Serum exosomal miR-125b is a novel prognostic marker for hepatocellular carcinoma

Figures & data

Table 1 Clinical characteristics of the participants

Variables	Cohort 1			Cohort 2
	CHB (n=30)	LC (n=30)	HCC (n=30)	HCC (n=128)
Gender
Male	27 (90.0%)	27 (90.0%)	27 (90.0%)	110 (85.9%)
Female	3 (10.0%)	3 (10.0%)	3 (10.0%)	18 (14.1%)
Age, years
≥50	21 (70.0%)	20 (66.7%)	22 (73.3%)	98 (76.6%)
<50	9 (30.0%)	10 (33.3%)	8 (26.7%)	30 (23.4%)
HBsAg
Positive	30 (100.0%)	27 (90.0%)	28 (93.3%)	121 (94.5%)
Negative	0 (0.0%)	3 (10.0%)	2 (6.7%)	7 (5.5%)
GGT, U/L
≥54	10 (33.3%)	8 (26.7%)	12 (40.0%)	58 (45.3%)
<54	20 (66.7%)	22 (73.3%)	18 (60.0%)	70 (54.7%)
AFP, ng/mL
≥20			18 (60.0%)	77 (60.2%)
<20			12 (40.0%)	51 (39.8%)
LC
No			9 (30.0%)	31 (24.2%)
Yes			21 (70.0%)	97 (75.8%)
Tumor size, cm
≥5			14 (46.7%)	55 (43.0%)
<5			16 (53.3%)	73 (57.0%)
Tumor number
Single			24 (80.0%)	97 (75.8%)
Multiple			6 (20.0%)	31 (24.2%)
MVI
No			16 (53.3%)	65 (50.8%)
Yes			14 (46.7%)	63 (49.2%)
Tumor encapsulation
Complete			12 (40.0%)	51 (39.8%)
None			18 (60.0%)	77 (60.2%)
Tumor differentiation#
I+II			16 (53.3%)	75 (58.6%)
III+IV			14 (46.7%)	53 (41.4%)
TNM stage
I			9 (30.0%)	48 (37.5%)
II+III			21 (70.0%)	80 (62.5%)

Notes:

# Tumor differentiation was assigned using the Edmondson’s grading system. The data is formatted as n (%).

Abbreviations: AFP, alpha-fetoprotein; CHB, chronic hepatitis B; GGT, gamma-glutamyl transferase; HCC, hepatocellular carcinoma; LC, liver cirrhosis; MVI, microvascular invasion; TNM, tumor-node-metastasis.

Figure 1 Characterization of exosomes isolated from the serum of HCC patients.

Notes: (A) Western blot analysis of the expression of proteins enriched in exosomes, including CD63 and CD9. As the control, GAPDH was not detected in exosomes. (B) Transmission electron microscopy analysis of the morphology of exosomes (scale bar: 200 nm). (C) Nanoparticle tracking analysis to quantify the size and concentration of exosomes.
Abbreviation: HCC, hepatocellular carcinoma.

Figure 2 The relative expression of miR-125b in the subjects.

Notes: (A) qRT-PCR was performed to measure miR-125b levels in exosomes, the whole serum, and exosome-depleted supernatant from patients of cohort 1, including CHB (n=30), LC (n=30), and HCC (n=30). (B) miR-125b relative expression in exosomes and the whole serum was detected in HCC patients of cohort 1 (n=30) and 2 (n=128). ** P<0.01.
Abbreviations: qRT-PCR, quantitative real-time polymerase chain reaction; CHB, chronic hepatitis B; LC, liver cirrhosis; HCC, hepatocellular carcinoma; NS, not significant.

Figure 3 Kaplan–Meier analysis of cumulative recurrence and overall survival in HCC patients.

Notes: After operation (n=128; cohort 2) according to miR-125b expression in exosomes (A and B) and serum (C and D). P-values were calculated by the log-rank test and P<0.05 denoted significance.
Abbreviation: HCC, hepatocellular carcinoma.

Table 2 Correlation between miR-125b expression and clinical characteristics of HCC (cohort 2, n=128)

Clinical characteristics	n	Serum miR-125b		P-value	Exosomal miR-125b		P-value
		Low (n=64)	High (n=64)		Low (n=64)	High (n=64)
Gender
Male	110	52	58	0.13	53	57	0.31
Female	18	12	6		11	7
Age, years
≥50	98	51	47	0.40	51	47	0.40
<50	30	13	17		13	17
HBsAg
Positive	121	61	60	0.70*	62	59	0.24*
Negative	7	3	4		2	5
GGT, U/L
≥54	58	32	26	0.29	29	29	1.00
<54	70	32	38		35	35
AFP, ng/mL
≥20	77	42	35	0.21	42	35	0.21
<20	51	22	29		22	29
Cirrhosis
Yes	97	53	44	0.06	50	47	0.54
No	31	11	20		14	17
Tumor size, cm
≥5	55	30	25	0.37	32	23	0.11
<5	73	34	39		32	41
Tumor number
Multiple	31	17	14	0.54	23	8	0.002
Single	97	47	50		41	56
MVI
Yes	63	35	28	0.22	35	28	0.22
No	65	29	36		29	36
Tumor encapsulation
Complete	77	33	44	0.0477	32	45	0.019
None	51	31	20		32	19
Tumor differentiation#
III+IV	53	26	27	0.86	36	17	0.001
I+II	75	38	37		28	47
TNM stage
II+III	80	41	39	0.72	47	33	0.011
I	48	23	25		17	31
Exosomal miR-125b
High	64	23	41	0.001	–	–	–
Low	64	41	23		–	–
Serum miR-125b
High	64	–	–	–	23	41	0.001
Low	64	–	–		41	23

Notes:

* Performed by the Fisher’s exact test.

