【作者】 金童；

【导师】 潘新良；

【作者基本信息】 山东大学 ， 耳鼻咽喉科学， 2009， 博士

【摘要】 头颈部鳞状细胞癌(Squamous Cell Carcinoma of Head and Neck,SCCHN)指发生于口腔、咽、喉、鼻腔、鼻窦、唾液腺、颈段气管及食管的鳞状细胞癌,其组织来源于上呼吸消化道的粘膜上皮细胞。2003年统计SCCHN每年在世界范围内大约有540,000例新发病例,其中可造成271,000例死亡病例,死亡率超过50%。由于SCCHN具有局部浸润生长、易原位复发及远处转移的特点,目前其治疗仍然是一个富有挑战性的临床课题,其发病率及死亡率较高。虽然外科手术、放射治疗及辅助化疗等治疗方法的综合应用在一定程度上提高SCCHN患者生存率,但大部分研究表明其晚期患者5年生存率仍仅在30%和40%之间。因此研究SCCHN发生过程中的分子生物学机制,对于SCCHN的早期诊断以及为SCCHN的治疗提供新靶点具有重要意义。表皮生长因子受体(Epidermal Growth Factor Receptor,EGFR)是一种分子量为170 kDa的糖蛋白,其编码基因位于染色体7p12,包含28个外显子,属于ErbB受体家族。EGFR的活化可以抑制细胞凋亡,促进细胞增殖和血管形成。其异常活化可导致细胞异常增殖及其他促进肿瘤增长的反应,并可增加肿瘤细胞恶性转移的潜能。研究表明在多种上皮源性肿瘤包括SCCHN中均存在EGFR的表达异常,其中大部分表现为EGFR的过表达,其表达可能与SCCHN的预后密切相关,因此EGFR基因被视为可能的SCCHN治疗的新靶点。已有研究表明,EGFR的表达受多种转录因子如p53、Sp1等的调控,探讨SCCHN中EGFR基因的表达调控对于进一步了解SCCHN的发生机制、为SCCHN治疗提供新的作用靶点具有重要意义。目前对EGFR基因的表达调控研究主要局限于EGFR 1号内含子及其上游几百碱基的范围,较大范围的调控机制少见于文献。近几年EGFR基因突变和单核苷酸多态性(Single Nucleotide Polymorphism,SNP)与肿瘤发生发展的关系也成为研究热点,目前的研究主要集中于该基因的18、19、20及21号外显子,这些外显子编码EGFR酪氨酸激酶活性区的ATP结合域,是影响EGFR激酶活性的关键区域。研究表明在非小细胞肺癌中约10～15%的患者存在上述关键区域的基因突变,该突变增强了非小细胞肺癌对某些EGFR抑制剂如gefitinib的治疗反应。这表明基因水平的突变引起该基因表达产物的变化,尤其是在某些关键区域的变化可以影响该区域的功能。因此可以预见,如果在SCCHN中检出上述EGFR基因关键区域的突变,对EGFR成为SCCHN治疗靶点,以及拓展gefitinib等EGFR抑制剂的治疗范围有重要意义。同时,EGFR基因的SNP和SCCHN的关系也引起了研究者的关注。SNP指的是DNA序列上发生的单个核苷酸碱基之间的变异,在人群中这种变异的发生频率至少大于1%,否则被认为是点突变。以往的研究表明基因多态性是一种可以用于预测SCCHN进程的标记。然而,到目前为止,有关中国SCCNH患者中EGFR基因酪氨酸激酶区域(18～21号外显子)突变或SNP的发生率及其与临床病理指标间的关系,尚无报道。因此,研究SCCHN中EGFR基因表达、关键外显子的突变或单核苷酸多态性与临床病理指标间的关系,并进一步分析其基因表达调控机制,有助于深入理解SCCHN发生的分子机制,对于SCCHN的分子靶向治疗有指导意义。本研①利用PCR技术检测了中国SCCHN患者新鲜组织标本中EGFR的表达改变,分析了EGFR表达与临床病理指标及预后的关系;②通过PCR及直接测序的方法证实在中国SCCHN患者中存在EGFR外显子的单核苷酸多态现象,并分析了其与临床病理指标及预后的关系;③克隆了人EGFR基因上游2875 bp的启动调控区片段,利用瞬时转染、报道基因分析、缺失突变分析、RT-PCR、Western blot等方法研究了该区域的启动子活性,并对该区域的顺式作用元件进行了初步分析。第一部分头颈部鳞状细胞癌中EGFR表达与临床病理指标及预后的关系【研究目的】研究头颈部鳞状细胞癌患者肿瘤组织中EGFR表达改变及其与临床病理指标及预后的关系。【研究方法】①收集96例原发SCCHN肿瘤组织,并收集距肿瘤切缘1 cm以上部位的形态学正常的粘膜组织作为对照。②TRIzol法分别提取肿瘤组织和正常粘膜组织中的总RNA,反转录成cDNA。根据EGFR基因组序列合成EGFR特异性引物,以5S RNA为内参照,通过PCR检测肿瘤组织和正常粘膜组织中EGFR表达水平的差异。③应用SPSS 13.0统计学软件进行统计学分析。卡方统计分析EGFR表达与临床病理指标间的关系,非参数统计应用于不适用卡方统计者;Kaplan-Meier生存率分析及Cox回归模型绘制生存率曲线并分析预后,无病生存期的计算采取自诊断之日起至明确证明发生局部复发、远处转移或死亡的时间。所有的统计分析均为双侧,取P＜0.05为有统计学意义。【结果】①在96例SCCHN临床标本中,74例(77.1%)肿瘤组织对比其临近的形态学正常的组织出现EGFR表达异常;其中47例(49.0%)过表达,27例(28.1%)低表达。②EGFR表达与性别、肿瘤大小(T分期)、颈部淋巴结转移(N分期)及饮酒无关(P＞0.05);与年龄(P=0.001)、肿瘤的原发部位(P=0.000)、组织分化程度(P=0.016)、临床分期(P=0.030)及吸烟有关(P=0.034)。③EGFR的过表达主要发生于年龄小于60岁的吸烟者、原发于口咽或喉咽部的肿瘤、中低分化的肿瘤及临床分期Ⅲ-Ⅳ期的SCCHN患者。④生存率分析表明EGFR表达与SCCHN的预后无关(P＞0.05),目前数据并不支持其作为SCCHN患者独立的预后因素(P＞0.05)。【结论】①在SCCHN患者肿瘤组织中存在EGFR的表达异常。②过表达EGFR的SCCHN多原发于喉咽及口咽部。③吸烟可能引起EGFR的表达增加。④EGFR是SCCHN发生及发展过程中的重要影响因子,起到了癌蛋白的作用;第二部分头颈部鳞状细胞癌中EGFR基因18～21号外显子单核苷酸多态性的研究【研究目的】研究中国头颈部鳞状细胞癌患者EGFR基因第18～21号外显子中是否存在突变或单核苷酸多态现象,并进一步研究及其与临床病理指标及预后的关系。【研究方法】①收集96例原发SCCHN肿瘤组织,并收集距肿瘤切缘1cm以上部位的形态学正常的粘膜组织作为对照。②提取肿瘤组织及正常粘膜组织中的基因组DNA。根据EGFR基因第18、19、20、21号外显子序列分别设计引物,进行PCR扩增,凝胶电泳回收上述四条外显子目的带,双向测序,检测其是否存在基因突变或单核苷酸多态现象。③应用SPSS 13.0统计学软件进行统计学分析。两样本率的比较采用u检验;卡方统计分析测序结果临床病理指标间的关系,非参数统计应用于不适用卡方统计者;Kaplan-Meier生存率分析及Cox回归模型绘制生存率曲线并分析预后,无病生存期的计算采取自诊断之日起至明确证明发生局部复发、远处转移或死亡的时间。所有的统计分析均为双侧,取P＜0.05为有统计学意义。