EXO1 overexpression is associated with poor prognosis of hepatocellular carcinoma patients

Figures & data

Figure 1. EXO1 gene copy number is increased in HCC as assessed by data-mining of the Oncomine database. Box-whiskers Plot of EXO1 gene copy number between normal and cancer tissue samples in (a) TCGA Liver, (b) Guichard Liver, and (c) Guichard Liver 2 data sets. B, blood; N, normal liver; T, HCC. Numbers below the horizontal axis indicate total cases in different groups. The line within each box represents the median value; lower and upper edges of each box represent the 25^th and 75^th percentiles, respectively; lower and upper whiskers represent 5^th and 95^th percentiles, respectively. P values were calculated by the Oncomine database.

Figure 2. EXO1 mRNA expression levels in HCC and paired adjacent non-tumorous liver tissues. (a) Semi-quantitative RT-PCR data for EXO1 in 20 liver cancer cases and paired normal tissues. (b) EXO1 mRNA levels in 143 paired HCC and adjacent non-tumorous liver tissues measured by the QuantiGene Plex assay. Statistical significance was determined by paired two-tailed Student’s t test. ****, P < 0.0001. Lines from the top represent the 75^th, 50^th, and 25^th of EXO1 mRNA values, respectively. N, normal liver; T, HCC.

Table 1. Analyses of the EXO1 gene in 143 HCC tissues.

Clinical character	variable	No. of patients	mRNA			χ^2	P value
			Low n (%)	High^a n (%)
Age (years)	< 55	100	33 (33.3)		67 (66.7)	0.565	0.452
	≥ 55	43	17 (39.5)		26 (60.5)
Gender	Male	131	45 (34.4)		86 (65.6)	0.259	0.611
	Female	12	5 (41.7)		7 (58.3)
Tumor size (cm)	< 5	31	18 (58.1)		13 (41.9)	9.288	0.002
	≥ 5	112	32 (28.6)		80 (71.4)
Tumor number	Single	115	40 (34.8)		75 (65.2)	0.009	0.926
	Multiple	28	10 (35.7)		18 (64.3)
HbsAg^b	Negative	25	10 (40.0)		15 (60.0)	0.338	0.561
	Positive	118	40 (33.9)		78 (66.1)
AFP^c (μg/l)	< 200	83	32 (38.6)		51 (61.4)	0.03	0.868
	≥ 200	68	18 (30.0)		42 (70.0)
Lymph node metastasis	No	128	48 (41.4)		78 (58.6)	4.567	0.033
	Yes	15	2 (13.3)		15 (86.7)
Edmonson grade	I	45	22 (48.9)		23 (51.1)	5.598	0.018
	II-III	98	28 (28.6)		70 (71.4)

High^a, high mRNA expression in HCC specimens was designed as ≥ 0.075

HbsAg^b, hepatitis B surface antigen;

AFP^c, alpha-fetoprotein

Figure 3. Prognostic value of EXO1 mRNA expression in HCC. (a) Distribution of EXO1 mRNA and serum AFP expression levels in 143 HCC patients. The numbers on the pie chart represent the percentages of various groups. EXO1 high, EXO1 mRNA levels ≥ 0.075 in HCC specimens; EXO1 low, EXO1 mRNA levels < 0.075 in HCC specimens; AFP high, AFP protein levels ≥ 200μg/l in peripheral blood; AFP low, AFP protein levels < 200μg/l in peripheral blood. (B to G) Kaplan-Meier analyses of EXO1 mRNA expression levels in 143 HCC patients (b), cases with serum AFP ≤ 20µg/L(c), patients with age ≤ 55 years (d), cases with single tumor (e), patients with no lymph node metastasis (f) and Edmonson stage I cases (G). High EXO1 expression was significantly correlated with poor prognosis (P < 0.05).

Table 2. Univariate and multivariate Cox regression analyses of EXO1 mRNA expression and clinical variables for overall survival in HCC patients.

Method	Variables	β^a	SE^b	Wald	Sig.	Hazard ratio	95%CI^c
							Lower	Upper
Univariate cox regression
	Age	−0.405	0.24	2.857	0.091	0.667	0.417	1.067
	Exo1 mRNA	0.591	0.234	6.379	0.012	1.805	1.141	2.854
	Grade	0.091	0.226	0.162	0.687	1.095	0.703	1.705
	Gender	1.52	0.716	4.513	0.034	4.573	1.125	18.591
	Tumor Size	0.744	0.301	6.099	0.014	2.105	1.166	3.799
	Tumor number	0.119	0.264	0.202	0.653	1.126	0.671	1.891
	HbsAg	−0.278	0.27	1.057	0.304	0.757	0.446	1.287
	AFP	0.326	0.213	2.347	0.126	1.386	0.913	2.103
	Transfer	−0.12	0.311	0.15	0.699	0.887	0.482	1.631
Multivariate cox regression
	Exo1 mRNA	0.491	0.238	4.243	0.039	1.633	1.024	2.605
	Gender	1.519	0.716	4.506	0.034	4.57	1.124	18.587
	Tumor Size	0.597	0.307	3.787	0.052	1.817	0.996	3.314

β^a, regression coefficient

SE^b, standard error

CI^c, confidence interval

Figure 4. EXO1 knockdown descreases the cell proliferation in HCC cells. (a) Cell growth curves of BEL-7404 and YY-8103. EXO1 proteins were detected by western blots. (b) Colony formation of BEL-7404 and YY-8103. All experiments were performed in triplicates and data are shown as mean ± SD. Statistical significance was determined by two-tailed Student’s t test. *, P < 0.05; **, P < 0.01.

Figure 5. EXO1 silencing dysregulates the cell cycle in HCC cells. (a) Cell cycle analysis by DNA content measured flow-cytometrically. (b) Cell cycle analysis 24h after ionizing radiation (4Gy). Cell cycle models were fit by the ModFit LT software (left). Average percentages of G0/G1, S and G2/M phase cells are shown on the right. All experiments were performed in triplicate, and data are mean± SD. Statistical significance was determined by two-tailed Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 6. EXO1 knockdown regulates the clonogenic survival, γ-H2AX locus formation and ATM accumulation in YY-8103 cells. (a) Clonogenic survival fractions of cells irradiated with a single dose of 0, 2, 4, 6 and 8 Gy X-rays. Survival curves were obtained by means of 3 standard colony formation assay. (b) γ-H2AX loci of cells irradiated with a single dose of 2 Gy. Quantitative analysis of γ-H2AX foci per cell averaged over 50 cells per data point. (c) ATM accumulation of cells irradiated with a single dose of 2 Gy revealed by western blots. Statistical significance was determined by two-tailed Student’s t test. *, P < 0.05; **, P < 0.01.