【作者】 谢红军；

【导师】 张东升；

【作者基本信息】 山东大学 ， 外科学（专业学位）， 2014， 博士

【摘要】 目的：头颈部鳞状细胞癌(Head and neck squamous cell carcinoma, HNSCC)主要影响口腔、舌咽、口咽、喉部及涎腺等,在常见的恶性肿瘤类型中位居第六,而口腔颌面部恶性肿瘤中约80%以上为口腔鳞状细胞癌(oral squamous cell carcinoma, OSCC)。尽管近几年我们在OSCC的治疗以及发病机制研究方面取得了一定的进展,但是在过去的几十年中其存活率仍没有明显改善,平均每年确诊的病例中仍有约50%的死亡率。蛋白酪氨酸磷酸酶Shp2(Src homology phosphotyrosyl phosphatase2)由PTPN11基因编码,广泛地表达于机体各种组织和细胞中,其含有2个SH2结构区域,分别为N-SH2、C-SH2,及一个PTP结构区域,其作为细胞因子、生长因子及其他胞外刺激因素的下游信号分子,参与细胞增殖、分化、迁移等诸多细胞重要的生命活动。研究表明,Shp2的编码基因PTPN11存在大量与人类疾病相关的遗传多态性与突变位点,作为重要的节点分子,Shp2在血液系统恶性肿瘤如白血病及实体肿瘤如乳腺癌、肺癌、胃癌、肝癌等的发生发展过程中发挥着重要的调控作用,是其肿瘤治疗的潜在分子靶点。目前国内外关于Shp2在口腔鳞癌中的表达及作用的研究鲜见报道。因此,本实验从临床标本、体外细胞和分子机制三个层面研究Shp2在口腔鳞癌中的表达及对其生物学行为的影响。首先通过临床病例标本检测口腔鳞癌组织及癌旁组织中Shp2的表达差异,并进一步分析其表达水平与患者的临床资料之间的关系,探讨Shp2在口腔鳞癌中的临床意义。其次,应用RNA干扰(RNA interference, RNAi)技术,构建Shp2慢病毒干扰载体,抑制舌鳞状细胞癌细胞SCC-4的Shp2基因表达,探讨Shp2基因沉默后对SCC-4细胞增殖、凋亡及侵袭能力的作用。同时研究Shp2基因沉默后对细胞凋亡相关蛋白的影响,初步阐明Shp2在口腔鳞癌细胞凋亡发生中的分子机制。本研究将为口腔鳞癌临床诊断、预后及治疗的新靶点提供新的思路和理论基础。方法：1.收集临床口腔鳞癌组织蜡块及临床有关资料,采用免疫组织化学染色方法观察Shp2蛋白在口腔鳞癌组织中的表达,并对免疫组化结果进行评分,评估Shp2的表达与患者的年龄、性别、烟酒暴露、肿瘤部位、肿瘤大小、临床分期、淋巴转移及病理分级等的相关性。2.收集18例临床手术中新鲜的口腔鳞癌及相应的癌旁正常组织,采用WesternBlot方法,进一步验证Shp2蛋白在口腔鳞癌组织及癌旁正常组织中的表达差异。3.于Shp2PTPN11基因编码区选择4个siRNA靶点合成4对shRNA干扰序列,分别与慢病毒载体连接,构建Shp2基因RNA干扰慢病毒载体,筛选出最佳干扰组,显微镜下观察Shp2慢病毒干扰载体感染目的细胞人舌鳞状细胞癌细胞SCC-4的效率。应用Real Time PCR及Western blot检测其干扰效率,筛选出最佳干扰组,建立稳定抑制Shp2基因表达的SCC-4细胞株,为探讨Shp2在口腔鳞癌发生中的作用提供细胞模型。4.采用四甲基偶氮唑盐(MTT)比色法观察Shp2基因沉默后对SCC-4细胞24h、48h、72h及96h增殖率的影响。5.应用流式细胞技术(Annexin V-APC/7-ADD染色法)及Westen Blot方法研究Shp2基因沉默后对SCC-4细胞凋亡的影响及其凋亡相关蛋白p53、Bax及Bcl-2的变化。6.采用Transwell小室侵袭实验,通过苏木精染色和细胞计数观察Shp2基因沉默后对SCC-4细胞体外侵袭能力的影响。结果：1.免疫组化结果发现,在口腔正常黏膜组织中,Shp2表达阴性或者呈弱表达,而在口腔鳞癌临床样本中高表达；进一步临床资料统计分析表明,在口腔鳞癌中Shp2蛋白的表达与患者的性别、年龄、是否吸烟、饮酒无关,与肿瘤的发生部位和体积大小亦无关。然而,临床分期在III/IV期的肿瘤Shp2的表达较Ⅰ/Ⅱ期的强,在有淋巴转移的病例中,Shp2的表达显著增加,统计学上均有显著性差异(p<0.05)。2.临床新鲜口腔鳞癌组织进一步证明,除一例以外,Shp2在口腔鳞癌组织中的表达明显高于癌旁正常组织中的表达(p<0.05),进一步验证了免疫组化结果。3. Real-time PCR和Western blot检测结果显示Shp2-shRNA3干扰效率最佳,SCC-4细胞中Shp2基因及蛋白表达明显降低,应用RNAi设计的慢病毒干扰载体具有较好的干扰效果。4.MTT法测定发现,Shp2基因沉默后,96h内SCC-4细胞的生长明显慢于对照组,出现明显的增殖抑制。5.采用nnexin-V-APC/7-ADD双荧光标记,流式细胞仪检测细胞凋亡发现,转染Shp2慢病毒于扰载体72h后的SCC-4细胞中,细胞凋亡率明显增加,同对照组正常细胞相比,差异具有显著性(p<0.05)。Western Blot结果显示,凋亡相关通路蛋白p53、Bax表达增加,Bcl-2表达下降。6.[ranswell小室侵袭实验结果显示,转染Shp2慢病毒干扰载体72h后的SCC-4细胞的侵袭能力与对照组相比较明显下降,差异具有显著性(p<0.05)。结论：Shp2在口腔鳞癌组织中的表达明显高于癌旁正常组织,过表达的Shp2与口腔鳞癌的临床分期及淋巴转移密切相关；Shp2基因对口腔鳞癌细胞的生长起促进作用,同时可提高肿瘤细胞的侵袭能力；Shp2能抑制口腔鳞癌细胞凋亡,凋亡相关蛋白p53、Bax及bcl-2可能参与其凋亡调控机制。以上结果提示,Shp2与口腔鳞癌的进展和转移有关,在口腔鳞癌可能发挥重要的促癌基因作用,是口腔鳞癌淋巴结转移的危险因子,可作为口腔鳞癌临床诊断、判断预后、诊疗方案制定及肿瘤靶向治疗的潜在生物标记物和分子指标。更多还原

【Abstract】 Objective:Head and neck squamous cell carcinoma （HNSCC） is a devastating disease, which accounts for3%f all malignancies and is the sixth most common cancer worldwide. It is a disease primarily affecting the oral cavity, hypopharynx, oropharynx, larynx, and salivary glands. More than80%f HNSCC are oral squamous cell carcinoma （OSCC）, which is one of the undertreated and understudied cancer types and remains a lethal disease in over50%of the cases diagnosed annually. Although we made appreciable advances in the treatment and understanding of the underlying molecular pathogenesis of OSCC, survival rates have not