The Research Of Epidermal Growth Factor Receptor Expression, Single Nucleotide Polymorphism And Gene Expression Regulation In Squamous Cell Carcinoma Of Head And Neck

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.