The Role And Mechanism Of Hsa_circ₀000051 In The Occurrence And Development Of Gastric Cancer

IntroductionGastric cancer（GC）is a common malignant cancer of the digestive systemwithhigh morbidity and mortality.Due to the lack of biomarkers for early diagnosis,most patients with gastric cancer are diagnosed at advanced stages.These patients always lose effective treatmentsand have a poor prognosis.Asa result,gastric cancer is a major problem that seriously endangers human life and health.The mechanism of gastric cancer is complicated,and environmental factors and genetic factors are involved.At present,some researches have been carried out on the mechanism and clinical treatment of gastric cancer,but the specific mechanism of the occurrence and development of gastric cancer has not yet been fully elucidated.Further clarifying the exact biological mechanism of the development of gastric cancer will contribute to the effective biomarker for early diagnosis and effective therapeutic targetsof gastric cancer.Circular RNA（circ RNA）is a novel non-coding RNA（ncRNA）.The unique closed structure of circ RNA protects it from being digested.Circular RNA is stable in tissues and cells.Circ RNA is versatile and can compete with mRNA for binding to RNA-binding protein（RBP）to regulate translation,to regulate selective splicing and gene transcription,or to regulate target gene expression as a sponge of miRNA.In recent years,more and more circRNAs have been revealed to function by regulating miRNAs,and participate in various physiological and pathological processes such as proliferation,apoptosis,invasion,differentiation,and aging.With rapid development of high-throughput sequencing technology and bioinformatics,a large number of differentially expressed circRNAs have been discovered and further confirmed to participate in the development of cancers.Studies have confirmed that circRNAs have been revealed to participate in the development and progression of various tumors such as lung cancer,liver cancer and breast cancer by competitively binding miRNA and then regulating downstream target genes.However,the functions of circRNAs in gastric cancer are still remains to be elucidated.As a result,it will be of great importance to explore the roles and possible mechanisms of circRNAs in gastric cancer.Objective1.To screen the abberrant circRNA expression profile in gastric cancer.2.To find the role of hsa_circ₀000051 in proliferation,colony formation,migrationapoptosisandcell cycle of gastric cancer cells.3.To verify the target miRNA of hsa_circ₀000051.4.To validate the role ofmiR-142-5p in proliferation,colony formation andmigrationof gastric cancer.5.To study how hsa_circ₀000051/miR-142-5p regulate target gene and the role of target gene in gastric cancer.Methods1.GEO database was used for screeningthe differentially expressed circRNAs,and GSE100170was obtained.2.Identification of the characteristics of hsa circ 0000051 by Sanger sequencing and RNase R digestion test.3.QRT-PCR was used to detect the expression of hsa_circ₀000051 in 34 gastric cancer tissues and adjacent tissues,and in gastric epithelial cells and gastric cancer cells.4.The hsa circ 0000051 siRNA and plasmidwere transfected into gastric cancer cell lines（AGS and SGC-7901）.The effects of hsa_circ₀000051 on cell proliferation,colony formation,cell migration,cell cycle and apoptosis were detected by CCK8,colony formation assay,Transwell and cell scratch assays and flow cytometry assay.5.The target miRNAs binding to circRNA was found by circinteractome,and then compared with the miRNA microarray analysis.QRT-PCRwas to verifythe miRNA expression regulated by hsa_circ₀000051.6.We used the database to predict the target geneof miR-142-5p,and analyzed the correlation of hsa_circ₀000051,miR-142-5p and target gene RHOA in gastric cancer tissues.7.miR-142-5p mimic and inhibitor were transfected into cells,and the effects of miR-142-5p on cell proliferation,colony formation and cell migration were detected by CCK8,colony