Elevated SIRT1 In Gastric Cancer Suppresses Migration And Invasion Of Cancer Cells Via STAT3/MMP-13 Signaling

Backgroud and Object:The prognosis of gastric cancer remains poor due to its strong tendency of metastasis.Constitutive activation of STAT3 is important to maintain the malignant behavior of gastric cancer.Acetylation of STAT3 has recently been shown to maintain the stability of phosphorylated STAT3 and enhance the transcriptional activity.Sirtl,the deacetylase of STAT3,could decrease the stability of phosphorylated STAT3 and inhibit its transcripitonal activity.However,the function of Sirtl has not been well understood in gastric cancer.The aim of this research is to study SIRT1 on the migration and invasion of human gastric cancer cells and the corresponding mechanism.Methods:1.We recruited 18 gastric cancer patients and examined the protein levels of SIRT1 using immunohistochemistry(IHC)in the cancerous and matched normal gastric specimens from these patients.2.Two human gastric cancer cells,AGS and HGC-27,were transfected with lentivirus to knockdown SIRT1 and use negative lentivirus transfected cells as control.CCK-8 assay was used to detect the proliferation ability of cells;Transwell chamber assay was used to detect the migration and invasion of transfected cells.Then we checked the protein expression of two markers,Vimentin and Snail,by Western blot.We transfected the mock vector(pECE),and the vector expressing wild-type SIRT1(pECE-SIRT1)into AGS and HGC27,transwell chamber was used to detect the migration ability of SIRT1-overexpression cells.Western blot was used to detect Vimentin and Snail protein levels.3.Western blot was used to detect the levels of acetylated STAT3 and phosphorylated STAT3 in SIRT1-depleted cells and SIRT1-overexpressing cells.JAK2/STAT3 inhibitor,AG490,was used to treat lentivirus-transfected cells,Transwell chamber assay was used to detect the migration and invasion of transfected cells;siRNA-STAT3 and the negative control(NC)-siRNA were designed and synthesized,treated SIRT1-depleted cells.Then transwell chamber assay was used to detect the migration and invasion of transfected cells.4.MMP-13 specific inhibitor CL-82198 was used to treat the cells,and the migration and invasion ability of cells were detected with transwell chamber assay.Two methods to inhibit STAT3 were used to treat cells.Western blot was used to detect the expression level of MMP-13 in cells.5.Western blot was used to detect the expression of MMP-13 in 14 fresh gastric cancer tissues and adjacent tissues.IHC staining of MMP-13 expression was carried out on stomach tissues,including 83 advanced gastric cancer(AGC),45 early gastric cancer(EGC),42 precancerous lesions(PL),and 38 normal gastric mucosa(NG)samples.Their relationship with lymph node metastasis and survival results were also analyzed.6.SIRT1-depleted HGC27 cells were transplanted subcutaneously into nude mice,observed tumor volume,then total proteins were extracted from the tumors.Western blot showed the protein expression of acetylated STAT3 and phosphorylated STAT3.The IHC staining was examined for the expression of MMP-13.Results:1.The expression level of SIRT1 was up-regulated in gastric cancer tissues.2.The AGS and HGC-27 gastric cancer cells knocked out of SIRT1 had significantly increased proliferation?migration and invasion,and increased Vimentin and Snail protein levels.SIRT1-Overexpression cells with impaired migration ability and reduced Vimentin and Snail protein levels.3.The level of acetylated STAT3 and phosphorylated STAT3 protein was increased after SIRT1 knockout;over-expression of SIRT1 decreased the level of acetylated STAT3 and phosphorylated STAT3 protein.Both methods inhibited STAT3 can attenuate the migration and invasion ability of SIRT1-depleted cells.4.The migration and invasion ability of cells were decreased by MMP-13 specific inhibitor CL-82198.As STAT3 levels were inhibited,MMP-13 expression levels are also decreased.5.MMP-13 protein expression was examined by Western blot,indicating elevated MMP-13 level in tumor tissues compared with matched non-cancerous tissues in 10 of 14 cases.Immunohistochemistry data demonstrated that enhanced MMP-13 expression led to a shorter survival outcome,possibly due to the elevated rate of metastasis induced by MMP-13.6.In the tumors of the SIRT1-knockout group,the levels of acetylated STAT3 and phosphorylated STAT3 protein were decreased,and the level of MMP-13 was increased.Conclusions:SIRT1 expression was upregulated in gastric cancer tissues compared with matched normal gastric mucosa.However,depletion of SIRT1 increased the migration and invasion of gastric cancer cells,and enhanced the protein expression of phosphorylated STAT3,acetylated STAT3,and MMP-13.Besides,siRNAs targeting STAT3,the JAK2/STAT3 inhibitor AG490,and the MMP-13 inhibitor CL-821983 all attenuated the enhancement of gastric cancer cell metastasis induced by depleted SIRT1.What's more,STAT3 silencing impeded the upregulation of MMP-13 expression triggered by knockdown of SIRT1.Notably,MMP-13 expression was associated with lymph node metastasis and poor survival outcome in gastric cancer patients.In vivo models also showed that depleted SIRT1 promoted gastric cancer growth via STAT3/MMP-13 axis.In conclusion,depletion of SIRT1 increases the ability of metastasis in gastric cancer cells via activating STAT3/MNP-13 signaling,indicating SIRT1 as a potential tumor suppressor.Upregulation of SIRT1 in gastric cancer patients might be a feedback to compensate the harmful STAT3 signaling.Activators of SIRT1 could be used as potentially preventive and therapeutic treatments for metastatic gastric cancer.