| Background:Gastric cancer is one of the most common digestive tract tumors.Influenced by clinical manifestations and detection methods,gastric cancer is often diagnosed at an advanced stage,resulting in distant metastasis and chemotherapy resistance in most patients.New diagnostic markers are urgently needed to solve clinical problems.treatment problems.The ETS transcription factor family can participate in the regulation of almost all cellular biological processes,and the abnormal expression of this family in tumor cells is closely related to the malignant progression of tumors.ELK3 is a special member of the ETS transcription factor family,which promotes transcription under the stimulation of Ras and Mitogen-Activated Protein Kinase(MAPK).In tumors,ELK3 can serve as an important therapeutic target,which can promote tumor cell proliferation,metastasis,drug resistance,and epithelial-mesenchymal transition(EMT).EMT is a crucial mechanism of cell morphological plasticity changes in embryonic development and tumor metastasis and plays an important role in tumor invasion,metastasis,and drug resistance.Studies have shown that EMT-producing cells can promote the chemoresistance of tumor cells through various mechanisms.Among them,the transforming growth factor-beta(TGF-β)signaling pathway,as the main factor inducing EMT,it’s can make cancer cells resistant to chemotherapeutic drugs.Greater resistance occurs.Reports have shown that ELK3 not only promotes TGF-β signaling but also promotes phenotypes such as EMT and drug resistance in tumor cells.However,whether ELK3 can induce the above process in gastric cancer,the specific mechanism and the relationship between them are still unclear.To solve these problems,this study firstly explored the expression and prognosis of ELK3 in gastric cancer and the potential mechanism of ELK3 affecting tumor cell proliferation,metastasis,and drug resistance based on bioinformatics;Secondly,in vitro and in vivo experiments were performed to verify whether ELK3 could promote EMT-induced chemotherapy resistance in gastric cancer by activating TGF-β signaling,and to analyze its specific mechanism.Thus,elucidating the regulatory mode of ELK3 affecting the progression of gastric cancer will be helpful for the precise treatment of gastric cancer and provide a theoretical basis for the development of more anticancer drugs with independent intellectual property rights in China.Materials and methods:1.Comprehensive analysis of the ETS transcription factor family(1)Integrate the sequencing data of gastric and gastric cancer from The Cancer Genome Atlas and Genotype Tissue Expression database,and analyze the differential expression of the ETS transcription factor family between gastric cancer and normal gastric tissues;Combined with clinical data,the expression level of the ETS transcription factor family in different clinicopathological characteristics were analyzed;(2)Use the copy number and single nucleotide polymorphism data of gastric cancer in The Cancer Genome Atlas database to analyze the possibility of genetic changes affecting the expression of ETS transcription factors;(3)Based on the expression data of the ETS transcription factor family in gastric cancer and the DNA methylation data of gastric cancer in The Cancer Genome Atlas database,the differences in the DNA methylation levels of the ETS transcription factor family were analyzed,and analyze the correlation between its m RNA expression and DNA methylation level;(4)Using the gastric cancer prognostic information in the database,univariate and multivariate Cox regression analysis was used to analyze the relationship between the ETS transcription factor family expression and prognosis;and the independent prognostic performance of ELK3 was analyzed combined with pathological characteristics.2.Prognostic verification of ELK3 expression and its effect on proliferation and metastasis of gastric cancer cells(1)Obtain gastric cancer expression profile microarray datasets in GEO and Array Express databases,extract paired samples and use paired samples t-test to verify the difference in ELK3 expression between gastric cancer and adjacent normal tissues;(2)Collect clinical gastric cancer samples,analyze the protein and m RNA expression differences of ELK3 in clinical gastric cancer samples and adjacent normal tissues by immunohistochemical staining,Western-blot,and real-time quantitative PCR,and analyze the relationship between ELK3 m RNA expression and clinicopathological characteristics;(3)The relationship between ELK3 and prognosis in gastric cancer Affymetrix platform microarray data set was analyzed on the Kaplan-Meier Plotter website;multidataset meta-analysis of gastric cancer expression profiling microarray datasets obtained from GEO and Array Express databases was used to further verify whether ELK3 is associated with poor prognosis;(4)Obtain the