| Background:Head and neck cancer is a major health problem in the world. Approximately 650000 cases are diagnosed each year, about 3 billion 500 million people died. In the past 30 years, the incidence of head and neck cancer increased year by year, so far, has become the world's fifth most common cause of tumor and tumor related deaths of the sixth. Head and neck cancer is a heterogeneous disease, with different histological and differentiation, including from breathing different anatomical sites (lip, oral cavity, nasal cavity, paranasal sinuses, nasopharynx, oropharynx, tongue and throat) of malignant tumor. This type of tumor can be subdivided into cancer, sarcoma, melanoma and lymphoma by histologic origin. The pathogenesis of head and neck squamous cell carcinoma (HNSCC) multi phase and multi factors of, accounting for about 85% of all malignant tumors of head and neck of the molecular pathogenesis of HNSCC in fully understanding the variety of carcinogenic factors leading to the root causes of HNSCC occurs and the development of targeted HNSCC treatment means great. Epithelial membrane protein 1(EPM1) is found to regulate epithelial cell proliferation and differentiation, and associated with the developments of several types of neoplasms. However it's necessary to clarify the potential role of EPM1 in the tumor genesis of HNSCC and the specific mechanism.In recent years, the rapid development of high-throughput microarray and sequencing technology, produced a large number of genetic data, the number of biological information in order to adapt to the rapid expansion of high-throughput gene expression data and growing people sharing data, various databases emerged, GEO database is a public database of the world's largest storage high-throughput molecular abundance data NCBI founded, involving more than 1 billion 600 million genes expression abundance data, covering a wide range of biological content, and is completely free to share, to analyze the specific gene expression and biological function of corresponding has become an important part of bioinformatics is currently using the GEO database, this study relates to the HNSCC on GEO data sets for screening analysis clear expression of EMP1 in HNSCC tissue changes, and analysis of the patients with HNSCC TNM stage and tumor The role of EMP1 in the pathogenesis of HNSCC and related molecular mechanisms were further elucidated.Methods:(1) we first retrieve the HNSCC data in the GEO database, and the classification and retrieval of tongue cancer, laryngeal cancer, oral cancer and nasopharyngeal carcinoma and other common HNSCC data sets, carefully screened information research object information platform and related data set, select the detection platform probe containing EMP1 gene data sets and data sets the study included HNSCC and normal tissue, HNSCC patients with TNM staging and tumor patients with HNSCC pathological classification information, statistical analysis of the data obtained by the expression of EMP1 gene in the database, the fabric and control differences, the expression of EMP1 gene in different TNM stage and tumor pathological grade in HNSCC tissues the expression of EMP1 gene in HNSCC group; (2) we selected HNSCC chip contains a variety of common head and neck cancers and normal tissues of the head and neck, free Immunohistochemical detection of EMP1 protein in HNSCC tissue and normal tissue expression differences of EMP1 gene in different TNM stage and tumor pathological grade in the HNSCC organization; (3) to construct EMP1 gene expression vector by biological engineering technology, with head and neck squamous cell carcinoma cell line, positive clones were screened and identified culture EMP1 gene expression is significantly increased, the eventual establishment of over expression of EMP1 in head and neck squamous cell carcinoma cell line; (4) the third part of the establishment of the detection of EMP1 expressing cells and control cells in cell proliferation and in vitro migration and cell biological behavior changes, then we analyzed and selected EMP1 co-expression genes in 1036 tumor cells in CCLE database, and the correlation between EMP1 gene and the expression levels of co-expression genes was analyzed in TCGA HNSCC database, GO analysis and KEGG pathway analysis were also used in analyzing EMP1 co-expression genes, finally Western blotting was used to detect the changes of EMP1 co-expression genes in HNSCC cells as EMP1 overexpression, clear expression of EMP1 gene in head and neck squamous cell carcinoma cell proliferation and migration, and early Step by step to explore the molecular mechanism.Results:(1) we selected objective data sets, analysis of EMP1 in HNSCC tissues and normal tissues the expression level and difference in different TNM stage, tumor grade HNSCC expression difference, found in the data set of GSE6631, GSE58911, HNSCC group and normal control pair the expression level of EMP1 in HNSCC tissues was significantly lower than that of normal control, P values were:<0.0001,<0.0001, GSE33205, GSE59102 in the data set, GSE51985 and GSE10774 in HNSCC tissues and normal tissues from different patients, the expression level of EMP1 in HNSCC tissues was significantly lower than the normal control group. The P values were: <0.0001,<0.0001,0.0508 and 0.0114. In the data set analysis of differences between the expression level of EMP1 in different TNM stage and tumor grade HNSCC in GSE30788 and GSE39366, results showed that the expression level of T EMP1 in different stages of HNSCC tissues had no significant difference. The expression of EMP1 in different N stages in the HNSCC organization focused on two data inconsistency in data in GSE30788, the expression level of NO is higher than the level in the HNSCC expression of EMP1 in N+patients with HNSCC tissues, while in the data set in GSE39366, NO and N+in HNSCC tissue, the expression level of EMP1 was no significant difference between the expression levels of EMP1 in different tumor pathological grade HNSCC tissues and found that with the degree of differentiation increased EMP1 gene expression levels gradually increased, the difference was statistically significant, P= 0.0001 and 0.0064; (2) immunohistochemical detection of EMP1 HNSCC in tissue microarray The expression levels revealed that the expression level of EMP1 protein in normal tissues was significantly higher than the expression level in HNSCC tissues, with significant statistical difference, T expression in different stages of HNSCC tissues had no significant difference, N expression in different stages of HNSCC tissues was no statistically significant difference in the expression level. Differentiated HNSCC tissues is higher than the expression in poorly differentiated HNSCC tissues, there is significant difference; (3) by gene engineering technique successfully constructed EMP1 expression vector, transfection of HNSCC cells with high expression, screening and identification of EMP1 HNSCC cells, the proliferation ability of HNSCC cells was found significantly decreased 48h, the absorbance value was only 50% in the control group, the difference was statistically significant, but also found that over expression of EMP1 and HNSCC cell lines In vitro migration capacity decreased significantly and Transwell assay 24 hours through the basement membrane of the cell number were:empl over expression group (34.8±10.6), blank control group (83.5±12.1) and negative control group (73.5±15.3), the difference was statistically significant; (4) we analyzed and selected EMP1 co-expression genes and analyzed inTCGA HNSCC database the correlation between EMP1 gene and the RNA expression levels of co-expression genes, it showed that the RNA expression levels of EPHA2? ITGA3 and ANXA1 were related to EMP1 expression and the difference was statistically significant, in the cell experiments the protein expression levels of EPHA2?ITGA3 and ANXA1 were significantly changed as EMP1 overexpression.Conclusions:(1) empl gene in HNSCC expression level was significantly lower than that in normal control tissues expression levels; (2) empl gene in HNSCC tissue expression level increased with the degree of differentiation increased and increased; (3) overexpression emp1, HNSCC cell proliferation and migration ability decreased and the possible molecular mechanism was related to the expression level changes of EPHA2? ITGA3 and ANXA1. |
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