【作者】 戴耀章；

【导师】 张欣；

【作者基本信息】 中南大学 ， 头颈外科， 2012， 博士

【摘要】 目的检测(?)Rab coupling protein (RCP) mRNA及蛋白在头颈部鳞状细胞癌(头颈鳞癌)组织、癌旁、癌前病变组织及细胞株中的表达,并探索其表达与头颈鳞癌临床病理特征和预后的关系。方法采用免疫组织化学技术检测RCP蛋白在95例头颈鳞癌、16例癌旁组织及18例声带白斑石蜡组织标本中的表达,并统计分析RCP蛋白在头颈鳞癌中的表达水平与其临床病理特征和预后之间的关系；另外,应用RT-PCR技术检测(?)RCP mRNA在10对喉癌新鲜标本及癌旁组织的表达水平；利用Western blot技术检测RCP蛋白在NP-69(鼻咽部永生化细胞株)及头颈鳞癌细胞株(Tu212、Tu686、M2、M4)中的表达水平。结果RCP mRNA在喉癌组织中表达明显升高,而在癌旁组织中表达微弱,差异具有统计学意义；RCP蛋白在癌旁组织、声带白斑(癌前病变)及头颈鳞癌组织中表达呈梯形依次升高,差异具有统计学意义；RCP蛋白在头颈鳞癌中的表达水平与头颈鳞癌的T分期(P=0.028)、临床分期(P=0.012)、淋巴结转移(P=0.004)和肿瘤复发(P=0.016)密切相关,而与患者年龄(P=0.383)、饮酒史(P=0.658)、抽烟史(P=0.811)、肿瘤类型(P=0.153)、分化程度(P=1.004)在统计学上无相关性；Kaplan-Meier生存分析显示头颈鳞癌患者RCP蛋白高表达组与低表达组5年总生存率(Overall survival)分别为40%和75%,5年无病生存率(Disease-free survival)分别为30.7%和64%,差异具有统计学意义(P均<0.05)；多因素Cox比例风险回归模型分析进一步显示,淋巴结转移(P=0.037)、肿瘤复发(P=0.000)和RCP蛋白表达水平(P=0.016)均为头颈鳞癌患者预后的独立影响因素。结论RCP在头颈鳞癌组织及细胞中高表达,而在癌旁组织及NP-69细胞株中表达较低,RCP可能与肿瘤的发生相关；RCP蛋白在头颈鳞癌中的表达水平与与头颈鳞癌患者的复发、转移及预后密切相关,且RCP蛋白表达是头颈鳞癌预后的独立影响因素。这些结果均提示RCP在头颈鳞癌的发生、发展中可能发挥了重要作用,并对头颈鳞癌复发、转移及预后具有评估价值。目的我们前期研究表明RCP蛋白在头颈鳞癌组织中显著上调,并与头颈鳞癌患者的临床分期、复发、转移及预后密切相关。因此,本部分将利用RNAi技术沉默RCP基因在头颈鳞癌细胞株CNE-2中的表达,并观察RCP基因沉默后对头颈鳞癌CNE-2细胞体外生长、迁移及侵袭能力等生物学行为的影响。方法利用慢病毒介导的RCP shRNA沉默头颈鳞癌CNE-2细胞中RCP基因表达,Western blot技术检验RCP基因的沉默效果；嘌呤霉素筛选,建立RCP基因稳定沉默的CNE-2细胞株；通过CCK-8法及平板克隆形成实验检测沉默RCP基因对CNE-2细胞增殖及克隆形成能力的影响；利用Transwell迁移侵袭实验观察RCP基因沉默后对CNE-2细胞迁移及侵袭能力的影响。结果慢病毒介导的RCP shRNA显著抑制了CNE-2细胞中RCP基因的表达。利用嘌呤霉素对转染后的细胞进行筛选,成功建立了CNE-2RCPRNAi+(RCP基因稳定沉默)(?)(?)CNE-2RCPRNAi-(空白对照)细胞株；与正常对照组CNE-2及空白对照组CNE-2RCPRNAi细胞比较,RCP干扰组CNE-2RCPRNAi+细胞增殖能力及克隆形成能力显著减弱。CCK-8实验中通过对OD450测得数据的统计学分析发现,CNE-2RCPRNAi+与CNE-2、CNE-2RCPRNAi-三组细胞生长到第2、3,4,5,6,7天,CNE-2RCPRNAi+与其他两组细胞的增殖能力差异具有统计学意义(P<0.05),而CNE-2、CNE-2RCPRNAi-细胞增殖能力无统计学差异；平板克隆形成实验结果表明,CNE-2RCPRNAi+细胞克隆形成数(均数±标准差)为7.3±3.02,而CNE-2RCPRNAi-、CNE-2细胞克隆形成数(均数±标准差)分别为18.0±3.43、20.1±4.09,独立因素T检验提示和其他两组相比,CNE-2RCPRNAi+克隆形成能力明显减弱,差别具有统计学意义(P<0.01),而CNE-2RCPRNAi-和CNE-2在克隆形成数上无统计学差异(P>0.05); Transwell迁移实验发现24小时后穿过Transwell小室聚碳酸酯膜的CNE-2RCPRNAi+细胞较两对照组(CNE-2和CNE-2RCPRNAi-)明显减少(173.67±8.08vs277.33±8.74,260.67±10.02)(P<0.01); Transwell侵袭实验发现48小时后穿过Transwell小室Matrigel胶的CNE-2RCPRNAi+细胞较两对照组(CNE-2和CNE-2RCPRNAi-)显著减少(94.00±6.56vs184.67±5.51,175.00±9.64)(P<0.01)结论RCP表达抑制后能明显减弱头颈鳞癌CNE-2细胞的体外增殖、迁移及侵袭能力,提示其在头颈鳞癌的恶性进展中发挥了重要作用。更多还原

【Abstract】 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.更多还原