【结果】①对EGFR基因18～21号外显子的DNA测序表明,在中国SCCHN患者中EGFR基因18～21号外显子未发现明显突变;22例(22.9%)患者的20号外显子存在单核苷酸多态性(SNP),其第78号位点的鸟嘌呤(G)被腺嘌呤(A)取代。密码由CAG变为CAA,相应的氨基酸(谷氨酰胺)并没有改变。这表明这是一个同义的单核甘酸多态(Synonymous Single Nucleotide Polymorphism,sSNP)。②上述22例患者EGFR基因20号外显子单核苷酸多态均为G/A杂合。③中国SCCHN患者中EGFR基因20号外显子G/A杂合sSNP出现率与GeneBank正常亚洲人的数据之间有统计学差异(P＜0.05)。④G/A杂合sSNP与组织分化程度、局部淋巴结转移无关(P＞0.05),而与原发部位(P=0.003)、T分期(P=0.002)及临床分期(P=0.001)有关。⑤G/A杂合sSNP主要存在于T1～2、临床分期Ⅰ～Ⅱ期及原发于喉咽的SCCHN中。⑥该sSNP与EGFR的表达无明显相关性(P＞0.05)。⑦生存率分析表明G/A杂合患者与G/G纯合患者的生存期差异无统计学意义(P＞0.05),目前的数据不能支持该sSNP为SCCNH独立的预后因素(P＞0.05)。【结论】①中国SCCHN患者中EGFR基因18～21号外显子发生突变的概率较低。②中国SCCHN患者中EGFR基因20号外显子第78号位点G/A杂合sSNP的发生率高于健康人群。③EGFR基因20号外显子第78位的sSNP可能成为一个有效的预测SCCHN,尤其是喉咽部鳞状细胞癌临床过程的指标。④EGFR基因20号外显子第78位的sSNP与EGFR的表达无关。⑤目前的数据并不支持EGFR基因20号外显子sSNP是中国SCCHN患者无病生存率的独立的预后因素。第三部分头颈部鳞状细胞癌中EGFR基因表达调控的初步研究【研究目的】克隆人EGFR基因5′上游2875 bp的启动调控区片段,对该区域可能存在的功能性顺式作用元件进行初步分析。【研究方法】①提取人基因组DNA,PCR扩增EGFR基因5′上游2875 bp(-2644～+231bp)启动调控区序列,插入pGL3-Basic,构建EGFR基因启动调控区-荧光素酶报道基因表达载体pGL3-EGFR-2875,酶切鉴定和测序分析证实插入片段是否正确。②pGL3-EGFR-2875瞬时转染喉癌细胞株Hep-2,荧光素酶报道基因分析检测启动子活性。③将C/EBPα等蛋白因子表达载体与pGL3-EGFR-2875共转染Hep-2细胞,报道基因分析观察C/EBPα等蛋白因子对启动子活性的影响。④应用RT-PCR和Western blot技术验证C/EBPα等蛋白因子的作用。⑤在pGL3-EGFR-2875基础上构建了3个较大范围的5′缺失突变体,与C/EBPα表达载体共转染Hep-2细胞,报道基因分析观察缺失突变对启动子活性的影响,并分析C/EBPα表达载体可能的作用区域。【结果】①构建了较大范围的EGFR基因5′上游启动调控区重组质粒pGL3-EGFR-2875,酶切鉴定及DNA测序结果正确。②pGL3-EGFR-2875在Hep-2细胞呈现很强的启动子活性。③共转染实验结果显示C/EBPα的表达载体明显抑制EGFR启动子活性,RT-PCR和Western blot进一步证实C/EBPα对EGFR表达的抑制作用。④pGL3-EGFR-2875的缺失突变分析显示,C/EBPα表达载体的作用区域可能位于-618～+231 bp内。⑤-1619～-871 bp区域的缺失可导致启动子活性下降72%,说明该区域可能存在功能性正调控元件。【结论】①正确克隆了人EGFR基因上游2875 bp(-2644～+231 bp)的启动调控区片段。②该2875 bp片段在喉癌Hep-2细胞中呈现了很强的启动子活性。③外源性C/EBPα过表达明显抑制EGFR启动子活性,并在mRNA和蛋白水平抑制EGFR表达,其抑制作用呈剂量依赖性。④C/EBPα的作用区域可能位于-618～+231 bp内。⑤EGFR基因启动调控区-1619～-871 bp区域可能存在功能性正调控元件。更多还原

【Abstract】 Squamous cell carcinoma of head and neck（SCCHN） was a sort of carcinoma which generated from the epithelium mucosae cell of oral cavity,pharynx,larynx, nasal cavity,nasal sinus,salivary gland,cervical trachea and esophagus.According to the documents in 2003,540,000 new SCCHN cases were reported worldwide, including 271,000 death cases.The death rate exceeded 50%.Because of the local infiltration,metastasis and local failure,SCCHN remained a challenging clinical problem and its incidence and death rate was still high.Though comprehensive therapies including surgery,radiotherapy and chemotherapy were applied to elevate the disease-free survival rote in recent years,the 5-years survival rate of advanced SCCHN was still between 30%and 40%.Therefore investigations in the field of molecule biology mechanism of the SCCHN process will have great significance to provide original ideas in early diagnosis and gene therapy strategy of SCCHN.Epidermal growth factor receptor（EGFR） is a glycoprotein of 170 kDa,encoded by EGFR gene located on chromosome 7p12 which had 28 exons.It belongs to the ErbB receptor family.The activation of EGFR leads to the inhibition of apoptosis, activation of cell proliferation and angiogenesis.EGFR abnormal activation can induce abnormal cell proliferation and enhancement of tumorous growth,as well as an increase in