| BackgroundOral squamous cell carcinoma（OSCC）is one of the most common malignant tumors in the head and neck,with a high incidence in Asia.Most OSCC patients are already in the advanced stages of the disease at the time of diagnosis.Although progress has been made in comprehensive treatments,including surgery,radiotherapy,and chemotherapy,the five-year survival rate for OSCC patients only remains at about 50%,and 25%-50%of patients will have local relapses and distant metastasis after treatment.Cetuximab,as a targeted therapy for OSCC,can improve oncologic outcomes in combination with conventional chemotherapeutic agents,but the overall response rate of patients is poor.Recently,immune checkpoint blockers have been approved for the treatment of patients with advanced and metastatic head and neck squamous cell carcinoma（HNSCC）to improve prognosis.However,the results of clinical trials have shown poor objective response rates in patients,possibly due to a deficiency of CD8+T cells present in the tumor microenvironment.Thus,it is necessary to elucidate the molecular mechanisms of OSCC development,and identify efficient therapeutic response and prognostic biomarkers and new therapeutic targets.The generation of massive omics data makes data-driven research possible.Highthroughput sequencing technologies as well as bioinformatics approaches have been applied as a significant tool in various aspects of tumor research and clinical treatment,including tumor heterogeneity,tumor drug resistance,tumor immune microenvironment,characterization and identification of tumor neoantigens,and biomarker discovery.Through mining and exploring a large number of clinical sample data from different independent cohorts,researchers can understand tumor development patterns at the molecular level.Based on a data-driven research paradigm,the purposes of this study are to:1.Explore new molecular mechanisms of occurrence and development of OSCC and find potential therapeutic targets.2.Investigate the interrelationship between OSCC and tumor microenvironment.3.Search for immunothrerapeutic response and prognostic biomarkers in OSCC.Part Ⅰ Histone lysine methyltransferase SMYD3 promotes tumorigenicity of oral squamous cell carcinoma through transcriptional enhancement of HMGA2 expressionTumorigenesis involves the reacquisition of developmental programs that give rise to cells with unlimited proliferative,self-renewing potential.In this process,the impact of epigenetic dysregulation,in addition to genetic level variation,cannot be ignored.Chromatin regulators can dynamically regulate chromatin structure to affect gene expression in an epigenetic manner in response to intra-and extracellular signaling.Aberrant alterations in these regulators may recode the chromatin epigenomic landscape,which in turn leads to tumorigenesis.Therefore,exploring the chromatin regulators that are abnormally altered during OSCC development and their mechanisms of action can provide theoretical support for the development of effective OSCC treatment strategies.We used machine learning and other methods to screen 429 chromatin regulators and found that histone lysine methyltransferase SMYD3 was significantly overexpressed in OSCC,and its overexpression was associated with poor patient prognosis.SMYD3 belongs to the histone methyltransferase family,which regulates the methylation levels of histones and non-histones,and influences the transcriptional activity of genes,playing an important role in tumorigenesis development.However,the biological functions and regulatory mechanisms of SMYD3 in OSCC development and malignant proliferation are still not elucidated.Research objectives1.To investigate the altered expression of SMYD3 in OSCC and its clinical significance.2.To analyze the molecular regulatory mechanism of SMYD3 in promoting OSCC development and malignant proliferation.3.To reveal the clinical transformation value of SMYD3 targeted inhibitor BCI-121 in OSCC treatment.Methods and results1.SMYD3 is identified as an OSCC hub gene and its high expression suggests poor prognosis of patientsWe performed differential gene expression analysis between OSCC and paraneoplastic tissues in the database to obtain differentially expressed chromatin regulators.Lasso logistic regression and Boruta machine learning algorithm were used to identify chromatin regulators that are closely related to tumorigenesis.Univariate and multifactorial Cox regression analyses as well as randomized survival forest results showed that SMYD3 had the greatest effect on patient survival outcomes.Then,we explored using K-M survival curves in different datasets and found that high SMYD3 expression was associated with shorter survival time in OSCC patients.Thus,SMYD3 was identified as a target gene for this study.2.SMYD3 