| Laryngeal cancer is one of common malignant tumors in head and neck squamous cell carcinoma, and the incidence accounts for about 1～5% in the whole-tumor. Laryngeal cancer ranks the third in the field of ENT except for nasopharyngeal carcinoma, the nasal cavity and paranasal sinus cancer. The etiology of laryngeal cancer remains unknown, which may be related to excessive long-term tobacco, alcohol, harmful chemical stimulation of the gas. Malignant tumor in throat, laryngeal cancer is the most, accounting for about 98.1%. Laryngeal squamous cell carcinoma is the largest, accounting for about 90%, adenocarcinoma accounts for about 2%, and the rest is sarcoma. The occurrence of laryngeal cancer is involved in a multi-gene, multi-step and multi-stage process, and the mechanism of the pathogenesis is complex and the treatment is difficult. Since 1980s, the treatment of laryngeal cancer has made great progress, combination with surgery, radiotherapy, chemotherapy and immunotherapy has significantly increased the 5-year survival rate of patients. However, surgery, radiotherapy and chemotherapy-induced syndrome and drug side effects are severe. It is obvious that the use of new techniques of molecular biology to find new molecular therapeutic targets and the better understanding of the pathogenesis of cancer from the molecular level is very important.Notch gene was found in Drosophila in vivo in 1919, and the study demonstrated that the deletion of part of the gene function in drosophila will result in notches of the edge of the wings, which is the origin of the name of Notch gene. At present, Notch1 signaling pathway in animal cells is one of the most extensive studies in signaling pathway, indicating the good prospects for clinical application, and is the intersection points of many important cell signaling pathways, suggesting that the signaling pathway in tumorigenesis appears an important position. Subsequent studies have shown that Notch1 is widely expressed in a variety of different species, and the structure of their family members are highly conservative, and that it plays an important role in the individual system development, cell fate decisions, differentiation of different cells, pattern formation, tumor development and neural physiological and pathological disorders process. At present, the study of the relationship between Notch1 and tumor is gradually expanding, and Notch1 signaling pathway plays an essential role in the occurrence and development of tumor, including: breast cancer, cervical cancer, colon cancer, pancreatic cancer, prostate cancer, brain cancer and esophageal cancer, etc. In addition, Notch1 signaling pathway in different tumors is not exactly the same role, and sometimes even the same tumor in different periods is also different. The most typical representative of the cancer research is cervical cancer, in which results show that Notch1 promotes the occurrence of tumor in the early stages of cervical cancer, and suppresses the occurrence of tumor in the late stage. These suggest that expression and function of Notch1 in different types of tumors exist evident difference, which may be due to the complexity of Notchl itself. Although the study of Notch1 signaling pathway is always expanding and its importance is obvious, so far there are no reports related to the relationship between Notch1 signaling pathway and laryngeal squamous cell carcinoma at home and abroad. Therefore, the author wandered if Notch1 signaling pathway plays an important role in occurrence and development of laryngeal squamous cell carcinoma. To solve the problem, in this study, the author first detected the Notch1 and downstream target gene Hes1 expressions by Real-time PCR, Western blotting and immunohistochemistry methods, and analyzed the relationship between expressions of Notch1 and Hes1 and clinicopathological features. Further, we constructed the Notch1 NICD retrovirual vector and green fluorescent protein eukaryotic expression vector, screened out Hep-2 cell strains with stably expressed NICD, as well as investigated the expressions of Notch1 and Hes1 genes by using semi-quantitative RT-PCR and Western blotting methods, and localized the Notch1 protein in Hep-2 cells by using inverted fluorescent microscopy. These studies aim at investigating the status of Notch1 signaling pathway in Hep-2 cells. Finally, effects of activated Notch1 signaling pathway on cell proliferation, cell cycle, cell apoptosis and cell migration and its related mechanisms were studied by CCK-8 cell proliferation experiment, flow cytometry, Boyden chamber, semi-quantitative RT-PCR and Western blotting methods. This study will provide feasible clinic basis for Notch1 signaling pathway target therapy of laryngeal squamous cell carcinoma.Part 1 Expressions of Notch1 and downstream target gene Hes1 in laryngeal squamous cell carcinoma tissue and their significanceMethods(1) Relative expression levels of Notch1 and downstream target gene Hes1 mRNAs were detected by Real-time PCR in fresh laryngeal squamous cell carcinoma tissues and vocal cord polyp tissues. (2) Relative expression levels of Notch1 and downstream target gene Hesl proteins were detected by Western blotting in fresh laryngeal squamous cell carcinoma tissues and vocal cord polyp tissues.(3) Expression levels of Notch1 and Hesl proteins were investigated by immunohistochemistry in 67 cases of laryngeal squamous cell carcinoma tissues,26 cases of vocal cord polyp tissues.(4) Statistics analysis:All data were analyzed by SPSS 13.0 software with X2 test, t test and analysis of variance, and P<0.05 was considered as significance.Results(1) The results of Real-time PCR and Western blotting demonstrated that expressions of Notchl and Hesl mRNAs and proteins in vocal cord polyp tissues were obviously higher than those in laryngeal squamous cell carcinoma tissues (P<0.05).