metastatic spread potential.Previous documents showed many epithelium tumors,including SCCHN had abnormal expression of EGFR,most of which was over-expression.So EGFR gene was regarded as a new target of anti-SCCHN therapy and its expression probably had intimate correlations with SCCHN prognosis.EGFR expression was regulated by a complicated mechanism. Several factors acted in this process,such as p53,Spl.It had important significance to investigate the EGFR gene expression regulation mechanism in SCCHN for further understanding the oncogenesis mechanism and finding new gene therapy.But now, the researches focused on EGFR gene regulation were limited in 5’-upstream hundreds bp.Few documents had reported the regulation mechanism in the extent of 5’-upstream 1000～3000 bp.Recently,the correlation between EGFR gene mutation or single nucleotide polymorphism（SNP） and tumorigenesis or progression became attractive.Existing researches focused on 18～21 exons of EGFR because they encoded adenosine triphosphate（ATP） banding area within EGFR tyrosine kinase domain,which was critical region to EGFR tyrosine kinase activity.It was reported that 10%to 15%of patients with non-small-cell lung cancer（NSCLC） had the mutations in exon 18～21,which enhanced the response to EGFR inhibitor like gefitinib.It shows gene mutations caused variations of gene products;especially variations in critical domain can influence its functions.These results suggest that EGFR-inhibitor treatment maybe perspective in SCCHN if the somatic mutation exists.On the other hand,researchers pay close attention to single nucleotide polymorphisms of EGFR.Single nucleotide polymorphism is genetic variation in a DNA sequence that occurs when a single nucleotide in a genome is altered.The frequency of this variation is greater than or equal to 1%,otherwise is regarded as point mutation.Previous researches indicated single nucleotid polymorphism was a symbol to prognosticate SCCHN process.However,few researches explored the incidence of EGFR mutations or SNP in tyrosine kinase domain（exon 18～21） and the relationship with clinical/histopathological parameters in Chinese SCCHN patients.In summary,investigations in EGFR gene expression,mutations or SNP in critical exons and gene expression regulation conduce to understand the molecule mechanism of SCCHN genesis and provide a new insight in gene therapy targeting EGFR in SCCHN.In this trial,we detected EGFR expression variation in SCCHN specimens by polymerase chain reaction（PCR） and electrophoretic technique and analyzed the correlations between EGFR expression and clinical/histopathological parameters and prognosis.We documented EGFR gene exon 20 had single nucleotid polymorphism in Chinese SCCHN patients by PCR and direct sequencing and analyzed the correlations between this SNP and clinical/histopathological parameters and prognosis. We cloned EGFR gene upstream 2875 bp priming regulatory domain by gene clone technique,analyzed promoter activity of this area by transient transfection assays and reporter gene assays,and primarily analyzed the