gastric cancer cell line dataset from the GEO database,integrate and analyze the expression of ELK3 in different gastric cancer cell lines,the expression of ELK3 in gastric cancer cell lines and normal gastric mucosal cell lines was verified by Western-blot,rt-q PCR,and cellular immunofluorescence;(5)The expression of ELK3 was changed in gastric cancer cell lines,and the effects of ELK3 on the proliferation,invasion,and migration of gastric cancer cells were analyzed by cell cycle detecting,cell plate clone formation,EDU staining,cell scratch,Transwell,and Western-blot.3.Preliminary analysis of the mechanism by which ELK3 promotes TGF-β signaling and affects gastric cancer progression(1)Gene set enrichment analysis was performed on the datasets with more than 200 gastric cancer samples,and Venn analysis was performed to identify signaling pathways that were co-enriched in samples with high ELK3 expression in different datasets;(2)The expression of ELK3 was changed in gastric cancer cells and combined with TGF-β active factor treatment,and the effect of ELK3 on the TGF-β signaling pathway and EMT phenotype of gastric cancer cells was analyzed by cell scratch,Transwell,and Western-blot.4.The mechanism of ELK3 regulating TGFBR2 to promote EMT in gastric cancer(1)Gene set enrichment analysis was performed on data sets with more than 200 gastric cancer and normal gastric tissues to understand whether Ras and MAPK signals were activated in gastric cancer samples,and to analyze whether ELK3 had transcriptional activation in gastric cancer;(2)Analyze whether TGF-β receptor 2(TGF-β Receptor 2,TGFBR2)is a downstream target gene of ELK3 by bioinformatics,and analyze the correlation between ELK3 and TGFBR2 in m RNA expression levels in multiple data sets;(3)The m RNA expression of TGFBR2 was analyzed in clinical samples,and the correlation between ELK3 and TGFBR2 expression in clinical samples was analyzed;the expression of ELK3 was changed in gastric cancer cells,and the expression changes of TGFBR2 were analyzed by Western-blot and real-time quantitative PCR;(4)Predict the binding site of ELK3 in the promoter region of TGFBR2 from public databases,and use chromatin immunoprecipitation and dual-luciferase reporter gene experiments to verify whether ELK3 directly regulates TGFBR2;(5)Altering the expression of ELK3 in gastric cancer cells and changing the expression of TGFBR2 in combination,using cell scratch,Transwell and Western-blot to analyze the changes of TGF-β signal transduction and EMT phenotype.5.In vitro and in vivo studies on the mechanism of ELK3-induced chemoresistance(1)Gastric cancer cells with altered ELK3 expression were treated with 5-fluorouracil(5-FU),and the number of apoptosis was detected by Tunel staining;(2)Gastric cancer cells with altered ELK3 expression were treated with different concentrations of 5-FU,the cell proliferation was detected by CCK-8,and the halfinhibitory concentration of 5-FU in different cells was analyzed;(3)Gastric cancer cells with altered ELK3 expression were treated with 5-FU and EMT inhibitor Rondomycin.The apoptosis rate was detected by flow cytometry,and the protein changes in EMT phenotype,drug resistance,and apoptosis were analyzed by Western-blot;(4)To establish a mouse tail vein metastasis model of stably altered ELK3 gastric cancer cells,and analyze the phenotype of metastasis and EMT;at the same time,a nude mouse tumorigenic model was constructed and treated with 5-FU to analyze the changes in the tumorigenic ability of different treatment groups.Results:1.Comprehensive analysis of the ETS transcription factor family in gastric cancer shows that ELK3 is a core gene(1)Differential analysis of 29 ETS transcription factors showed that in gastric cancer,22 were up-regulated,6 were down-regulated,and the expression of 23 genes was correlated with clinicopathological features;(2)The DNA methylation levels of 12 ETS transcription factors are out of balance,and the m RNA expression of 11 genes is negatively correlated with the DNA methylation level,suggesting that there may be the possibility of DNA methylation regulating expression;the expression levels of all genes are lowly correlated with changes in genetic status;(3)The expression of 4 ETS transcription factors was associated with overall survival and 4 with progression-free survival.Among them,ELK3 could predict both prognostic outcomes and was an independent prognostic factor for gastric cancer patients.2.ELK3 acts as an oncogene to promote the malignant progression of gastric cancer cells(1)Bioinformatics analysis was performed on 15 gastric cancer data sets,15 pairs of frozen clinical gastric cancer samples were analyzed by immunohistochemical staining,and 81 pairs of fresh gastric cancer samples were analyzed by Western-blot and real-time quantitative PCR.The expression of ELK3 was significantly higher than that in adjacent normal