Relationship Between BTB/POZ Domain-containing Protein 7 And Fibronectin In Invasion And Metastasis Of Salivary Adenoid Cystic Carcinoma

Objective:Salivary adenoid cystic carcinoma(SACC)is a common malignant tumor in the salivary gland.SACC is highly invasive and it can metastasize along the peripheral nervous tissues and through blood circulation.However,there is no reliable biomarker for prediction of invasion and metastasis patients.Patients with invasive and distant metastatic SACC usually have a poor prognosis.The poor prognosis has not been improved over the past several years due to the lack of powerful markers forprediction of invasion and metastasis.Discovery of new regulators for proliferation,migration and metastasis of SACC may be of significance in prediction of metastasis and development of therapeutic strategies for intervention of SACC.During the process of distant metastasis,SACC cells can enter the blood vessels and migrate into distant organs,where they interact with extracellular matrix(ECM)and adapt to new environment.The metastasis of SACC is regulated by the expression and modification of ECM,which can promote the process of epithelial-mesenchymal transition(EMT)in tumor cells.Aberrant EMT has been reported to be associated with poor prognosis in a variety of malignancies,such as breast cancer,lung cancer,liver cancer and so on.Fibronectin(FN)is a component of the ECM and basement membrane.Its expression and glycosylation levels are crucial for its biological functions.FN can mediate cell adhesion to the matrix and affect cell growth,differentiation and movement,which are important for the migration and metastasis of tumor cells.BTB/POZ domain containing protein(BTBD)7 is one of the spliced variants of BTBD,containing binding sites for notable transcription factors.Functionally,BTBD7 regulates epithelial cell dynamics and branching morphology.Previous studies have shown that BTBD7 promotes invasion and metastasis of lung cancer and hepatocellular carcinoma(HCC).Our recent study indicates that BTBD7 is expressed in SACC tissues,and correlated with Slug,a downstream gene of the BTBD7,expressio.Interestingly,downregulation of BTBD7 expression inhibits FN expression in HCC.Furthermore,a previous study has indicated that FN induces BTBD7 expression at the cleft base.However,the relationship between FN and BTBD7 expression in SACC has not been explored.In this study,the BTBD7 and FN expression in SACC and non-tumor salivary tissues as well as SACC cells were characterized by immunohistochemistry and immunofluorescence.Furthermore,we explored the impact of FN or BTBD7 silencing on the BTBD7 and FN expression in SACC cells,respectively.Finally,we examined the effect of BTBD7 silencing on the proliferation,migration and invasion of SACC cells.by wound healing and transwell invasion assays.Methods:1.Paraffin-embedded blocks of 70 SACC and 16 non-tumor salivary gland tissues were obtained and immunohistochemical method was used to detect the expression of BTBD7 and FN in the.tissue specimens.Based on the score of immunochemistry,the correlation between the expression of BTBD7 and various clinicopathologic parameters(such as:age,gender,TNM staging,organization types and distant metastasis)were investigated.Using the statistical software to evaluate the association between BTBD7 and FN。2.Using the Kaplan-Meier method to evaluate the overall survival in the BTBD7-positive expression group to the BTBD7-negative expression group.3.Using the cell immunofluorescence test to detect the expression of BTBD7 and FN in SACC-83 cell lines and SACC-LM cell lines4.Using the small RNA interference technology to silence the gene of BTBD7 in SACC-LM cell lines.Quantitative real-time PCR and western blot analysis were used to investigate the expression of BTBD7and FN.5.Using the small RNA interference technology to silence the gene of FN in SACC-LM cell lines.Quantitative real-time PCR and western blot analysis were used to investigate the expression of FNand BTBD7.6.Transwell invasion assay and wound healing assays were used to examine the effect of BTBD7 silencing on the proliferation,migration and invasion of SACC-LM cell lines.Results:1.Immunohistochemistry results showed that the percentage of BTBD7 expression increased from the normal cases to the SACC:the former is 12.5%while the latter 55.71%.the percentage of FN expression decreased from the normal cases to the SACC:the former is 87.5%while the latter 27.14%.The difference in expression rates were considered statistically significant(P<0.05).Stratification analysis indicated that the positive rates of BTBD7 expression were significantly associated with the higher tumor stages,solid type and distant metastasis of SACC.while the positive rates of FN expression were significantly associated with lower tumor stages,non-solid type and non-metastatic SACC.Hence,there was a reverse relationship in the BTBD7 and FN expression between the SACC and non-tumor salivary gland tissues,and BTBD7 expression was associated with aggressiveness of SACC.2.The average follow-up period was six years(range 12-89 months).During the follow-up period,29 patients died of SACC and 9 patients were lost at the end of the study.The remaining 32 patients were still alive.The Kaplan-Meier survival curves demonstrated that the overall survival in the BTBD7-positive group was statistically shorter than the BTBD7-negtive.group3.The levels of BTBD7 and FN expression in SACC-83 and SACC-LM cells were characterized by immunofluorescence.Strongly fluorescent anti-BTBD7 signals and anti-FN signals were detected both in the cytoplasm of SACC-LM cells.Hence,higher levels of BTBD7 and FN expression were detected in SACC-LM cells.4.Downregulate BTBD7 expression of SACC-LM cellst significantly upregulated the levels of FN both in mRNA and protein5.Downregulate FN expression of SACC-LM cellst,it did not affect the relative levels of BTBD7 mRNA and protein in SACC-LM cells.6.Knockdown of BTBD7 expression significantly inhibited proliferation and invasion of SACC-LM cells in vitro.Conclusions:The data indicated that the positive rates of BTBD7 expression were significantly associated with the higher tumor stages,solid type and distant metastasis of SACC.while the positive rates of FN expression were significantly associated with lower tumor stages solid type and distant metastasis of SACC.BTBD7 was inversely associated with FN expression in SACC.BTBD7 may inhibit FN expression,but promote proliferation,migration and invasion of SACC-LM cells.Hence,BTBD7 may be associated with metastasis and a new therapeutic target for intervention of SACC.