A Preliminary Study Of DOCK6 Promoting The Migration And Invasion Of Oral Squamous Cell Carcinoma Cells

Objective: Oral squamous cell carcinoma(OSCC)is one of the common head and neck malignancies.Although surgery combined with radiotherapy and chemotherapy has greatly improved the prognostic survival of patients,tumor invasion and metastasis are still important causes for the death of patient with OSCC and the related molecular mechanisms are still unclear.Dedicator of cytokinesis 6(DOCK6),could act as an atypical guanine nucleotide exchange factor to activate Rho GTPase to regulate cell morphology,cell proliferation,apoptosis,adhesion,migration and other biological behaviors.Although studies have shown that DOCK6 is related to the occurrence of congenital defects and is highly expressed in gastric cancer,acute myeloid leukemia and other tumors to play as an oncogene.While its expression and potential function in oral squamous cell carcinoma are still not very clear.Therefore,this study intends to use molecular biology techniques to explore the expression differences and potential functions of DOCK6 in oral squamous cell carcinoma and the regulation of DOCK6 expression and function by MYCT1,in order to understand the occurrence,development and clinical treatment of oral squamous cell carcinoma.Provide basis for the formulation of measures and the evaluation of prognosis.Materials and methods: 1.Experimental materials: oral squamous cell carcinoma cell lines SCC9,SCC15,CAL27;oral keratinous immortal cell line Ha Cat;oral squamous cell carcinoma tissue and normal gingival tissue paraffin sections;oral squamous cell carcinoma and pairing Normal tissue specimen.2.Experimental methods: Real-time PCR and Western blotting assay were used to detect the m RNA and protein expression levels of MYCT1 and DOCK6 respectively;Public databases was used to analyze the differential expression of DOCK6 in tumors and normal tissues;Immunohistochemical staining was used to explore DOCK6 expression in patients with OSCC;Wound scratching assay and Transwell assay were used to explore the effecting of DOCK6 on oral squamous cell carcinoma;ELISA assay was used to determine the level of IL8 secretion by oral squamous cell carcinoma after knocking down the DOCK6.3.Statistical methods: independent sample t-test analysis or paired t-test analysis was used for difference analysis,and expressed as mean ± sem;Chi-square test or Fisher’s exact test was used to analyze the relationship between clinical parameters and protein expression;Kaplan-Meier method and univariate and multivariate COX model were used to analyze the prognostic risk factors of patients with oral squamous cell carcinoma.The P value less than 0.05 was considered to be statistically significant.Result: 1.Real-time PCR and Western blot results showed that,consistent with the previous chip results,MYCT1 could significantly negatively regulate DOCK6 pathways and translation levels(P<0.05).2.Analysis of the public databases TCGA and GEO results showed that DOCK6 m RNA expression in oral squamous cell carcinoma was higher than that in normal tissues(P<0.05).3.Real-time PCR and Western blot results showed that the m RNA and protein expression of DOCK6 in oral squamous cell carcinoma were higher than those in the adjacent tissues(P<0.05).4 The results of immunohistochemical staining showed that compared with normal gingival tissue,DOCK6 was highly expressed in oral squamous cell carcinoma samples,and it indicated a shorter overall survival time for patients(P<0.05).5.Using small interfering RNA technology to target interference DOCK6,the results showed that small interfering RNA significantly reduced DOCK6 m RNA and protein expression levels(P<0.05).6.The results of the cell scratching assay and the Transwell assay showed that compared with the control group,knocking down DOCK6 could effectively inhibit the migration and invasion of oral squamous cell carcinoma cells.7.Bioinformatics prediction results showed that there are more than 10 interacting proteins with DOCK6,and they were closely related to the coagulation process,activation of small GTPase,and cell adhesion.8.The results of DOCK6 gene pathway enrichment analysis showed that signal pathways such as TGFβ and PI3K-AKT-m TOR were significantly enriched at high DOCK6 levels.9.The secretion level of IL-8 was dramatically repressed by si DOCK6 in OSCC cells.10.The results of cell scratching assay and Transwell assay showed that rh IL-8 could significantly rescue the inhibition of the migration and invasion of oral squamous cell carcinoma cells by si DOCK6(P<0.05).Conclusion: 1.MYCT1 inhibits DOCK6 m RNA and protein expression.2.DOCK6 is highly expressed in oral squamous cell carcinoma,and it suggests a poor prognosis in patients with oral squamous cell carcinoma.3.Silencing DOCK6 can inhibit the migration and invasion of oral squamous cell carcinoma cells.4.During the occurrence and development of oral squamous cell carcinoma,DOCK6 may play a role in promoting cancer through multiple signaling pathways such as TGFβ and PI3K-AKT-m TOR.5.IL-8 mediates the migration and invasion of oral squamous cell carcinoma cells promoted by DOCK6.