The Research Of The Expression And Function Of Beclin1and LC3in Hypopharyngeal Squamous Cell Carcinoma

Hypopharyngeal squamous cell carcinoma (HSCC) is one of the most common head and neck malignant cancer, and about5%of all head and neck carcinomas. The main symptoms of HSCC are pharyngeal foreign body sensation, deglutation barrier, odynophagia and dysphagia, tumor infringing laryngeal cavity can cause hoarseness and dyspnea. Hypopharyngeal cancers are about95%squamous cell carcinomas and usually poor differentiation. With the characters of high infiltration and easy submucosal diffusion, symptoms are not obvious in early stage. Therefor, the majority of patients are advanced at clinical diagnosis stage, often involving the tongue, larynx and cervical esophagus, and usually occur lymph node metastasis. In recent years, with the development of treatment technology of head and neck caner. Despite several comprehensive therapies including surgery, radiotherapy and chemotherapy currently available, The5-year survival rate is estimated to be at25%to40%, lack of diagnostic and prognostic markers hampers the management of this dismal disease. Therefor, there is a need to acquire deeper investigation of the biological characteristics of HSCC, the search for predictive molecular markers and novel methods of diagnosis and treatment based on molecular biology become an urgent issue to be resolved.Autophagy, a type of non-apoptotic cell death, is characterized by the delivery of cytosolic materials and organelles to lysosomes for bulk degradation. This evolutionally conserved process includes three major intracellular pathways in eukaryotic cells, macroautophagy, microautophagy and chaperone-mediated autophagy (CMA), we usually refers to macroautophagy. It is implicated in tumor growth and progression, and has been explored as a potential therapeutic target. The role of autophagy and its underlying molecular mechanism is still controversial in cancer, especially in tumor progression. Autophagy occurs at a basal level and can be induced to promote cell survival under conditions of nutritional deprivation, thus acting as a cytoprotective role. However, autophagy may also cause cell death when activated by certain stimuli, making it a potentially deleterious process.Approximately30genes have been identified to regulate autophagy in yeasts, with16homologues in humans. Among these, Beclin1and LC3(microtubule-associated protein1A/1B-light chain3) play important roles in autophagy in mammalian cells. Beclin1is a mammalian orthologue of the yeast Apg6/Vps30gene, and Beclin1functions as a scaffold for the formation of the PI3K complex, one of the first components recruited during the development of autophagosomes. Liang et al. reported reduced Beclin1expression in human breast cancer tissues, Beclin1stable transfected human MCF7cells, the down-regulation of expression of Beclin1inhibited cell proliferation in vitro and tumorigenesis in nude mice. Beclin1play a tumor suppressor role in human breast cancer. Clelia et al. revealed that Beclin1was significantly decreased in human malignant melanoma cells. Furthermore, Beclin1down-regulation linked to autophagy defect may be associated with malignant phenotype and poor prognosis of hepatocellular carcinoma. The loss of Beclin1expression was detected in ovarian cancer, lung cancer and brain cancer. In contrast, overexpression of Beclin1has been reported to correlate with progression of gastric and colorectal cancer, Beclin1has a carcinogenic effect.LC3(microtubule-associated protein1A/1B-light chain3) is a mammalian homologue of yeast Atg8. It is activated and processed by an ubiquitination-like reaction, and is regulated by Atg3and Atg7. LC3consists of a soluble form, LC3-I, with a molecular weight of18KD and a lipidated form, LC3-II, with a molecular weight of16KD, and has three isoforms, LC3A, LC3B, and LC3C in mammalian cells. LC3-II and the LC3B isoenzymes are implicated in autophagy. Various stressors, such as hypoxia, starvation and chemotherapy, upregulate LC3expression and promote the binding of cytosolic LC3-I to phosphatidylethanolamine to form autophagosome-specific LC3-II, LC3-II is localized on autophagosomes. Therefore, LC3, especially the LC3-II is considered a reliable marker of autophagy. Aoki et al. reported that Anti-LC3B can be used as an effective tool to detect glioblastoma cells and tissues. Recent studies revealed that reduced LC3-II expression in lung cancer and cervical squamous cell carcinoma, but LC3expression is increased in esophageal and gastrointestinal neoplasms. We therefore speculate that the status and role of LC3expression vary across tumor types.However, the expression of Beclin1and LC3has not been characterized in hypopharyngeal squamous cell carcinoma (HSCC). In the current study, we examined the expression of Beclin1and LC3at both the mRNA and protein levels in hypopharyngeal squamous cell carcinoma tissues and paired non-cancerous tissues and further studied whether Beclin1and LC3expression correlated with patient clinicopathological characteristics and prognostic factors. The down regulation of Beclin1expression in FaDu cells was established by Beclin1-siRNA, then test the effects of Beclin1on the apoptosis and proliferation of hypopharyngeal squamous cell carcinoma (HSCC) FaDu cells. Part1Aberrant expression of Beclin1and LC3correlates with poor prognosis of human hypopharyngeal squamous cell carcinomaObjective:To investigate the expression of the autophagic genes Beclin1and LC3in hypopharyngeal squamous cell carcinoma (HSCC) and their correlation with patient clinical characteristics and prognosis.Methods:1. Eighty-two surgical hypopharyngeal squamous cell carcinoma specimens and fifty-four adjacent non-cancerous