formation,Transwell and cell scratch assays.8.We constructed wild-type and mutant plasmids of hsa_circ₀000051 and RHOA,and then co-transfected with miR-142-5p mimics and negative control,fluorescence intensity was measured then;9.RNA-binding protein immunoprecipitation assaywas used to confirm the binding ability of hsa_circ₀000051 with miR-142-5p.10.QRT-PCR and western blot were used to measure the protein and mRNA expression change in the groups of hsa_circ₀000051 siRNA,hsa_circ₀000051 siRNA+miR-142-5p inhibitor,miR-142-5p mimics,miR-142-5p mimics+RHOA.11.Co-transfection of miR-142-5p inhibitor was used to observe the proliferation,colony formation and migration of gastric cancer cells promoted by hsa_circ₀000051.12.Co-transfection of target geneplasmid（RHOA）was used to observe proliferation,colony formation and migration inhibition of miR-142-5p.Results1.By searching GEO database,we obtained GSE100170,which contains 62,998 circRNAs,207 differentially expressed circRNAs,57 of which are up-regulated and 150 were down-regulated circRNAs,and hsa circ 0000051 was selected for the following studies.2.hsa_circ₀000051was resistant to RNase R treatment in contrast to the linear control RNAs.3.QRT-PCR indicated that the expression of hsa_circ₀000051 in gastric cancer tissues and gastric cancer cells was significantly higher than that in adjacent tissues and gastric epithelial cells.4.Overexpression of hsa_circ₀000051 promoted cell proliferation,colony formation,migration and cell cycle.Overexpression of hsa_circ₀000051 also inhibited cell apoptosis.Downregulation of hsa_circ₀000051 inhibited cell proliferation,colony formation migrationand cell cycle.Downregulation of hsa_circ₀000051 promoted cell apoptosis.5.Bioinformatics suggested thathsa_circ₀000051 bound to miR-142-5p,miRNA microarrays suggested low expression of miR-142-5p in gastric cancer,and miR-142-5p expression could be regulated by hsa_circ₀000051.6.RHOA was the target gene of miR-142-5p.The expression of miR-142-5p was negatively correlated with RHOA and hsa_circ₀000051.In addition,hsa_circ₀000051 was positively correlated with RHOA in gastric cancer tissues.7.Transfection of miR-142-5p plasmid inhibited proliferation,colony formation and migration of gastric cancer cells.Transfection of miR-142-5p siRNA promoted proliferation,colony formation and migration of gastric cancer cells.8.Fluorescence was significantly attenuated in cells co-transfected with miR-142-5p mimics and pGL3-RHOA or pGL3-hsa_circ₀000051 wild-type plasmids.There was no significant change in fluorescence activity in cells co-transfected with miR-142-5p mimics and pGL3-RHOA or pGL3-hsa_circ₀000051 mutants.9.RNA-binding protein immunoprecipitation assay showed that hsa_circ₀000051 was significantly higher in miR-142-5p mimics-treated cells inAGO2 protein-bindinggroup than in control group.10.miR-142-5p inhibitor reversed the down-regulation of RHOA expression by hsa_circ₀000051 siRNA.RHOA over-expression plasmid reversed the down-regulation of RHOA expression by miR-142-5p mimics.11.Transfection of hsa_circ₀000051 siRNA inhibited cell proliferation,colony formation and cell migration.Co-transfection of miR-142-5p inhibitor alleviated the inhibition of hsa_circ₀000051 siRNA on cell proliferation,colony formation and cell migration.12.Transfection of miR-142-5p mimics inhibited cell proliferation,colony formation and cell migration.Co-transfection of RHOA overexpression plasmid reversed the inhibition of proliferation,colony formation and cell migration caused by miR-142-5p mimics.Conclusions1.hsa_circ₀000051 is up-regulated in gastric cancer.2.hsa_circ₀000051 promotedproliferation,colony formation,migration and cell cycle,and inhibited apoptosis of gastric cancer cells.3.miR-142-5p bound to hsa_circ₀0000514.miR-142-5p inhibited proliferation,colony formation and migration of gastric cancer cells5.hsa_circ₀000051 competitively bond to miR-142-5p and participated in the development and progression of gastric cancer by regulating the target gene RHOA.