Comprehensive evaluation of microRNA as a biomarker for the diagnosis of hepatocellular carcinoma

Table 1 Overview of the 26 selected known and putative microRNA candidates from next-generation sequencing and real-time quantitative PCR

Known miRNA	Fold change	AUC	P value (Wilcoxon)	Method	Comparison
hsa-miR-185-5p	2.01	0.77	0.000099c	NGS	All HCC vs CLD
hsa-miR-320a1	1.52	0.74	0.000335c
hsa-miR-423-5p	-1.22	0.69	0.004126b
hsa-miR-664a-5p	1.38	0.69	0.005695b
hsa-miR-203a	-2.73	0.67	0.01167a
hsa-miR-320d	2.56	0.66	0.017151a
hsa-miR-21-5p1	1.34	0.66	0.020486a
hsa-miR-28-5p	-1.65	0.80	0.0004c	RT-qPCR
hsa-miR-21-5p1	1.59	0.79	0.0006c
hsa-miR-103a-3p	-1.65	0.78	0.012a
hsa-miR-301a-3p	-1.50	0.78	0.013a
hsa-miR-30b-5p	-1.63	0.75	0.0033b
hsa-miR-495-3p	-1.50	0.73	0.0068b
hsa-miR-320a1	1.37	0.72	0.0093b
hsa-miR-30a-3p	-1.60	0.72	0.008145b	NGS	Early HCC vs CLD
hsa-miR-25-3p	1.75	0.71	0.01019a
hsa-miR-7706	1.67	0.69	0.02002a
hsa-miR-186-5p1	1.68	0.68	0.02364a
hsa-miR-15b-5p	4.21	0.97	< 0.0001c	RT-qPCR
hsa-miR-339-3p	-2.73	0.87	0.0006c
hsa-miR-10b-5p	2.81	0.86	0.0007c
hsa-miR-151a-5p	-2.25	0.85	0.0011b
hsa-miR-652-3p	-2.22	0.85	0.001b
hsa-miR-32-5p	2.03	0.85	0.001b
hsa-miR-221-3p	-2.96	0.83	0.0021b
hsa-miR-486-5p	2.43	0.82	0.0034b
hsa-miR-186-5p1	1.46	0.75	0.0215a
Putative miRNA	Fold change	AUC	P value (Wilcoxon)	Method	Comparison
put-miR-6	3.63	0.68	0.001956b	NGS	All HCC vs CLD
put-miR-99	1.83	0.6	0.188		Early HCC vs CLD

¹MicroRNAs detected in both next-generation sequencing and real-time quantitative PCR.

^aP < 0.05.

^bP < 0.01.

^cP < 0.001.

AUC: Area under the curve; CLD: Chronic liver disease; HCC: Hepatocellular carcinoma; miRNA: MicroRNA; NGS: Next-generation sequencing; RT-qPCR: Real-time quantitative PCR; miR: MicroRNA; put-miR: Putative microRNA.

Table 2 Individual standard deviation and accumulated standard deviation for consecutive microRNA reference genes

miRNA	SD	SDacc
hsa-let-7i-5p	0.49	0.49
hsa-miR-222-3p	0.57	0.37
hsa-miR-23a-3p	0.63	0.33
hsa-miR-30e-5p	0.65	0.29
hsa-miR-191-5p	0.79	0.28

The lower the standard deviation/accumulated standard deviation, the more applicable microRNA/microRNA combination was as reference gene. These miRNAs were used as endogenous controls for the normalization of real-time quantitative PCR data. SD: Standard deviation; SD_acc: Accumulated standard deviation; miRNA: MicroRNA; miR: MicroRNA.

