| Background:RhoGTPase activation protein(RhoGAP)promotes the hydrolysis of GTP-bound RhoGTPase,thereby inactivating RhoGTPase,which is involved in many biological processes such as cytoskeleton recombination,cell regulation and the occurrence of malignant tumors.To explore the mechanism of the occurrence and development of gastric cancer,the gene expression profiles of 16 pairs of gastric cancer and paired normal tissues were analysed in this study.We identified upregulated genes in gastric cancer tissues,among which ARHGAP11 A in the RhoGAP family showed significantly high expression.However,the role of ARHGAP11 A in the occurrence and development of gastric cancer and its regulatory mechanism need to be further studied.Objective:This project will investigate the effects of ARHGAP11 A on the proliferation,migration and invasion of gastric cancer cells,explore the role of ARHGAP11 A in the biological behaviour of gastric cancer cells,and identify the proteins that interact with ARHGAP11 A.The mechanism of ARHGAP11 A involvement in the development and progression of gastric cancer was explored from the perspective of its regulation of actin filaments formation to provide a basis for the study of the mechanisms of gastric cancer and the development of potential new strategies for targeted therapy.Methods:1.Bioinformatics analysis conducted to evaluate the expression of ARHGAP11 A in gastric cancer tissues and normal tissues in the GEPIA database,and the protein and m RNA expression levels of ARHGAP11 A in gastric cancer and normal tissues were detected by Western blotting,immunohistochemistry and quantitative real-time PCR(q RT-PCR).Moreover,the relationship between the expression level of ARHGAP11 A and clinical data was studied,and Kaplan-Meier survival analysis was performed on the relationship between the expression level of ARHGAP11 A and the prognosis of gastric cancer patients.2.The effect of ARHGAP11 A on cell proliferation was observed by a high-content cell imaging analysis system,Ed U detection kit,and plate clone formation assay.The scratch test and Transwell assay were used to detect the effects of ARHGAP11 A on cell migration and invasion.The levels of the invasion-related proteins E-cadherin,N-cadherin and MMP9 were detected by Western blotting.A cell senescence detection kit was used to detect the effect of ARHGAP11 A on cell senescence.Immunofluorescence analysis was performed to detect the changes in intracellular stress fibres after ARHGAP11 A was knocked out.Tumor growth was observed by subcutaneous tumorigenesis experiments in nude mice.3.Flow cytometry was used to analyse the cell cycle and apoptosis after ARHGAP11 A knockout.Western blotting was used to detect the protein levels of Cyclin A2,Cycli B1,Cyclin D1,P27,PTEN and AKT in each group.The m RNA expression levels of Cyclin A2,Cyclin B1,and Cyclin D1 were measured by q RT-PCR.4.To study the relationship between ARHGAP11 A and RhoGTase members,we constructed constitutively active mutants RhoA-Q63 L,Rac1-Q61 L,Cdc42-Q61 L of RhoA,Rac1,and Cdc42,and dominant negative mutants RhoA-T19 N,Rac1-T17 N and Cdc42-T17 N.Immunoprecipitation was performed to detect the interaction between ARHGAP11 A and RhoA,Rac1,and Cd C42.Comprehensive IP assays were performed to confirm that the protein interacts with ARHGAP11 A through mass spectrometry identification and bioinformatics analysis,and the interaction between ARHGAP11 A and the interacting protein was detected in 293 T and AGS cells by co-immunoprecipitation experiments.A truncation experiment was used to study the interaction domain between ARHGAP11 A and interacting protein.5.ARHGAP11 A was overexpressed,and interacting protein was knocked out in AGS and HGC27 cells with stable and high expression of ARHGAP11 A.Scratch test,Transwell test and immunofluorescence staining were used to study the specific mechanism of AHGAP11 A regulating the formation of stress fibres through interacting proteins,and then affecting the migration and invasion of gastric cancer.Results:1.High expression of ARHGAP11 A is associated with invasion,metastasis and poor prognosis of gastric cancerBioinformatics analysis in the GEPIA database showed that ARHGAP11 A expression was significantly increased in gastric cancer.Western blotting,q RT-PCR,immunohistochemical staining,and tissue immunofluorescence staining showed that ARHGAP11 A was highly expressed in gastric cancer tissues compared with normal tissues.In addition,the high expression of