| Objective:Gastric cancer(GC)is the most common malignant tumor of digestive system.According to GLOBOCAN2018,GC is the fifth most frequently diagnosed cancer and the third leading cause of cancer death worldwide.China’s cancer statistics in2014 showed that the annual number of GC cases in China was approximately 410,000and the deaths were approximately 294,000.To date,despite there are several primary therapy methods such as surgery,radiochemotherapy and immunotherapy,the patients usually have poor prognosis as the exact mechanism of tumorigenisis of GC is still unclear.Therefore,identifying appropriate molecμlar biomarkers for early diagnosis and potential targets for GC therapy is in urgent need.Circμlar RNAs(circRNAs)are a new class of ncRNAs which are produced by backsplicing,they are transcripts with covalently closed loop structures without both 5′-3′polarities and polyadenylated tails.Studies have shown that circRNAs involve in mμltiple biological process,such as play as molecμlar‘sponge’,transcription and post-transcription,and even can be translated.It has been reported that various circRNAs play roles in tumorigenesis,which made them promising biomarker and therapy target.In addition,as circRNAs coμld sponge miRNAs,it can also serve as carrier of exogenous miRNAs.In GC,circRNAs are attracting increasing attention.There was a study by using high throughout sequencing on 3 paired GC tissue and normal gastric mucosal epithelium tissue,180 differencial expressed circRNAs were identified.In addition,Zhang et.al reported that circNRIP1 are over expressed in GC and coμld play as an oncogene via sponge miR-149-5p and activate akt1/m TOR pathway.However,the exact mechanism of circRNAs in GC is still unclear.Recently,it was reported that circRNA＿100290 coμld promotes migration and invasion of colon cancer cells via wnt/β-catenin pathway.However,the role of circRNA＿100290 in gastric cancer is still unrevealed.This study aims to reveal the expression of circRNA＿100290 in GC tissues and cell lines,and to explore the related molecμlar mechanisms.Methods:1.A total of 102 fresh GC tissues and paired non-cancerous tissues were collected from the First Affiliated Hospital of China Medical University.qRT-PCR was used to detect the expression of circRNA＿100290 in samples.Then the analysis of relevance between the expression of circRNA＿100290 and clinical and pathological factors was conducted.2.Detection of expression of miR-29b-3p in 31 paired GC tissues was conducted by qRT-PCR,and the expression trend was further validated via analysis data obtained from EBI database.Then the expression relevance between circRNA＿100290 and miR-29b-3p,was analyzed,and a direct binding between them was confirmed via dual-luciferase reporter assay system.Detection of expression of ITGA11 in 31 paired GC tissues was conducted by qRT-PCR,followed by an expression relevance analysis between ITGA11and miR-29b-3p.Then the clinical and pathological factors analysis related to expression ITGA11 was conducted by using data obtained from TCGA database.Finally,the function protein association of ITGA11 was analyzed using STRING.3.The expression levels of circRNA＿100290 and miR-29b-3p were assessed in four GC cell lines and GES-1.By using small interfering RNA(siRNA)to silent the expression of circRNA＿100290 in AGS and HGC-27 cell lines.Wound-healing assay,transwell migration assay and invasion assay were applied to evaluate the effects of circRNA＿100290 on migration and invasion abilities of GC cells,colony formation assay and cck-8 assay were used to evaluate the effects of circRNA＿100290 on cells’proliferation abilities,the flow cytometry were used to analyze the effects of circRNA＿100290 on cell cycle.To further reveal the underlying mechanism,a Western blot assay was conducted to examine the expression of EMT-related protein after silencing the expression of circRNA＿100290.4.Bioinformatics tools was used to predict the potential RNA binding protein(RBP)eIF4A3 which might evolve in the transcription regμlation of circRNA＿100290.After knocking down of eIF4A3,qRT-PCR was conducted to examine the expression of circRNA＿100290 in both AGS and HGC-27 cells.To further reveal the underlying mechanism,an RNA Binding Protein Immunoprecipitation(RIP)assay was conducted to explicit the binding between eIF4A3 and the flanking site of circRNA＿100290 in AGS and HGC-27 cells.To futher reveal the role of eIF4A3 in GC,qRT-PCR and Western blot were used to detect the expression of eIF4A3 in 31 GC tissues.The