Metallophosphoesterase 1, a novel candidate gene in hepatocellular carcinoma malignancy and recurrence

Figures & data

Figure 1. SNVs potentially associated with recurrent HCC were identified using high-throughput whole-exome sequencing, PCR-MassArray, and Sanger DNA sequencing. (a) Frequency of gene mutations in the genome of a single HCC patient. Data from five exomic SNP profiles were combined. Nucleotide transversions in (b) primary HCC (n = 87) and (c) recurrent HCC (n = 34) tissue samples. (d) Illustration showing the presence of 70 verified SNVs in 69 genes and their chromosomal locations. (e) Mutant genes occurring with the highest frequency in primary HCC, recurrent HCC, and benign liver tumor tissues

Table 1. Associations of mutation frequency with the risk of primary HCC or HCC recurrence

Gene	chr SNV	A1	F_A	F_U	A2	OR (95% CI)	P value
Recurrent HCC vs control
SPON1	chr11_14284477	A	0.044	0.023	G	1.89 (0.31–11.66)	0.48
NOX5	chr15_69325606	C	0.044	0.023	A	1.94 (0.31–11.94)	0.46
TP53	chr17_7579801	C	0.41	0.55	G	0.55 (0.25–1.25)	0.15
MPPE1	chr18_11897016	C	0.18	0.035	T	5.93 (1.60–21.97)	0.003
MYBPC2	chr19_50967640	A	0.20	0.16	G	1.31 (0.57–3.02)	0.52
Primary HCC vs control
SPON1	chr11_14284477	A	0.075	0.036	G	0.48 (0.067–3.49)	0.46
NOX5	chr15_69325606	C	0.057	0.023	A	2.56 (9.5486–11.96)	0.21
TP53	chr17_7579801	C	0.45	0.55	G	0.68 (0.3–1.38)	0.28
MPPE1	chr18_11897016	C	0.080	0.034	T	2.42 (0.68–8.66)	0.16
MYBPC2	chr19_50967640	A	0.15	0.16	G	0.92 (0.45–1.86)	0.80

A1, mutated; F-A, frequency of this mutated allele A1 in cases; A2, wild type; F_U, frequency of this mutated allele A1 in control. Statistical analysis performed with the Chi-square test.

Table 2. Logistic regression analysis of the associations of gene mutations with the risk of primary HCC or HCC recurrence

Gene	Chr SNV	A1	Test	OR (95% CI)	FDR_BH	FDR_BY	STAT	P value
Recurrent HCC vs control
SPON1	chr11_14284477	A	ADD	1.94 (0.30–12.3)	0.6377	1	0.6997	0.48
NOX5	chr15_69325606	C	ADD	1.98 (0.31–12.6)	1	1	0.7262	0.47
TP53	chr17_7579801	C	ADD	0.57 (0.26–1.28)	1	1	−1.354	0.17
MPPE1	chr18_11897016	C	ADD	6.05 (1.57–23.36)	1	1	2.613	0.009
MYBPC2	chr19_50967640	A	ADD	1.33 (0.56–3.12)	1	1	0.6467	0.51
Primary HCC vs control
SPON1	chr11_14284477	A	ADD	5.39 (0.58–49.72)	1	1	1.485	0.14
NOX5	chr15_69325606	C	ADD	2.24 (0.69–7.30)	1	1	1.338	0.18
TP53	chr17_7579801	C	ADD	0.71 (0.37–1.39)	1	1	−0.991	0.32
MPPE1	chr18_11897016	C	ADD	2.56 (0.69–9.43)	1	1	1.41	0.16
MYBPC2	chr19_50967640	A	ADD	0.91 (0.45–1.86)	1	1	−0.2439	0.80

A1, mutated; Test, the additive model of analysis; FDR_BH, false discovery rate BH analysis; FDR_BY, false discovery rate BY analysis; STAT, T value

Table 3. Associations between the MPPE1 mutation and the clinicopathological features of HCC disease and prognosis

