Screening Of MicroRNAs Associated With The Invasion And Metastasis Of Human Laryngeal Carcinoma And Identification Of Its Candidate Tumor Biomarkers

Cancer of the larynx is one of the most common malignant tumor of head and neckcancer. Laryngeal cancer is the second most common cancer of the respiratory tract and squamous cell carcinoma accounts for about95%of all laryngeal cancer. With the growing industrialization andair pollution in China, the prevalence has gradually increased by about25%. Although the improvement of surgical and radiation therapy techniques as well as the application of chemotherapy and targeted medicine in the treatment of laryngeal carcinoma in the recent30years, its survival rate has not improved and even tends to decrease (about50%overall,30-40%in advanced cases). The therapeutic effect remains unsatisfactory.It is generally believed that the poor prognosis of laryngeal cancer is related to late stage diagnosis, recurrence and metastasis of disease. So far, the study results about the factors related to the poor prognosis of the laryngeal carcinoma varies. Better understanding of those factors affecting the survival of laryngeal carcinoma after surgical treatment would help to evaluate the prognosis of the patients and the individual therapeutic protocol making. Early diagnosis and detection of the recurrence as well as early application of the effective interference is the key. Although the advance of the imaging technique and the universe usage of the high definition endoscope provide useful tools for the clinical doctors to make early diagnosis of the disease, there are limitations for the traditional pathological diagnosis which only demonstrated the biological change at the cellular level. As the growing of the molecular biology, studies have found that tumors have different subtypes at the molecular level. These molecular patterns may associate with the different therapeutic response and prognosis of tumors. In order to improve the survival, it is pivotal to study the molecular mechanism of laryngeal carcinoma and to find out the characteristic target for treatment.microRNAs (miRNAs) are20to22nucleotides non-coding RNAs that incompletely bind the3’UTR of multiple target mRNAs, enhancing their degradation and inhibiting translation. miRNAs account for about2%of the whole human genom but more than30%of the gene are regulated by them. Accumulating evidence show that miRNAs play an important role in tumors.Each microRNA can regulate the hundreds of gene’s expression. The specific miRNAs can function as an oncogene or tumour suppressor and dysregulation of miRNAs have been linked to the aetiology, progression, invasion, metastasis and angiogenesis of cancer. Some studies have shown that miRNAs may play a critical role in carcinogenesis and progression of head and neck cancer, suggesting that miRNAs may have a potential clinical value of early diagnosis, treatment and prognosis in laryngeal cancer.It could be a novel biomarker and therapeutic target of laryngeal cancer.Latest techniques involved in the microRNA study such as microarray and some advance molecular biology technique provide very helpful tools for the study. The outstanding advantages of these techniques have been well documented in many of tumor research. The study of microRNA in laryngeal carcinoma is at the initial stage and has lots of shortcomings such as mostly a combination of all squamous cell cancer of head and neck rather a single individual cancer of them. Thus the results are vary significantly. Studies suggest the application of the modern advance techniques such as microarray and so on may help to find out the microRNAs and their possible target genes related to recurrence and metastasis of laryngeal carcinoma, and ultimately to develop novel monitoring system against early recurrence and metastasis of the tumor. Aimed at the problem mentioned above, this study was undertaken as following six parts.Chapter I Establishment and management of laryngeal carcinoma biobank for clinical and molecular biologic researchObjective:To establish a standardized laryngeal cancer biobank and provide high-quality specimens for laryngeal cancer studies and setup an information management system.Methods:The laryngeal cancer specimens were collected and preserved. Total RNA of a few specimens were extracted for quality control regularly. All patients were followed up. A systematic management of information of patients and collected specimens were conducted.Results:147Specimens of primary cancer and adjacent normal tissue,80plasma and serum samples were collected from patients with laryngeal cancer. The samples are high-quality. The SOP of collection, processing, storage of laryngeal cancer bio-specimens and information management system were setup and improved.Conclusion:A well-developed laryngeal cancer biobank is critical for research. It can preserve precious medical resources and provide high-quality specimens for laryngeal cancer studies. Chapter II Retrospective analysis of prognostic factors in205patients with laryngeal squamous cell carcinoma who underwent surgical treatmentObjectives:To investigate the most important factors affecting the prognosis of the patients with squamous cell carcinoma (SCC) of the larynx.Method:Based on the clinical and follow-up data,205patients with SCC of the larynx receiving total laryngectomy, partial laryngectomy, CO2laser surgery in GuangDong General Hospital were retrospectively analyzed. A survival analysis was performed by the Kaplan-Meier methodand a multivariable analysis of prognostic factors was carried out using the Cox proportional hazard model.Results:Subtypes of carcinoma included69.8%glottic and30.2%supraglottic. Most patients were in NO stage (77.6%), and22.4%patients were in N1-N3stage. Over half of the patients were in T1-T2stage (55.1%),20.0%in T3, and24.9%in T4. Mean follow-up duration was49.2months. The survival rates1,2, and3years after the surgery were99.0%,91.7%, and81.5%, respectively. The survival rate for those patients with clinical stage IV was significantly lower than for those with clinical stage I and II (p<0.001and p=0.013, respectively). Thedisease-free progression rates1,2, and3years after the surgery