SIRT1 Suppresses The Migration And Invasion Of Gastric Cancer By Regulating ARHGAP5 Expression

Background:Gastric cancer is one of the most common malignant tumors and is the most common cause of tumor death.It is reported that the incidence and mortality of gastric cancer have been declining,but its prevention and treatment is still a worldwide concern.For lack of early effective diagnosis of cancer,patients often miss the best time to treat.Currently,the preferred treatment of gastric cancer patients is surgical resection along with radiotherapy and chemotherapy.However,the survival and prognosis after operation can almost not live up to our expectations.At present,infiltration metastasis is the bottleneck of current tumor treatment.Once the lesion reaches the advanced or metastatic stage,our present therapeutic strategies are largely ineffective.Thus,it is urgent to make a better understanding of the molecular pathogenesis of gastric cancer metastasis to improve patient outcomes.SIRT1 is a member of the sirtuin family and is a deacetylase,which relies mainly on NAD+ to work and the substrates of which include not only histone but also nonhistone proteins.It's mammalian homolog of silent information regulator 2(Sir2)in Saccharomyces cerevisiae which is the first evolutionarily conserved gene acting as regulators of longevity in yeast.By regulating different targets,SIRT1 has shown exciting connections between protein deacetylation and biological or pathobiological processes including energy metabolism,autophagy,inflammation and cancer.However,the effects of SIRT1 on cancer development are controversial which seem to be dependent on the tumor type and signaling pathway involved.Our previous study has revealed an inhibitory role of SIRT1 in the development of GC via NF-?B/cyclin D1 signaling.In this study SIRT1 induces gastric cancer cells G1 stagnation by inhibiting the expression of cyclin Dl,thus inhibiting the development of gastric cancer.In the past,a number of studies have shown that SIRT1 is closely related with different kinds of tumor development.In addition to proliferation,SIRT1 also participates in the invasion and metastasis process of cancer.By deacetylating smad4,SIRT1 repressed the effect of TGF-? signaling on MMP7,a smad4 target gene and consequently cell migration and tumor metastasis in breast cancer and oral squamous cell carcinoma.In lung cancer and ovarian cancer,SUMO E3 ligase PIASy suppressed SIRT1 expression by reducing Spl occupancy on the promoter of SIRT1 and thus promoted metastasis.It is also reported that SIRT1 promoted proteasomal degradation of oncogenic transcription factor FOXM1 and thus reversed the nutrient sensor O-linked-?-N-acetylglucosamine(O-GIcNAc)transferase(OGT)mediated invasion and metastasis of breast cancer.However,the effect of SIRT1 on the invasion and metastasis of gastric cancer and the involved regulatory mechanism is largely unknown.In the present study,we explored the role and related target genes of SIRT1 in the migration and invasion of gastric cancer.We found that SIRT1 suppressed the migration and invasion of gastric cancer in vitro and in vivo.Next,we used mRNA microarray to identify the downstream targets that are regulated by SIRT1.The results of mRNA microarray were verified in several gastric cancer cells.The regulatory mechanism of SIRT1 on the target gene was further investigated.Objective:We explore the function and regulation mechanism of SIRT1 in gastric cancer invasion and metastasis to provide a new target for treating gastric cancer.Methods:1.We used stably lentivirus-infected SIRT1-overexpression and SIRT1-silencing gastric cancer cell lines(AGS,BGC-823,MGC-803,HGC-27,and SGC-7901)to evaluate the effect of SIRT1 on gastric cancer cell metastasis.We found an inhibitory role of SIRT1 on migration and invasion of gastric cancer cells in vitro by wound healing assay and transwell assay without or with matrigel.2.We injected stably lentivirus-infected SIRT1-overexpression,SIRT1-silencing and their corresponding control BGC cells in immunodeficient nude mice of each group(5×105 cells/mouse)by tail vein.Four weeks later,mice were