Expression Of Long Non-coding RNA MYOSLID In Head And Neck Squamous Cell Carcinoma And Mechanism Of Promoting Metastasis

Background:Head and neck squamous cell carcinoma(HNSCC)is one of the most common malignancies of epithelial origin in the world(Chi AC et al.,2015).Local recurrence and distant metastasis were one of the main reasons that leads to death in about 80%-90%advanced HNSCC.The epithelial to mesenchymal transition(EMT)program has been considered play an essential role in cancer metastasis.Recently,scholars have observed that cancer cells at the leading edges of primary tumor in aggressive HNSCC only acquire partial traits of mesenchymal cells but still retain the traits of epithelium cells,which is referred to as partial EMT(P-EMT).Many lncRNAs have been found played important roles in regulating the EMT program.However,no lncRNAs were founded that were associated with the P-EMT program.Therefore,this study will focus on searching for key lncRNAs based on the key gene SLUG of the P-EMT program,expecting to provide more evidences for further clarify the nature of the P-EMT program and developing targeting strategies.Part I.Screening of Long Non-Coding RNA Associated with Partial Epithelial-Mesenchymal Transformation in Head and Neck Squamous Cell Carcinoma.Objective:To identify key key lncRNAs involved in the regulation of P-EMT using bioinformatics methods,and analyzing their biological functions and clinical significance.Methods:In this study,RNA-sequencing(RNA-Seq)profiles of Asian HNSCC were downloaded from the TCGA and GEO database,and differentially expressed genes(DEGs)or non-coding RNAs were screened out using the R language.Based on the gene expression matrix of HNSCC obtained from TCGA database,we then screened out SLUG associated lncRNAs with the absolute value of Pearson correlation coefficient is greater than 0.4.There were 9 overlapping lncRNAs between the DEGs or lncRNA from the TCGA database,GEO datasets and SLUG associated lncRNA.We then performed a survival analysis for the 9 overlapping lncRNAs.Gene Ontology(GO)function annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis for the MYOSLID associated mRNAs were performed.The clinical value of MYOSLID expression in HNSCC was also analyzed based on the clinical information download from the TCGA database.Results:9128 DE-lncRNAs or DEGs were screened out from the TCGA database,and 819 DE-lncRNAs or DEGs were screened out from the GEO database,and 54 lncRNAs related to SLUG were identified.Among the 9 lncRNAs,only MYOSLID was proved to be associated with the survival prognosis of HNSCC patients.The results of GO terms and KEGG pathways analysis show that MYOSLID is a lncRNA with powerful biofunctions,and most of the associated DEGs mainly enriched in the pathway that in charge of invasion and metastasis and biological phenotype of cancer cells a.Abnormal MYOSLID expression is related to the TNM stage and advanced clinical stage,and can act as an independent prognostic factor according to the results of univariate and multivariate regression analysis based on the clinical data downloaded from the TCGA database.Conclusions:MYOSLID was identified as a long-noncoding RNA that associated with the P-EMT program.Part II.Long Non-Coding RNA MYOSLID Expression in Head and Neck Squamous Cell Carcinoma and Its' Clinical Values.Objective:To verify the differentially expression of MYOSLID in HNSCC tissue samples and cell lines,and explore its' clinical values and correlation with key molecules involved in the P-EMT program.Methods:Real-time fluorescent quantitative PCR(RT-qPCR)was used to detect the expression level of MYOLID in fresh head and neck squamous cell carcinoma tissues and paired adjacent mucosal tissues.In site hybridization(ISH)was used to evaluate MYOSLID expression in our constructed HNSCC microarray.Univariate and multivariate analyses were used to analyze its' relationship with clinicopathological parameter and prognostic value.SLUG,PDPN and LAMB3 expression were detected by immunohistochemistry(IHC),and correlations were analyzed using Pearsons'correlation analysis.Conclusions:The high expression of MYOLID in human HNSCC may be a biomarker for predicting higher risk of metastasis.MYOLID expression in HNSCC is correlated with the P-EMT program.Part ?.The Biological Function of Long Non-Coding RNA MYOLID and Mechanism of Promoting Metastasis in Head and Neck Squamous Cell Carcinoma.Objective:To study the effects of MYOSLID on the metastasis of human HNSCC cell lines and explore potential mechanisms.Methods:We evaluated the presence of P-EMT in Cal27,SCC4,SCC9 and TCA81134 HNSCC cell lines and selected out the ones closest to the P-EMT status for further study.The invasion and metastasis ability were evaluated after knockdown MYOSLID with siRNA in Cal27 and SCC4 cell line by wound-healing and transwell assay.The changes of epithelial marker E-cadherin,mesenchymal marker Vimentin,and P-EMT markers SLUG,PDPN and LAMB3 expression levels were detected with Western blot.The siRNA MYOSLID-homo-323's sequence with the highest knockdown efficiency was selected to design lentiviral vectors containing short hairpin RNA(shRNA)of MYOSLID.The lentivirus vector of MYOSLID-shRNA was stably transfected into Cal27 cell line and constructed MYOSLID stably knocked out cell Cal27 cell line.Subsequently,Cal27 cells with MYOSLID knockdown and controls were injected into nude mice through vein,then the numbers of metastatic nodules on the surface and hematoxylin-eosin(HE)staining tissue section of liver and lung were counted.At last,MYOSLID expression was detected in Cal27 and SCC4 cell line after treated with PI3K signaling pathway activator 740 Y-P.Results:After knockdown MYOSLID,the invasion and migration ability of HNSCC cells were significantly reduced,and the expression levels of SLUG,PDPN and LAMB3 was significantly downregulated,while the E-cadherin and Vimentin remain unchanged.MYOSLID stably knockout Cal27 cell line was successfully constructed.The result of HE staining of liver and lung tissue showed that the amount of tumor metastases in the shRNA-MYOSLID group was significantly less than that of the control group.The MYOSLID expression level in the Cal27 and SCC4 cell lines increased significantly after treated with PI3K pathway activator 740 Y-P.Conclusions:MYOSLID affect the distant metastasis of HNSCC by regulating the P-EMT program.Upregulation of MYOSLID might is associated with aberrant activation of the PI3K signal pathway.