tissues;the study of the clinical cohort showed that the expression of ELK3 was correlated with the degree of tissue differentiation,T stage,N stage,and pathological stage;(2)Kaplan-Meier analysis of dataset on the Affymetrix platform showed that ELK3 was associated with poor prognosis;prognostic meta-analysis of multiple datasets showed that gastric cancer patients with high expression of ELK3 had poor prognosis;(3)Down-regulation of ELK3 expression in Hs746 t gastric cancer cells with high ELK3 expression inhibited cell proliferation,invasion,and migration;up-regulation of ELK3 in HGC27 gastric cancer cells with low ELK3 expression increased the proliferation,invasion,and migration capacity of ELK3 cells.3.ELK3 promotes EMT in gastric cancer through canonical TGF-β signaling(1)Gene set enrichment analysis was performed on 6 data sets,and Venn analysis was performed on the obtained results,and a total of 16 co-enriched signaling pathways including Epithelial-mesenchymal transition and TGF-β were obtained;(2)Knockdown of ELK3 in Hs746 t cells can inhibit cell invasion,migration,TGF-β signaling,and EMT phenotype;overexpression of ELK3 in HGC27 cells promotes these signals and cellular phenotypes;TGF-β factor treatment further promotes the functional phenotype and signal transduction of these cells.4.ELK3 transcriptionally regulates TGFBR2 and activates TGF-β signaling and promotes EMT in gastric cancer(1)Through bioinformatics analysis,11 co-enriched core genes including TGFBR2 were identified in the TGF-β signaling pathway co-enriched in 6 datasets,and ELK3 was positively correlated with TGFBR2 expression in multiple datasets;(2)In fresh gastric cancer samples,the expression of ELK3 was significantly positively correlated with TGFBR2,and changing the expression of ELK3 in gastric cancer cells resulted in corresponding changes in the expression of TGFBR2;(3)The gene set enrichment analysis showed that both Ras and MAPK signaling pathways were significantly enriched in gastric cancer samples,suggesting that ELK3 can act as a transcriptional activator;the public database predicts that there are three ELK3 binding sites in the promoter region of TGFBR2,and chromatin immunoprecipitation and dual-luciferase reporter gene experiments show that two of these sites are transcriptionally active;(4)Up-regulation of TGFBR2 in Hs746 t cells with stably knocked down ELK3 reversed the inhibition of cell invasion,migration,TGF-β signaling,and EMT phenotype;down-regulation of TGFBR2 in HGC27 cells with stably overexpressing ELK3 inhibited the aforementioned functions,signal transduction and phenotype of the cells.5.ELK3 induces chemoresistance in gastric cancer by promoting EMT(1)Inhibiting the expression of ELK3 can reduce the half inhibitory concentration of 5-FU on gastric cancer cells,and overexpression can increase the half inhibitory concentration of 5-FU on gastric cancer cells and promote chemotherapy resistance;(2)In Hs746 t cells,inhibiting the expression of ELK3 combined with the 5-FU group increased the apoptosis level and decreased the expression of EMT-promoting and drug resistance-related proteins compared with the 5-FU group alone;in HGC27 cells,the apoptotic rate of promoting ELK3 expression combined with 5-FU group was lower than that of 5-FU group alone,and the expressions of EMT-promoting and drug resistance-related proteins were up-regulated;after treatment with Rondomycin,the apoptosis level in each group increased correspondingly,and the expression of EMTpromoting and drug-resistant proteins decreased;(3)The results of the nude mouse tail vein model showed that down-regulation of ELK3 inhibited the in vivo metastasis ability and EMT phenotype of gastric cancer cells,while over-expression of ELK3 promoted cell metastasis and EMT;(4)The results of the subcutaneous tumorigenic model in nude mice showed that down-regulation of ELK3 inhibited the tumorigenic ability of gastric cancer cells,while overexpression of ELK3 increased the tumorigenic ability;the tumorigenic ability of down-regulated ELK3 combined with 5-FU group was lower than that of 5-FU alone group,and the tumorigenic ability of overexpressed ELK3 combined with 5-FU group was higher than that of 5-FU alone group.Conclusions:1.ETS transcription factor ELK3 is highly expressed in gastric cancer tissue and is associated with a poor prognosis of gastric cancer2.ELK3 promotes the proliferation and migration of gastric cancer cells and acts as an oncogene in gastric cancer;3.ELK3 promotes epithelial-mesenchymal transition in gastric cancer cells by regulating TGF-β signaling;4.ELK3 transcriptionally activates the expression of TGFBR2 and promotes TGF-β signaling to induce epithelial-mesenchymal transition in gastric cancer;5.ELK3 promotes chemoresistance in gastric cancer cells through epithelial-mesenchymal transition. |
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