| ObjectivesGastric cancer(GC)has become the fifth incidence and the third mortality rate all over the world.In China,the GC incidence and the resultant mortality rates account for 50%of those worldwide and have become the highest cancer mortality in men.The etiology of GC has been confirmed,but the exact pathogenesis is not fully understood,genomic instability as a potential mechanism of gastric carcinogenesis has received increased focus in recent years.In the process of gastritis,intestinal metaplasia,dysplasia even GC,the degree of DNA mutations was associated with gastric epithelial damage cascade,and thus DNA damage and DDR in gastric epithelial cells are meaningful in the pathogenesis of GC.DNA damage followed DNA damage response(DDR)will repair the damaged genome to ensure genome integrity and prevent cell canceration,the impaired DDR is a crucial cause of genomic instability.Homologous recombination repair(HR)is a high-fidelity repair of DDR and uses the sister chromatid as a template to repair in an error-free fashion.If HR inhibited intracellularly,the cells will tend to select the error-prone non-homologous end joining(NHEJ)to complete the repair,thereby causing more abnormal DNA occurs in cells,resulting in increased genomic instability.Cyclic GMP-AMP synthase(cGAS)is a DNA sensor located in the cytoplasm,it can activate the innate host immune reaction by the sensing abnormal DNA,and also,the cGAS-independent response can be triggered by-products of Genomic instability and tumor-derived DNA,such as endogenous DNA from dead tumor cells,micronucleus,cytoplasmic chromatin fragments,free telemeric DNA,inducing cellular senescence,inflammatory and anti-tumor immune,autophagy and DNA repair response,resulting in different effects in a variety of tumorigenesis.According to new research,cGAS inhibits HR repair in Lewis lung cancer cell line and promotes tumorigenesis.Due to the different regulatory roles of cGAS in the various tumors,and no relevant study in GC,our study explores the expression of cGAS in GC;and to identify the role-played of cGAS in gastric carcinogenesis process,we used RNA interference technology to knockdown gene expression of cGAS in AGS and MKN45 cell lines,and observed impaction of differential expression of cGAS in GC cell lines by cell functional experiments;and detected the changes of HR key proteins in GC cell lines to explore the possible mechanisms.Materials and Methods1.The TCGA(Human Cancer Genome Atlas)database was used for analyzing the differential expression of cGAS in 384 GC patients’ tumors and 37 health people’s normal stomach tissues;immunohistochemistry experiment of 41 cases GC patients’tumours and adjacent tissues was detected expression of cGAS,and analyzed correlation between expression of cGAS and clinicopathological features of GC patients.The relative expression of cGAS in "normal" gastric epithelial cell line GES-1 and three common gastric cancer cell line MKN45,NCI-N87 and AGS were analyzed by real-time quantitative qPCR.2.RNA interference(RNAi)was used for silence the expression of cGAS in MKN45 and AGS cell lines,and CCK-8 cell proliferation assay,cell migration assay,wound healing and apoptosis assay were performed to evaluate cGAS knockdown on the proliferation ability,invasiveness and apoptosis rate of GC cell lines in vitro.3.The stable packaging sh-cGAS lentiviral-MKN45 cell line was constructed by cell transfection and puromycin screening,and a nude mouse xenograft model was acted to observe the cGAS knockdown on the proliferation ability in vitro.4.The KEGG and GO pathway analysis about the first 200 genes co-expressed with cGAS in 384 GC patients in the TCGA database were performed at the DAVID platform,and found out the significant biological function of cGAS.Comet assay was completed to verify the relationship between cGAS and genomic instability.5.The Microarray Data Analysis Based on Clustering Algorithms was work by using R(Pheatmap)and R(corrplot)software package to explore the highest correlation genes with cGAS in 384 GC patients in TCGA database.6.Western blot assay was adopted to explore the expression of DNA damage marker protein y-H2AX and homologous recombination repair key protein RAD51,CtIP,pCtIP,MRE11,pMRE11 in sh-cGAS AGS cell line,and immunofluorescence was implemented to observe the localization of y-H2AX and RAD51 in the cell,which would research the influence of cGAS on HR repair of DNA damage.7.Kaplan-Meier Plotter database was exerted to judge the relationship between the expression of cGAS and prognosis of GC.Results1.TCGA database showed that cGAS is over-express in 384 GC patients(P<0.0001);the IHC experiment also confirmed the high expression of cGAS in GC tumor tissues than adjacent gastric tissues(P<0.001).and also,the qRT-qPCR experiment indicated that The mRNA expression of cGAS in MKN45,NCI-N87 and AGS were higher than in GES-1.2.Cell functional experiments demonstrated that compared with the control group,proliferation ability,invasiveness and wound healing repairability are reduced,but the apoptosis is increasing in si-cGAS AGS and MKN45 cell lines in vitro;and tumor xenograft model of cGAS knockdown MKN45 cells displayed decreased growth rate and tumor mass in vivo.3.GO function and KEGG pathway enrichment analysis indicated that cGAS concentrate on the cell division,mitotic nuclear separation,DNA replication,DNA repair and other 10 biological functions in GC tissues,and is mainly involved in 10 signal pathways,such as DNA replication.And also,comet assay proved that GES-1 cells with overexpressed cGAS have increasing genome instability.4.Western blot experiments exhibited that response to the DNA injury agent etoposide treated,the DNA damage marker protein y-H2AX was down-regulated,while HR repair key proteins RAD51,pCtIP,pMRE11 expression were down-regulated in sh-cGAS AGS cells;and immunofluorescence results confirmed RAD51 foci increased in DNA damage sites.5.There was no correlation between the high expression of cGAS and sex(P=0.691),age(P=0.523),differentiation degree(P=0.123),invasion depth(P=0.648),lymph node metastasis(P=0.383),tumor stage(P=0.849),However,K-M survival analysis displayed that GC patients with low expression of cGAS correlated to improved overall survival.ConclusionscGAS is overexpression in GC,which may promote the malignant transformation of GC cells by inhibiting the HR repair response under conditions of genomic stress and is expected to become potential of a new target for GC therapy. |