functional element in this area by cotransfection assays,deletion mutation assays,reporter gene assays,reversed transcriptive polymerase chain reaction（RT-PCR） and Western blot.PART ONE:THE RELATIONSHIP IN EGFR EXPRESSION, CLINICAL/HISTOPATHOLOGICAL PARAMETERS ANDPROGNOSIS IN SCCHNObjective:To investigate the relationship in EGFR expression alteration,clinical/ histopathological parameters and prognosis in SCCHN.Methods:①We collected 96 cases of primary SCCHN tumor tissue,and morphologically normal mucosa adjacent to the carcinomas（1 cm from the tumor margin） as control.②RNA of tumor tissues and morphologically normal mucosas were extracted by TRIzol and were reverse transcribed to cDNA by M-MLV.EGFR specific primer was synthesized base on EGFR genome sequence and 5s RNA was synthesized as endo-reference.We detected the EGFR expression difference between tumor tissue and morphologically normal mucosa by PCR and electrophoretic technique.③SPSS 13.0 was used for statistical analysis.The Chi-square test was used to test equal proportion between EGFR expression and clinical and/or histo -pathological parameters in two-way contingency tables.Kraskal-Wallis test was used to test samples that were not fit for Chi-square test.Disease-free survival time was calculated from the date of diagnosis to the date of first documentation of a local recurrence or death for regional or distant failure. Survival curves were computed according to Kaplan-Meier method and Cox’s regression model was used for the multivariate analysis of prognostic factors. Prognostic significance was assessed using log-rank test.All statistical tests are two sided,and a value of P＜0.05 was considered statistically significant.Results:①In 96 cases of SCCHN,74（77.1%） tumor tissues had the abnormal EGFR expression comparing with the morphologically normal mucosa adjacent to the carcinoma,including 47（49.0%） tumors with an increased expression and 27 （28.1%） with a decreased expression.②There was no significant correlation between the abnormal EGFR expression and gender,tumor size（T stage）,local lymph nodes metastasis（N stage） and alcohol drinking status（P＞0.05）.However,the abnormal EGFR expression were correlated significantly with age（P=0.001）,primary localization（P=0.000）, histopathological differentiation（P=0.016）,clinical stage（P=0.030） and smoking status（P=0.034）.③The EGFR over-expression mainly occurred in the patients who were less than 60 years old smokers,primary oropharyngeal or hypopharyngeal carcinoma, moderately-poorly differentiation and stageⅢ～Ⅳ,which was consistent with previous research.④Prognosis analysis showed EGFR expression had no statistically significant with prognosis and was not an independent prognosis factor in SCCHN（P＞0.05）.Conclusions:①SCCHN tumor tissue had EGFR abnormal expression. ②EGFR over-expression mainly occurred in the carcinoma generating from hypopharynx or oropharynx.③Smoking might induce EGFR over-expression.