euthanized and the lungs were resected,weighed,taken pictures,fixed in 4%formaldehyde and performed HE staining for metastasis examination.We found SIRT1 can inhibit gastric cancer metastasis to their lungs in vivo.3.To screen the genes that are regulated by SIRT1 in gastric cancer cells,we performed mRNA profile microarray using stably lentivirus-infected SIRT1-overexpression,SIRT1-silencing and their corresponding control BGC-823.The result of mRNA profile microarray was verified by qRT-PCR and western blot in stably lentivirus-infected BGC-823 cells and other different gastric cancer cell lines with stable lentivirus transfection,then ARHGAP5 was chosen as the negatively regulated target of SIRT1 for further study.4.We analyzed the promoter of ARHGAP5 with JASPAR database and found putative binding sites of c-JUN and RELA.Then small interference RNAs(siRNAs)targeting the above two transcription factors were transfected into gastric cancer cells.Interference efficiency of transcription factor was verified by qRT-PCR and western blot,then proving that it is transcription factor c-JUN not RELA involved in the expression of ARHGAP5 in gastric cancer cells.Furthermore,the upregulation of ARHGAP5 in gastric cancer cells by SIRT1 depletion was reversed via silencing of c-JUN.These datas give us further confirmation that c-JUN participates in the expression of ARHGAP5 regulated by SIRT 1.To further examine whether c-JUN was involved in SIRT1 regulated transcription of ARHGAP5,we performed dual-luciferase assays in SIRT1 stable lentivirus-infected gastric cancer cells.lt was found that SIRT1 inhibited the activity of luciferase,that is to say,SIRT1 inhibited the expression of ARHGAP5 at the transcription level.Moreover,the luciferase experiment found that the binding site of transcription factor was c-JUN but not RELA.Then we performed co-IP experiments in AGS cells.The result indicates SIRT1 and transcription factor c-JUN can form complex precipitation.In order to evaluate the effect of SIRT1 on c-JUN,we performed co-IP experiments in SIRT1 stable lentivirus-infected AGS cells.We found the acetylation level of c-JUN is increased in SIRT1 silencing AGS cells and is reduced in SIRT1 overexpression AGS cells,suggesting that SIRT1 not only physically interacts with but also deacetylates c-JUN.The results of ChIP experiment in AGS gastric cancer cells showed that SIRT1 was recruited to occupy the promoter of ARHGAP5 at the same binding site as the transcription factor c-JUN.The qPCR experiments after ChIP experiment in stable SIRT1-silencing AGS cells observed a sharp reduction of SIRT1 but a large accumulation of transcription factor c-JUN.These results confirmed that SIRT1 is recruited to ARHGAP5 promoter,physically cooperates with and deacetylates c-JUN and thus suppresses transcription activity of c-JUN and expression of ARHGAP5.5.By analyzing the Oncomine database,we found that the expression of ARHGAP5 in different types of gastric cancer is higher than that in the normal tissues.Data from TCGA database indicated that the expression of ARHGAP5 in gastric cancer is also higher than that in the normal tissues.Data from the NCBI GEO database(GSE63089 and GSE13195)showed higher ARHGAP5 levels in gastric cancer tissues compared to their corresponding adjacent normal mucosa.To determine the protein levels of ARHGAP5,we carried out IHC staining using the tissue array including 90 pairs of paraffin-embedded gastric cancer and matched normal tissues.Staining results showed that ARHGAP5 expression was low in adjacent normal mucosa with 4 of which is no staining at all.But in gastric cancer tissues,levels of ARHGAP5 are significantly enhanced.The above data prove that ARHGAP5 is markedly upregulated in gastric cancer.The data also showed that ARHGAP5 expression is positively correlated with tumor size(p=0.003),depth of tumor infiltration(T stage,p<0.001),local lymph node metastasis(N stage,p=0.003)and clinical stage(TNM stage,p<0.001).Using univariate Cox regression analyses,we found that tumor size(p=0.003),depth of tumor infiltration(T stage,p=0.004),local lymph node metastasis(N stage,p=0.002),clinical stage(TNM