RhoA和Ezrin基因表达与喉鳞状细胞癌浸润转移的相关性研究

发布时间：2018-08-21 13:12

【摘要】：喉癌是头颈部最常见的肿瘤之一,其中以喉鳞状细胞癌(laryngeal squamous cell carcinoma,LSCC)为主,约占喉癌的96%~98%。最新研究显示喉癌的总生存率有下降趋势,而影响喉癌预后的关键因素是癌细胞的侵袭性和转移性,因此进一步了解喉癌侵袭转移的相关分子机制,可为喉癌患者的预后评估及个体化治疗提供必要的依据。肿瘤的侵袭转移是一个动态且复杂的过程,是多种蛋白分子参与多个步骤共同作用的结果。Rho A作为“信号通路分子开关”介导了多种信号转导通路而参与调节细胞的活动。近年研究发现RhoA的表达水平与肿瘤的发展密切相关。Ezrin为膜细胞骨架连接蛋白,在多种恶性肿瘤中呈高表达,被认为是肿瘤转移的关键调节因子,在肿瘤的浸润转移中发挥着重要作用。活化的Rho A可通过激活ROCK-1进而促进Ezrin的磷酸化,增强Ezrin与细胞骨架的联系,从而改变细胞骨架结构并参与细胞的运动过程。本研究旨在通过检测Ezrin及RhoA在喉鳞癌细胞中的表达情况,以了解这些参与肿瘤侵袭转移的基因表达在喉鳞状细胞癌发生发展的作用及其相关性,进一步探讨喉癌细胞局部浸润及淋巴结转移的作用机制。一、RhoA和Ezrin在新鲜喉癌组织及新鲜喉粘膜组织中的表达本实验应用免疫组织化学染色(Immunohistochemistry,IHC)、免疫蛋白印迹(Western blot,WB)及实时荧光定量聚合酶链反应(Quantita-ti ve reverse transcription polymerase chain reaction,q RT-PCR)法分别检测30例喉鳞状细胞癌患者手术切除新鲜癌组织及30例相对应正常喉粘膜组织中RhoA和Ezrin的蛋白表达量及mRNA的基因表达水平,并结合临床病理特征进行统计学分析。应用SPSS22统计分析软件进行统计学处理,P0.05时差异具有统计学意义。免疫组化结果显示在LSCC组织中RhoA和Ezrin的阳性表达率(60%和70%)明显高于正常喉粘膜组织(13.3%和16.7%);Western blot结果显示RhoA和Ezrin在LSCC组织中的蛋白表达量均明显高于正常喉粘膜组织;qRT-PCR结果显示RhoA和Ezrin在LSCC组织中的m RNA表达量也均高于正常喉粘膜组,差异均具有统计学意义(P0.05)。两者的表达与淋巴结的转移、临床分期密切相关(P0.05);而与肿瘤分化程度、年龄、性别、吸烟程度无关(P0.05)。同时一致性检验结果显示RhoA和Ezrin在喉鳞状细胞癌细胞中的表达呈正相关(Ka ppa=0.638)。二、回顾性分析RhoA和Ezrin在喉鳞癌组织及正常喉粘膜组织中的表达本实验应用免疫组织化学染色方法检测近10年的喉癌组织石蜡块68例及正常喉粘膜组织石蜡块35例中RhoA和Ezrin的表达情况,并结合临床病理特征进行统计学分析。结果显示LSCC组织中RhoA和Ezrin的阳性表达率(80.9%和82.4%)明显高于正常喉粘膜组织(28.5%和25.7%),差异具有统计学意义(P0.05);且两者的表达量与淋巴结的转移、临床分期密切相关(P0.05);而与分化程度、年龄、性别、吸烟程度无关(P0.05)。一致性检验结果显示RhoA和Ezrin在喉鳞状细胞癌细胞中的表达呈正相关(Kappa=0.363),与上述新鲜组织中RhoA和Ezrin的表达情况基本一致,进一步验证结果的可靠性。综上所述,RhoA和Ezrin在LSCC组织中不论从细胞分子水平还是mRNA的基因表达水平较正常喉粘膜组织均呈高表达,并同时与喉癌患者淋巴结转移、临床分期密切相关,且两者在喉癌细胞中的表达呈正相关,提示两者可能共同参与喉癌细胞的浸润转移过程。免疫组化结果显示RhoA分布于细胞浆及细胞膜,Ezrin主要分布于细胞膜,提示Ezrin可能通过与细胞膜蛋白结合使肌动蛋白与细胞膜相连从而改变细胞骨架结构,调节细胞结构及粘附性而参与肿瘤细胞的浸润转移过程,两者之间表达的一致性提示RhoA可能作为Ezrin上游信号通路促进Ezrin的活化后参与肿瘤的浸润转移过程。为喉癌的早期诊断和预后评估提供新的依据。
[Abstract]:Laryngeal squamous cell carcinoma (LSCC) is one of the most common tumors in the head and neck, accounting for 96%-98% of the total laryngeal cancer. Recent studies have shown that the overall survival rate of laryngeal cancer is declining, and the key factors affecting the prognosis of laryngeal cancer are the invasiveness and metastasis of cancer cells. Invasion and metastasis of laryngeal carcinoma is a dynamic and complex process, which is the result of multiple protein molecules participating in multiple steps. Rho A, as a "molecular switch of signal pathway", mediates a variety of signal transduction pathways. Recent studies have shown that the expression of Rho A is closely related to the development of tumor. Ezrin, a membrane cytoskeleton connexin, is highly expressed in many malignant tumors. Ezrin is considered to be a key regulator of tumor metastasis and plays an important role in tumor invasion and metastasis. Activated Rho A can activate ROCK-1 by activating ROCK-1. The aim of this study was to investigate the expression of Ezrin and RhoA in laryngeal squamous cell carcinoma (LSCC) cells and to understand the role of these genes involved in tumor invasion and metastasis in the development of LSCC. The expression of RhoA and