④EGFR can be evaluated as a valuable influential factor in SCCHN genesis, which acted as an oncoprotein.PART TWO:THE INVESTIGATION OF EGFR GENE EXON 18～21 SINGLE NUCLEOTIDE POLYMORPHISM IN SCCHNObjective:To investigate whether or not the mutation or single nucleotide polymorphism was existed in EGFR gene exon 18～21 and its relationship with clinical/histopathological parameters and prognosis in Chinese SCCHN.Methods:①We collected 96 cases of primary SCCHN tumor tissue,and morphologically normal mucosa adjacent to the carcinomas（1 cm from the tumor margin） as control.②Genomic DNA was extracted from 50 mg fresh frozen tumors tissue and morphologically normal mucosa adjacent to the carcinomas.Four sets of primers corresponding to exons 18～21 were designed and synthesized individually.Exons 18～21 were amplified by PCR from genomic DNA samples individually.The DNA fragments were purified by agarose gel and sequenced to detect the mutation or SNP.③SPSS 13.0 was used for statistical analysis.The u-test was used to compare two sample proportion.The Chi-square test was used to test equal proportion between sequencing results and clinical and/or histopathological parameters in two-way contingency tables.Kraskal-Wallis test was used to test samples that were not fit for Chi-square test.Disease-free survival time was calculated from the date of diagnosis to the date of first documentation of a local recurrence or death for regional or distant failure.Survival curves were computed according to Kaplan-Meier method and Cox’s regression model was used for the multivariate analysis of prognostic factors.Prognostic significance was assessed using log-rank test.All statistical tests are two sided,and a value of P＜0.05 was considered statistically significant.Results:①DNA sequencing in exons 18,19,20 and 21 of EGFR showed that no mutations were found in exons 18～21 and exon 20 revealed a single nucleotide polymorphism in 22（22.9%） patients.The 78^th site was changed from guanine （G） to adenine（A）.The code was changed from CAG to CAA.However,the corresponding amino acid（glutamine） was not changed.This means that it was a synonymous single nucleotide polymorphism（sSNP）.②22 EGFR gene exon 20 78^th sSNPs were all G/A heterozygosis.③There was statistical significance between our finding and GeneBank documents （P＜0.05）.④There was no significant association between the sSNP and histopathological differentiation and local lymph nods metastasis（P＞0.05）.Nevertheless,the correlation between the sSNP in exon 20 and primary localization（P=0.003）,T stage（P=0.002） and clinical stage（P=0.001） had statistical significance.⑤G/A heterozygosis mainly occurred in the patients who were T1～2,stageⅠ～Ⅱand primary hypopharyngeal carcinoma.⑥Further statistical analysis showed 78^th sSNP in exon 20 did not influence the EGFR expression（P＞0.05）.⑦In prognosis analysis,Patients who were G/G homozygosis did not experience statistically significant poorer disease-free survival compared with patients who were G/A heterozygosis（P=0.719）.Results at present did not support that this sSNP was an independent prognostic factor in disease-free survival of SCCHN.Conclusions:①The incidence of mutation in EGFR exon 18～21 was low in Chinese SCCHN.