| Background: Gastric cancer?GC?is one of the most common malignant tumors worldwide.It is also a major disease that causes cancer deaths in China and worldwide.Although the diagnosis and comprehensive treatment strategies for gastric cancer have improved significantly,the5-year overall survival rate for patients with advanced gastric cancer is only about 30%.The prognostic factors of patients with gastric cancer are related to malignant biological behaviors such as rapid proliferative capacity,lymphatic/blood-derived metastasis,and direct invasion.The molecular mechanisms that cause malignant biological behaviors such as gastric cancer occurrence,development,and recurrence and metastasis are not fully understood.Circular RNA?circRNA?,as a kind of non-coding RNA with a circular structure,has recently been reported in a variety of tumors as a diagnostic biomarker and therapeutic target.Based on this,combining our team's circRNA chip and the gastric cancer circRNA chipin the GEO database,we screened and found significantly differently expressed hsacirc0000390?Note: It is derived from chr12: 32760890 |32764217 and consists of exons 8,9,and 10 of the parental gene FGD4,the size is 345 bp,named crcFGD4?as the research molecule.This project intends to explore the expression of circFGD4 in gastric cancer,its biological characteristics,and the potential mechanisms involved in regulating gastric cancer.It is hoped that this study can provide new ideas and new evidence for the diagnosis and treatment of gastric cancer.Objectives: To elucidate the biological function of circFGD4 in gastric cancer and its molecular mechanism that may regulate gastric cancer.Methods:?1?Our team's circRNA chip and the circRNA chip in the GEO database jointly were used to screen for the differentially expressed circFGD4 molecules in gastric cancer.The qRT-PCR assay was used to detect the expression of circFGD4 and the parental gene FGD4 in human gastric cancer and adjacent tissues.The correlation between circFGD4 expression and clinical cases in gastric cancer patients was analyzed.The association between the expression of circFGD4 and FGD4 in human gastric cancer tissues was analyzed.qRT-PCR and FISH assays were used to explore the expression and localization of circFGD4 in human gastric cancer cells.?2?In vitro experiments were performed to construct gastric cancer cell lines that overexpressed or silenced circFGD4.CCK-8,EdU,colony formation,Transwell,wound healing,WB,and IF assays were used to validate the effects of circFGD4 on gastric cancer cell proliferation,migration,and EMT.In vivo experiments were performed using gastric cancer cell lines overexpressing circFGD4 to establish subcutaneous tumor formation and lung metastasis models in nude mice,and qRT-PCR and IHC assays were used to detect proliferation and EMT markers.Bioinformatics analysis,qRT-PCR,FISH,and dual-luciferase assays were used to identify the competitive binding of circFGD4 and miR-532-3p.The rescue experiment showed that after interfering with circFGD4 and miR-532-3p molecules,CCK-8,colony formation,Transwell,and WB assays were used to confirm the effects of circFGD4 on gastric cancer proliferation,migration,and EMT through miR-532-3p.?3?Bioinformatics analysis screened miR-532-3p downstream molecules and signal pathways,and qRT-PCR,WB,and dual-luciferase assays were used to detect the relationship between miR-532-3p and APC in gastric cancer cells.CircFGD4 was overexpressed/silencing in gastric cancer cells,and APC expression was detected by WB and IF assays.The rescue experiment showed that after interfering with circFGD4 and miR-532-3p,and APC expression was detected by WB and IF assays.After overexpression or silencing with circFGD4 in gastric cancer cells,wedetected TOP / FOP changes and ?-catenin expression and localization.Rescue experiments revealed that after interfering with circFGD4 and miR-532-3p,we then detected TOP / FOP changes and ?-catenin expression and localization.Results:?1?CircFGD4 was relatively low expressed in human gastric cancer tissues.Low expression of circFGD4 was related to poor tumor differentiation and lymphatic metastasis in patients with gastric cancer.CircFGD4 was relatively low expressed in human gastric cancer cells and was mainly located in the cytoplasm of gastric cancer cells.There was no significant correlation between circFGD4 expression and the parental gene FGD4 expression in gastric cancer.?2?In vitro experiments showed that circFGD4 inhibited gastric cancer cell proliferation,colony formation,migration,and induced EMT.In vivo experiments showed that circFGD4 inhibited subcutaneous tumor formation and lung metastasis in nude mice,accompanied by changes in indicators such as proliferation and EMT.CircFGD4 and miR-532-3p had a competitive binding relationship in gastric cancer.CircFGD4 inhibited gastric cancer cell proliferation,colony formation,migration,and induced EMT by competitively binding to miR-532-3p.?3?Bioinformatics analysis showed that the targeted molecules?including APC?predicted by miR-532-3p were closely related to cell migration and the ?-catenin signaling pathway.CircFGD4 and miR-532-3pcould regulate APC expression in gastric cancer,respectively,and rescue experiments displayed that circFGD4 competitively bound with miR-532-3p to regulate APC expression.CircFGD4 could regulate the?-catenin pathway in gastric cancer,and rescue experiments revealed that circFGD4 competitively bound miR-532-3p to regulate the ?-catenin pathway,thereby causing that circFGD4 inhibited gastric cancer cell proliferation,migration and induced EMT.Conclusion: CircFGD4 competitively binds to miR-532-3p in gastric cancer,and then regulates APC expression to inactivate the ?-catenin signaling,thereby inhibiting gastric cancer cell proliferation,migration,and inducing changes in EMT. |
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