(2) Expression of Notchl protein was mainly localized in the cytoplasm of laryngeal squamous cell carcinoma, partly appeared in cell nuclear of laryngeal squamous cell carcinoma, and expression of Hesl protein was mainly localized in the cell nuclear of laryngeal squamous cell carcinoma. The results of immunohistochemistry demonstrated that expressions of Notchl and Hesl proteins in vocal cord polyps tissues were obviously higher than those in laryngeal squamous cell carcinoma tissue (P<0.05).(3) Expression of Notchl and Hesl proteins was closely related to differentiation, tumor TNM stage and lymph node metastasis (P<0.05), and the degree of differentiation was lower, the expressions of Notchl and Hesl proteins was also accordingly lower. Part 2 Construction of Notch1 intracellular domain retroviral vector and preliminary study of its functionMethods(1) Cloning of full-length of Notch1 NICD and sequencing:Total RNA was isolated from human placenta tissue, and cDNA the first strand was synthesized. The full-length of Notch1 NICD was amplified by using cDNA template with PCR method and sequenced.(2) Construction of pCLXSN-Notchl and pEGFP-C1-NICD vectors:NICD fragments with restriction enzyme sites were ligated to retroviral vector pCLXSN and eukaryotic expression vector pEGFP-C1 to produce the pCLXSN-Notchl and pEGFP-C1-NICD vectors.(3) Screening of Hep-2 cell strains with stably expressed NICD:Virus supernatant fluid and polybrene were mixed and added to Hep-2 cells, screening of Hep-2 cell strains with stably expressed NICD was performed by using G418 (400μg/ml).(4) Identification of Hep-2 cell strains with stably expressed NICD:Relative expression levels of Notch1 and downstream target gene Hesl mRNAs and proteins were detected by Real-time PCR and Western blotting methods.(5) Localization of protein was observed by inverted fluorescent microscopy to study the activated status of Notch1 signaling pathway in laryngeal squamous cell carcinoma cell line Hep-2 cells.(6) Statistical analysis:The results of semi-quantitative RT-PCR and Western blotting were analyzed by using Gene Tools software. All experiments results were from at least three independently repeated experiments. The data were carried out with one-way analysis of variance using SPSS version 13.0, as described previously. In all statistical analyses, a P value<0.05 was considered as statistically significance. Results(1) Notch1 NICD retroviral vector pCLXSN-Notch1 and green florescent protein eukaryotic expression vector pEGFP-C1-NICD were successfully constructed.(2) Hep-2 cell strains with stably expressed Notch1 NICD were successfully screened out.(3) The result of RT-PCR demonstrated that expressions of Notchl and Hesl mRNAs were markedly up-regulated in Hep-2 cell strains with stably expressed Notch1 NICD.(4) The result of Western blotting revealed that expressions of Notchl and Hesl proteins were markedly up-regulated in Hep-2 cell strains with stably expressed Notch1 NICD.(5) Introducing of NICD fragment could obviously enhance the expression level of downstream target gene Hes1 mRNA and protein of Notch1 signaling pathway, which suggested that foreign NICD could activate Notch1 signaling pathway in Hep-2 cells.(6) Green florescence was observed mainly in cell nuclear of Hep-2 cells after transfected with pEGFP-C1-NICD, which further suggested that Notch1 signaling pathway was activated.Part 3 Effect of up-regulation of Notch expression on cell biology behavior of laryngeal squamous cell carcinoma cell line Hep-2 cellsMethods(1) Effect of activated Notch1 signaling pathway on cell proliferation was investigated by using new-style CCK-8 kit.(2) Effect of activated Notch1 signaling pathway on cell cycle and cell apoptosis was detected by Flow cytometry.(3) Effect of up-regulation of Notchl expression on cell migration ability was analyzed by using Boyden chamber experiment.(4) Expressions of cell cycle related genes, cell apoptosis related genes and cell migration related genes were analyzed by semi-quantitative RT-PCR and Western blotting methods.(5) Statistical analysis:The results of semi-quantitative RT-PCR and Western blotting were analyzed by using Gene Tools software. All results were from at least three independently repeated experiments. The data were carried out with one-way analysis of variance by using SPSS version 13.0, as described previously. In all statistical analyses, a P value<0.05 was considered as statistical significance.Results(1) Activation of Notch1 signaling pathway obviously inhibitted the proliferation of Hep-2 cells.(2) Activation of Notch1 signaling pathway arrested Hep-2 cell cycle at G0/G1 phase, which was closely related to the down-regulation of cyclin D1, cyclin E and cdk2 expressions and up-regulation of p53 expression.(3) Activation of Notch1 signaling pathway induced Hep-2 cell apoptosis, which was closely associated with the up-regulation of caspase-3 and caspase-9 expressions and down-regulation of Bcl-2 expression.(4) Activation of Notch1 signaling pathway decreased the Hep-2 cell migration ability, which was closely associated with the down-regulation of MMP-2 and MMP-9 expressions.Conclusion(1) Notch1 and Hesl may play an important role in the occurrence and development of laryngeal squamous cell carcinoma.(2) Overexpression of Notchl can arrest cell cycle, induce cell apoptosis and inhibit cell migration, which may be closely associated with changes of related genes and proteins expressions.(3) Regulation of Notchl gene may provide the theoretical basis for molecular target of laryngeal squamous cell carcinoma.
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