| BackgroundHypopharyngeal cancer is common in head and neck malignant tumors, patients in early stage of this disease often feel no obvious symptoms. By the time of diagnosis, primary tumors often have involved theadjacent organs including the base of tongue, larynx, cervical esophagus, etc. Cervical lymph node metastases are often observed. Most patients have late clinical stages in their first visits.Surgical management of advanced hypopharyngeal cancer may affect pronunciation and swallowing functions thus severely compromise survival quality of patients.Chemo-radiotherapy is needed in case of some patients with unresectable tumor invasion of cervical arteries. At present, treatment of hypopharyngeal cancer is a comprehensive modes of surgery combined with radiotherapy and chemotherapy. Most patients need radiotherapy. However, decrease of radiosensitivity in tumor cells greatly limits the prognosis of radiotherapy.Radiosensitivity or resistance of tumor is regulated by some genes in vivo, thecell killing effect of radiation on tumor cells is mainly by ionizing radiation-induced DNA damage and apoptosis related gene and protein changes, by which apoptosis of tumor cells is started.radiation sensitivity differences in tumor cells depend on cytological types, cell differentiation, and expression of the regulatory genes.Analysis of differentially expressed genes may provide some theoretical basis for this disease.ObjectiveThrough the study of different doses of FaDu cells after X-ray irradiation, the radiation sensitivity analysis, and screened with radiosensitivity cell lines, provide experimental basis and experimental materials for the next experiment. Tumor cells differing responses to radiation is mainly due to the radiation sensitivity of different decisions, the radiation sensitivity and radiation induced gene expression and gene regulation. Therefore, the use of gene expression spectrum differences can be found with hypopharyngeal cancer radiosensitivity is closely related to the base for through the intervention of these genes, making pharyngeal individualized treatment regimens for cancer patients. Research with the whole human genome oligo poly nucleotide acid chip has been selected to have difference in radiosensitivity of FaDu cell lines were detected by the accept profiles of gene expression before and after the irradiation of the screened genes with differential expression and carries on the preliminary verification, to find and hypopharyngeal cancer radiosensitivity closely related gene.MethodsApplication of gradient ray dose (2Gy,4Gy,6Gy,8Gy,10Gy-each dose 2 times) to establish multiple irradiation of hypopharyngeal carcinoma radioresistant cell line FaDu-RS. By calculating the clone formation rate, difference analysis of FaDu cells and FaDu-RS cells by flow cytometry technology. Determine FaDu and FaDu-RS cell line difference in radiosensitivity by Beijing Boao crystal code Biotechnology Co., Ltd. Boao Jingxin lncRNA human gene expression microarray V4.0,4180K chip detection in FaDu cell lines and FaDu-RS cell lines exposed to line the gene expression profile changes, compared with the two radiation inducible gene expression differences. The use of Agilent Feature Extraction (v10.7) software for hybrid image analysis and data extraction. And then analyze the normalization and difference of the data using Agilent GeneSpring software. Looking for genes associated with hypopharyngeal carcinoma radiosensitivity, explore the molecular mechanism of radiation resistance.Results1. Clone formation experiment showed FaDu-RS and FaDu there was a significant difference in radiation sensitivity. FaDu-RS resistance to radiation significantly enhanced.2. The chip data have 1714 differentially expressed lncRNAs (compared with the FaDu,759 lncRNAs in FaDu-RS expression upregulated significantly,955 lncRNAs were significantly downregulated in the FaDu-RScell); in addition, have 2521 differentially expressed mRNAs (in FaDu-RS have 1089 mRNAs expression upregulated significantly,1432 mRNAs expression significantly downregulated). Combined with 0 hour,2 hours of chip analysis results for mRNA and lncRNA expression FaDu and FaDu-RS were different in time, we found that 212 mRNA common difference,85 lncRNA have similar significant differences.3. Application KOBAS (KEGG orthology based annotation system) prediction analysis, pathway analysis indicated that the biological functions of differentially expressed mRNAs were related to 44 cellular pathways that may be associated with radiosensitivity. GO analysis revealed that 247 mRNAs involved in biological processes,15 mRNAs involved in cellular components, and 41 mRNAs involved in molecular functions. In addition, we found 132 lncRNA and mRNA were identified as coregulated transcripts.Conclusions1.The radiosensitivity of FaDu-RS cells compared with FaDu cells with low radiation resistance.2.LncRNA and mRNA in FaDu and FaDu-RS in response to radiotherapy are differentially expressed, FaDu-RS radiosensitivity of cells molecular mechanism is extremely complex, is the result of multiple genes and pathways synergistically relates to cell communication cell, tumor necrosis factor pathway, post-translational modification of protein and small molecule RNA transcription silencing, p53 signaling pathway of various genetic and biological change.3.Cluster analysis of differentially expressed genes may be related to FaDu cell radiosensitivity is closely related. To have significant differences in the expression of genes and pathways regulated by further research will be helpful to understand pharyngeal cancer radiation tolerance in molecular biology mechanism, in order to enhance the sensitivity of radiotherapy provide new targets and strategies. |
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