The Study Of Tumor-derived Small Extracellular Vesicles And FOXD1 In The Development Of Oral Squamous Cell Carcinoma & Case Report

Part 1.Study on the role and mechanism of oral squamous cell carcinoma-derived small extracellular vesicles on normal epithelial cells Objective: To investigate the role and mechanism of OSCC-derived sEVs on normal epithelial cells.Methods: Differential ultracentrifugation was used to isolated OSCC-derived sEVs.Transmission electron microscopy(TEM),dynamic light scattering(DLS)and Western blot were used to identify the shape,particle size and protein markers of the extracted sEVs.HIOEC uptake of sEVs from OSCC was observed by immunofluorescence.Then,wound healing,Transwell migration assay,CCK-8 and Ed U were used to analyze the effects of sEVs on migration,invasion and proliferation of HIOEC.HIOEC treated with sEVs was observed the changes in the size and morphology of spheres in 3D culture conditions.Next,the expression of Ki67 and E-cadherin of spheres frozen section were detected by immunofluorescence staining.The changes in apoptosis in HIOEC treated with sEVs were analyzed by flow cytometry.Western blot was used to detect apoptosis related markers PARP,cleaved-PARP,caspase3,cleaved-caspase3,BCL-2,BAX and EMT-related markers E-cadherin,N-cadherin,vimentin of HIOEC after stimulation of sEVs.The morphology and ultrastructure of HIOEC after stimulation of sEVs were observed by microscope and TEM.The expression of p53 and PTEN in HIOEC treated with sEVs was detected by Western blot and immunofluorescence.The p53-targeting miRNA was predicted by bioinformatics analysis,and the miRNA content of sEVs,HIOEC and OSCC cells were screened out by q RT-PCR.Dual-luciferase was used to test whether miRNA could target p53.After transfection of HIOEC with miRNA mimics and inhibitors,the regulatory effect of p53 in sEVs of OSCC was verified,and the changes of EMT and apoptosis related markers of transfected cells were detected by Western blot.Results: The results of TEM,DLS and Western blot showed that sEVs were successfully extracted and purified.The results of wound healing and Transwell migration assay showed that OSCC-derived sEVs promoted the migration and invasion of HIOEC.Moreover,CCK-8 and Ed U assays showed that the proliferation ability of sEVs stimulated cells were enhanced.Compared with the control group,sEVs-treated HIOEC formed larger and irregular spheres under 3D culture condition.Immunofluorescence of frozen sections showed increased expression of Ki67 and decreased expression of E-cadherin in sEVs-treated HIOEC.The results of flow cytometry showed that the apoptosis rate of HIOEC was decreased when treated with sEVs.Meanwhile,the results of Western blot showed that sEVs reduced apoptosis and promoted EMT process of HIOEC.In addition,the morphology of HIOEC transformed from oval into spindle.The nuclear-cytoplasmic invaginations and multiple nucleoli were observed in the treated epithelial cells which are typical characteristics of premalignant oral lesions.Mechanistically,OSCC-derived sEVs down-regulated the expression of tumor suppressor genes p53 and PTEN.Bioinformatics analysis and q RTPCR results demonstrated that miR-let-7c may be involved in sEVs caused phenotype transformation of HIOEC.Dual-luciferase assay showed that miR-let-7c could target and inhibit the expression of p53.Mi R-let-7c mimics inhibited the apoptosis and promoted the EMT process of HIOEC while miR-let-7c inhibitor played the opposite roles.Conclusion: OSCC-derived sEVs promoted the malignant transformation of normal oral epithelial cells,in which miR-let-7c/p53/PTEN axis played important roles.Part 2.Study on the role and mechanism of FOXD1 in oral squamous cell carcinoma Objective: To explore the role and mechanism of FOXD1 in the progression of OSCC.Methods: Western blot and q RT-PCR were used to detect the expression of FOXD1 in HIOEC,malignant transformation HIOEC,OSCC cell lines CAL27,SCC25 and HN4.GEPIA web tool was used to analyze the survival analysis and the expression of FOXD1 of OSCC patients and normal patients.Immunohistochemical staining was used to detect the expression of FOXD1 in 60 OSCC tissues and 8 normal mucosal specimens.After knockdown of FOXD1 by sh RNA and overexpression of FOXD1 by lentivirus,the changes of EMT and cell stemness in tumor cells were analyzed.Finally,FOXD1 overexpressed and knockdown CAL27 cells were prepared and xenotransplantation mice model was used to further study the role of FOXD1 in tumorigenicity.Results: In this study,we found that FOXD1 was upregulated in OSCC when compared with normal samples.Patients with a higher FOXD1 expression had a poorer overall survival and disease-free survival.Immunohistochemical staining results showed that FOXD1 expression was related to the clinical stage and relapse status of OSCC patients,but not to the gender,age and pathological stage.When FOXD1 was knocked down in CAL27 and SCC25 cells,the migration,invasion,colony formation,sphere formation,and proliferation abilities were decreased.Moreover,the EMT and stemness-related markers changed remarkably,indicated that the EMT process and cell stemness were inhibited.Conversely,overexpression of FOXD1 promoted EMT and cell stemness.Further study demonstrated that FOXD1 could bind to the promoter region and activate the transcription of SNAI2.Knockdown of SNAI2 in CAL27 and SCC25 inhibited EMT and cell stemness.The in vivo study showed that FOXD1-overexpressing CAL27 cells possessed stronger tumorigenic ability.Conclusion: FOXD1 promotes EMT and cell stemness in OSCC through transcriptional activation of SNAI2.