Identification of recurrence-related serum microRNAs in hepatocellular carcinoma following hepatectomy

Figures & data

Table 1 Clinicopathologic features in 10 HCC patients

	Recurrence group (n = 5)	Non-recurrence group (n = 5)	P value
Age (years)	53.80±11.30	60.00±9.41	0.373
Gender (Male/Female)	4/1	5/0	1.000
Tumor size (cm)	5.96±2.90	4.48±2.76	0.432
Microvascular invasion (Present/Absent)	5/0	1/4	0.048
Differentiation (Well/Moderate/Poorly)	0/3/2	3/1/1	0.115
Satellite nodules (Present/Absent)	2/3	0/5	0.444
Envelope invasion (Present/Absent)	5/0	2/3	0.167
AFP(≤400u/>400u)	0/5	5/0	0.000
GGT	44.2±12.30	57.2±37.40	0.481
Recurrence (months)	<12	>24	-

Figure 1. Flow chart of the screening process. TaqMan Real-time PCR microRNA Array (Card A) (Applied Biosystems, CA) representing 381 mature miRNAs was used to identify differentially expressed miRNAs from 10 tumor tissue samples (5 from recurrence group vs. Five from non-recurrence group) and the matched serum samples. Of the 40 miRNAs that differentially expressed (fold2- altered) both in tissue and in serum samples, 15 miRNAs have the same variation tendency. And, 4 of the 15 miRNAs were differentially expressed with a p value below 0.10 between recurrence group and non-recurrence group in tissue samples. Four candidate miRNAs were further validated in 116 independent serum samples and 20 tumor tissues

Table 2 Fifteen differentially expressed microRNAs with the same variation tendency in serum and tumor tissue between recurrent and non-recurrent patients

	In serum	In tissue
miRNAs	Fold change (R/NR)*	Fold change (R/NR)*	P value
miR-125b	0.482	0.3917	0.0617
miR-139–5p	0.3856	0.2193	0.0233
miR-422a	0.0875	0.1836	0.0874
miR-486–5p	0.1652	0.3516	0.0302
miR-142–5p	0.3234	0.4685	0.4261
miR-145	0.4918	0.4854	0.3675
miR-199a-3p	0.362	0.3856	0.3626
miR-204	0.2926	0.3893	0.2697
miR-212	0.1584	0.4347	0.1676
miR-214	0.0768	0.4106	0.2441
miR-218	0.1263	0.4308	0.2864
miR-449a	0.3155	0.4109	0.4856
miR-486–3p	0.189	0.2217	0.119
miR-511	0.2505	0.4982	0.2298
miR-627	0.2639	0.484	0.5443

R, recurrence; NR, non-recurrence; miR, microRNA.

Figure 2. The levels of serum miRNAs were associated with relapse free survival. (A) Patients with low or high expression of miR-486–5p. (B) Patients with low or high expression of miR-422a. (C) Patients with low or high expression of miR-125b. (D) Patients with low or high expression of miR-139–5p

Table 4 Univariate and Multivariate Cox proportional hazards regression analysis of miRNAs and clinical parameters in relation to postoperative recurrence of HCC

	Overall survival
	Univariate analysis		Multivariate analysis
Variable	HR(95%CI)	p value	HR(95%CI)	p value
miR-486–5p	0.789 (0.698–0.892)	0.000	0.789 (0.698–0.892)	0.000
miR-422a	0.899 (0.838–0.965)	0.003
miR-125b	0.717 (0.581–0.886)	0.002
miR-139–5p	0.837 (0.634–1.105)	0.209
Microvascular invasion	5.287 (2.947–9.487)	0.000	4.754 (2.620–8.625)	0.000
Tumor size	1.113 (1.044–1.186)	0.001
Statellite nodules	3.229 (1.550–6.728)	0.002
AFP	1.150 (1.048–1.261)	0.003	1.105 (1.003–1.217)	0.043
Capsule invasion	2.577 (1.337–4.970)	0.005
Differentiation	1.824 (1.083–3.073)	0.024
BCLC	1.884 (1.054–3.367)	0.033
GGT	1.004 (0.999–1.009)	0.117
Hepatitis	0.685 (0.371–1.262)	0.225
Cirrhosis	1.657 (0.652–4.214)	0.289
Tumor multiplicity	1.334 (0.565–3.148)	0.511
Gender	0.997 (0.482–2.063)	0.993
Age	1.000 (0.975–1.026)	0.996

HR, hazard ratio; CI, confidence interval; miR, microRNA.

Table 5. Correlation between the expression of 4 miRNAs and clinical parameters of 116 HCC patients underwent hepatectomy

Parameters	microRNAs	P value
Gender	None
Age (years)	None
Preoperative GGT	miR-139–5p	0.020
Preoperative AFP	None
Viral hepatitis	None
Tumor size	None
Tumor multiplicity	None
Satellite nodules	miR-139–5p	0.002
Differentiation	None
Microvascular invasion	miR-125b	0.042
Envelop invasion	None
Cirrhosis	miR-486–5p	0.040
BCLC	None
Recurrence (<12 months)	miR-486-5p	0.000
	miR-125b	0.002
	miR-422a	0.003

Figure 3. Positive correlations between the expression levels in tumor tissues and the paired serum samples

Figure 4. Receiver operating characteristic curve analysis for predicting prognostic accuracy of postoperative recurrence of HCC patients. (A) ROC curve for miR-486–5p yielded area under the curve (AUC) of 76.79%, the sensitivity of 81.58% and specificity of 65.38% in predicting prognosis. (B) ROC curve for AFP and microvascular invasion yielded AUC of 75.98%, the sensitivity of 85.90% and specificity of 63.13% in predicting prognosis. MVI: microvascular invasion. (C) ROC curve for combination of AFP, MVI and miR-486–5p yielded AUC of 88.02%, the sensitivity of 69.20% and specificity of 92.10% in predicting prognosis

Table 3. Clinicopathologic features in 116 HCC patients underwent hepatectomy

Parameters	Patients with HCC (n=116)
Gender
Female	94(81.0%)
Male	22(19.0%)
Age(years)
≤60	78(67.2%)
60	38(32.8%)
Preoperative GGT
≤55u	71(61.2%)
>55u	45(38.8%)
Preoperative AFP
≤400u	89(76.7%)
>400u	27(23.3%)
Viral hepatitis
Negative	17(14.7%)
Positive	99(85.3%)
Tumor size
≤5cm	78(67.2%)
>5cm	38(32.8%)
Tumor multiplicity
Single	104(89.7%)
Multiple	12(10.3%)
Satellite nodules
Absent	104(89.7%)
Present	12(10.3%)
Differentiation
Well or Moderate	99(85.3%)
Poorly	17(14.7%)
Microvascular invasion
Absent	82(70.7%)
Present	34(29.7%)
Envelop invasion
Absent	49(42.2%)
Present	67(57.8%)
Cirrhosis
Absent	17(14.7%)
Present	99(85.3%)
BCLC
A	79(68.1%)
B	37(31.9%)
Recurrence (<12 months)
Yes	38(32.8%)
No	78(67.2%)