mucosal epithelial tissues were obtained.2. Beclin1and LC3-II expression was examined by immunohistochemistry in82HSCC specimens and54adjacent non-cancerous tissues.3. Total RNA was isolated from54tumor tissues and paired non-cancerous mucosal epithelium tissues, and was reverse transcribed to cDNA. The mRNA ranscript levels of Beclin1and LC3-II were measured in HSCC specimens and adjacent non-cancerous tissues by real-time RT-PCR.4. Proteins were extracted in RIPA lysis buffer, the protein level of Beclin1and LC3-II were measured in HSCC specimens and adjacent non-cancerous tissues by Western blotting assays.5. Statistical analysis was performed using the GraphPad Prism5.0(GraphPad Software Inc., USA) and SPSS16.0(SPSS Inc., USA). Wilcoxon matched pairs test was used to compare mRNA and protein of Beclin1and LC3in tumor vs. non-tumor tissues. The relationship between categorical variables was analyzed by Chi-square test or Fisher’s exact test. Spearman’s test for non-normal correlation coefficients was used to evaluate the correlation between Beclin1and LC3expression. Overall survival curves were drawn using Kaplan-Meier method, and differences between the curves were analyzed by the log-rank test. Univariate and multivariate analyses of prognostic factors were performed by the Cox proportional hazards regression model. For all tests, a two-sided P value<0.05was considered statistically significant.Results:1. We examined the expression of Beclin1and LC3in hypopharyngeal squamous cell carcinoma tissues by immunohistochemistry. Beclin1and LC3were observed mainly in the cytoplasms or cytomembranes of hypopharyngeal squamous cell carcinoma cells and adjacent normal squamous epithelial cells. In addition, LC3was occasionally found in the nuclei of cancer cells and normal epithelial cells. Beclin1was positively expressed in42.7%(35/82) of HSCC specimens (adjacent non-cancerous tissues,79.6%,43/54; P<O.0001). Furthermore,41.5%(34/82) of HSCC specimens exhibited high LC3immunoreactivity (adjacent non-cancerous tissues,74.1%,40/54; P=0.0002).2. Beclin1and LC3-II mRNA transcript levels were significantly lower in HSCCs than in paired non-cancerous tissues (P<0.0001, P=0.0001, respectively). A positive correlation was observed between the mRNA transcript levels of Beclin1and LC3-II in HSCCs (r=0.51, P<0.0001;95%CI:0.273to0.689).3. Similarly, western blotting assays showed that Beclin1and LC3-II were markedly decreased in HSCCs (P=0.02, P=0.004, respectively).4. Significant correlation was found between negative Beclin1expression and advanced clinical stage (r=0.299, P=0.005), increasing T stage (r=0.365, P=0.0004), poor differentiation (r=0.392, P=0.0006) and cervical lymph node metastases (r=0.302, P=0.004). Similarly, LC3level decreased in hypopharyngeal squamous cell carcinoma tissues of patients with advanced T stage (r=0.245, P=0.022), poor differentiation (r=0.504, P<0.0001) and cervical lymph node metastases (r=0.275, P=0.01).5. The survical rates of Beclin1negative tumors were significantly lower than of patients with beclin-1positive tumors (P<0.0001). Patients with low LC3level had a significantly poorer prognosis than high LC3patients (P=0.0145). Univariate Cox regression analysis showed that clinical stage (P=0.0009), T stage (P=0.0038), lymph node metastasis (P=0.0007), Beclin1expression (P<0.0001) and LC3level (P=0.018) were significantly associated with overall survival. In the multivariate Cox regression analysis, Beclin1expression was an independent prognostic factor.Conclusion:Beclin1and LC3-II are downregulated in HSCCs and their aberrant expression correlates with patient clinical characteristics and poor prognosis of HSCCs. Beclin1expression was an independent prognostic factor. The autophagic genes Beclin1and LC3-II could be novel therapeutic target for further treatment of HSCC. Part2Beclin1knockdown effects on apoptosis and proliferation of hypopharyngeal squamous cell carcinoma FaDu cellsObjective:To investigate the effects of Beclin1knockdown on the cell proliferation, apoptosis and invasion of hypopharyngeal squamous cell carcinoma (HSCC) FaDu cells.Methods:1. Beclin1-siRNA was transfected in FaDu cells by using lipofectamine2000, inhibition expression of beclin1was tested by Real-time PCR and western blotting analysis.2. Cell apoptosis were measured with flow cytometry after transfenction.3. Cell proliferation was determined by MTT after transfection treatment with Beclin1-siRNA.4. Transwell assay was used to evaluate the invasion and migration of the treated cells.Results:1. Real-time PCR and western blot analysis demonstrated successful silence of Beclin1after Beclinl-siRNA was transfected into FaDu cells. The mRNA and protein levels of LC3-II were significantly lower in the Beclin1-siRNA group than the control group (P<0.05). Compared with the control group, LC3-II protein levels were markedly decreased in Beclin1-siRNA group (F=0.003).2. Compared to the control group, downregulation of Beclin1increased cell apoptosis (P=0.001). The percentage of apoptosis was17.11±0.46%for treated groups and4.773±0.3%for negative groups.3. There was no significant influence on cell proliferation by knockdown of Beclin1after24h,48h,72h and96h (P>0.05).4. Transwell assay showed that the transfer rate of FaDu cells was1.844±0.035、1.918±0.029and3.306±0.362for the control group, the negatitive group and si-RNA group, respectively. Knockdown of Beclin1bviously increased cell invasiveness (P=0.02).Conclusion:Inhibiton of autophagy by Beclin1knockdown promotes HSCC FaDu cells apoptosis, migration and invasion. But there was no significant influence on the FaDu cells proliferation although Beclin1inhibition. Our findings may have important implications for the treatment of HSCC patients.