Variables		MPPE1 mutation
	N (%)	Yes n (%)	No n (%)	P-value
Gender				1.00
Male	114 (94.2)	23 (95.8)	91 (93.8)
Female	7 (5.8)	1 (4.2)	6 (6.2)
Age (yrs.)				0.65
≤50	53 (43.8)	12 (50.0)	41 (42.3)
>50	68 (56.2)	12 (50.0)	56(57.7)
Number of tumor nodules				0.45
1	48 (39.7)	7 (29.2)	41 (42.3)
2–3	48 (39.7)	12(50.0)	36 (37.1)
≥4	25(20.6)	5 (20.8)	20 (20.6)
Tumor size (cm)				0.18
≤5	98(81. 0)	15 (62.5)	83 (85.6)
>5	23(19.0)	9 (37.5)	14 (14.4)
Microvascular invasion				0.61
Absent	87 (71.9)	16 (66.7)	71 (73.2)
Present	34 (28.1)	8 (33.3)	26 (26.8)
TNM stage				0.002
T1	45 (37.2)	4 (16.7)	41 (42.3)
T2	61 (50.4)	12 (50.0)	49 (50.5)
T3	15 (12.4)	8 (33.3)	7 (7.2)
Tumor differentiation				0.52
I (well)	5 (4.1)	2 (8.3)	3 (3.1)
II (moderate)	108 (89.3)	21 (87.5)	87 (89.7)
III (poor)	8 (6.6)	1 (4.2)	7 (7.2)
AFP level (ng/ml)				0.60
≤400	69 (57.0)	14 (58.3)	55 (56.7)
>400/≤ 2000	19 (15.7)	5 (20.8)	14 (14.4)
>2000	33 (27.3)	5 (20.8)	28 (28.9)
Child–Pugh score				0.04
A(5–6)	66 (54.5)	16 (66.7)	50 (51.5)
B(7–9)	39 (32.2)	3 (12.5)	36 (37.1)
C(10–15)	16 (13.2)	5 (20.8)	11 (11.3)
HBV DNA				0.43
Negative (≤10^3 IU/ml)	90 (74.4)	16 (66.7)	74(76.3)
Positive (>10^3 IU/ml)	31 (25.6)	8 (33.3)	23(23.7)
Antitumor therapy pre-operation				0.65
No	61 (50.4)	11 (45.8)	50 (51.5)
Yes	60 (49.6)	13 (54.2)	47 (48.5)
Tumor recurrence post-operation				0.16
No	68 (56.2)	58 (59.8)	10 (41.7)
Yes	53 (43.8)	39 (40.2)	14 (58.3)

Figure 2. Kaplan–Meier curves showing the association between tumor recurrence rate and the MPPE1 mutation. n = 121 HCC patients stratified by presence/absence of the MPPE1 mutation

Figure 3. MPPE1 expression in HCC tumor samples and adjacent nontumor tissues based on data extracted from the GEO and TCGA (a) GSE22058, (b) GSE144520, (c) GSE63898, (d) TCGA_LIHC (**** P-value < 0.0001, *** P-value < 0.001, **P < .01)

Figure 4. Functional analysis of MPPE1 knockdown in Huh-7 and HepG2 cells. (a) Knockdown of MPPE1 expression significantly inhibited cell growth in a CCK-8 assay (n = 5). (b) Knockdown of MPPE1 expression significantly increased the proportion of HuH-7 cells and HepG2 in the G0/G1 phase and significantly decreased the proportion of HuH-7 cells and HepG2 cells in the S phase. The DNA content of HCC cells was analyzed using PI staining and flow cytometry (n = 4). (c) Knockdown of MPPE1 expression significantly increased the percentage of apoptotic HCC cells, detected by flow cytometry, and increased the level of cleaved PARP, identified with Western blotting analysis. Tubulin was used as an internal control. (d) Knockdown of MPPE1 expression inhibited cell invasion in a Transwell assay. Invasive cells were counted under a microscope (200×) as the mean of ten randomly selected fields of view. (e) Knockdown of MPPE1 expression inhibited cell migration in a Transwell assay. Migrated cells were counted under a microscope (200×) as the mean of ten randomly selected fields of view. Error bars represent the SD of the mean value. Statistical analysis was performed by the two-tailed least-significant difference (LSD) t-test. *, P < .05, **P < .01, ***P < .001

Data availability

The datasets generated and analyzed during the present study are available from the corresponding author on reasonable request.