BTBD7 Through Slug Inducing Epithelial Mesenchymal Transition In Human Salivary Gland Adenoid Cystic Carcinoma Research

Objective:Salivary adenoid cystic carcinoma(SACC)is a rare malignant tumor,accounts for only 1%of all head and neck cancer.It is characterized by a high local invasion and distant metastasis to the lung,liver and bone,survival rate of patients with distant metastasis is lower than whom without distant metastasis.At present,there is no effective solution in the treatment of adenoid cystic carcinoma,the treatment is still predominantly of comprehensive treatment of surgical adjuvant radiotherapy or chemotherapy,the long-term survival rate is low and with poor outcomes after treatment.Because of the lack of specific tumor markers,therapeutic method of SACC is limited.With the deepening research of the biological behavior and molecular mechanism about SACC,more specific tumor markers for the treatment of salivary gland adenoid cystic carcinoma is of great significance.Epithelial-mesenchymal transition(EMT)is an important physiological process,which of transfer of epithelial cells into mesenchymal cells and causes most of the recurrence and metastasis of malignant tumor.Slug,with zinc finger structure,is one of EMT transcription facto and play an important role in the migration of EMT,cancer cell invasion metastasis.In the process of perineural invasion of SACC,Slug silencing can downregulate the expression of EMMPRIN,meanwhile upregulate the expression of E-cadherin,ultimately inhibited the EMT.BTB domain is exists in eukaryotic protein interaction motif.BTB/POZ domain-containing protein 7(BTBD7),with conservative BTB/POZ protein interaction motif,contains binding sites for notable transcription factors including alpha-fetoprotein(AFP)-1,CAAT enhancer binding protein beta,GATA,and activator protein.Recently,BTBD7 was recognized as a regulatory gene that promoted epithelial tissue remodeling and formation of branched organs.Highly focal expression of BTBD7 can lead to local regulation of Slug,E-cadherin,and epithelial cell motility.In addition,BTBD7 was also involved in the process of invasion and metastasis of lung cancer and in the EMT of hepatocellular carcinoma.However,the role of BTBD7 in primary salivary adenoid cystic carcinoma(SACC)has not been identified.In this study,in order to investigate the possible functions of BTBD7 in SACC,BTBD7 protein and Slug protein were detected in the tissue of SACC using the method of immunohistochemical method,then the relationship between the BTBD7 expression with clinical pathological parameters,and with the prognosis of patients were analyzed.By gene silencing technology,assays were performed to survey cell migration,invasion and proliferation capabilities with BTBD7 silencing in high invasive SACC-LM cells,furthermore,the expression levels of Slug and EMT-related genes were detected in both gene and protein levels by qRT-PCR and Western bolt analysis for providing new targets for the biological treatment of SACC.Methods:1.66 paraffin embedded tissue specimens from primary SACC patients and 15 cases of normal salivary gland tissue specimens were collected and immunohistochemical method was performed to detect BTBD7 and Slug protein expression in SACC and normal salivary gland tissue,the relationship between BTBD7 and Slug in SACC as well as the relevance of the patient's gender,age,tumor size,TNM staging,organization types and distant metastasis were investigated.2.Using Kaplan-Meier survival analysis investigate the differences overall survival between patients with positive-BTBD7and negative-BTBD7.Using COX multivariate proportion of risk analysis to investigate the influence factors influencing the survival of patients with SACC.3.Cell immunofluorescence test was used to detect the expression ofBTBD7 and Slug in SACC-LM cells and SACC-83 cells.4.Using the small RNA interference technology to silence gene expression of BTBD7 in SACC-LM cells,qRT-PCR and Western bolt technique were used to detect the changes of BTBD7,Slug and EMT related genes mRNA and protein expression.5.Scratch-wound assay,matrigel invasion assay and MTT assay were used to survey cell migration,invasion and proliferation capabilities with BTBD7 silencing.Results:1.The expression of BTBD7 and SLUG were much higher in salivary gland adenoid cystic carcinoma tissues than normal salivary gland tissues which with lower expression or without expression.Expression of BTBD7 and Slug was significantly associated with the TNM stage,tissue typing and distant metastasis but no correlations with gender,age,and tumor size.In salivary gland adenoid cystic carcinoma tissues,BTBD7 expression was significantly correlated with Slug expression in SACC.The positive BTBD7 expression rate was 70.2%(33/47)in patients accompanied by Slug-positive expression.2.According to the follow-up results of the 66 patients with salivary gland adenoid cystic carcinoma,the Kaplan-Meier survival curves revealed that the overall survival in BTBD7-positive group was statistically shorter than the BTBD7-negtive expression group.Multivariate Cox proportional hazard analysis revealed that BTBD7-positive expression,Slug-positive expression and distant metastasis were independent risk factors for survival.The risk of survival in patients with BTBD7-positive expression was 3.287 times greater than that in those with BTBD7-negitive expression.3.BTBD7 and Slug Immunofluorescence staining were detected in two cell lines.Positive BTBD7 expression was mainly in the cytoplasm of two cell lines,positive Slug expression was both in the nuclear and cytoplasm of two cell lines.4.Downregulate BTBD7 expression of SACC-LM cells,Western bolt analysis demonstrated significantly upregulated E-cadherin protein expression nevertheless downregulated Slug and MMP9 protein expression in SACC-LM cells.5.Downregulate BTBD7 expression of SACC-LM cells,the migration and invasion abilities of cells were obviously suppressed but with no influence on cell proliferation.Conclusions:In the salivary gland adenoid cystic carcinoma tissues,the survival rate of patients with BTBD7positive expression was low,the prognosis were poor,it was predicted that BTBD7 may play an important role in the process of the department of SACC.BTBD7 silencing down-regulated the expression of Slug and MMP9 meanwhile up-regulated the expression of E-cadherin in SACC-LM cells,the migration and invasion abilities of cells were obviously suppressed but with no influence on cell proliferation.The findings illustrated that BTBD7 might be the upstream gene to regulate Slug and EMT process,and also suggest that BTBD7 may serve-as a new biological therapeutic target and potential prognostic marker for the diagnosis and prognosis of SACC.