# Tumor differentiation was assigned using the Edmondson’s grading system. The bold values indicate P-value <0.05.

Abbreviations: AFP, alpha-fetoprotein; CHB, chronic hepatitis B; GGT, gamma-glutamyl transferase; HCC, hepatocellular carcinoma; LC, liver cirrhosis; MVI, microvascular invasion; TNM, tumor-node-metastasis.

Table 3 Univariate and multivariate analysis of prognostic factors in HCC (cohort 2, n=128)

Variables	TTR		OS
	HR (95% CI)	P-value	HR (95% CI)	P-value
Univariate analysis
Gender, male vs female	1.59 (0.58–4.37)	0.364	3.08 (0.71–13.8)	0.133
Age, years, ≥50 vs <50	1.82 (0.80–4.16)	0.155	1.76 (0.69–4.49)	0.238
HBsAg, positive vs negative	1.41 (0.32–6.19)	0.652	2.42 (0.65–9.02)	0.186
GGT, U/L, ≥50 vs <50	1.03 (0.50–2.13)	0.935	1.42 (0.69–2.89)	0.342
AFP, ng/mL, ≥400 vs <400	1.74 (0.87–3.45)	0.116	1.79 (0.77–4.16)	0.176
Cirrhosis, yes vs no	1.11 (0.48–2.53)	0.812	1.37 (0.55–3.41)	0.500
Tumor size, cm, ≥5 vs <5	1.55 (0.83–2.90)	0.168	1.95 (0.95–4.03)	0.040
Tumor number, multiple vs single	1.37 (0.57–3.29)	0.476	1.10 (0.45–2.73)	0.833
MVI, yes vs no	4.95 (2.23–11.01)	0.000	4.46 (1.69–11.78)	0.003
Tumor encapsulation, none vs complete	1.12 (0.59–2.16)	0.726	1.14 (0.56–2.35)	0.718
Tumor differentiation, III-IV vs I–II	1.02 (0.54–1.93)	0.958	1.60 (0.82–3.14)	0.171
TNM stage, II+III vs I	2.06 (0.91–4.67)	0.085	2.10 (0.88–5.02)	0.095
Serum miR-125, high vs low	0.51 (0.25–1.05)	0.067	0.61 (0.29–1.26)	0.178
Exosomal miR-125, high vs low	0.14 (0.06–0.33)	0.000	0.35 (0.16–0.77)	0.009
Multivariate analysis
Tumor size, cm, ≥5 vs <5			1.97 (1.02–3.81)	0.025
MVI, yes vs no	3.82 (2.02–7.20)	0.000	4.76 (2.13–10.61)	0.000
Exosomal miR-125b, high vs low	0.14 (0.07–0.29)	0.000	0.36 (0.18–0.74)	0.005

Notes: Cox proportional hazards regression model. The bold values indicate P-value <0.05.

Abbreviations: AFP, alpha-fetoprotein; GGT, gamma-glutamyl transferase; HCC, hepatocellular carcinoma; MVI, microvascular invasion; OS, overall survival; TNM, tumor-node-metastasis; TTR, time to recurrence.

Figure 4 ROC curve analysis for MVI and exosomal miR-125b in HCC patients (n=128; cohort 2).

Notes: ROC curves for predicting the recurrence of MVI (A), exosomal miR-125b (B), and combination of exosomal miR-125b and MVI (C). ROC curves for predicting the survival of MVI (D), exosomal miR-125b (E), and combination of exosomal miR-125b and MVI (F).
Abbreviations: MVI, microvascular invasion; HCC, hepatocellular carcinoma; AUC, area under the ROC curve.

Table 4 ROC curve analysis for predicting prognostic accuracy of recurrence in HCC patients (cohort 2; n=128)

Variables	AUC (95% CI)	Sensitivity	Specificity	P-value
Recurrence
MVI	0.716 (0.624–0.809)	0.766	0.667	0.003#
Exosomal miR-125b	0.739 (0.648–0.830)	0.830	0.679	0.048#
Exosomal miR-125b + MVI	0.807 (0.731–0.883)	0.787	0.716
Survival
MVI	0.688 (0.589–0.786)	0.750	0.625	0.032#
Exosomal miR-125b	0.702 (0.602–0.802)	0.825	0.534	0.042#
Exosomal miR-125b + MVI	0.765 (0.682–0.848)	0.800	0.625

Notes:

# Compared with Exosomal miR-125b + MVI, and performed with MedCalc software. The bold values indicate P-value <0.05.

Abbreviations: HCC, hepatocellular carcinoma; AUC, area under the ROC curve; MVI, microvascular invasion.