stage,p=0.002)and ARHGAP5 levels(p<0.001)are significantly associated with patients' survival.Furthermore,multivariate Cox regression analysis further confirmed the depth of tumor infiltration(T stage,p=0.013),local lymph node metastasis(N stage,p=0.008)and ARHGAP5 levels(p=0.001)as independent predictors of the overall survival of gastric cancer patients.Survival curves were plotted to compare patients' outcomes according to the expression levels of ARHGAP5.The overall survival of gastric cancer patients with high ARHGAP5 levels is markedly worse than that of gastric cancer patients with low ARHGAP5 expression.Analysis of gastric cancer samples in TCGA database showed that overall survival periods are shorter among patients with higher ARHGAP5 levels in the tumor.Data from Kaplan-Meier plotter also indicated that lower expression of ARHGAP5 in the tumor results in a longer survival period.Furthermore,when we included lymph node metastasis in overall survival analysis in Kaplan-Meier plotter,the influence of ARHGAP5 remains significant.Taken together,above results uncovered a potential link between increased ARHGAP5 levels and gastric cancer progression.6.Specific siRNAs targeting ARHGAP5 were transfected into AGS and BGC-823 gastric cancer cells and the results of qRT-PCR and Western blot showed dramatical reduction of ARHGAP5 at both mRNA and protein levels.Transwell assays without or with matrigel indicated that gastric cancer cell migration and invasion were significantly repressed with the downregulation of ARHGAP5.Then we transfected siRNAs against ARHGAP5 into stably lentivirus-infected SIRT1-silenced and their corresponding control gastric cancer cells(AGS and BGC-823).Western blot experiments were used to validate the efficient knockdown of ARHGAP5 again.Transwell assays without or with matrigel confirmed that the inhibition of ARHGAP5 suppressed the migration and invasion of gastric cancer cells,thus effectively rescued the promotion of migration and invasion of gastric cancer cells induced by SIRT1-deletion.Conclusions:SIRT1 can restrain the migration and invasion of gastric cancer cells in vitro and can also inhibit the metastasis of gastric cancer cells in vivo.The downstream target genes of SIRT1 is ARHGAP5 which is selected by mRNA profile microarray.We found that SIRT1 can physically cooperates with and deacetylates c-JUN and be recruited to ARHGAP5 promoter,thus suppresses transcription activity of c-JUN and expression of ARHGAP5 in gastric cancer cells via double luciferase experiments,immunoprecipitation experiments and ChIP experiments The data analysis from several databases and the IHC staining results of tissue array indicates that ARHGAP5 expression in gastric cancer is markedly upregulated.Statistical analysis showed that the expression of ARHGAP5 is significantly correlated with the clinicopathological characters:tumor size,depth of tumor infiltration,local lymph node metastasis and clinical stage.Using Cox regression analyses,we found that the expression levels of ARHGAP5 are significantly associated with patients'survival and confirmed as independent predictors of the overall survival of gastric cancer patients.In addition,survival analysis datas from databases and data statistics of clinical experimental samples showed that high expression of ARHGAP5 is not good for the patients' survival.Datas from both databases and clinical data statistics show the potential link between the increased ARHGAP5 levels and gastric cancer progression.The results of functional experiments showed that the migration and invasion of gastric cancer cells are inhibited because of interference with ARHGAP5 expression in the gastric cancer cells.Moreover,we found that downregulation of ARHGAP5 in stably lentivirus-infected SIRT1-silenced and their control gastric cancer cells significantly suppresses cell migration and invasion of gastric cancer cells and clearly reverses the promotion of migration and invasion induced by SIRT1-depletion.Taken together,the mechanism of SIRT1 regulating ARHGAP5 expression may provide a new target for the prevention and treatment of gastric cancer in the future.