GIT1 overexpression promotes epithelial-mesenchymal transition and predicts poor prognosis in hepatocellular carcinoma

Figures & data

Figure 1. GIT1 expression in human cancers

(a–c) The mRNA level of GIT1 in gastric and liver cancers exceeded that of normal tissues based on oncomine database. According to the TCGA database, (d) the expression of GIT1 was different between HCC and normal liver tissues, and (e) was correlated significantly with the clinical stage. (f) Overall survival (OS) curve of HCC patients with high and low expression of GIT1.

Figure 2. Expression pattern of GIT1 in HCC tissues

(a) GIT1 protein expression in randomly selected HCC tissues and matched adjacent tissues. GAPDH was used as the house-keeping gene. (b) The mRNA expression level of GIT1 in 28 pairs of HCC tissues and their matched peritumor tissues. GIT1 expression was normalized to that of GAPDH. (c) Immunohistochemical staining for GIT1 expression in randomly selected 54 pairs of HCC tissues from the TMA. (d) Representative images of HCC tissues and adjacent matched normal tissues (magnification: 40 ×, 200 ×). Scale bar: 50, 250 μm; **p < 0.01; ***p < 0.001.

Figure 3. GIT1 overexpression promoted HCC cell invasion, proliferation, and migration

(a) Successful transfection of lentiviral vector into Huh7 and MHCC97-H cell lines as determined by western blot. GAPDH was used as an internal control. (b) CCK-8 and (c) colony-forming assays were used to assess the ability of cell proliferation. (d) Determination of the migratory ability of HCC cells by wound-healing assay. Scale bar = 100 μm. (e) Measurement of cell invasion ability by Transwell assay. Scale bar = 100 μm. **p < 0.01; ***p < 0.001.

Figure 4. GIT1 promoted HCC tumorigenesis in vivo

(a, b) Representative pictures of tumors derived from nude mice implanted with the MHCC97H-NC, MHCC97H-shRNA2, Huh7-NC, and Huh7-GIT1 cell lines (n = 3). Tumor volume (c, d) and weight (e-f) revealed that GIT1 significantly promoted tumor growth. *p < 0.05.

Figure 5. GIT1 promoted EMT in HCC cells through the ERK1/2

(a) Expression of Vimentin, β-catenin, and E-cadherin, in HCC cells with different GIT1 expression was determined using western blotting assays. (b) Protein levels of p-ERK1/2, ERK1/2, AKT, and p-AKT were analyzed by western blot. (c-d) SCH772984, the inhibitor of ERK1/2, inhibited ERK1/2 signaling and reversed the GIT1-induced EMT.Abbreviations: ERK1/2, extracellular regulated kinase 1/2; p-, phosphorylated.

Figure 6. The expression of GIT1 and its prognosis value in 158 HCC patients

(a) Representative graphs of immunohistochemical and H&E staining for GIT1 in HCC samples: (+++) strong; (++) moderate; (+) low; (-) absence; Scale bar: 50, 250 μm. (b–c) Prognostic significance of GIT1 expression in patients with HCC are illustrated.Abbreviations: H&E, hematoxylin and eosin; OS, overall survival.

Table 1. Correlations between GIT1 with clinicopathologic features in 158 HCC patients

Variable		Number of patients		P-value
		GIT1^low	GIT1^high
Gender	Men	79	54	0.019
	Women	21	4
Age, years	≧53	50	25	0.403
	<53	50	33
HBsAg	Positive	77	48	0.391
	Negative	23	10
HCV	Positive	14	4	0.176
	Negative	86	54
Serum TB, μ mol/L	≧17	30	19	0.718
	<17	70	39
Serum ALB, g/dL	≧3.5	99	55	0.140^a
	<3.5	1	3
Serum ALT, U/L	≧75	15	9	0.930
	<75	85	49
Serum AFP, ng/mL	≧20	60	40	0.260
	<20	40	18
Cirrhosis	YES	91	51	0.538
	NO	9	7
Tumor size (diameter, cm)	≧5	35	31	0.023
	<5	65	27
Tumor number	Multiple	15	10	0.710
	Solitary	85	48
Embolus	YES	15	21	0.002
	NO	85	37
Capsulation	YES	54	27	0.367
	NO	46	31
Tumor differentiation	ⅠⅠⅠ/Ⅳ	31	12	0.160
	Ⅰ/Ⅱ	69	46

Notes: a, Fisher’s exact test. GIT1^high≥50% staining; GIT1^low<50% staining. Statistically significant values are shown in bold.

Abbreviations: HBsAg, hepatitis B surface antigen; HCV, hepatitis C virus; AFP, α-fetoprotein; TB, total bilirubin; ALB, albumin; ALT, alanine aminotransferase;

Table 2. Univariate and multivariate analyses of factors associated with recurrence and survival

	Overall survival			Cumulative recurrence
Variable	Univariate	Multivariate		Univariate	Multivariate
	P-value	HR (95% CI)	P-value	P-value	HR (95% CI)	P-value
Gender (men vs women)	0.140		NA	0.059		NA
Age, years (≧53 vs, <53)	0.500		NA	0.498		NA
HBsAg (positive vs negative)	0.770		NA	0.613		NA
HCV (positive vs negative)	0.978		NA	0.852		NA
AFP, ng/ml (≧20 vs, <20)	0.170		NA	0.133		NA
Serum TB, μmol/L (≧17 vs, <17)	0.920		NA	0.983		NA
Serum ALB, g/dL (≧3.5 vs, <3.5)	0.693		NA	0.224		NA
Serum ALT, U/L (≧75 vs, <75)	0.347		NA	0.467		NA
Cirrhosis (yes vs no)	0.388		NA	0.275		NA
Tumor size (diameter, cm) (≧5 vs, <5)	<0.001	2.454(1.393-4.322)	0.002	0.003	2.013(1.237-3.276)	0.005
Tumor number (multiple vs solitary)	0.042		NS	0.066		NA
Embolus (yes vs no)	0.021		NS	0.118		NA
Capsulation (yes vs no)	0.362		NA	0.011	1.790(1.127-2.844)	0.014
Tumor differentiation (ⅠⅠⅠ/Ⅳ vs, Ⅰ/Ⅱ)	0.700		NA	0.831		NA
GIT1 density (<50% vs, ≧50%)	<0.001	3.794(2.119-6.794)	<0.001	<0.001	1.943 (1.185-3.188)	0.009

Abbreviations: HBsAg, hepatitis B surface antigen; HCV, hepatitis C virus; AFP, α-fetoprotein; TB, total bilirubin; ALB, albumin; ALT, alanine aminotransferase; NS, not significant; NA, not adopt.