improved significantly over the last several decades. Therefore, there is increasing concern about determining novel molecular targets for the treatment of OSCC. Cellular activities, such as cell survival, proliferation, and differentiation, are tightly controlled by intracellular signaling processes initiated by extracellular signals, in which protein phosphorylation （conducted by protein kinases） and dephosphorylation （conducted by protein phosphatases） are necessary events. Src homology phosphotyrosyl phosphatase2（Shp2） encoded by the PTPN11gene in humans, is a ubiquitously expressed protein tyrosine phosphatase （PTP） with two N-terminal Src homology2（SH2） domains （N-SH2, C-SH2, respectively） and a catalytic （PTP） domain that acts as an important transducer of the MAPK pathway by growth factors, cytokines, and hormones. Autosomal-dominant mutations in the human gene PTPN11have been detected in nearly50%of patients with Noonan syndrome who have higher risk of suffering juvenile myelomonocytic leukemia （JMML）, and somatic mutations constitutively activating Shp2have also been detected in several types of leukemias. Meanwhile, high level of Shp2expression has been reported in several types of cancers, such as breast cancer, gastric cancer, cervical cancer, prostate cancer, and melanomas, which indicated that Shp2functions as a proto-oncogene. Interestingly, the opposite effect of Shp2on tumorigenesis was found in hepatocellular carcinoma, implying that Shp2acted as a tumor suppressor. However, so far there is a lack of information concerning the expression and clinical significance of Shp2in OSCC. Therefore, this study investigated the clinical significance of Shp2protein expression in OSCC and elucidated the effect of Shp2gene on biological behaviours of in vitro. The correlation between the expression level and clinical parameters in OSCC tissues was analyzed. The biological significance in OSCC cells was elucidated preliminarily.Methods:1. Seventy paraffin-embedded specimens were retrospectively recruited and immunohistochemical method was used to detect the expression of Shp2in OSCC. Based on the score of immunochemistry, the correlation between the expression of Shp2and various clinicopathologic parameters （age, sex, tumor size, tumor location, clinical stage, histological grade and lymphatic metastasis） was investigated.2. Eighteen cases of cancerous and normal tissues were obtained from patients with OSCC. These samples were histologically confirmed by frozen sections to have OSCC and used for Western blot analysis of Shp2expression.3. To knockdown Shp2expression in cell lines, we designed and synthesized Shp2shRNAs according to Shp2cDNA sequences （Gen-Bank accession NM₀02834） from Shanghai Genomeditech Co. Ltd. These oligonucleotides were then subcloned into a lentiviral vector （pGMLV-SB1RNAi） and after estimating a multiplicity of infection （MOI） using a standard procedure, these viruses were used to infect SCC-4cells using an oligofectamine transfection reagent. Thereafter, the cells were observed under a fluorescence microscope and harvested on the4th,7th and10th