The Expression Of Rab Coupling Protein And Its Relationship With Invasion, Metastasis And Prognostic Value In Squamous Cell Carcinoma Of The Head And Neck

ObjectiveThe role of Rab coupling protein (RCP) has not been previously investigated in squamous cell carcinoma of the head and neck (SCCHN). The aim of this study was to explore RCP protein expression and its clinicopathological significance in SCCHN.MethodsRCP mRNAs were detected in10of laryngocarcinoma tissues and its corresponding adjacent tissues by RT-PCR. RCP protein expression in95SCCHN samples,18corresponding adjacent epithelia and16leukoplakia epithelia samples was analyzed by immunohistochemistry and correlated with clinicopathological parameters and patient outcome. Furthermore,4SCCHN cell lines and an immortal cell line from nasopharynx (NP-69) were evaluated for RCP expression by RT-PCR. Statistical analyses were performed using the SPSS statistical software version17.0(SPSS Inc., Chicago, IL, USA). Statistical significance between the expression of RCP protein and clinicopathological parameters was compared by the χ2test. Survival analyses were undertaken using the Kaplan-Meier method and curves were compared by the log-rank test. Identification of relevant prognostic factors was performed by the univariate and multivariate Cox regression analysis. Tests were two-sided, and P<0.05was considered to indicate a statistically significant difference. Results1. The expression of RCP mRNAs in laryngocarcinoma tissues was statistically up-regulated than corresponding adjacent tissues.2. RCP was statistically detected in SCCHN cell lines over expressed than that of NP-69by western blot.3. Our data indicated that corresponding adjacent epithelia, leukoplakia epithelia and SCCHN showed a gradual increase in the expression of RCP protein.4. RCP overexpression was significantly associated with T classification (p=0.028), clinical staging (p=0.012), lymph node metastasis (p=0.004) and recurrence (p=0.016).5. The multivariate analysis revealed that RCP had independent prognostic effects on the overall survival rate of the patients with SCCHN.6. Survival analysis revealed that a high RCP expression was significantly correlated with shorter overall survival and disease-free survival.ConclusionRCP protein may contribute to the malignant progression of SCCHN, and serve as a novel prognostic marker of the recurrence, metastasis and prognosis in patients with SCCHN. ObjectiveOur previous studys have demonstrated that RCP was statistically up-regulated in SCCHN than that of corresponding adjacent tissues and NP-69cell line, and RCP protein overe-xpression was related to tumor recurrence, metastasis and poorer survival in patients with SCCHN. These suggested RCP may serve as a novel prognostic marker of the recurrence, metastasis and prognosis in SCCHN. The aim of this study was to explore the impact of RCP on the proliferation, cloning efficiency, migration and invasion of the squamous cellcarcinoma of the head and neck in vitro.MethodsRCP shRNA lentiviral particles were used to knockdown RCP gene expression in SCCHN cell line CNE-2. Western blotting was estimated the gene silencing efficiency of RCP. Stable transfected cell lines were obtained by puromycin screening. CCK-8assay and Flat cloning formation experiment were carried out to assess the effect of RCP inhibition on the proliferation and cloning efficiency of CNE-2. Invasion and migration assay were used to observe the variation of the migration and invasion of CNE-2when RCP knocked out.Results1. RCP shRNA lentiviral particles efficiently decreased the transcription and translation level of RCP in CNE-2cell line.2. By puromycin screening, the stable transfected cell lines were obtained:CNE-2RCPRNAi+(RCP gene knocked out) and CNE-2RCPRNAi-(Blank control).3. The cell proliferations of CNE-2R PRNAi+cells were much slower than that of the control cells (CNE-2, CNE-2RCPRNAi-) by CCK-8assay.4. The proliferation and cloning efficiency of CNE-2RCPRNA+cell was significantly down-regulated than that of both control groups (CNE-2and CNE-2RCPRNAi-). Specifically, the quantity of the cell cloning of the CNE-2RCPRNAi+was7.3±3.02(x±s), while that of the CNE-2and CNE-2RCPRNAi-was20.1±4.09,18.0±3.43respectively, P<0.05.5. RCP silence led to statistically decreased migration ability of CNE-2. Specifically, the quantity of the cells through the transwell membrane24hours after treatment in CNE-2RCPRNAi+was173.67±8.08(x±s), while that of the CNE-2and CNE-2RCPRNAi-were277.33±8.74,260.67±10.02respectively, P<0.01.6. RCP silence led to statistically decreased invasion ability of CNE-2. Specifically, the quantity of the cells through the transwell matrigel membrane48hours after treatment in CNE-2RCPRNAi+was94.00±6.56,while that of the CNE-2and CNE-2RCPRNAi-were184.67±5.51,175.00±9.64respectively, P<0.01.ConclusionSilencing the expression of RCP led to the downregulation of tumor growth, cloning efficiency, migration and invasion in vitro. These suggest that RCP promotes the aggressive behavior of SCCHN, indicating RCP may be a promising targeted gene to block SCCHN progression.