Expression And Function Study Of ATAD2/RRM2 In Oral Squamous Cell Carcinoma&Case Report

Oral squamous cell carcinoma(OSCC)is the most common malignant tumor of the head and neck.Early-stage tumors with surgery or radiation have a significantly better prognosis than advanced tumors.The current focus of OSCC research is to elucidate its pathogenesis at the molecular level,search for effective screening indicators,further understand the relevant molecular mechanisms,and provide new molecular targets for treatment.Previous studies have shown that ATAD2 is involved in chromatin dynamics,transcription and apoptosis regulation,thereby promoting tumor cell proliferation and inhibiting their differentiation.ATAD2 is overexpressed in several malignancies,and its overexpression usually indicates poor prognosis.However,the expression and clinical significance of ATAD2 in OSCC have not been reported.Therefore,this study will explore the possibility of ATAD2 protein molecule as a clinical prognostic indicator of OSCC patients,and further explore the effects of ATAD2 on the biological behavior of OSCC,expecting to provide more evidence for molecular targeted treatment of OSCC.Part oneExpression and clinical significance of ATAD2 in oral squamous cell carcinomaObjective:To investigate the expression level of ATAD2 protein molecule in human oral squamous cell carcinoma tissues,and to explore the correlation between the expression level of ATAD2 protein and clinicopathological parameters.To explore the possibility of ATAD2 protein molecule as a clinical prognostic indicator in patients with oral squamous cell carcinoma.To clarify the correlation between ATAD2 protein molecule and tumor microenvironment related molecules of oral squamous cell carcinoma.Methods:Custom made OSCC tissue microarray was performed by collecting oral squamous cell carcinoma specimens,oral mucosa specimens adjacent to OSCC and oral epithelial dysplasia specimens.Kaplan-Meier survival analysis was performed to explore the correlation between the expression of ATAD2 protein molecule and the clinical prognosis of oral squamous cell carcinoma patients.The correlation between ATAD2 protein molecule and pathological grade,tumor size,lymph node status,smoking,alcohol consumption and TPF chemotherapy in patients with oral squamous cell carcinoma was explored by immunohistochemical staining analysis.Correlation analysis and cluster analysis were used to clarify the correlation between ATAD2 protein expression and tumor microenvironment related molecules.Immunohistochemical results showed that the expression level of ATAD2 was closely related to lymph node metastasis and HPV status,but not significantly related to pathological grade,tumor size and TPF chemotherapy.Correlation analysis showed that the expression of ATAD2 protein was correlated with EMT marker Slug,as well as with inhibitory immune checkpoints PD-L1 and B7-H4.Results:The results of tissue microarray immunohistochemistry showed that the expression level of ATAD2 was significantly increased in oral squamous cell carcinoma tissues compared with normal oral mucosal tissues.Kaplan-Meier survival analysis showed that higher levels of ATAD2 expression predicted poorer survival in patients with oral squamous cell carcinoma.Immunohistochemical results showed that the expression level of ATAD2 was closely correlated with lymph node metastasis and HPV status,but not with pathological grade,tumor size and TPF chemotherapy.Correlation analysis showed that the expression of ATAD2 protein was correlated with EMT marker Slug,as well as with inhibitory immune checkpoints PDL1 and B7-H4.Conclusions:The expression level of ATAD2 in oral squamous cell carcinoma is significantly increased,and the protein molecule of ATAD2 might be used as a clinical prognostic indicator in patients with oral squamous cell carcinoma.The abnormal expression level of ATAD2 protein may play an important role in the occurrence and development of oral squamous cell carcinoma.Part twoEffect of ATAD2 on proliferation and metastasis of oral squamous cell carcinoma Objective:To investigate the effects of ATAD2 on the proliferation,cycle,apoptosis,metastasis and EMT of oral squamous cell carcinoma cells by bioinformatics and in vitro experiments.To investigate the effect of ATAD2 on the progression of oral squamous cell carcinoma by in vivo experiments.Methods:The RNA sequence data and related clinical data of 502 cases of head and neck squamous cell carcinoma and 44 cases of paracancerous normal tissues were downloaded from TCGA database.The mRNAs with Pearson correlation coefficient greater than 0.3 were included in GO and KEGG pathway enrichment analysis.The function and signaling pathway with strong correlation with ATAD2 were searched.Human oral squamous cell carcinoma cell line CAL27 with down-regulated ATAD2 gene was obtained by transfection of small interfering RNA(siRNA).Cell clone formation assay and CCK8 assay were used to detect the effect of down-regulated ATAD2 expression on the proliferation of oral squamous cell carcinoma