②The incidence of EGFR exon 20 78^th G/A heterozygosis was higher in SCCHN patients than in health people in Chinese.③EGFR exon 20 78^th sSNP could be a useful predictor of the SCCHN clinical course,especially in hypopharyngeal squamous cell carcinoma.④EGFR exon 20 78^th sSNP did not influence EGFR expression.⑤Results at present did not support that this sSNP was an independent prognostic factor in disease-free survival of SCCHN.PART THREE:THE INVESTIGATION OF EGFR GENE EXPRESSION REGULATION IN SCCHNObjective:To clone 2875 bp priming regulatory domain upstream of EGFR gene and primarily analyze the existing functional element in this area.Methods:①We extracted human genome DNA.2875 bp（-2644～+231 bp） regulatory domain upstream of EGFR gene was amplified by PCR and inserted into pGL3-Basic to form 2875 bp EGFR promoter-luciferase reporter plasmid （pGL3-EGFR-2875）.This result was proved to be right by restriction enzyme digestion and DNA sequencing.②pGL3-EGFR-2875 transient transfected laryngeal cancer cell line Hep-2.The promoter activity was detected by luciferase reporter gene assay.③pGL3-EGFR-2875 was cotransfected into laryngeal cancer cell line Hep-2 with expression vectors of C/EBPα,p53,SP1,myc and PTEN.The change of promoter activity was observed by luciferase reporter gene assay.④The effect of C/EBPαwas verified by RT-PCR and Western blot.⑤Three 5’ deletion mutants-luciferase reporter plasmids of 2875 bp promoter were constructed based on pGL3-EGFR-2875,which were cotransfected into laryngeal cancer cell line Hep-2 with expression vectors of C/EBPαindividually.The effect of deletion mutation on promoter activity and the possible reaction area of C/EBPαexpression vector were analyzed by luciferase reporter gene assay.Results:①We constructed the EGFR gene 5’-upstream regulatory domain recombinant plasmid（pGL3-EGFR-2875）,which was proved to be right by the restriction enzyme digestion and DNA sequencing.②pGL3-EGFR-2875 presented a strong promoter activity in Hep-2 cell line.③The results of cotransfection experiments demonstrated that C/EBPαexpression vector inhibited obviously the EGFR promoter activity.This inhibitory was confirmed by RT-PCR and Western blot.④The analysis of pGL3-EGFR-2875 deletion mutants demonstrated the reaction area of C/EBPαexpression vector was in -618～+231 bp area.⑤The deletion of-1619～-871 bp area induced promoter activity to descend for about 72%,which demonstrated a functional positive-regulatory element located in this area.Conclusions:①The 2875 bp（-2644～+231 bp） regulatory domain upstream of EGFR gene was cloned and a recombinant plasmid（pGL3-EGFR-2875） was constructed successfully.②This 2875 bp fragment presented a strong promoter activity in Hep-2 cell line.③C/EBPαexpression vector inhibited obviously the EGFR promoter activity and EGFR expression in the level of mRNA and protein.This inhibitory was dose dependent.④The reaction area of C/EBPαexpression vector was in -618～+231 bp area.⑤A functional positive-regulatory element probably located in EGFR gene 5’-upstream -1619～-871 bp area.更多还原