were83.9%,74.6%, and71.2%, respectively.Furthermore, those patients with a Charlson score of1to2and3had higher risk of mort-ality than those with Charlson score of0(hazard ratios of1.8and2.41p=0.042and p=0.008). Multivariable analysis revealed that clinical stage, surgical margin, and comorbiditywere significantly associated with both mortalityand disease-free progression.Conclusion:The surgical resection margin, clinical stage, and comor-bidity were independent factors affecting the laryngeal cancer prognosis. The survival rates werelower for patients with advanced laryngeal cancer, positive surgical margins, or severe comorbidity, suggesting the importance of early diagnosis, early treatment, negative surgical margins, and conditions of comorbidity. Chapter III Screening the differentiated expression microRNA of laryngeal carcinoma using microarray chip technologyobjective:To investigate the differential microRNA expression Profiling between laryngeal cancer and adjacent normal laryngeal mucosa.Methods:10paired laryngeal cancer tissue and adjacent normal laryngeal mucosa tissue were collected. Total RNA of the samples were extracted by miRNeasy mini kit and Trizol method, the RNA were tested by the fluorospectrophotometer to check quality and integrity. Using miRCURYTM Hy3TM/Hy5TM Power labeling kit to label RNA and hybridized. Axon GenePix4000B microarray scannerand was used to scan.All the data was analyzed with GenePix Pro6.0(Axon) software. The microRNA whose ratios of signal intensity were higher than2.0or lower than o.5were considered as remarkable differential expression. SAM software was used for data adjustment.Results:2662differentiated expression microRNAs were obtained by the microarray chip technology. After deletion of the non-human and hardly detectable microRNAs,780microRNAs remained, among which277were up-regulated and503were down-regulated. Using SAM software analyzed with FDR0.05, the data were adjusted. After adjustment11up-regulated and114down-regulated significantly differentiated expression microRNAs were obtained.Conclusion:1. It is feasible to use microarray chip technology for micro-RNA screening.2. To avoid statistic error during the data analysis, data adjustment using SAM software is necessary.3. There could be some false positive results of microarray test, further validation should be considered. Chapter IV Validation of the differentiated expression microRNAs from microarray chip screen by qRT-PCRObjective:To test the reliability of results from microarray chip.Method:32pairs laryngeal carcinoma and adjacent normal tissue were entered into this study. Firstly, total RNA was extracted from tissues by one-step Trizol method. let-7f-5p、miRNA-10a-5p、miRNA-125a-5p、miRNA-144-3p、 miRNA-195-5p、miRNA-203were selected to test and U6was served as endogenous control. Invitrogen RT-PCR kit was used to amplify and quantify each transcript microRNA and SYBR Green as fluorescent stain. Bio-Rad CFX96Managersoftware was used to data analysis, GraphPad software was used to plot the figures.Results:The results of qRT-PCR for the selected microRNAs showed the same trend as that in microarray test. let-7f-5p、miRNA-10a-5p、miRNA-125a-5p、 miRNA-144-3p、miRNA-195-5p、miRNA-203were significantly lower expression than their adjacent normal tissues (p<0.05). Among them miRNA-125a-5p、 miRNA-144-3p and miRNA-203showed the most significant changes.Conclusion:The results of qRT-PCR for the selected microRNAs were consistent with microarray’s. The results of microarray are reliable. Chapter V The effect of mir-144-3p on laryngeal carcinoma cell proliferation, invasion and migrationObjiective:To study the effect of mir-144-3p on cell proliferation, invasion and migration in laryngeal carcinomasquamous cell line Hep-2and search for its potential target gene.Methods:1.Base on the bioinformatics tool to search for mir-144-3p’s potential target gene.2. With cell transfection technique, the effect of mir-144-3p on Hep-2cell proliferation was observed by MTT assay, clony formation assay,and cell cycle analysis.3. The effect of mir-144-3p on Hep-2cell invasion and migration was detected by transwell assay,3D-culture assay and wound healing assay.4. A combination of qRT-PCR, Western blot, GFP reporter assay, and dual luciferase reporter assay were used to detect the effect of mir-144-3p on its possible target gene.Results:1.ETS-1could be the target gene of mir-144-3p according to the bioinformatics tools.2. mir-144-3p suppressed the Hep-2cell proliferation, invasion and migration.3. Mir-144-3p can inhibit the expression of ETS-1.4. Mir-144-3p enhanced the expression of some epithelial biomarkers such as, E-Cadherin and a-catenin and suppressed the expression of some interstitial biomarkers such as, Fibronectin and Vimentin.Conclusion:1. Down expression of mir-144-3p in the laryngeal carci-noma tissue suggested mir-144-3p might act as a antioncogene.2. Mir-144-3p inhibited the laryngeal carcinoma cell invasion and migration through targeted to ETS-1. Also, mir-144-3p could suppress laryngeal carcinoma cell prolif-eration.3. Mir-144-3p inhibited the laryngeal carcinoma cell invasion and migration through its action on the epithelia-mesenchymal transition (EMT). Chapter VI Expressions of ETS-1in Iaryngeal cancer and their prognostic significanceObjective:To investigate the expression and prognostic significance of transcription factor ETS-1(E26transformation-specific-1) in laryngeal carcinoma (LC).Methods:The expression levels of ETS-1was examined by immunohistochemisty in54cases of LC and adjacent normal tissue,34cases of atypical hyperplasia tissues.8cases of fresh frozen tissues were validated by qRT-PCR and Western blot.Results:The positive rates of ETS-1wwas significantly higher in laryngeal carcinoma than those in adjacent normal tissue respec-tively (P<0.001). The expressions of ETS-1was significantly related with the clinical stage, T stage, throid cartilage invasion and pathological differentiation (P<0.05). Inaddition, higher expression of ETS-1was correlated with worse survival situation (P<0.05)Conclusion:High expression of ETS-1in laryngeal carcinoma may be correlated with tumor progression and prognosis. But its mechanism is still unclear and needs further investigation.