cells.Wound healing assay and Transwell assay were used to detect the change of migration ability of oral squamous cell carcinoma cells after down-regulation of ATAD2 expression.Flow cytometry was used to detect the effect of down-regulated ATAD2 expression on the cycle and apoptosis of oral squamous cell carcinoma cells.Western blot was used to detect the effect of ATAD2 down-regulation on the expression of tumor progression-related genes in oral squamous cell carcinoma cells.Subcutaneous tumorigenesis experiment in nude mice was used to detect the effect of ATAD2 knockout on the progression of oral squamous cell carcinoma.Lung metastasis assay was used to detect the effect of ATAD2 knockdown on oral squamous cell carcinoma metastasis.Results:Enrichment analysis based on TCGA database showed that the main functions of ATAD2-related mRNAs were chromatin binding,transcriptional coregulatory factor activity,ATPase activity and DNA catalytic activity.The main signaling pathways of ATAD2-related mRNAs were cell cycle,RNA transport,DNA replication and cell senescence.SiRNAs can effectively down-regulate the expression of ATAD2 in oral squamous cell carcinoma cell line CAL27.When the expression of ATAD2 gene was down-regulated,the proliferation ability of oral squamous cell carcinoma cells was decreased.The apoptosis ability of oral squamous cell carcinoma cells was improved and the cell cycle progression was decreased after the silencing of ATAD2 gene.Compared with the control group,the down-regulation of ATAD2 gene expression inhibited the metastasis of oral squamous cell carcinoma cells and affected the EMT process.The results of subcutaneous tumorigenesis in nude mice showed that the silencing of ATAD2 gene inhibited the growth of oral squamous cell carcinoma in vivo compared with the control group.In addition,lung metastasis experiments showed that ATAD2 knockdown could attenuate the lung metastasis ability of oral squamous cell carcinoma.Conclusion:The down-regulation of ATAD2 gene expression can reduce the proliferation ability of oral squamous cell carcinoma cells,increase the apoptosis,slow the cell cycle process,inhibit the cell migration ability,and inhibit the EMT process.It has been proved from the side that the role of ATAD2 as oncogene in the occurrence and development of oral squamous cell carcinoma is to promote cell proliferation,inhibit cell apoptosis,accelerate cell cycle,increase cell migration ability and promote EMT.In addition,ATAD2 downregulation can also inhibit the progression of oral squamous cell carcinoma in vivo.Part threeEffect of RRM2 on proliferation and metastasis of oral squamous cell carcinoma cellsObjective:To investigate the correlation between RRM2 and ATAD2 in oral squamous cell carcinoma.To explore the expression level of RRM2 protein in oral squamous cell carcinoma and its correlation with clinical prognosis.To study the effect of RRM2 on the biological behavior of oral squamous cell carcinoma cells.Methods:Bioinformatics,tissue microarray staining and Western blot were used to verify the correlation between RRM2 and ATAD2.Immunohistochemical staining was performed on the tissue microarray to detect the expression level of RRM2 protein molecules in each tissue specimen.Kaplan-Meier survival analysis was performed to explore the correlation between RRM2 protein expression and clinical prognosis in patients with oral squamous cell carcinoma.The effect of RRM2 on the proliferation,apoptosis and metastasis of oral squamous cell carcinoma cells was studied by cell function experiment.Results:The results of bioinformatics and tissue microarray immunohistochemistry indicated that there was a significant positive correlation between ATAD2 and RRM2 in gene and protein levels.Western blot results showed that RRM2 protein content was significantly decreased after the ATAD2 gene was knocked out.When RRM2 gene was knocked out,the protein content of ATAD2 showed no significant trend change.The results of tissue microarray immunohistochemical analysis showed that RRM2 was highly expressed in human oral squamous cell carcinoma,and was associated with overall survival.When RRM2 gene expression was down-regulated,it could inhibit the proliferation and migration of oral squamous cell carcinoma cells and promote cell apoptosis.Conclusion:RRM2 is positively correlated with ATAD2 gene,and RRM2 is located downstream of ATAD2 gene.The expression of RRM2 in oral squamous cell carcinoma was significantly higher than that in normal tissues.In addition,the high expression of RRM2 in oral squamous cell carcinoma is associated with poor prognosis of patients,and RRM2 may be used as a reference indicator for clinical prognosis of oral squamous cell carcinoma.The down-regulation of RRM2 gene expression can inhibit the proliferation and migration of oral squamous cell carcinoma cells,and promote cell apoptosis.RRM2 may act as a coregulator of ATAD2 to assist ATAD2 in the effect on the biological behavior of oral squamous cell carcinoma cells,but further studies on the molecular mechanism are needed.