ARHGAP11 A was highly correlated with the level of tumor differentiation in gastric cancer,and Kaplan-Meier survival analysis further showed that it was related to the poor prognosis of patients.2.ARHGAP11 A promotes gastric cancer cell proliferation,migration and invasion and regulates stress fibres formationARHGAP11A knockout significantly reduced cell proliferation.However,the knockout of ARHGAP11 A did not have significant effects on the number of senescent cells.Flow cytometry analysis showed that the knockout of ARHGAP11 A induced cell cycle arrest in G1 phase but did not affect apoptosis.After ARHGAP11 A knockout,the migration and invasion abilities of gastric cancer cells were significantly reduced,the expression level of E-cadherin increased,and the expression level of N-cadherin and MMP9 decreased.The number and length of stress fibres in the ARHGAP11 A knockout group were significantly reduced,and the stress fibres became thinner.The number of branches,branch length and branch complexity of stress fibres in the ARHGAP11 A knockout group were significantly reduced.3.Subcutaneous tumorigenesis in nude mice showed that ARHGAP11 A promotes tumor formationTo further study the role of ARHGAP11 A in vivo,tumor growth of gastric cancer cells after ARHGAP11 A knockout was observed in a subcutaneous tumorigenesis model.The tumor corresponding to the ARHGAP11 A knockout group was significantly smaller than that of the control group.Immunohistochemical methods were used to detect the expression of ARHGAP11 A and Ki67 in the tumor tissues of the two groups.The results showed that the expression levels of ARHGAP11 A and Ki67 in the knockout group were significantly lower than those of the control group.4.ARHGAP11 A interacts with TPM1The results of co-immunoprecipitation assay showed that there was no interaction between ARHGAP11 A and RhoA,Rac1 and Cdc42 with wild type,dominant negative mutants and constitutively active mutants.Protein–protein interactions were identified by mass spectrometry combined with bioinformatics analysis,and it was found that the actin-binding protein TPM1 interacted with ARHGAP11 A.The interaction between ARHGAP11 A and TPM1 was confirmed in both 293 T and AGS cells by coimmunoprecipitation assay,and the interacting domains were 517-1024.5.TPM1 plays a central role in the malignant transformation of gastric cancer induced by ARHGAP11 A.To further confirm that ARHGAP11 A promotes the invasion and migration of gastric cancer through TPM1,the wound healing experiment and the Transwell assay were used to detect the cell migration and invasion abilities.Compared with the control group,the ARHGAP11 A overexpression group promoted the migration and invasion ability of gastric cancer cells.In addition,we simultaneously knocked out TPM1 in AHGAP11A-overexpressing cell lines,and the migration and invasion abilities were significantly lower than those of the ARHGAP11 A overexpression group,indicating that the ability of ARHGAP11 A to promote migration and invasion is dependent on TPM1 expression.6.ARHGAP11 A promotes the malignant progression of cells by regulating the formation of stress fibresImmunofluorescence analysis showed that the complexity and the number of stress fibres in the OE-ARHGAP11A+KO-TPM1 group were significantly lower than those in the OE-ARHGAP11 A group.In addition,the total length of stress fibres,trees,branches,and the number of branches in the OE-ARHGAP11A+KO-TPM1 group were significantly lower than those in the OE-ARHGAP11 A group,suggesting that the function of ARHGAP11 A in regulating stress fibre formation was dependent on TPM1.Conclusion:1.The expression level of ARHGAP11 A in gastric cancer tissues is significantly higher than that in normal tissues,and the high expression of ARHGAP11 A is highly correlated with the differentiation level of gastric cancer patients,which is a risk factor for poor prognosis of gastric cancer patients.2 ARHGAP11 A can promote the proliferation of gastric cancer cells in vivo and in vitro.The inhibition of ARHGAP11 A can cause cell arrest in G1 phase.3.ARHGAP11 A regulates the number and complexity of stress fibre branching and promotes cell migration and invasion.4.The actin-binding protein TPM1 is the interacting protein of ARHGAP11 A,and the interaction domain between the two is 517-1024.5.ARHGAP11 A regulates stress fibre formation through TPM1 to promote cell migration and invasion of gastric cancer cells. |
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