expression is upregulated in OSCC and correlates with tumor malignancyBioinformatics analysis,real-time quantitative PCR（real-time quantification polymerase chain reaction,RT-qPCR）,Western blotting,and immunohistochemical staining were performed by public databases and collected clinical samples,and it was found that in comparison with paraneoplastic tissues,tumor tissues SMYD3 expression was found to be abnormally high in tumor tissues compared with paraneoplastic tissues,and SMYD3 expression was gradually upregulated with the increase of tumor malignancy.Moreover,SMYD3 expression tended to be higher in male patients,patients with a history of smoking and drinking,and tumor tissues with higher tumor stage,TP53 mutation,classical epithelial subtype,and hypomethylation subtype OSCC tissues,and in addition,SMYD3 copy number tended to be amplified in OSCC tissues.The results of immunofluorescence experiments showed that SMYD3 was mainly distributed in the nucleus and cytoplasm of OSCC cells.3.SMYD3 is associated with OSCC stemness maintenance and cell proliferationUsing single-cell sequencing data and the ssGSEA algorithm,we found that SMYD3 expression was positively correlated with stemness score and proliferation score at the cellular level as well as at the tissue level.Subsequently,we performed RNA-seq sequencing of CAL-27 cells transfected with the NC group and SMYD3 siRNA group,analyzed the two groups of samples for differential gene expression,and performed GO and KEGG analysis of the differentially expressed genes obtained,which showed that they were mainly enriched in gene transcriptional regulation,cell differentiation,cell proliferation regulation,stem cell differentiation,cell growth factor,cell metastasis,and epithelial-mesenchymal transition（EMT）and tumor-related signaling pathways.Finally,the data obtained from RNA-seq sequencing were again validated for cell function and pathways using GSEA algorithm.4.SMYD3 promotes OSCC stemness maintenance and cell proliferationThe results of stemness sphere formation assay showed that SMYD3 interference significantly impaired the ability of tumor stemness sphere formation.The results of clone formation and EdU assays revealed that SMYD3 interference inhibited OSCC cell proliferation.western blotting assays showed that SMYD3 deletion decreased tumor stem cell markers such as c-MYC,BMI1 and NANOG,and CCND1 expression.In addition,we found that reduced SMYD3 expression suppressed H3K4me3 levels,while overexpression of SMYD3 promoted stemness spheroidogenesis and cell proliferation ability of OSCC cells.Stable transfected CAL-27 cell line was injected subcutaneously into both sides of the back of nude mice to construct a subcutaneous tumorigenic model,and the results showed that SMYD3 interference impaired tumor formation and inhibited tumor weight and volume in mice.Immunohistochemical staining showed that the expression of Ki67 protein,a proliferation-associated molecule,was reduced in the tumor tissues of SMYD3-interfered mice.5.SMYD3-targeted inhibitor BCI-121 inhibits OSCC cell growth and proliferationIn OSCC cells,BCI-121,an inhibitor of SMYD3,was able to inhibit SMYD3 methyltransferase activity without altering its expression level.In vitro experiments showed that BCI-121 significantly inhibited stemness maintenance,growth and proliferation of OSCC cells,and reduced the expression of tumor stem cell markers such as c-MYC,BMI1 and NANOG,as well as CCND1.Intratumoral injection of BCI121 in nude mice effectively suppressed OSCC tumor cell growth and Ki67 protein levels in vivo.6.HMGA2 is a potential key effector molecule for SMYD3-mediated functionThrough ChIP-seq sequencing,self-organizing mapping neural network,protein mutual network construction,and MCODE algorithm,we finally identified three potential genes that could be directly regulated by SMYD3:HMGA2,CCND1,and SNAI1.Through correlation analysis,we found that SMYD3 was co-expressed with HMGA2 in OSCC and in pan-cancer.As a result,we hypothesized that HMGA2 is a potential effector molecule for SMYD3-mediated function.7.HMGA2 is highly expressed in OSCC and associated with poor prognosisMeta-GEO dataset analysis indicated that HMGA2 expression was significantly upregulated in tumor tissues compared to normal tissues.K-M survival curves showed that patients with high HMGA2 expression had poor prognosis compared to those with low expression.We examined HMGA2 expression in clinical samples collected by RTqPCR,Western blotting and immunohistochemical staining,and confirmed that HMGA2 was overexpressed in tumor tissues,and the expression level increased with the increase of OSCC malignancy.In addition,SMYD3 was positively correlated with HMGA2 at the RNA level as well as at the protein level.Immunofluorescence experiments confirmed the