| Objective: Gastric cancer is a malignant tumor of the digestive tract with high morbidity and mortality.Its diagnosis and treatment still have certain limitations.The objective of this study was to find circular RNA(Circ RNA)derived from plasma small extracellular vesicles(sEVs)with good clinical value in the progression of gastric cancer.Through screening and identification,we determined hsa＿circ＿0012172(circ12172)as the research object,and then evaluated its clinical value in the progression of gastric cancer,explored its specific mechanism of action on the development of gastric cancer,and provided new ideas for the diagnosis and treatment of gastric cancer.Methods: Plasma was collected from age and sex matched healthy people,gastritis patients(chronic atrophic gastritis),and gastric cancer patients.Then,sEVs were extracted.The differential circ RNA expression profiles were obtained by ce RNA chip sequencing,and circ12172 was identified as the target molecule.RNase R assay and sanger sequencing were used to verify its stability.Nanoparticle tracking analysis(NTA),transmission electron microscopy(TEM)and protein characterization were used to evaluate the quality of plasma and cell-derived sEVs.q RT-PCR was used to detect the expression of circ12172 in normal gastric epithelial cells(GES-1)/gastric cancer cells and plasma sEVs of healthy people/gastritis patients/gastric cancer patients,and the ROC curve was used to evaluate its diagnostic efficacy.Fluorescence in situ hybridization(FISH)and nuclear-cytoplasmic separation assay were used to determine the localization of circ12172 in gastric cancer cells.Overexpression plasmid and si RNA were constructed to overexpress and knockdown circ12172.The biological function of circ12172 in gastric cancer cells was revealed through CCK8,colony formation,migration,invasion,and Western blot.The protein,miRNA and its downstream target molecules that may bind to circ12172 were predicted by bioinformatics,then screened and confirmed by RIP,q RT-PCR and dual luciferase reporter gene experiments.The supernatant of circ12172 overexpressing cells was collected,and sEVs were extracted by ultracentrifugation and co-cultured with AGS cells.The biological effect of sEVscirc12172 was explored by cell functional experiments.Results: Circ12172 was down-regulated in the plasma of sEVs in patients with gastritis(p<0.01)and gastric cancer(p<0.001)compared with healthy people,and was upregulated after surgery in patients with gastric cancer(p<0.05).The diagnostic efficacy of circ12172 in gastric cancer was 0.7133,and clinical correlation analysis was related to age(p=0.032)and depth of invasion(p=0.047).Circ12172 was significantly downregulated in gastric cancer cells and their sEVs.Circ12172 was localized in the nucleus and cytoplasm and could withstand RNase R digestion.Overexpression of circ12172 could inhibit the proliferation,migration and invasion of gastric cancer cells,while knockdown of circ12172 could reverse the effect.RIP experiment showed that circ12172 could bind to Ago2.The results of bioinformatics prediction showed that miR-6799-3p could bind to circ12172,which was also proved by dual luciferase reporter gene experiment.Mi R-6799-3p was significantly up-regulated in gastric cancer cells compared with GES-1,which could enhance the proliferation,migration and invasion of gastric cancer cells,and reverse the inhibitory ability of circ12172 on the proliferation and metastasis of gastric cancer cells.As a downstream target of miR-6799-3p,MXI1 further affected the expression of C-myc and Cyclin D1.The expression of sEVs in plasma of gastric cancer patients was positively correlated with circ12172,which could inhibit the progression of gastric cancer.In addition,sEVs-circ12172 inhibited the proliferation,migration and invasion of gastric cancer cells and participated in intercellular communication.Conclusion: Circ12172 is significantly down-regulated in gastric cancer cells,plasma sEVs of gastric cancer patients and gastritis patients.Circ12172 can play a tumor suppressor role through miR-6799-3p/MXI1/C-myc/Cyclin D1 axis,and can be enriched in sEVs and participate in intercellular communication.Circ12172 is expected to be a new target for gastric cancer progression. |
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