The Experimental Study Of Autophagy Gene Beclin1and Chemosensitivity To Cisplatin In Laryngeal Neoplasms

Background&ObjectiveLaryngeal cancer is one of the common malignant tumors in head and neck, anda serious threat to the health of patients. It is urgent to find effective treatment oflaryngeal cancer in clinical practice. Surgery, chemotherapy and radiation therapy areconventional treatment for laryngeal carcinoma, and gene therapy is regard as a newtreatment strategy for laryngeal carcinoma.Autophagy plays a critical role in removing damaged or surplus organelles inorder to maintain cellular homeostasis. For example, by removing damagedorganelles, autophagy may limit the exposure of cellular DNA to genotoxic-stressessuch as free radicals. The removal of damaged organelles through autophagicdegradation would thus decrease the basal mutation rate and suppress oncogenesis.Beclin1, the first identified mammalian autophagy gene, is essential to formation ofautophagosome. Some studies reported that Beclin1expression was down-regulatedin several carcinomas, and loss of Beclin1would contribute to an increased incidenceof cancer, such as heptocellular carcinoma, lung adenocarcinoma and lymphoma. It isunclear that correlation of autophagy gene Beclin1to tumorigenesis anddevelopment of laryngeal cancer.In the study, immunohistochemistry was employed to determine the expressionof Beclin1and explored its clinical significance in laryngeal cancer. We constructedthe eukaryotic expression vector pcDNA3.1/Beclin1and shRNA expression vectorpSUPER/Beclin1, transfected into Hep-2cells, to find the effect of Beclin1overexpression and downexpression on the growth, autophagy and apoptosis oflaryngeal cancer cell line Hep-2in vitro, and to investigate the role of the autophagyin the regulation of chemosensitivity to cisplatin in laryngeal cancer Hep-2cells, sowe explore the feasibility of induce of autophagy in treatment of laryngeal cancer. Methods1. The expression of Beclin1was detected with SP immunohistochemistry inspecimens of laryngeal carcinoma and normal laryngeal tissue. Correlations ofexpression of Beclin1gene to clinicopathologic factors of laryngeal carcinoma werestatistically analyzed;2. The eukaryotic expression vector pcDNA3.1/Beclin1and shRNA expressionvector pSUPER/Beclin1were constructed;3. The vectors were transfected into Hep-2cells via lipofectamine. Theexpression levels of Beclin1, LC3and p62mRNA and protein were detected byreal-time RT-PCR, Western Blot analysis in transfected cells. The autophagy,apoptosis and cell proliferations of Hep-2cells were measured with flow cytometry,Hoechs dye and MTT method after transfection;4. Hep-2cells were treated with cisplatin and the autophagysonmes weredetected by MDC analysis. The vectors, pcDNA3.1/Beclin1and pSUPER/Beclin weretransfected into Hep-2cells, then, treated with cisplatin, and cell proliferations ofHep-2cells were measured with MTT method.Results1. Beclin1protein expression in tumor cells were down-regulation in laryngealcancer, significantly lower than that in normal laryngeal tissue (P <0.05); theexpression of Beclin1has no correlation with clinic types (P>0.05), and correlationwith T stage, lymph nodes metastases and tumor histological grade (P <0.05);2. PCR analysis and DNA sequencing confirmed that the eukaryotic expressionvector pcDNA3.1(+)/Beclin1and shRNA expression vectors pSUPER/Beclin1wereconstructed successfully;3. The eukaryotic expression vector pcDNA3.1/Beclin1significantly improvedthe expressin of mRNA and protein of Beclin1and LC3,and decreased theexpressin of mRNA and protein of p62in Hep-2cells (P <0.05), the cellproliferations of Hep-2cell were inhibited, while more apoptosis cells and moreautophagy cells were identified in these cells; 4. The shRNA expression vectors pSUPER Beclin1significantly inhibited theexpressin of mRNA and protein of Beclin1and LC3,and increased the expressin ofmRNA and protein of p62in Hep-2cells (P <0.05), and the cell proliferations ofHep-2cell were improved, while less apoptosis cells and less autophagy cells wereidentified;5. Cisplatin significantly improved the expression of Beclin1and LC3protein((P <0.05)) and the pSUPER/Beclin1increased the sensitibity to cisplatin of Hep-2cell.ConclusionsBeclin1expression was down-regulated in laryngeal cancer, which may becorrelated with the occurrence and development of laryngeal cancer. Beclin1overexpression can induce autophagy and apoptosis in Hep-2cell, inhibittumorigenesis of Hep-2cell in vitro, and defective autophagy may contribute to thesensitibity to cisplatin of Hep-2cell.