intranuclear localization of HMGA2 in OSCC cells.8.SMYD3 promotes HMAG2 transcriptional expression by increasing H3K4me3 modification in the promoter regionSMYD3 interference meaningly inhibited HMGA2 expression at the RNA level and protein level,while overexpression of SMYD3 promoted HMGA2 expression.Similarly,HMGA2 expression was suppressed by adding BCI-121 inhibitor.Subsequently,we confirmed that SMYD3 regulates HMGA2 expression by affecting the histone lysine methylation level in the promoter region of HMGA2 through ChIPqPCR and dual luciferase reporter assays.Rescue experiments exposed that HMGA2 inhibition reversed the promotion of stemness spheroidogenesis in OSCC cells by SMYD3.Conclusions and implicationsIn this study,we utilized a multidimensional research approach including bioinformatics,machine learning and biological experiments to find that high expression of the chromatin regulator SMYD3 in OSCC promotes tumor stemness maintenance and cell proliferation,which is associated with poor patient prognosis.Furthermore,SMYD3 promotes tumorigenesis in OSCC by transcriptionally regulating HMGA2 expression through histone methylation modification,and its targeted inhibitor BCI-121 can effectively inhibit cell tumorigenicity,which has potential clinical application.This study provides an experimental basis for further elucidating the molecular mechanism of OSCC development and finding new targets for OSCC treatment.Part Ⅱ SNARE protein YKT6 mediates cell invasive and metastasis and CD8+T cell infiltration in oral squamous cell carcinoma as a potential biomarker for prognosis and immunotherapyIn the first part of this dissertation,we analyzed the molecular mechanism of OSCC occurrence and malignant proliferation,which may be one of the reasons for poor patient prognosis.In addition,OSCC is highly aggressive and usually as a results of cervical lymph node metastasis,while the level of immune cell infiltration and immunogenicity of OSCC tend to be poor and the response rate to immunotherapy is low,leading to poor patient prognosis.Due to above reasons,there is an urgent need to investigate the molecular mechanisms of OSCC invasive metastasis and to explore potential immunotherapeutic response and prognostic biomarkers.Multi-omics analysis based on clinical samples can reflect tumor heterogeneity and is vital for identifying immunotherapeutic response and prognostic biomarkers.In this study,we combined HNSCC/OSCC large sample cohort data with weighted gene co-expression network construction and survival analysis methods to uncover the potential value of SNARE protein YKT6 for predicting immunotherapy response and patient prognosis.YKT6 has been reported to be involved in intracellular vesicle transport,exosome production and release,and autophagic vesicle and lysosomal fusion,but its function in tumor progression,especially in antitumor immunity,still unclear.Research objectives1.To investigate the abnormal expression level of YKT6 in OSCC and its clinical significance.2.To reveal the role of YKT6 in the malignant development of OSCC and the molecular regulatory mechanism.3.To investigate the relevance of YKT6 to the immune microenvironment in OSCC.Methods and results1.Weighted gene co-expression network identifies red gene module as an OSCCrelated hub moduleWe used the CIBERSORT algorithm in combination with gene expression data from OSCC patients to calculate CD8+T cell infiltration levels,and then used the weighted gene co-expression network to identify red module genes as highly correlated with pN staging and CD8+T cell infiltration levels.KEGG pathway analysis showed that these genes were associated with extracellular matrix-receptor interactions and cell adhesion.2.YKT6 is identified as a hub gene affecting OSCC prognosisAnalysis of gene expression differences between OSCC and paraneoplastic tissues in the database yielded 946 genes with upregulated expression.The 100 genes with the highest connectivity in the red module were intersected and 29 candidate genes were obtained.The truncation values were then obtained using the R language "survminer"package for K-M survival analysis and log-rank test.Finally,by using Ranger random forest and sliding window method,we determined that high YKT6 expression was associated with unfavorable survival outcome,and defined YKT6 as the target gene.By univariate and multifactorial Cox regression analysis,we found that high YKT6 expression was a significant risk factor for patient survival.Furthermore,we integrated YKT6 expression with tumor stage,two independent predictors of survival,to draw a nomogram model.The results of area under the subject working curve showed that the model was able to predict patient survival excellently.3.YKT6 expression is upregulated in malignant HNSCC/OSCCIn the