day after infection. The efficiency of Shp2knockdown in SCC-4cells was confirmed by using real time PCR and Western blot.4. The proliferation rate of SCC-4cells at24h,48h,72h and96h after Shp2-shRNA3lentivirus vector and empty vector infection was investigated by MTT dection.5. Cells were collected after lentivirus vector and empty vector infection for72h and then5μl PE Annexin V and5μl7-ADD were added into cell solutions and incubated in the dark for30min. The apoptotic rate was detected by a flow cytometer. P53, Bax and Bcl-2, were detected for further demonstrating the mechanism involved in apoptosis.6. SCC-4cells were transfected with Shp2lentiviral vector and empty vector for investigating the effect of Shp2on cell invasion ability by tumor cell transwell invasion assay.Results:1. Immunohistochemistry results showed that in the adjacent normal epithelia, Shp2protein was either absent or weakly expressed, whereas the average total score of Shp2protein levels in OSCC tissues was significantly higher than that in adjacent normal epithelia. Furthermore, there was significant association of Shp2expression with tumor clinical stage and lymph node metastasis.2. The expression of Shp2protein in the fresh OSCC tissues was higher than that in the adjacent non-tumor tissue, except in one sample.3. We produced lentiviruses carrying Shp2shRNA to knockdown Shp2expression in SCC-4cells. Real-time PCR data showed that the levels of Shp2expression in Shp2shRNA1,2, and3subgroups were remarkably reduced by39%,47%, and75%, respectively. Western blot analysis revealed that the levels of Shp2protein expression in these three subgroups were also significantly reduced.4. The effect of Shp2knockdown on regulation of SCC-4cell viability was determined by using MTT assay. The result showed that the Shp2shRNA3lentivirus-infected cell had a reduction in cell viability compared with the control cells （p<0.05）.5. Annexin V/7-ADD staining by flow cytometry showed that the reduced cell viability was due to induction of apoptosis （p<0.05）. The western blot results showed that the expression of p53protein was increased upon Shp2knockdown in SCC-4cells. Meanwhile, Shp2knockdown was able to induce increase in expression of Bax protein, but decreased the expression of Bcl-2protein.6. Knockdown of Shp2expression on SCC-4cells significantly reduced tumor cell invasive capacity compared to the control cells （p<0.05）.Conclusions:Expression of Shp2protein was significantly upregulated in OSCC tissues compared to the normal tissues and Shp2overexpression was associated with advanced tumor clinical stages and lymph node metastasis in vivo. Knockdown of Shp2expression in vitro inhibited OSCC cell viability and invasion but induced apoptosis by regulating expression of the apoptosis-related proteins. In summary, these results demonstrated that Shp2may be an oncogene and promote OSCC lymph node metastasis. These data also suggest that Shp2might be further evaluated as a biomarker for prediction of OSCC progression and that target of Shp2may be a novel therapeutic strategy for clinical control of OSCC in future.更多还原