| Generalized oral squamous cell carcinoma(OSCC)is one of the major pathological types in head and neck cancer(HNC).Although it is relatively easy to find,but there are still a lot of OSCC cases diagnosed until late clinical stage.In addition,despite some progress in its diagnosis and comprehensive treatment,OSCCs still have a poor overall prognosis.The identification of effective biomarkers and therapeutic targets,as well as a better understanding of the mechanisms underlying the occurrence and progression of OSCC,is currently the top priority.Long non-coding RNAs(lncRNAs)are RNA transcripts>200 nt but do not coding proteins.As a research hotspot,lncRNAs have been proved to be differentially expressed in various cancer types such as OSCC,involved in a variety of regulatory pathways,and have influences on a number of cell biological functions.Therefore,lncRNAs are expected to become biomarkers for tumor diagnosis and prognosis,and have the potential to become new targets for tumor therapy.To study the differential expression of lncRNAs in OSCC in transcriptome level,to better understand the roles lncRNAs play in OSCC carcinogenesis and development,and to explore its mechanism,this study first discovered a set of differentially expressed lncRNAs in OSCC using public database mining,then the lncRNA LINC02487 with the highest expression difference was selected for in-depth study,to explore the function and the regulatory mechanism of its influence on cell migration and invasion in OSCC cell lines.This thesis is divided into four parts:Part Ⅰ Screening and validation of differentially expressed lncRNAs in OSCCObjective:To screen differentially expressed lncRNAs in OSCC in the whole transcriptome level,and to further analyse and validate the lncRNAs with large differential expression.Methods:1.The chip data from the public database were searched,the training data set was screened out,and the probe sets mapped to lncRNAs were matched to compare the differential expression profiles of lncRNAs between OSCC and the control group;2.Independent data sets was used for verification,to screen out the stably expressed lncRNAs with large differences in expression;3.Online interactive database and bioinformatics analysis tools(DAVID)were used for target prediction and pathway enrichment analysis of lncRNALINC02487,which is the most differentially expressed lncRNA.Results:1.Between 167 OSCCs and 45 healthy oral mucosa controls,there were 790 probe sets(representing 658 lncRNAs)significantly differentially expressed;Fourteen lncRNAs were differentially expressed in all validation data sets,and LINC02487 was 14 times down-regulated;2.The functional cluster analysis based on computer target prediction showed that LINC02487 may be involved in biological processes such as cell movement,ion channel signaling and ubiquitination modification,which provided a reference for further study of its biological functions.Conclusion:A group of differentially expressed lncRNAs were found in OSCC,among which LINC02487 was the most differentially expressed.The function prediction in silico was performed.Part Ⅱ The effects of lncRNA LINC02487 on the growth,migration and invasion of OSCC cellsObjective:To test the expression level of LINC02487 in OSCC clinical samples and cell lines,and to preliminarily explore its biological function in OSCC cell lines.Methods:1.The relative expression levels of lncRNA LINC02487 in OSCC tissue samples and cell lines were detected by RT-qPCR,and the relationship between LINC02487 and clinical and pathological features was analyzed;2.Lentivirus transfection was used to construct LINC02487 overexpressing OSCC cell lines;3.Cell function changes were observed by cell proliferation,clone formation,cell cycle,apoptosis,wound healing,migration,and invasion detection.Results:1.Compared with the matched paratumor control,the expression of LINC02487 was significantly down-regulated in 50 OSCC samples from the Chinese population,and its expression level was correlated with lymph node metastasis.Meanwhile,compared with immortalized normal oral epithelial cells,the expression of LINC02487 was also significantly down-regulated in 6 OSCC cell lines;2.Overexpression of LINC02487 significantly inhibited cell proliferation ability,clone formation ability,wound healing ability,cell migration and invasion ability of OSCC cells,and increased the proportion of cells in G1 phase,but had no significant effect on cell apoptosis.Conclusion:The expression of LINC02487 was decreased in OSCC samples and cell lines.Overexpression of exogenous LINC02487 can inhibit the proliferation,migration and invasion of OSCC cells.Part Ⅲ Study on the mechanism of lncRNA LINC02487 inhibiting cell migration and invasion of OSCC cellsObjective:To explore the mechanism of LINC02487 affecting the migration and invasion of OSCC cells,and to further clarify its role in the metastasis of oral cancer.Methods:1.The localization of LINC02487 in cells was determined by RNA fluorescence in situ hybridization;2.The binding protein of LINC02487 was identified by Chirp-MS,and confirmed by RNA immunoprecipitation assay.The intracellular position of LINC02487 and USP17 was observed using dual fluorescence co-localization experiment;3.The expression of EMT-related proteins after overexpression of LINC02487 was detected by Western Blot assay;4.Transwell assay and Western Blot assay were used to explored the effect of silencing LINC02487 and silencing USP17 on cell migration and invasion,and rescue experiment was conducted to further verify.Results:1.RNA FISH results showed that LINC02487 was localized in the cytoplasm and clustered around the nuclear membrane.2.The CHIRP-MS assay showed that LINC02487 was directly bound to the deubiquitination enzyme USP17,and the RIP assay verified it.The double fluorescence co-localization assay showed that the intracellular localization of LINC02487 and LINC02487 was highly overlapped.3.After the overexpression of LINC02487,the epithelial marker E-cadherin protein level was increased compared with control,while the mesenchymal marker N-cadherin and Vimentin protein levels were decreased,and the key EMT regulatory factor SNAI1 protein level was decreased;4.Silencing LINC02487 increased the ability of cell migration and invasion,while silencing USP17 decreased them.Silencing LINC02487 and USP17 simultaneously restored the ability of cell migration and invasion to the control level.Western Blot results showed the protein levels changed accordingly.Conclusion:LINC02487 directly binds to USP 17,thereby affecting the EMT process and cell phenotype.It may acts as a tumor suppressor by regulating USP 17-SNAI1 axis.Part Ⅳ The observation of lncRNA LINC02487 inhibit the experimental lung metastasis of OSCCObjective:To further clarify the effect of LINC02487 on the experimental lung metastasis of OSCC cells in vivo.Methods:1.Stably LINC02487 overexpresseion OSCC cell line HN30-OE was used to construct an experimental lung metastasis model using tail vein injection in nude mice,and the changes of activities,food intake and body weight of nude mice were observed and recorded.2.Gross specimens and HE slides were observed to evaluate the effect of overexpression of LINC02487 on tumor-forming ability of experimental lung metastases in nude mice.Results:1.OSCC cells overexpressed LINC02487 group of nude mice was not significantly emaciated,body weight increased significantly compared with the control group;2.The lung surface of the LINC02487 overexpression group was nodular,while the lung of the control group showed significant solid nodular fusion,and the lung weight of the former group was significantly lower than that of the control group.Lung HE slides showed that the tumor in the control group showed a tumor fusion growth,and the tumor area percentage was higher than that of the overexpression group.Conclusion:Overexpression of LINC02487 inhibits tumorigenesis ability of experimental lung metastases in nude mice,suggesting that LINC02487 inhibits distant metastasis of OSCC. |
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