public database,we found that YKT6 was expressed higher in tumor tissues than normal control tissues by statistical methods such as single-cell sequencing data analysis,chi-square test,and Fisher’s exact test,and that YKT6 overexpression was associated with HNSCC/OSCC progression,recurrence,immune dysfunction,tumor purity,and poor prognosis.The results of genomic data analysis revealed that altered copy number and DNA methylation levels may be potential mechanisms for the upregulation of YKT6 expression.Subsequently,we performed Western blotting and immunohistochemical staining experiments using the collected clinical samples,which showed that YKT6 expression was higher in OSCC tissues than in paraneoplastic tissues and was upregulated with increasing pN stage.4.YKT6 is involved in OSCC cancer-related signaling pathwaysPerforming GSVA analysis in OSCC dataset,we found that EMT,TGF-βsignaling pathway,hypoxia,tumor signaling pathway,protein secretion pathway,TNFa signaling pathway,and NF-κB signaling pathway were significantly enriched in the YKT6 high expression group.In contrast,T-cell and B-cell receptor signaling pathways were enhanced in patients in the YKT6 low expression group.5.YKT6 promotes OSCC cell invasion and migration in vitroThe results of cellular wound healing assay showed that YKT6 knockdown inhibited OSCC cell migration.transwell assay showed that cell invasion and migration ability were reduced when YKT6 was disturbed.western blotting assay showed that MMP9 protein expression decreased in OSCC cells as YKT6 expression decreased.In addition,ELISA experiments showed that TGF-β1 protein secretion was reduced when YKT6 expression was downregulated.6.High YKT6 expression indicates low level of CD8+T cell infiltration in OSCCTIMER2 database analysis showed that high YKT6 expression was significantly negatively correlated with the level of CD8+T cell infiltration in HNSCC patients.Meanwhile,YKT6 gene copy number alteration affects the infiltration level of CD8+T cells.We calculated the tumor immune microenvironment-related scores of OSCC patients using CIBERSORT,EPIC,and ssGSEA algorithms,and found that YKT6 expression was negatively correlated with the level of CD8+T cell infiltration,immunerelated indexes,and the expression of chemokines such as CCL5,CCR5,CCR2,CXCR3,and CXCL9.Immunohistochemical staining verified the negative correlation between YKT6 and CD8+T cell infiltration level in OSCC.7.YKT6 predicts response to immune checkpoint blocker therapyIn the HNSCC and OSCC cohorts,YKT6 was negatively correlated with PD-1,CTLA-4,IDO1,BTLA,LAG-3,TIM-3,TIGIT and VISTA expression,while tumor mutational load was weakly associated with YKT6.By ssGSEA as well as correlation analysis,we found that immune cytolytic activity,tumor-infiltrating lymphocyte ratio,immune checkpoint level,human leukocyte antigen abundance,immunomodulatory level,antigen-presenting cell co-stimulation and T-cell co-stimulation levels decreased with increasing YKT6 expression in OSCC samples.Further,through TIDE,IPS,and Submap algorithms,computational analysis revealed that OSCC patients in the YKT6 low expression group may be more sensitive to immune checkpoint blocker treatment than the YKT6 high expression group.Subsequently,we confirmed the above results by immunotherapy dataset.8.Overview of YKT6 in human pan-cancerFinally,we performed a pan-cancer analysis using TCGA samples.The results showed that YKT6 showed high expression in several tumor types and its copy number underwent amplification.In most tumors,including hepatocellular carcinoma,YKT6 expression upregulation was negatively correlated with immune score,activated CD8+T cell level,activated B cell level,effector memory CD8+T cell level,eosinophil level,and immune cytolytic activity,while it was positively correlated with high tumor purity,extracellular matrix-receptor interaction,TGF-β signaling pathway,and EMT2 level.One-way Cox regression analysis was performed to verify the prognostic value of YKT6 expression on patients.Conclusions and implicationsIn this study,we found that YKT6,a member of the SNARE protein family,was upregulated in OSCC by multi-omics data analysis combined with biological experimental validation,and its high expression was related with shorter survival in OSCC patients.Meanwhile,YKT6 promoted OSCC cell invasion and migration,and participated in the remodeling of tumor immune microenvironment,thus suggesting that YKT6 may be a potential biomarker for OSCC prognosis,immunotherapeutic response,and therapeutic target.The above study can help to elucidate the new mechanism of malignant transformation in OSCC and provide an experimental basis for the development of effective OSCC biomarkers and discovery of new targets. |
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