The Research On Functions And Regulatory Mechanisms Of MicroRNA-203in Squamous Cell Carcinoma Of Head And Neck

【Background】Squamous cell carcinoma of head and neck(SCCHN) is the most common type of head andneck cancers which also include laryngeal carcinoma, oral carcinoma, nasopharyngealcarcinoma, hypopharyngeal carcinoma, paranasal sinus carcinoma, etc. Early-stageSCCHN is often curable by surgery or radiotherapy. However, the outcome for mostpatients with advanced disease has not improved much in the last20years, despitetherapeutic advances. Hence, elucidation of the mechanisms underlying the initiation andprogression of SCCHN is important for improving the prognosis and treatment options forpatients with this disease.MicroRNAs (miRNAs) are a class of small single-stranded non-coding RNAs. EachmiRNA consists of20-25ribonucleotides. miRNAs regulate gene expression at thepost-transcription level by binding with3’-untranslated regions (3’-UTRs) of targetmRNAs. A large number of miRNAs that are aberrantly expressed in cancer tissues havebeen functionally characterized as either tumor suppressive miRNAs or oncomirs by gain-of-function and loss-of-function experiments. Previous studies have shown thatmiR-203acts as a tumor suppressive miRNA in various cancers, but its roles in SCCHNremain elusive. We aim to investigate the biological functions and regulatory mechanismof miR-203in SCCHN.【Aims】1. To identify potential miR-203target genes using bioinformatic miRNA targetprediction tools and determine the regulation of the target genes by miR-203in SCCHNcells.2. To investigate the functions and underlying molecular mechanisms of miR-203in theproliferation of SCCHN cells.3. To determine the roles and underlying molecular mechanism of miR-203on themigration of human SCCHN cells.4. To investigate the effects and underlying molecular mechanisms of miR-203on theradiation sensitivity of human SCCHN cells.【Methods】1. We searched for potential miR-203target genes using bioinformatic miRNA target predictiontools and KEGG database, and identified a large number of candidates. We selected three genes（survivin, MAP3K1and DNA-PKcs）for further investigation. These genes are all known toplay roles in cell cycle progression, tumor metastasis and tumor’s radiation resistance.2. We analyzed miR-203’s binding sites in the target genes’ mRNA3′UTR by luciferasereporter assays. Next, the protein expression of these three genes(surviving, MAP3K1andDNAPKcs）in Hep-2-Lv-miR-203and Fadu-Lv-miR-203cells was determined by Westernblot assay. The regulation of these three genes’ mRNA expression by miR-203wasanalyzed by qRT-PCR.3. The correlation between miR-203and survivin expression was analyzed in40laryngeal carcinoma tissues by qRT-PCR. Colony formation assay, MTT assay, and cellcycle analysis by flow cytometry were performed to explore the regulatory effect andmechanism of miR-203on laryngeal carcinoma cell proliferation by targeting survivin.4. Transwell cell migration assays were performed to confirm the impact of miR-203on hypopharyngeal carcinoma metastasis. The interplay between MAP3K1and SRC-3inregulating hypopharyngeal carcinoma cell migration and metastasis was investigated byimmunofluorescence assay and pulse-chase assay and transwell cell migration/invasionassays.5. The roles of miR-203and DNA-PKcs on laryngeal carcinoma cell survival after γ-rayradiation was determined by cell apoptosis analysis using flow cytometry afterFITC-AnnexinⅤ/PI staining.【Results】1. Survivin, MAP3K1and DNAPKcs are the target genes of miR-203. Theexpression level of endogenous miR-203is negatively correlated with the mRNA andprotein expression levels of Survivin, MAP3K1and DNAPKcs in SCCHN cells.2. Ectopic expression of miR-203significantly reduced cell growth and colonyformation of Hep-2cells. Cell cycle distribution analysis demonstrated thatmiR-203-overexpressing Hep-2cells were blocked in G1phase, with a correspondingreduction in the percentage of S and G2/M phase.3. Transwell migration assay results showed that miR-203overexpression inhibitedhypopharyngeal carcinoma cell migration ability. Further, Both MAP3K1and SRC-3promote hypopharyngeal carcinoma cell migration ability. MAP3K1overexpressionincreased SRC-3protein level. MAP3K1increased SRC-3protein stability in akinase-activity dependent manner.4. Either miR-203overexpression or DNA-PKcs depletion enhancedγ-radiation-induced cell apoptosis.【Conclusions】1. Survivin, MAP3K1and DNA-PKcs are the direct targets of miR-203. MiR-203negatively regulates their expression.2. miR-203inhibits the proliferation of laryngeal carcinoma cells by directlytargeting survivin.3. miR-203expression is negatively correlated with cell migration ability inhypopharyngeal carcinoma cells. 4. MAP3K1can promote the metastasis of hypopharyngeal carcinoma cells byinhibiting SRC-3degradation.5. miR-203plays a role in laryngeal carcinoma cell’s sensitivity to radiation throughDNA-PKcs.6. Together, our results suggest that miR-203is an important tumor suppressor inhuman squamous cell carcinoma of head and neck.