CRBP-1 over-expression is associated with poor prognosis in tongue squamous cell carcinoma

Abstract

Background: Tongue squamous cell carcinoma (TSCC) is one of the most common malignancies of oral squamous cell carcinomas. Cellular retinol binding protein-1 (CRBP-1) as a carrier protein transports retinol from the liver storage site to peripheral tissue. Up-regulated expression of CRBP-1 is associated with some tumor types such as prostate cancer, breast cancer and ovarian cancer as reported, but its role in TSCC remains uncertain.

Methods: In this study, an integrated bioinformatics analysis based on the multiple cancer microarray data sets available from Oncomine database was conducted to view the differential expression of CRBP-1 between TSCC and the adjacent non-tumorous tissues. Quantitative real-time polymerase chain reaction (qRT-PCR), western blotting (WB) and immunohistochemical (IHC) assays were performed to investigate CRBP-1 expression in 101 paraffin-embeded TSCC tissues and 48 pairs of freshly frozen tissues. Kaplan-Meier curve and univariate and multivariate Cox-regression analysis were used to estimate the association between CRBP-1 expression and patients' prognosis. Then western blotting, MTT, transwell migration and invasion assays were performed in TSCC cell lines to investigate the effects of CRBP-1 on cellular proliferation and invasion.

Results: Compared with the matched adjacent non-tumorous tissues, the expression of CRBP-1 was significantly up-regulated in TSCC tissues, which correlated with the differentiation state (P = 0.003), N classification (P = 0.048), the clinical stage (P = 0.048) and death (P = 0.001). The Kaplan-Meier curve showed that TSCC patients with higher CRBP-1 expression levels had lower overall survival rates than those with lower CRBP-1 expression levels. A univariate and multivariate analysis demonstrated that CRBP-1 was an independent prognostic factor (P < 0.05). Furthermore, we knocked down CRBP-1 expression and observed that TSCC cell proliferation and invasion in vitro were significantly blocked, as determined by MTT and transwell assays.

Conclusions: Up-regulated expression of CRBP-1 is associated with poor prognosis in TSCC, so it might potentially serve as an additional prognostic marker, and the inhibition of CRBP-1 might provide new therapeutic approaches for TSCC.

Keywords: CRBP-1; Expression; Invasion; Knockdown; Prognosis; Proliferation; Tongue squamous cell carcinoma.

Fig. 1

Expression of CRBP-1 mRNA in human TSCC tissues and matched adjacent non-tumorous tissues. a The relative CRBP-1 mRNA expression levels significantly increased in TSCC tissues (T) compared with those of the matched adjacent non-tumorous tissues (ANT)(n = 48, P < 0.001). The mRNA expression level of CRBP-1 was measured by qRT-PCR. b CRBP-1 is overexpressed in human TSCC tissues(T) than the adjacent normal tissues(N) in multiple cancer microarray data sets available from Oncomine. c CRBP-1 is overexpressed in HNSCC tissues(T) than the adjacent normal tissues(N) in multiple cancer microarray data sets available from Oncomine. Data is presented as mean ± S.D. **P < 0.01 versus the corresponding control groups

Fig. 2

Expression of CRBP-1 protein in human TSCC tissues and matched adjacent non-tumorous tissues. a The relative CRBP-1 protein expression in 7 pairs of TSCC tissues (T) and the matched adjacent non-tumorous tissues (ANT). GAPDH was measured as the loading control. b Immunohistochemical staining of CRBP-1 in three pairs of representative TSCC tissues (T) and the adjacent non-tumorous tissues (ANT)(× 100)

Fig. 3

Correlation between CRBP-1 expression in TSCC and clinicopathological features. a Increased expression of CRBP-1 in advanced stages of TSCC. Representative IHC analysis of CRBP-1 expression in TSCC specimens (T) and the matched adjacent non-tumorous tissues (ANT) of different clinical stages. b CRBP-1 expression in TSCC tissues with different differentiation states

Fig. 4

Kaplan-Meier survival curves for TSCC patients with high CRBP-1 expression (red line) versus low CRBP-1 expression (blue line). The overall survival of patients with high or low CRBP-1 expression

Fig. 5

Knockdown of CBRP-1 reduced the cell proliferation and invasion ability in CAL-27 and SCC-25 cells. a The relative expression levels of CRBP-1 in TSCC cell lines were evaluated by western blotting. b Immunoblotting analysis of CRBP-1 in CAL-27 and SCC-25 cells transduced with two specific shRNA. c Growth curves of CAL-27 and SCC-25 cells with knockdown of CRBP-1. Scramble (sc): the lentiviral vector with a scrambled sequence that does not target any mRNA. d Images (upper panel) and quantification (lower panel) of invaded TSCC cells treated with melatonin for 24 h were analyzed in a transwell matrix penetration assay. Scar bar: 100 μm. Data is presented as mean ± S.D. **P < 0.01 versus the corresponding control groups