Overexpression of MPC1 inhibits the proliferation, migration, invasion, and stem cell-like properties of gastric cancer cells

Figures & data

Figure 1 The protein and mRNA levels of MPC1 were significantly decreased in GC tissues and cell lines.

Notes: (A) Representative images of immunohistochemistry (IHC) staining of MPC1 in GC and adjacent normal tissues. Scale bar, 50 μm. (B) IHC scores of MPC1 of adjacent normal tissues and cancerous tissues in 152 paired GC specimens (P<0.001). (C) MPC1 mRNA levels in 15 pairs of fresh GC specimens and adjacent normal tissues (P<0.01). (D) Analyses of MPC1 mRNA levels in GSE27342 and GSE26942 (P<0.001, P=0.0016, respectively). (E) Western blotting analysis of MPC1 in two pairs of GC and adjacent normal tissues. (F) Detection of MPC1 in GC cell lines (SGC7901, MGC803, BGC823, and AGS) and gastric epithelium cell line (GES-1) by Western blotting analysis. (G) Detection of MPC1 in GC cell lines (SGC7901, MGC803, BGC823, and AGS) and GES-1 by qRT-PCR analysis. *P<0.05, **P<0.01.
Abbreviations: GC, gastric cancer; IHC, immunohistochemistry.

Table 1 MPC1 expression in GC and adjacent normal mucosa

Tissue type	MPC1^low	MPC1^high	χ^2	P-value
Carcinoma	78	74	31.594	0.000
Adjacent normal mucosa	31	121

Abbreviation: GC, gastric cancer.

Figure 2 MPC1 expression was closely correlated with TNM stage in patients with GC.

Notes: (A) Representative images of MPC1 staining in normal gastric mucosa and GC samples at different infiltration depths. Scale bar, 50 μm; magnification ×200. (B) IHC scores of MPC1 in patients with TNM III/IV tumors were significantly lower compared with TNM I/II tumors (P=0.005). (C) Analysis of NCBI GEO dataset GSE26942 showed that the mRNA levels of MPC1 in TNM III/IV cases were obviously lower than those of TNM I/II cases (P=0.016).
Abbreviations: TNM, tumor node metastasis; GC, gastric cancer; IHC, immunohistochemistry.

Table 2 The relationship between MPC1 expression and clinicopathological features of GC patients

Clinicopathological features	N	MPC1
		Low	High	P-value
Age (years)				0.688
≥60	60	32	28
<60	92	46	46
Sex				0.257
Male	100	48	52
Female	52	30	22
Tumor site				0.120
Proximal gastric	61	36	25
Distal gastric	91	42	49
Histological grade				0.486
G1 + G2	43	24	19
G3	109	54	55
T stage				0.000
T1–T2	49	13	36
T3–T4	103	65	38
N stage				0.037
N0	61	25	36
N1–N3	91	53	38
TNM stage				0.028
I/II	89	39	50
III/IV	63	39	24

Abbreviations: GC, gastric cancer; TNM, tumor node metastasis.

Figure 3 Kaplan–Meier analysis of overall survival according to the expression of MPC1 in patients with GC.

Notes: (A) Kaplan–Meier analyses of 152 patients with GC showing MPC1^high patients (n=74) have longer overall survival as compared with MPC1^low patients (n=78) (P<0.001). (B) Kaplan–Meier analyses of 380 GC patients from NCBI GEO database showing that the overall survival rates for MPC1^high patients (n=173) are significantly higher than MPC1^low patients (n=207, P=0.0036).
Abbreviation: GC, gastric cancer.

