CircPTPN22 Regulates Autophagy In Gastric Cancer Cells Through MiR-6788-5p/PAK1 Axis

Objectives 1.To detect the relative expression levels of autophagyrelated CircPTPN22 in plasma of gastric cancer patients and explore the potential of plasma CircPTPN22 as a biomarker for gastric cancer.2.To investigate the upstream regulatory mechanisms of CircPTPN22 and its effects on the biological functions of gastric cancer cells such as autophagy.3.To investigate the binding regulatory relationship between miR-6788-5p and CircPTPN22 and their effects on the biological progression of gastric cancer.4.To clarify the mechanism by which the CircPTPN22/miR-6788-5p/PAK1 axis regulates autophagy in gastric cancer cells.Methods 1.Immunohistochemistry to detect autophagy levels in gastric cancer tissues;whole transcript sequencing and RT-q PCR experiments to screen CircPTPN22 associated with autophagy and detect its expression level.2.ROC curve to evaluate the diagnostic performance of CircPTPN22 and Kaplan-Meier curve to analyze the prognosis of gastric cancer patients.3.Bioinformatics tools to predict upstream regulated transcription factors and binding proteins of CircPTPN22,and verify the binding using CHIP and RIP experiments.4.Transmission electron microscopy and m RFP-GFP-LC3 dual fluorescence autophagy indicator system experiments to detect the changes of cellular autophagy levels.Verification of CircPTPN22 regulation of gastric cancer biological progression using CCK-8 assay,cell colony formation,Ed U,Transwell,flow cytometry,Western blot and in vivo nude mouse assay.5.Dual luciferase reporter gene assay to verify the binding of CircPTPN22 to miR-6788-5p and miR-6788-5p to PAK1.6.Functional reversion assay was used to verify whether CircPTPN22 regulates PAK1 expression through competitive binding of miR-6788-5p and thus affects autophagy and other biological functions in gastric cancer cells.Results Autophagy levels in gastric cancer tissues were heterogeneous.The autophagy-associated CircPTPN22 was upregulated in gastric cancer tissues and plasma,and its expression was significantly different among gastric cancer patients,gastritis patients and healthy subjects with positive correlation with metastasis.ROC curve analysis revealed that CircPTPN22 could effectively distinguish gastric cancer patients from healthy subjects and gastric cancer patients from gastritis patients.Analysis of clinicopathological data revealed that CircPTPN22 expression correlated with tumor size(P = 0.001),lymph node metastasis(P < 0.0001)and TNM stage(P < 0.0001).Kaplan-Meier analysis showed that patients in the group with high CircPTPN22 expression had poorer survival.The transcription factor RUNX1 binds to the promoter region of PTPN22 and negatively regulates the expression of PTPN22 and CircPTPN22.FUS and ELAVL1 bind to the pre-m RNA of CircPTPN22 and positively regulate the expression of CircPTPN22.Interfering with CircPTPN22 promoted autophagy and apoptosis and inhibited proliferation and metastasis of gastric cancer cells.In vivo tumorigenic experiments in nude mice revealed that interference with CircPTPN22 inhibited tumor growth.CircPTPN22 is mainly localized in the cytoplasm and can bind to miR-6788-5p.In addition,miR-6788-5p was down-regulated in gastric cancer tissues and negatively correlated with CircPTPN22 expression.miR-6788-5p had an inhibitory effect on gastric cancer progression.The addition of miR-6788-5p inhibitor could effectively reply to the effect of interfering with CircPTPN22 on the ability of gastric cancer cells such as autophagy.MiR-6788-5p was able to bind to the 3’-UTR of PAK1,which was upregulated and negatively correlated with miR-6788-5p expression and positively correlated with CircPTPN22 expression in gastric cancer tissues.In vitro cell function experiments revealed that overexpression of PAK1 could effectively reply to the effect of interfering with CircPTPN22 on autophagy in gastric cancer cells.In addition,interference with CircPTPN22 reduced the phosphorylation levels of AKT and ERK,which could be reverted by adding miR-6788-5p inhibitor or by overexpression of PAK1.Conclusions The expression of CircPTPN22 associated with autophagy is upregulated in gastric cancer tissues and plasma and can be used as a potential biomarker for gastric cancer;the expression of CircPTPN22 is regulated by the transcription factor RUNX1 and the binding proteins FUS and ELAVL1;CircPTPN22 can act as an oncogene through the miR-6788-5p/PAK1 axis to activate AKT and phosphorylation of ERK,which regulates autophagy and thus accelerates the progression of gastric cancer cells.