The Role And Mechanism Of Hsa＿circ＿0007991 In Gastric Cancer Diagnosis And Progression

Objective:CircularRNAs(circRNAs)are critically involved in the development and progression of human cancer.However,the roles of circRNAs in gastric cancer(GC)and its underlying mechanism have not been fully understood.This study aims to screen and identify the differentially expressed hsa＿circ＿0007991(circ7991)in gastric cancer,establish its detection method,clarify its correlation with the clinicopathological characteristics of gastric cancer,and provide a new biomarker for the prognosis and efficacy judgment of gastric cancer.Moreover,future research for circ7991 is to help elucidate its biological function,molecular regulatory mechanism involved in the malignant progression of gastric cancer and provide new therapeutic targets for gastric cancer.Methods:1.Bioinformatics analysis was performed using the results of gastric cancer tissue and plasma circRNA chips in the GEO database to screen out abnormally expressed circRNAs in gastric cancer tissues.Hsa＿circ＿0007991(named as circ7991)that is differentially expressed in gastric cancer tissues was verified by real-time quantitative PCR.The expression distribution of circ7991 in gastric cancer cells was detected by q RT-PCR after nucleocytoplasmic isolation andRNA fluorescence in situ hybridization,and circ7991 was characterized and statistically evaluated for the correlation with clinicopathological features of gastric cancer patients.2.After constructing a plasmid overexpressing circ7991 and knocking down circ7991 small interferingRNA,the expression efficiency of circ7991 overexpression and knockdown was detected by real-time quantitative PCR in gastric cancer cell lines.Growth curves,clone formation and Transwell experiments were used to determine the effect of circ7991 on the proliferation and invasion of gastric cancer cells.The apoptotic and cyclic effect of circ7991 on gastric cancer cells were performed via flow cytometry.To determine the effect of circ7991 on the proliferation and metastasis of gastric cancer cells in vivo,a subcutaneous and metastasis model by subcutaneous and intraperitoneal injection into BALB/c nude mice were established.Immunohistochemical analysis was applied to determine the expression of related indexes in tumor tissues.3.The target protein bound to circ7991 was screened by TRAP experiment,mass spectrometry analysis combined with co-immunoprecipitation experiment.RNA immunoprecipitation experiments was verified the interaction between circ7991 and the binding protein δ-catenin.The expression of δ-catenin protein in gastric cancer tissues was detected by Western blot.After gastric cancer cells overexpressing circ7991 were pretreated with proteasome inhibitor MG132 and protein synthesis inhibitor cycloheximide CHX,the effect of protein expression level,half-life and ubiquitination level of δ-catenin protein were detected by Western blot and ubiquitination experiments.The effects of the interaction between TRIM25 and circ7991/δ-catenin were identified by co-immunoprecipitation and mass spectrometry analysis.4.Several bioinformatics softwares such as Circbank,Circ Interactome,mi RDB and Star Base V2.0 were used to predict miRNA molecules that might bind to circ7991.The circ7991 dual-luciferase reporter gene vector was constructed,miRNA mimics were synthesized,and the target miRNA was screened by luciferase reporter gene technology.The circ7991 reporter gene vector with miRNA binding site mutation was constructed to further verify the binding site of circ7991 and miRNA.After cotransfection of circ7991 overexpression plasmid and miRNA mimics into gastric cancer cell lines,growth curve,clone formation and transwell assay were used to investigate whether there are an interaction between the two and the changes in gastric cancer cell function.5.Target genes that may act on miRNAs were predicted byRNA sequencing and multiple bioinformatics analysis,and the signal pathways that circRNAs may be involved in are analyzed byRNA sequencing enrichment.The dual-luciferase reporter gene assay,Western blot and cytology-related experiments were used to further study and analyze the targeting effect of miRNA on target genes.Results:1.The GEO database selected circRNA molecules that were differentially expressed in cancer tissues,and finally determined circ7991 as our follow-up research object based on molecular characterization and expression in gastric cancer tissues and cells.Circ7991 was expressed at a low level in gastric cancer tissues and serum.The results of survival curve analysis showed that circ7991 with high expression in gastric cancer patients had better overall survival.Circ7991 is formed by reverse splicing of the linear transcript of exons 3-5 of EIF4G3 gene.The results ofRNA-FISH and nucleocytoplasmic separation experiments appeared that circ7991 was distributed in both cytoplasm and nucleus.2.Growth curve,plate colony formation and Transwell assay showed that high expression of circ7991 inhibited the proliferation,migration and invasion of gastric cancer cells.The results of flow cytometry showed that overexpression of circ7991 promoted the apoptosis of gastric cancer cells.In the nude mouse tumor formation test,overexpression of circ7991 reduced subcutaneous tumor volume.3.TRAP experiments,mass spectrometry analysis combined with RIP experiments were used to screen and identify the specific binding of circ7991 to the candidate protein δ-catenin,and RIP was used to reversely verify the binding of circ7991 to δ-catenin in gastric cancer cells.Overexpression of circ7991 did not affect the expression of δ-catenin mRNA,but reduced the level of δ-catenin protein.At the same time,MG132 and CHX experiments revealed that circ7991 may mediate the degradation of δ-catenin by inhibiting the ubiquitination level of δ-catenin protein.Coimmunoprecipitation experiments combined with mass spectrometry were used to screen and identify the E3 ligase that TRIM25 plays a key role in the ubiquitination and degradation of δ-catenin.4.The results of RIP experiments showed that circ7991 could bind to the AGO2 protein,the core component of the RISC complex.The existence of a binding site between circ7991 and mi R-4449 was confirmed by multiple bioinformatics analyses and luciferase reporter experiments.Cell complementation experiments showed that mi R-4449 mimics could partially reverse the inhibitory effect of circ7991 overexpression on gastric cancer cells.5.The related genes involved in the β-catenin pathway in the circ7991 overexpression sequencing results combined with the target genes predicted by bioinformatics,showed that SIK1 may be regulated by mi R-4449.The dual-luciferase reporter gene results confirmed the existence of a binding site between mi R-4449 and SIK1.The expressions of circ7991 and SIK1 were positively correlated in gastric cancer tissues.Cell proliferation and transwell experiments showed that downregulation of SIK1 expression could partially compensate for the gastric cancer inhibitory effect caused by overexpression of circ7991.Conclusions:The low expression of circ7991 in gastric cancer is closely related to the poor prognosis of gastric cancer patients.The results of in vitro and in vivo experiments indicate that overexpression of circ7991 in gastric cancer cells can inhibit their proliferation,migration and invasion.The mechanism follow:(1)circ7991 specifically binds to δ-catenin protein through E3 ubiquitin ligase TRIM25 and promotes its ubiquitination and degradation.(2)At the same time,circ7991 can act as a molecular sponge for mi R-4449 to upregulate the expression of SIK1,resulting in inactivation of the β-catenin pathway and affecting tumors progress.Our findings suggest that circ7991 has the potential to be a new prognostic marker and therapeutic target.