MicroRAM-106b Regulates The Tumor Suppressor Gene RUNX3in Laryngeal Carcinoma

Laryngeal carcinoma is a common head and neck malignancy with highincidence as it accounts for approximately2.4%of new malignancies worldwideevery year. Although most early-stage laryngeal cancer could be cured via surgeryor radiotherapy, the overall prognosis for advanced stage Laryngeal carcinoma isstill poor. To further improve the survival and cure rate, the carcinogenicmechanisms of laryngeal carcinoma need to be elucidated.Despite the fact thatconsiderable effects have been made in rencent years, the molecular mechanisminvolved in the initation and progression of laryngeal carcinoma remains largelyunknown.MicroRNA(miRNA) are recently characterised, endogenous phylogeneticallyconserved, small RNA molecules of approximately21-25nucleotides encoded in thegenomes of plants and animals in length. They can regulate target gene expressionby pairing homologous sequences within the3-untranslated region (3’UTR) ofmessenger RNAs, thus preventing or impairing their translation or promoting RNAdegradation. MiRNAs may control the majority of all human genes that are involvedin a wide variety of biological processes, such as cell development, proliferation,death, and metabolism. Emerging evidence has been indicated that miRNA plays animportant role in a variety of pathogenic conditions, including cancer development.Using the miRNA microarray, we first performed the miRNA profiles in ten pairedlaryngeal carcinoma and normal laryngeal tissues. Statistical analysis identified29miRNAs that were differentially expressed in all ten tumor tissues compared withthe matched non-neoplastic controls, such as miR-106b. We focused on the role of miR-106b in the development of laryngeal carcinoma.Part MiR-106b expression in laryngeal tissuesObjectiveTo examine the difference of miR-106b expression in laryngeal tissues.MethodsIsolation of the RNA of14paired laryngeal carcinoma tissues and the adjacentnormal tissues samples. The expression of miR-106b in laryngeal tissues weremeasured with stem-loop RT-PCR technology.ResultsMiR-106b were overexpressed in laryngeal carcinoma tissues compared to thepaired adjacent normal tissues.ConclusionHigh expression of miR-106b may function as an oncomir and also correlatewith laryngeal carcinoma development. Part Role of miR-106b in laryngeal carcinoma cellsObjectiveTo determine the role of miR-106b in laryngeal carcinoma cells in vivo and invitro.MethodsWe transfected the cells with miR-106b ASO or the miR controls. Then theexpression level of miR-106b in laryngeal carcinoma cells (Hep-2and TU212) weremeasured using stem-loop RT-PCR. The cell proliferation was measured by colony formation assay, and the invasion ability were detected by the Transwell chamber kit.In vivo proliferation experiment was performed in nude mice.ResultsThe expression level of miR-106b was decreased approximately80%in cellstransfected with ASO-miR-106b when compared with that in miR control cells. Aftertransfected with miR-106b ASO, The cell viability, colony formation and theinvaison abilities was decreased in vivo and in vitro.ConclusionMiR-106b was overexpressed in laryngeal carcinoma cells. MiR-106b mayfunction as an oncomir and also correlate with tumor development and progression.MiR-106b promoted the cell proliferation and invaison abilities. Part Runx3is a direct target of gene miR-106bObjectiveTo determine if Runx3is a direct target of miR-106b.MethodsSelection of candidate miRNAs for binding to RUNX3were identified usingthree web-based bioinformatic algorithms. To determine which miRNA have themost effect on regulation of RUNX3, a luciferase reporter assay was performed.Western blot was used to detect the expression level of target gene RUNX3. Tofurther determine whether miR-106b directly regulates RUNX3, anotherluciferase-reporter assay was performed to validate the target site in the RUNX3UTR.Results 1.11candidate miRNAs were identified.2.MiR-106b has the most effect on the down-regulation of the luciferaseintensity of pGL3/Luciferase-RUNX3-3’UTR.3.The RUNX3protein level was increase approximately3fold in Hep2cells and2fold in TU-212cells transfected with ASO-miR-106b when compared with that inmiR control transfected cells.Meanwhile, the RUNX3protein level was decreasedapproximately40%in Hep2and70%in TU-212cells transfected with miR-106bmimics when compared with that in control cells.4.The intensity of luciferase in cells transfected with miR-106b mimics wassignificantly decreased compared to that in control cells,whereas the intensity ofluciferase in cells transfected with ASO-miR-106b was significantly increased.Incontrast, the luciferase intensity from the reporter vector containing the mutatedRUNX3-3’UTR was not affected by transfection with miR-106b mimics or miR-106bASO.ConclusionRUNX3was a direct target gene of miR-106b. Part IV RUNX3is regulated by miR-106b inlaryngeal carcinomaExperiment1Relationship between RUNX3hypermethylation inpromoter region and its protein expression in laryngeal carcinomaObjectiveTo investigate the relationship between Runx3methylation and its protein expression, and its possible relationship with genesis of carcinoma Larynx.MethodsMethylation-specific PCR was used to detect methylation of RUNX3at itspromoter region. The expression level of RUNX3protein in these unmethylated andmethylated laryngeal carcinoma tissues was measured by Western Blot. The RUNX3protein of expression in the laryngeal carcinoma irrespective of its methylation statusin CpG islands compared to the normal laryngeal epithelium and the adjacent tumortissues were performed by immunhistochemistry assay.Results41.67%tumors were fully methylated at the RUNX3CpG islands, RUNX3wasdownregulated in larygngeal carcinoma whether or not it was methylated.ConclusionRUNX3was downregulated in laryngeal carcinoma, and imply us there may beanother mechanism that may attributed to its downregulation. Experiment2Role of RUNX3in laryngeal carcinoma cellsObjectiveOverexpression of RUNX3to study the role of RUNX3in laryngeal carcinomacells growth and invasion.MethodsRUNX3was overexpressed using an RUNX3expression plasmid (pCMV6/RUNX3). Transfected the pCMV6/RUNX3plasmid into laryngeal carcinoma cells,the expression of RUNX3was measured by Western Blot, the proliferation and invasion abilities were measured.ResultsOver-expression of RUNX3inhibited the cell growth and invasion of laryngealcarcinoma cells.ConclusionOverexpression of RUNX3could partly reverse the cell viability,colonyformation and invasion abilities in laryngeal carcinoma cells. Experiment3miR-106b targets and regulates RUNX3expression in the larygngeal carcinoma cell lines.ObjectiveTo validate the ability of miR-106b to promote the proliferation and invasion ofHep2and TU-212cells by regulating RUNX3.MethodsHep2and TU-212cells were co-transfected with miR-106b ASO and RUNX3siRNA plasmid, the cell colony formation and invasion abilities were measured. Theexpression level of RUNX3protein was measured by western blot.ResultsWhen Hep2and TU-212cells were cotransfected with ASO-miR-106b andRUNX3-siRNA, the inhibition rate of colony formation and cell invasion viabilitycaused by transfection of ASO-miR-106b was counteracted by the expression ofRUNX3-siRNA but not by transfection with a control vector.ConclusionMiR-106b promoted the cell proliferation and invaison abilities by the suppression of RUNX3.