Circular RNA Circ-0002570 Accelerates Cancer Progression By Regulating VCAN Via MiR-587 In Gastric Cancer

Objective: Circular RNAs(CircRNAs)are closely associated with the occurrences and progress of gastric cancer(GC).We aimed to delve into the function and pathological mechanism of Circular RNA-0002570(Circ-0002570)in GC progression.Methods: 1.Bioinformatics techniques were used to screen out differentially expressed CircRNAs in GC tissues.The tumor tissues and adjacent normal tissues of24 GC patients confirmed by the Pathology Department in The First Hospital of Lanzhou University from January 2018 to July 2019 were collected.qRT-PCR was used to detect the expression of CIRC-0002570 in GC samples and common GC cell lines(AGS,MKN28,MKN45,SGC7901,HGC27,BGC23)owned by our research group.Kaplan-meier curve survival analysis was performed to explore the correlation between cir C-0002570 expression and prognosis of GC patients.2.In order to further study the role of Circ-0002570 in the function of GC cells,the GC cell model with knockdown Circ-0002570 expression was constructed by lentivirus transfection in vitro,and verified by qRT-PCR.CCK8,EDU and clonogenesis assay were used to detect the effect of Circ-0002570 on the proliferation of GC cells.The effect of Circ-0002570 on GC cell metastasis was detected by cell scratch,Transwell migration and invasion assay.TUNEL assay was used to detect the effect of Circ-0002570 knockdown on apoptosis of GC cells.The xenograft tumor model of nude mice was constructed,and the effect of Circ-0002570 on the growth ability of GCcells in vivo was detected.3.The localization of Circ-0002570 in gastric cancer cells was detected by RNA FISH in situ hybridization.Mi RNA binding sites of Circ-0002570 were predicted by CircBank and CircInteractome databases.The expression of Circ-0002570 in gastric cancer cells after overexpression of miR-587 or miR-935 was further verified by qRT-PCR.Circ-0002570 reporter vector and miR-587 mimics were co-transfected.Luciferase detection kit detected luciferase activity.qRT-PCR was used to detect the overexpression and knockout of Mir-587 in Circ-0002570 gastric cancer cells,and RIP assay was further used to determine the relationship between Circ-0002570 and miR-587.4.Screening and identification of miR-587 targets by bioinformatics analysis,luciferase reporter assay and Western blot.qRT-PCR and western blot assay were used to detect the expression of in gastric cancer tissues.Survival analysis based on VCAN expression was performed using TCGA STAD queues and KM online tools.KEGG and GO enrichment analysis was further used to predict the clinical significance of VCAN.Finally,VCAN expression was detected by qRT-PCR and Western blot.The effects of Circ-0002570-miR-587-VCAN on biological functions of gastric cancer cells were verified by CCK-8,Edu,clonogenesis assay and Transwell assay.Results: 1.Bioinformatics analysis showed that Circ-0002570 was one of the most up-regulated CircRNAs in gastric cancer.Circ-0002570 was high expression in GC tissues.Compared with human normal gastric mucosa cells(GSE-1),the expression of Circ-0002570 was also significantly increased in gastric cancer cell lines.Meanwhile,GC patients with higher Circ-0002570 expression had poor prognosis.2.The knockdown expression model of Circ-0002570 was successfully constructed in vitro.CCK-8,EdU and clonogenesis experiments showed that the knockdown of Circ-0002570 inhibited the proliferation of gastric cancer cells.Scratch assay and Transwell assay showed that the invasion and migration of gastric cancer cells transfected with Circ-0002570-SH were inhibited.Knockdown the expression of Circ-0002570 significantly promoted apoptosis.According to the in vivo experiment results of xenograft tumor model,we found that tumor growth rate of cir C-0002570 nude mice was not only slower than that of NC-sh group,but also significantly decreased tumor volume and weigh.Immunohistochemistry showed that silencing Circ-0002570 reduced the positive expression of Ki-67.Circ-0002570 enhanced the carcinogenic effect of gastric cancer cells to a certain extent.3.Circ-0002570 was expressed in both the nucleus and cytoplasm.Online database analysis and qRT-PCR results showed that after overexpression of miR-587 in gastric cancer cells,the expression of Circ-0002570 was decreased.Luciferase activity was measured and it was observed that co-transfection of Luciferase activity was significantly reduced in cells co-transfected with miR-587 mimics and Circ-0002570-WT.In addition,overexpression of Circ-0002570 inhibited the expression of miR-587,while silencing of Circ-0002570 promoted the expression of miR-587.RIP assay also confirmed that Circ-0002570 can be used as a sponge to competitively bind miR-587.4.Database bioinformatics analysis combined with luciferase activity determination confirmed that VCAN was the target of miR-587.After miR-587 was overexpressed in gastric cancer cells,the expression levels of VCAN RNA and protein were significantly decreased.Further Western blot analysis confirmed the above regulation.Salvage experiments showed that the significant inhibition of VCAN expression by Circ-0002570-sh was reversed by miR-587 inhibitor.The expression of VCAN in gastric cancer tissue is significantly increased.The expression of VCAN was negatively correlated with prognosis.VCAN is significantly associated with multiple tumor-related pathways.The results showed that Circ-0002570 promoted the progression of GC by competitively binding miR-587 to increase VCAN expression.Conclusion: CirC-0002570 is overexpressed in gastric cancer samples,and the increase of Circ-0002570 predicts poor prognosis of gastric cancer patients.The Circ-0002570-miR-587-VCAN regulatory pathway promoted the proliferation,migration and invasion of GC cells.The results of this study provided potential new targets for the diagnosis and therapy of GC.