Ezrin in fresh laryngeal carcinoma tissues and fresh laryngeal mucosa was studied by immunohistochemical staining (IHC), Western blot (WB) and real-time quantitative polymerase chain reaction. Quantita-ti ve reverse transcription polymerase chain reaction (q RT-PCR) was used to detect the expression of RhoA and Ezrin proteins and mRNA in 30 fresh laryngeal squamous cell carcinoma tissues and 30 normal laryngeal mucosa tissues, respectively. The positive expression rates of RhoA and Ezrin in LSCC tissues (60% and 70%) were significantly higher than those in normal laryngeal mucosa tissues (13.3% and 16.7%). The expression of RhoA and Ezrin in LSCC was significantly higher than that in normal laryngeal mucosa (P 0.05). The expression of RhoA and Ezrin was closely related to lymph node metastasis and clinical stage (P 0.05), but not to tumor differentiation, age, sex and smoking (P 0.05). Conformity test showed that the expression of RhoA and Ezrin in laryngeal squamous cell carcinoma cells was positively correlated (Ka ppa=0.638). Second, the expression of RhoA and Ezrin in laryngeal squamous cell carcinoma and normal laryngeal mucosa was retrospectively analyzed. Immunohistochemical staining was used to detect paraffin masses in 68 cases of laryngeal carcinoma and normal laryngeal mucosa in the past 10 years. The expression of RhoA and Ezrin in 35 cases of paraffin-embedded lesions was statistically analyzed. The results showed that the positive rates of RhoA and Ezrin in LSCC tissues (80.9% and 82.4%) were significantly higher than those in normal laryngeal mucosa tissues (28.5% and 25.7%). The difference was statistically significant (P 0.05). The results of consistency test showed that the expression of RhoA and Ezrin in laryngeal squamous cell carcinoma cells was positively correlated (Kappa = 0.363), which was basically consistent with the expression of RhoA and Ezrin in the above fresh tissues, and further verified the reliability of the results. The expression of RhoA and Ezrin in LSCC tissues was higher than that in normal laryngeal mucosa at both cellular and molecular levels. The expression of RhoA and Ezrin was closely related to lymph node metastasis and clinical stage of laryngeal carcinoma. The expression of RhoA and Ezrin in laryngeal carcinoma cells was positively correlated, suggesting that both of them might participate in the invasion and metastasis of laryngeal carcinoma cells. Immunohistochemistry showed that RhoA was distributed in cytoplasm and cell membrane, and Ezrin was mainly distributed in cell membrane, suggesting that Ezrin might participate in the invasion and metastasis of tumor cells by binding to actin and cell membrane proteins to alter cytoskeleton structure, regulate cell structure and adhesion. These results suggest that RhoA may be an upstream signaling pathway of Ezrin to promote the activation of Ezrin and participate in the invasion and metastasis of laryngeal carcinoma.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.65

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