Table 3 Univariate analysis of real-time quantitative PCR results for comparison

miRNA	Wilcoxon Q-value	AUC	Specificity at 90% sensitivity
All HCC vs CLD
hsa-miR-21-5p	0.000003a	0.71	0.24
hsa-miR-320d	0.010872b	0.63	0.25
hsa-miR-423-5p	0.046214b	0.61	0.26
hsa-miR-221-3p	0.099403	0.60	0.27
hsa-miR-320a	0.109399	0.60	0.28
hsa-miR-339-3p	0.154338	0.58	0.29
hsa-miR-301a-3p	0.160690	0.57	0.30
hsa-miR-652-3p	0.160690	0.56	0.31
hsa-miR-30b-5p	0.160690	0.56	0.32
hsa-miR-25-3p	0.186436	0.57	0.33
Early HCC vs CLD
hsa-miR-21-5p	0.113812	0.64	0.20
hsa-miR-320d	0.302365	0.61	0.16
hsa-miR-652-3p	0.382238	0.60	0.23
hsa-miR-320a	0.432100	0.58	0.01
hsa-miR-423-5p	0.432100	0.56	0.06
hsa-miR-301a-3p	0.432100	0.57	0.09
hsa-miR-15b-5p	0.432100	0.57	0.07
hsa-miR-186-5p	0.432100	0.57	0.15
hsa-miR-221-3p	0.432100	0.56	0.24
hsa-miR-25-3p	0.432100	0.56	0.15
Early HCC vs cirrhosis
hsa-miR-652-3p	0.038792b	0.69	0.34
hsa-miR-221-3p	0.094025	0.66	0.29
hsa-miR-151a-5p	0.201979	0.63	0.15
hsa-miR-21-5p	0.201979	0.63	0.24
hsa-miR-15b-5p	0.217324	0.61	0.10
hsa-miR-25-3p	0.217324	0.61	0.15
hsa-miR-320a	0.306457	0.59	0.00
hsa-miR-423-5p	0.306457	0.59	0.07
hsa-miR-320d	0.306457	0.59	0.15
hsa-miR-28-5p	0.306457	0.59	0.20

^aQ-value < 0.001.

^bQ-value < 0.05.

AUC: Area under the curve; CLD: Chronic liver disease; HCC: Hepatocellular carcinoma; miRNA: MicroRNA; miR: MicroRNA.

Table 4 Multivariate analysis of real-time quantitative PCR results for comparison

Marker	AUC	Specificity at 90% sensitivity	Comparison
PIVKA-II	0.83	34	All HCC vs CLD
PIVKA-II + miRNAs	0.87	48
PIVKA-II	0.70	15	Early HCC vs CLD
PIVKA-II + miRNAs	0.74	29
PIVKA-II	0.64	12	Early HCC vs cirrhosis
PIVKA-II + miRNAs	0.71	42

Area under the curve and specificity at 90% sensitivity for Protein induced by vitamin K absence/antagonist-II alone and in combination with the 26 selected miRNAs, resulting from the logistic regression model. AUC: Area under the curve; CLD: Chronic liver disease; HCC: Hepatocellular carcinoma; miRNA: MicroRNA; PIVKA-II: Protein induced by vitamin K absence/antagonist-II.

Table 5 Fractions of characteristics (feature) selected during internal cross-validation for evaluating the two-marker combination performance

Feature	Fraction selected in cross-validation
All HCC vs CLD
PIVKA-II	1.00
hsa-miR-21-5p	0.86
hsa-miR-320d	0.10
hsa-miR-339-3p	0.02
hsa-miR-320a	0.01
hsa-miR-423-5p	0.00
Early HCC vs CLD
PIVKA-II	1.00
hsa-miR-21-5p	0.60
hsa-miR-320d	0.18
hsa-miR-652-3p	0.16
hsa-miR-339-3p	0.02
hsa-miR-221-3p	0.01
hsa-miR-30b-5p	0.01
hsa-miR-10b-5p	0.00
hsa-miR-15b-5p	0.00
Early HCC vs cirrhosis
PIVKA-II	1.00
hsa-miR-652-3p	0.64
hsa-miR-221-3p	0.26
hsa-miR-21-5p	0.05
hsa-miR-423-5p	0.04
hsa-miR-320a	0.01
hsa-miR-339-3p	0.01
hsa-miR-320d	0.00

CLD: Chronic liver disease; HCC: Hepatocellular carcinoma; PIVKA-II: Protein induced by vitamin K absence/antagonist-II; miR: MicroRNA.

Table 6 Univariate analysis of real-time quantitative PCR results for comparison

miRNA	Wilcoxon Q-value	AUC	Specificity at 90% sensitivity	Comparison
hsa-miR-21-5p	0.00025a	0.65	0.17	All HCC vs CLD
hsa-miR-423-5p	0.037b	0.59	0.11
hsa-miR-320d	0.078	0.57	0.11
hsa-miR-652-3p	0.25	0.59	0.13	Early HCC vs cirrhosis

^aQ-value < 0.001.

^bQ-value < 0.05.

AUC: Area under the curve; CLD: Chronic liver disease; HCC: Hepatocellular carcinoma; miRNA: MicroRNA; miR: MicroRNA.