r2	0.498	0.429	0.024	0.1807	0.427	0.672	0.049
	P	< 0.001a	< 0.001a	0.005	< 0.001a	< 0.001a	< 0.001a	0.009a

Relationship was evaluated through a linear regression analysis adjusted by body mass index, sex, age, high-density lipoproteins, and triglycerides, using SPSS software.

^aP < 0.05. GLU: Glucose; HbA1c: Glycosylated hemoglobin; HIRI: Hepatic insulin resistance index; HOMA-IR: Homeostasis model assessment-insulin resistance; ISI: Insulin sensitivity index; T2DM: Type 2 diabetes mellitus.