The Role And Mechanism Of CircDIDO1 In Gastric Cancer Progression

Objective:To study the expression of circRNA in tissue samples of gastric cancer,verify the differentially expressed circRNA,and screen out the target circRNA.To establish the detection method of circDIDO1,clarify its correlation with clinical features in patients with gastric cancer,and provide new molecular markers for prognosis and efficacy of gastric cancer.To study the effect of circDIDO1 on the proliferation,migration,invasion and apoptosis of gastric cancer cells.To explore the exact molecular mechanism of circDIDO1 involved in the development of gastric cancer,and provide a new therapeutic target for gastric cancer.Methods:1.Through bioinformatics analysis of circRNA microarrays of tissue and serum samples of gastric cancer in the GEO database,we screened out the differentially expressed circDIDO1 molecule in gastric cancer tissues.The real-time quantitative PCR technology was used to verify the selected circDIDO1 molecule.Identification analysis and clinicopathological analysis of the selected circDIDO1 were carried out.2.Lentiviral vector was used to transfect gastric cancer cells for circDIDO1 overexpression experiment.Small interfering RNA was used to transfect gastric cancer cells for circDIDO1 gene knockdown experiment.Cell growth curve assay was conducted to detect the effect of circDIDO1 on the proliferation ability of gastric cancer cells.Flow cytometry was performed to detect the effect of circDIDO1 on apoptosis of gastric cancer cells.Transwell migration experiment was used to detect the effect of circDIDO1 on the migration ability of gastric cancer cells.Matrigel invasion test was used to detect the effect of circDIDO1 on the invasion of gastric cancer cells.Subcutaneous tumor model and metastatic tumor model were conducted to detect the effects of circDIDO1 on gastric cancer cell growth and metastasis in vivo.Immunohistochemical experiments were conducted to detect the effects of circDIDO1 on the expression of gastric cancer cell proliferation and migration.3.RNA FISH was used to detect the location of circDIDO1 in gastric cancer cells.TRAP experiment and mass spectrometry analysis were used to detect key protein molecule bound by circDIDO1.FISH/IFC technology was used to detect the colocalization of circDIDO1 and protein.Overexpression and knockdown experiments were used to detect the cellular biological functions of interacting protein.qPCR and Western blot were used to detect the effect of circDIDO1 on the expression level of binding protein.We used catRAPID to predict the binding domain of circDIDO1 and protein,and constructed truncated vectors to verify the binding site.The effects of overexpression and knockdown of circDIDO1 on the signaling pathway downstream of the binding protein were examined by Western blot.4.RIP experiment was used to detect whether circDIDO1 could bind to AGO2 protein.CircInteractome and StarBase V2.0 were used to predict miRNA molecules that may bind to circDIDO1.We constructed the circDIDO1 dual luciferase reporter gene vector,and synthesized miRNA mimics,and then co-transfected 293 T cells with circDIDO1 reporter gene vector and miRNA mimics to detect the regulatory effect of miRNA on reporter gene activity.The circDIDO1 reporter gene vector with miRNA binding site mutation was constructed to further verify the binding site of circDIDO1 and miRNA.The gastric cancer cells were transfected with miRNA mimics to detect the effect of miRNA on the biological function of gastric cancer cells and verified by a rescue experiment.Results:1.circDIDO1 was significantly down-expressed in gastric cancer tissues,and the low expression of circDIDO1 was associated with tumor size,distant metastasis and poor prognosis.circDIDO1 is formed by reverse splicing of the linear transcript of exons 2-6 of DIDO1 gene.When using circDIDO1 specific primers for PCR experiments,PCR products could only be amplified from the cDNA template,but not from the gDNA template.circRNA characterization analysis results showed that circDIDO1 was resistant to RNase R digestion.2.The results showed that circDIDO1 overexpression by lentivirus-mediated gene transfer inhibited the proliferation and clonal formation of gastric cancer cells.In addition,overexpression of circDIDO1 could also inhibite the migration and invasion of gastric cancer cells.In contrast,siRNA-mediated knockdown of circDIDO1 expression in gastric cancer cells promoted gastric cancer cell proliferation,clonal formation,migration and invasion.The results of nude mice subcutaneous and metastatic tumor models in vivo showed that overexpression of circDIDO1 inhibited the growth and metastasis of gastric cancer.3.The result of RNA in situ hybridization showed that circDIDO1 was distributed in the cytoplasm and nucleus.TRAP experiments and mass spectrometry results showed that circDIDO1 could specifically bind to PRDX2.Overexpression of circDIDO1 could downregulate PRDX2 protein level without changing PRDX2 mRNA level.The result of FISH/IFC showed that circDIDO1 and PRDX2 were co-localization in gastric cancer cells.circDIDO1 promoted the ubiquitination degradation of PRDX2 protein.After treatment with proteasome inhibitor MG-132,PRDX2 protein degradation was inhibited in gastric cancer cells.Using cycloheximide(CHX)to inhibit protein synthesis,the result showed that the half-life of PRDX2 protein in gastric cancer cells overexpressing circDIDO1 was significantly shorter.4.RIP experiment result showed that circDIDO1 could bind to AGO2 protein.CircInteractome and CIRC explorer software were used to predict the miRNA molecules bound by circDIDO1.The luciferase reporter gene experiment result showed that miR-1307-3p had a strong inhibiting effect on the circDIDO1 luciferase reporter gene activity.qRT-PCR result showed that miR-1307-3p overexpression inhibited the expression level of circDIDO1,suggesting that the low expression of circDIDO1 in gastric cancer may be related to miR-1307-3p-mediated gene silencing.In addition,miR-1307-3p overexpression promoted gastric cancer cell proliferation,migration and invasion.Conclusions:The low expression of circDIDO1 in tissues of patients with gastric cancer is associated with tumor size,distant metastasis and poor prognosis of gastric cancer,and circDIDO1 can be used as a molecular marker for prognosis of gastric cancer;circDIDO1 inhibits the activation of ERK and β-catenin signaling pathways downstream of PRDX2 by promoting the ubiquitination and degradation of PRDX2,thereby inhibiting the transcription of oncogenes such as c-Myc and CyclinD1;circDIDO1 is regulated by upstream miR-1307-3p,and the highly expressed miR-1307-3p in gastric cancer downregulates the expression of circDIDO1.