Table 3 Univariate and multivariate analyses of overall survival in GC patients

Prognostic variables	Univariate analysis		Multivariate analysis
	HR (95% CI)	P-value	HR (95% CI)	P-value
Age (years)	1.243 (0.819–1.886)	0.307	1.189 (0.764–1.852)	0.481
Sex	1.022 (0.667–1.565)	0.921	0.984 (0.630–1.538)	0.842
Histological grade	0.698 (0.451–1.1080)	0.106	0.733 (0.466–1.154)	0.131
T stage	2.387 (1.463–3.895)	0.000	1.279 (0.703–2.325)	0.709
N stage	2.218 (1.420–3.464)	0.000	2.034 (1.300–3.183)	0.069
TNM stage	2.200 (1.456–3.325)	0.000	1.037 (0.607–1.770)	0.462
Low MPC1 expression	0.285 (0.184–0.442)	0.000	0.268 (0.172–0.419)	0.000

Abbreviations: GC, gastric cancer; HR, hazard ratio; CI, confidence interval; TNM, tumor node metastasis.

Figure 4 Overexpression of MPC1 attenuated the proliferative, migrating, and invasive abilities of human GC cells.

Notes: (A) The overexpression of MPC1 in MPC1-FLAG and control GC cells (SGC7901 and MGC803) was tested by Western blotting analysis. (B) Representative images of colonies formed in control and MPC1-FLAG GC cells. (C) Histograms showing the number of colonies. The means ± standard deviations from three independent experiments were shown. (D) The growth rate of control and MPC1-FLAG GC cells (SGC7901 and MGC803) was determined by cell counting kit-8 (CCK-8) assay. (E, F) Transwell chamber migration and Matrigel-invasion assays for MPC1-FLAG and control GC cells (SGC7901 and MGC803). **P<0.01, ***P<0.001. Scale bar, 50 μm; magnification ×200.
Abbreviation: GC, gastric cancer.

Figure 5 Overexpression of MPC1 attenuated the stem cell-like properties of human GC cells.

Notes: (A) Protein levels of “stemness” markers in MPC1-FLAG and control GC cells (SGC7901 and MGC803). (B) mRNA levels of “stemness” markers in MPC1-FLAG and control GC cells (SGC7901 and MGC803). (C, D) Representative images and quantitative analysis showed that MPC1-FLAG GC cells (SGC7901 and MGC803) formed fewer tumorspheres. (E) Xenograft tumors formed by MPC1-FLAG and control GC cells. (F) Analysis of the weight of xenograft tumors. *P<0.05, **P<0.01, ***P<0.001. Scale bar, 50 μm; magnification ×200.
Abbreviation: GC, gastric cancer.

Figure S1 (A) H&E staining of GC samples. (B) The intensity of MPC1 in GC cells. Scale bar, 50 μm; magnification ×200. (C) The relative risk plot of each IHC score generated by X-tile software to identify the optimal cutoff value.

Abbreviations: GC, gastric cancer; IHC, immunohistochemistry; Pt, patient.

Table S1 Clinical features of patients with GC

Clinical characteristic	Number	Percentage (%)
Age
≥60	60	39.47
<60	92	60.53
Sex
Male	100	65.79
Female	52	34.21
Histology grade
G1	6	3.95
G2	37	24.34
G3	109	71.71
T stage
T1	31	20.39
T2	18	11.84
T3	74	48.68
T4	29	19.08
N stage
N0	61	40.13
N1	41	26.97
N2	38	25.00
N3	12	7.89
TNM stage
I	39	25.66
II	50	32.89
III	47	30.92
IV	16	10.53

Abbreviations: GC, gastric cancer; TNM, tumor node metastasis.

Table S2 Sequences of primers used for qRT-PCR in this study

Gene	Forward	Reverse
MPC1	5′-TCAGTGGGCGGATGACATTTG-3′	5′-CAGATGCCGTTTTAGTCATCTCG-3′
Nanog	5′-TGAACCTCAGCTACAAACAG-3′	5′-TGGTGGTAGGAAGAGTAAAG-3′
Sox-2	5′-GTATCAGGAGTTGTCAAGGCAGAG	5′-TCCTAGTCTTAAAGAGGCAGCAAAC-3′
Oct-4	5′-AGCGAACCAGTATCGAGAAC-3′	5′-TTACAGAACCACACTCGGAC-3′
β-actin	5′-TTGCGTTACACCCTTTCTTG-3′	5′-CACCTTCACCGTTCCAGTTT-3′

Abbreviation: qRT-PCR, quantitative reverse transcription polymerase chain reaction.