Screening, Identification And Molecular Mechanism Of Migration Adhesion Of Collagen Ⅵ In Laryngeal Cancer

Introduction: Laryngeal squamous cell carcinoma(LSCC)is one of the most common malignant tumors in Otorhinolaryngology,accounting for about 20% of all head and neck malignancies.In China,especially in the northeast,the incidence of laryngeal cancer increased by.The malignant degree of Laryngeal Carcinoma is high,and it is easy to metastasize and recur.It is generally believed that its occurrence is related to such factors as smoking,drinking,environmental pollution,virus infection and occupation.Studies have shown that EGFR,Cyclin D1,COX-2,EGF,STAT3,C-MYC,p53,P16 and JAB1 are involved in the development and progression of Laryngeal Carcinoma.However,the molecular mechanism of laryngeal carcinoma is still unclear.Up to now,there are no ideal molecular markers for the diagnosis and treatment of Laryngeal Carcinoma.The study of the molecular mechanism of tumorigenesis and metastasis,including laryngeal carcinoma,is still the key and difficult point in tumor research.Therefore,the study of the molecular mechanism of Laryngeal carcinogenesis is helpful to find the molecular markers of diagnosis,treatment and prognosis of Laryngeal Carcinoma,which has potential clinical significance.Weighted Gene Co-Expression Network Analysis(WGCNA)is an efficient and accurate method for bioinformatics data mining.In practical applications,co-expression network analysis can identify highly expressed gene modules as an effective method for mining and presenting gene expression patterns in different samples The hub genes(genes that are most closely related to other genes in the gene network and play a key role in the gene network)can be used to extract information from the modules The relationship between the key genes in a gene module or module and the characteristics of the sample concerned by researchers is discussed.Collagen Ⅵ is a widely distributed extracellular matrix protein and is highly expressed in many cancers.It is beneficial to the growth and development of tumors.Extracellular matrix is often released from regulation and disorganization in the tumor microenvironment,thereby contributing directly to cancer progression by promoting cancer cell growth and metastasis and indirectly by educating other microenvironmental components.Type Ⅵ collagen is a major ECM protein,composed of three main polypeptide chains α1(Ⅵ),α2(Ⅵ)and α3(Ⅵ),which are encoded by different genes COL6A1,COL6A2 and COL6A3 respectively.The expression of COL6A1,COL6A2 and COL6A3 transcripts and the level of type Ⅵ Collagen in tumor tissues were much higher than that in normal tissues.This study suggests that MYCT1 mediates the expression of FOXP3 and Collagen Ⅵ in laryngeal carcinoma to exert the potential function of tumor suppressor genes.Based on the above research,I intend to answer the following questions under the guidance of my tutor: 1 how to use WGCNA to analyze the data of throat cancer related sequencing and chip to obtain the gene set with research potential? What is the level of expression of Collagen Ⅵ related genes in Laryngeal Carcinoma? how and to what extent does Collagen Ⅵ-related genes influence the biological behavior of Laryngeal Carcinoma? Collagen Ⅵ-related genes are regulated by MYCT1 to exert their biological function in laryngeal carcinoma cells?In this study,we intend to use the LIMMA and WGCNA packages in R to analyze and validate the published chip and sequencing results.Then,bioinformatics was used to identify the potential diagnostic molecular biomarkers of Laryngeal carcinogenesis and progression,and validated in vitro.The aim of this study was to investigate the effect of Collagen Ⅵ gene on the biological behavior of laryngeal Carcinoma Cells.Materials and Methods: 1.Materials: human laryngeal squamous cell carcinoma cell line Hep-2,GSE6631,TCGA-HNSCC.2.Methods: data acquisition(GEO,TCGA)and R studio analysis,overexpression of MYCT1 in laryngeal carcinoma cells third generation RNA-seq,cell culture,transient gene transfection method,Real-time PCR assay,cell adhesion assay,Western blot method,Luciferase reporter assay,Transwell and wound healing assay and statistical analysis.Results:The COL6A1,COL6A2 and COL6A3 genes related to Collagen Ⅵ were successfully enriched in LSCC,and their functions and pathways were also enriched.We hypothesized the role of Collagen Ⅵ in the regulation of Laryngeal Carcinoma.The expression quantity and biological function of the related genes were verified.The differentially expressed genes were extracted by Lipma package and the weighted network was enriched by WGCNA package.In GSE6631,DEGs were 903 and enriched into 7 modules,while in TCGA-HNSCC,DEGs were 843 and enriched into 9 modules.After cross-comparison,the functions and channels are enriched by using David and Cytoscape plug-in ClueGO.The results were confirmed by LSCC sequence data GSE123275,and the genes of COL6A1,COL6A2,COL6A3 were identified.To construct the overexpression vector of MYCT1 gene,RNA interference(RNAI)and the Corresponding Control Short fragment(NC)as the control,and transfect Hep-2 cells respectively,and detect the expression of MYCT1 gene and protein level to test the transfection efficiency Collagen Ⅵ protein and related genes were detected to verify the data analysis results.REAL-TIME PCR and Western blot results showed that the expression level of Collagen 6a1,Collagen 6a2,Collagen 6a3(HEP-2)in laryngeal carcinoma cells was significantly lower than that in Hep-2 cells(p 0.05).There was a negative correlation between them.The results of TargetScan and Pictar show that there are FoxP3 binding sites in the 3’-utr region of COL6A1,COL6A2 and Col6a3,which suggests that COL6A1,COL6A2 and Col6a3 may be potential FoxP3 target genes.The results of luciferase reporter assay indicated that FOXP3 can bind to the promoter region of COL6A2 and COL6A3 to enhance its transcriptional activity.REAL-TIME PCR and Western blot results showed that MYCT1 Sirna transfected into Hep-2 cells decreased significantly the level of MYCT1 Mrna and protein(p 0.05)48 hours after transfection,suggesting that specific Sirna could effectively knock down the expression of MYCT1 gene.Transwell and wound healing assays showed that MYCT1 overexpressed in Hep-2 cells was significantly decreased in MYCT1 Sirna transfected group(p 0.05)The heteroadhesion assay showed that MYCT1 overexpressed in Hep-2 cells decreased significantly in MYCT1 Sirna transfected group(p 0.05)Conclusions:1.Gene modules determined by WGCNA based on database were biologicall y meaningful.WGCNA can be used as a tool to detect the molecular mechanisms of laryngeal cancer and to explore potential biomarkers.2.Collagen Ⅵ is highly expression in Laryngeal carcinoma,negatively correlated with MYCT1 expression,promotes heterogeneous adhesion and migration,suggesting that it plays an oncogenic role in laryngeal cancer.3.FoxP3 acts as a transcription factor that binds to the COL6A2 and COL6A3 promoter region and promotes its transcription.4.FoxP3 is highly expression in Laryngeal carcinoma,positively correlated with Collagen Ⅵ but negatively correlated with MYCT1 expression,promotes heterogeneous adhesion and migration,suggesting that it plays an oncogenic role in laryngeal cancer.5.MYCT1 partially inhibits the expression of Collagen Ⅵ by inhibiting the expression of FoxP3,and suppressed the adhesion and migration of laryngeal cancer cells.