Expression Of USP9X In Nasopharyngeal Carcinoma Tissues And Its Effect On The Biological Behavior Of Nasopharyngeal Carcinoma Cells

Objective:To analyze the expression of ubiquitin specific peptidase 9X(USP9X)in tumor tissues of nasopharyngeal carcinoma patients and its clinical significance.And explore the changes of biological behaviors such as cell proliferation,migration,invasion after USP9X knockdown in nasopharyngeal carcinoma cells.Methods:1.In the experimental group,we collected 99 cases of nasopharyngeal biopsy tissue samples from nasopharyngeal carcinoma patients admitted to the Affiliated Hospital of North Sichuan Medical College from June 2016 to November 2019,and the pathological type was poorly differentiated squamous cell carcinoma.In the control group,34 cases of nasopharyngeal biopsy tissue samples were collected,and the pathological type was chronic nasopharyngeal inflammatory tissue.Immunohistochemical staining was used to detect the expression of USP9X protein in the experimental group and control group.At the same time,the complete clinical data of nasopharyngeal carcinoma patients were collected and analyzed,and the relationship between the expression of USP9X in nasopharyngeal carcinoma tissues and clinicopathological factors of the patients was analyzed.92 patients with nasopharyngeal carcinoma were followed up by telephone and outpatient service,and the factors affecting the survival and prognosis of patients were analyzed.2.Human nasopharyngeal carcinoma poorly differentiated squamous cell line CNE2 was selected as the template,and Public Protein/shared Library was commissioned to synthesis LV-shrNA-USP9X short hairclip RNA sequence(LV-SHRNA-USP9X)specifically blocking down USP9X expression,and the sequence was packaged into lentivirus.LV-shRNA-USP9X lentivirus was transfected into CNE2 cells and CNE2 cell with low USP9X expression were obtained.q-PCR and Western blotting were applied to determine the inhibition efficiency on USP9X expression of LV-shRNA-USP9X lentivirus transfected CNE2 cells.CCK-8 assay,tablet cloning assay,trans well assay was applied to confine the affection of USP9X expression inhibition on the proliferation,migration,invasion behaviors of CNE2 cells.Results:1.Immunohistochemical staining results showed that USP9X positive staining was brownish yellow with diffuse distribution in cytoplasm(partly expressed in the cell membrane).The positive expression rate in nasopharyngeal carcinoma tissues and chronic nasopharyngeal inflammation tissues was 73.7%and 23.7%(P<0.01).The expression of USP9X was correlated with T stage(local tumor range)and clinical stage of NPC patients(P<0.05).Univariate log-rank analysis showed that age,M stage(distant metastasis),clinical stage and USP9X expression were correlated with prognosis(P<0.001).Kaplan-Meier survival curve indicated that the overall survival rate of patients with USP9X positive expression was significantly lower than that of patients with USP9X negative expression.Cox regression analysis further indicated that USP9X positive expression was an independent risk factor affecting the prognosis of Nasopharyngeal carcinoma(P<0.05),and the death risk of patients with USP9X positive expression was 1.624 times higher than that of patients with negative expression.Positive expression of USP9X may be an adverse prognostic factor for nasopharyngeal carcinoma.2.After successful transfection of LV-shRNA-USP9X lentivirus into nasopharyngeal carcinoma CNE2 cells,PCR(q-PCR)and Western blotting determine the expression of USP9X mRNA and protein was decreased.Compared with the blank CNE2 cell line(CON group)and the negative control group(NC-shRNA lentivirus transfection group)transfected with meaningless sequence,the biological behaviors of CNE2 cells in knockdown group(LV-shRNA-USP9X lentivirus transfection group)were changed,such as proliferation,migration,and invasion.CCK8 results indicated that cell growth rate in LV-shRNA-USP9X lentivirus transfection group was significantly lower than that in CON group,and cell proliferation was inhibited.The results of tablet cloning assay indicated that compared with the CON group,the relative clone formation rate of CNE2 cells in NC-shRNA lentivirus transfection group was 77.55%,and that of CNE2 cells in LV-shRNA-USP9X lentivirus transfection group was 42.64%,the single cell clone formation decreased after USP9X knockdown.The difference was statistically significant(P<0.001).In Trans well migration experiment,the relative mobility of NC-shRNA lentivirus transfection group was 93.76%Compared with CON group,and that of LV-shRNA-USP9X lentivirus transfection group was 58.89%,suggesting that the migration ability of USP9X knockdown decreased,and the difference was statistically significant(P<0.001).In the invasion experiment,the relative invasion rate of cells in NC-shRNA lentivirus transfection group was 86.19%compared with that in the CON group,and that in LV-shRNA-USP9X lentivirus transfection group was 56.06%,the invasion ability of CNE2 cells decreased after the USP9X knockdown,and the difference was statistically significant(P<0.001).Conclusion:1.USP9X is highly expressed in nasopharyngeal carcinoma tissues and is related to tumor local scope and clinical stage.Positive expression of USP9X may be an adverse prognostic factor of nasopharyngeal carcinoma.2.After specific inhibition the expression of USP9X in CNE2 cells,the cell proliferation,migration,and invasion ability were decreased.USP9X is related to the malignant biological behavior of nasopharyngeal carcinoma CNE2 cells and may be an effective target for molecular therapy of nasopharyngeal carcinoma.