SIRT7 Inhibits The Epithelial-to-mesenchymal Transition And Metastasis Through The Deacetylation Of SMAD4 In Oral Squ Amous Cell Carcinoma

BackgroundOral squamous cell carcinoma(OSCC)is a common malignant tumor in the head and neck,which has high incidence rate and mortality in the world.Although surgical treatment,chemotherapy and radiotherapy can effectively prolong the life of patients with OSCC,surgical treatment has limitations for patients with lymph node metastasis of OSCC.Moreover,OSCC has the characteristics of high malignancy,easy metastasis,easily recurrence and so on,most patients have poor prognosis,and the quality of life after operation is significantly decreased,even can affect daily interpersonal communication.Studies have found that the prognosis and survival rate of OSCC patients can effectively improve through early clinical diagnosis and treatment.Therefore,it is more and more important to study the occurrence,development and metastasis of OSCC.SIRT7 is one of the main members of sirtuins protein family,and research shows that SIRT7 not only participates in the transcription of RNA polymerase,but also catalyzes the deacetylation of SMAD4,p53 and PAF53,so as to regulate the process of cell aging,moreover SIRT7 can also regulate the stress response of cells.More and more.research found that SIRT7 is involved in the regulation of the evolution of most malignant tumors,which plays a dual role in both carcinogenic and antitumor effects.SMAD4 belongs to a family of signal transduction proteins which is a tumor suppressor gene.Some studies also showed that SMAD4 can regulate the occurrence and development of tumor.Now it has not been reported that SIRT7 can regulate the progression of OSCC by regulating the expression of SMAD4.The epithelial-to-mesenchymal transition(EMT)is a reversible transition between epithelial cells and mesenchymal cells.What is more,EMT can make epithelial cells gain the ability of invasion and regulate the evolution and recurrence of tumor.However,EMT is one of the most important factors of tumor metastasis.Related studies have shown that SIRT7 can regulate EMT,while the TGF-β signaling pathway regulated by SMAD4 is an important regulator of EMT.Therefore,we speculate that SIRT7 inhibits the epithelial-to-mesenchymal transition and metastasis through the deacetylation of SMAD4 in oral squamous cell carcinoma.ObjectiveRecently,there are studies have shown that SIRT7 is related to the evolution of tumor,and EMT plays a crucial role in tumor metastasis of OSCC.However,the potential effect of SIRT7 in EMT and metastasis of OSCC has not been reported.We speculate that SIRT7 can regulate the metastasis of OSCC by regulating EMT.We speculate that SIRT7 may control the metastasis of OSCC by regulating the epithelial-to-mesenchymal transition.In order to prove this hypothesis,we analyzed the deacetylation of SMAD4,which plays a significant role in EMT pathway,so as to reveal the molecular mechanism of SIRT7 in OSCC of metastasis.Methods1.To detect the expression of SIRT7 in OSCCThe expression level of SIRT7 in OSCC tissues and cells was detected by immunohistochemistry(IHC),Western Blot(WB)and quantitative real-time PCR(RT-qPCR),and the role of SIRT7 in the evolution of OSCC was analyzed.The effects of SIRT7 on the invasion of HSC3 and OECM1 cells were observed by constructing SIRT7 stable overexpression and silencing cell lines,so as to explore the role of SIRT7 in OSCC and provide the theoretical support for the diagnosis and treatment of OSCC.Firstly.HOK,HSC3,OECM1,OC3,SCC4 and SCC25 cell lines were cultured by cell culture technology.In HSC3 and OECM1 cell lines,SIRT7 overexpression or knockout was obtained by lentiviral vector and cell transfection.SIRT7 overexpression group was recorded as OE-SIRT7 group,and SIRT7 knockout group was recorded as shSIRT7 group.Then the expression of SIRT7 in normal oral cell line HOK and human OSCC cell lines HSC3,OECM1,OC3,SCC4 and SCC25 were detected by IHC,WB and RT-qPCR.Finally,wound healing assay and Transwell invasion test were used to verify the effect of SIRT7 on the migration and invasion of OSCC,so as to explore the expression of SIRT7 in OSCC and verify the effect of SIRT7 expression on the invasion and migration of OSCC cells.2.To verify the effect of SIRT7 on the migration and invasion of OSCC,and to preliminarily verify the interaction relationship between SIRT7 and EMT.Western Blot,immunofluorescence assay and RT-qPCR were used to test the effects of SIRT7 overexpression and knockout on EMT.Moreover,these experimental methods were also used to prove the molecular mechanism of SIRT7 in OSCC.We verified the expression of E-cadherin,N-cadherin,vimentin and MMP-7 protein and the level of EMT by Western Blot,immunofluorescence assay and RT-qPCR when SIRT7 was overexpressed or knocked out lentiviral vector and cell transfection,in order to find that SIRT7 can reduce the migration and invasion of OSCC by inhibiting EMT.3.SIRT7 inhibits the epithelial-to-mesenchymal transition and metastasisthrough the deacetylation of SMAD4 in in oral squamous cell carcinomaWestern Blot,Transplanted tumors in animal models and immunofluorescence assay were used to detect the effect of SMAD4 deacetylation on the epithelialto-mesenchymal transition d4 uring SIRT7 overexpression and knockout,so as to provide a new target for the treatment of OSCC.WB and immunofluorescence assay were used in our experiment to verify the expression levels of SMAD4 and Ac-SMAD4 when SIRT7 was overexpressed or knockout.Transplanted tumors in animal models were used to verify that SIRT7 can inhibit EMT of OSCC through the deacetylation of SMAD4.ResultsThe expression of SIRT7 was down regulated in OSCC cell lines and OSCC tissue with lymph node metastasis.However,the expression of SIRT7 in HSC3,OECM1,OC3,SCC4 and SCC25 was lower than the expression of SIRT7 in HOK cells.According to wound healing assay,we came to the conclusion if SIRT7 was overexpressed,the healing rate in the OE-SIRT7 group was significantly lower when compared with the control group;However,when SIRT7 was knocked out the healing rate of cells in the shSIRT7 group was higher when compared with the control group;Transwell invasion test also showed similar results,in other words,the invasion rate of OE-SIRT77 group was lower,and the invasion rate of shSIRT7 group was higher when they were compared to the control group(P<0.05).In vitro,SIRT7 overexpression significantly decreased the proliferation and invasion of OSCC cells,as well as SIRT7 knockout significantly increased the growth and invasion of OSCC cells.Moreover,high expression of SIRT7 can up regulate the expression of E-cadherin and down regulate the expression of related mesenchymal markers,and also reduce the protein expression levels of ac-SMAD4 and Matrix Metallopeptidase 7(MMP-7).SIRT7 overexpression can also reduce the acetylation level of SMAD4 in OSCC cells.When silencing SMAD4,SIRT7 knockout can significantly reduce the effect of ac-smad4 expression and EMT.In addition,SIRT7 overexpression significantly inhibited OSCC lung metastasis in vivo.Conclusions1.The expression of SIRT7 is related to the progression of OSCC.The expression level of SIRT7 in OSCC is lower than that in normal tissues.When OSCC with lymph node metastasis is compared to OSCC without metastasis,the expression level of SIRT7 is lower.2.SIRT7 inhibits the metastasis and invasion of OSCC by inhibiting EMT.3.SIRT7 inhibits EMT by promoting SMAD4 deacetylation.