The Biological Role And Mechanism Of Circular RNA CircREPS2 In Gastric Cancr

Background: Gastric cancer(GC)is one of the most common cancers worldwide,ranking fifth and fourth in morbidity and mortality,respectively.According to the latest domestic cancer data report,more than 478,000 people in China were diagnosed with GC,and about 373,000 people died of GC.The reason may be due to the special dietary structure and eating habits of the Chinese people,including their preference for pickled foods,shared tableware,etc.,as well as a large number of people infected with Helicobacter pylori.Due to the insidious onset of GC,atypical symptoms,and lack of effective early biomarkers,once detected,it is often in the middle and late stages of diagnosis,which brings great challenges to the prevention and treatment of GC.Although the current comprehensive treatment methods are mainly surgery,chemotherapy and immunotherapy,the metastasis and recurrence of advanced gastric cancer often led to the final treatment failure.Early gastric cancer and advanced gastric cancer have completely different prognosis.Therefore,it is an urgent problem to find effective early biomarkers and related intervention targets to inhibit and reduce the metastasis and recurrence of GC.Circular RNAs(circ RNAs)are covalently closed endogenous RNA molecules with a cyclic structure in eukaryotes,with tissue and cell expression specificity,and their origin is regulated by specific cis-acting elements and trans-acting factors.CircRNAs are abundant in vivo,evolutionarily conserved,and highly stable due to their special ring structure.And because of its detectability in liquid samples such as plasma,saliva and urine,it is expected to become an emerging tumor biomarker.Functionally and mechanically,circ RNAs can act as molecular sponges for micro RNAs(miRNAs)and proteins,and they also play important biological functions by regulating protein function or self-translation.Current studies have found that circ RNAs are involved in the physiological and pathological processes of many diseases,including GC,and have been reported as early biomarkers and therapeutic targets in GC.Based on this,our group previously detected the expression of 5 pairs of circ RNAs in GC and its adjacent tissues by circ RNA tissue microarray chip,screened and found a significantly differentially expressed circular RNA hsa＿circ＿0139996(derived from the parental gene REPS2,hence named circREPS2).This study will explore the biological function of circREPS2 in GC and its regulatory mechanism network,in order to find early biomarkers and new therapeutic targets for GC,and to provide the basis for clinical molecular targeted therapy of GC.Objective:(1)To explore the correlation between the expression of circREPS2 in GC and clinicopathological features.(2)To analyze the effect of circREPS2 on the malignant biological behavior of GC cells.(3)To clarify the mechanism of circREPS2 regulating GC cell function.Methods:(1)The expression and identification of circREPS2.Convergent primers and divergent primers were designed and amplified for circREPS2,and Sanger sequencing was used to confirm the reverse splicing site of circREPS2.q RT-PCR was used to detect the expression of circREPS2 in GC and adjacent tissues,as well as in normal gastric epithelial cells and GC cell lines.Chi-square test was used to analyze the correlation between its expression and clinicopathological characteristics of patients.Fluorescence in situ hybridization(FISH)was used to observe the localization of circREPS2 in GC cells.(2)Cell function experiments to verify the effect of circREPS2 on GC cells.The stable overexpressed circREPS2 cell lines were constructed and the expression of circREPS2 was silenced by transfection of small interfering RNA(si RNA).The overexpression and silencing efficiency were detected by q RT-PCR.The effects of circREPS2 on proliferation,invasion and migration of GC cells was verified by CCK8,clone formation,Ed U,wound healing and transwell experiments.Western blot(WB)was used to detect the regulation of circREPS2 on epithelial-mesenchymal transition(EMT)-related indicators(E-cadherin,N-cadherin and vimentin).A nude mouse model of subcutaneous tumorigenesis and spleen-liver metastasis was constructed,and the effect of overexpression of circREPS2 on the occurrence and liver metastasis was evaluated.(3)Explore the potential downstream miRNAs of circREPS2.The possible downstream miRNAs of circREPS2 were analyzed and predicted by bioinformatics.RNA-pulldown,RNA immunoprecipitation(RIP),FISH and dual-luciferase experiments were used to verify the interaction between circREPS2 and miR-558.And through CCK8,clone formation and transwell rescue and recovery experiments confirmed that circREPS2 can reverse the tumor-promoting effect of miR-558.(4)Detect the effect of circREPS2 on downstream target genes and pathways.Bioinformatics analysis screened the downstream target genes and signaling pathways of miR-558.WB and dual luciferase verified the targeted binding relationship between miR-558 and the 3’-UTR of RUNX3 in GC cells.WB experiment was used to verify the regulatory effect of circREPS2 and miR-558 on RUNX3.The expressions of RUNX3,β-catenin and other related proteins were detected by WB when Wnt/β-catenin agonists were used and RUNX3 overexpressed plasmids were transfected.Results:(1)The Sanger sequencing results confirmed the head-to-tail reverse splicing site sequence of circREPS2.q RT-PCR verified that circREPS2 was relatively low expressed in GC cells,and its expression correlated with tumor size and TNM stage.Circ REPS2 was mainly localized in the cytoplasm of GC cells.(2)Cell functional experiments showed that circREPS2 could significantly inhibit the proliferation,migration and invasion of GC cells,and WB results showed that circREPS2 could inhibit the EMT process;similar results were obtained in vivo experiments,which were overexpressed in subcutaneous tumorigenesis and spleen-liver metastasis models circREPS2 can inhibit subcutaneous tumorigenesis and spleen-liver metastatic ability.(3)Through bioinformatics analysis,circREPS2 was found to have 7potential target miRNAs,and RNA-pulldown experiments confirmed that circREPS2 may bind to miR-558 and miR-615-3p.Dual luciferase experiments confirmed miR-558,but not miR-615-3p is the direct target of circREPS2.FISH experiments also verified that circREPS2 and miR-558 have a colocalization relationship.Furthermore,rescue and recovery experiments also verified that circREPS2 can reverse the tumor-promoting effect caused by miR-558.(4)Through bioinformatics prediction analysis,dual luciferase experiments confirmed that RUNX3 was a downstream target gene of miR-558.WB confirmed the negative regulation of miR-558 on RUNX3.Circ REPS2/miR-558 can regulate the expression of RUNX3 in GC,and rescue and recovery experiments found that miR-558 can inhibit the up-regulation of RUNX3 caused by circREPS2.WB experiments showed that circREPS2/miR-558 can inhibit the expression of RUNX3 and thus inhibit Wnt/β-catenin signaling axis.WB experiments showed that circREPS2/miR-558 could inhibit the Wnt/β-catenin signaling axis by affecting the expression of RUNX3.Conclusion:(1)Circ REPS2 forms circ RNA through reverse splicing in GC,and it is significantly low expressed in GC tissues and cells.In GC patients,the low expression level of circREPS2 is related to tumor size and TNM stage.(2)Functionally,overexpression of circREPS2 can significantly inhibit the malignant biological behavior of GC cells,and vice versa.(3)Mechanistically,circREPS2 inhibits the function of GC cells by competitive binding to miR-558,thereby regulating the expression of RUNX3 and inhibiting β-catenin signaling.