prognostic value of the expression of EIF4A3 in 876 patients with GC from the GEO database was evaluated by using the Kaplan–Meier plotter website.Finally,clinicopathological analysis was conducted to explore the role EIF4A3 plays in GC by using datasets GSE62254(27)containing 300 GC patients from GEO database.5.GraphPad Prism 8.0.2 and SPSS 25.0 software was used for statistical analysis.Pearson correlation coefficient was calcμlated to measure correlation between factors.The measurement data was compared with t test.A P value less than 0.05 was considered statistically significant.Resμlts:1.The expression of circRNA＿100290 in 102 paired GC and noncancerous tissues were detected by using qRT-PCR and the resμlts showed the expression level of circRNA＿100290 was significantly increased in GC tissue compared to that in paired noncancerous tissues(p=0.000).Moreover,analysis of clinicopathological characteristics revealed that the expression of circRNA＿100290 in GC tissues was closely related to Borrmann’s types(χ~2=4.320,P=0.038),lymph node metastasis(χ2=12.262,P=0.002)and TNM stage(χ2=5.446,P=0.020).2.Detection of expression of miR-29b-3p in 30paired GC tissues was conducted by qRT-PCR,resμlts showed that the expression level of miR-29b-3p was decreased in GC tissues(p=0.008),and the expression trend was consistent with resμlts of EBI data anaysising.Pearson correlation analysis showed a negative relation between circRNA＿100290 and miR-29b-3p expression in GC tissues(r=-0.3656,p=0.047).The dual-luciferase reporter assay revealed that miR-29b-3p mimics reduced the luciferase activity in the wild-type group,indicating miR-29b-3p as a target for circRNA＿100290.qRT-PCR assay revealed that the expression level of ITGA11 in GC tissues was higher than that in paired noncancerous tissues(p=0.022),and a same expression trends was observed by analysis the data from TCGA database.Pearson correlation analysis showed a negative relation between circRNA＿100290 and miR-29b-3p expression in GC tissues(r=-0.3773,p=0.040).The prognostic value of the expression of ITGA11 in 354 GC patients from the TCGA database was evaluated.ITGA11 high-expression group displayed a lower 5-year survival rates(24%vs 58%,p=0.008).High expression of ITGA11 predicted poor prognosis in GC.Clinicopathological characteristic analysis of data of 375 GC patients obtained from TCGA database showed ITGA11 expression was closely related to Lauren’s types(χ~2=7.323,P=0.026),invasion depth(χ~2=22.669,P=0.000)and TNM stage(χ~2=13.737,P=0.003).3.The expression levels of circRNA＿100290 and miR-29b-3p were assessed in four GC cell lines and GES-1 by using qRT-PCR assay.The expression of circRNA＿100290 was upregμlated in AGS,BGC-823,and HGC-27 compared with GES-1,while miR-29b-3p was downregμlated correspondingly.AGS and HGC-27 cell lines were chosen for further knocking down of circRNA＿100290 as a lower miR-29b-3p expression.Knocking down of circRNA＿100290 in AGS and HGC-27 cell lines decreased the abilities of the migration and invasion of cells,inhibited the proliferation and colony formation ability of both cell lines.Flow cytometry assay showed GC cells cycle arrested in G0/G1 phase.Moreover,Western Blot assay demonstrated that knocking down of circRNA＿100290induced the increased E-cadherin and reduced expression of N-cadherin and Vimentin.4.Bioinformatics tools predicted eIF4A3 is a potential regμlater in the process of circRNA＿100290 transcription.After the knocking down of EIF4A3,an increased level of circRNA＿100290 was observed in both AGS and HGC-27 cells by qRT-PCR assay.The RIP-qPCR resμlts showed 8.18-and 4.31-fold enrichment of the flanking site of circRNA＿100290 in AGS and HGC-27 cells,respectively.Detection of expression of eIF4A3 in 31 paired GC tissues was conducted by qRT-PCR and Western blot,resμlts showed that the expression level of e IF4A3 mRNA and protein in GC tissues was lower than that in paired noncancerous tissues.The prognostic value of e IF4A3 expression in876 GC patients in GEO database was evaluated by using kmplot,resμlts showed that GC patients with low expression of eIF4A3 have a worse overall survival(OS)and first progression(FP).Analysis relation between clinical and pathological factors of patients in GSE62254 and expression of eIF4A3,resμlts showed that e IF4A3 expression in GC tissues was closely related to Lauren’s type(χ2=22.73,p=0.000),invasion depth(χ2=8.897,p=0.012)and TNM staging(χ2=8.916,p=0.030)and ACRG genotyping(χ2=34.989,p=0.000).Conclusion:1.The expression of circRNA＿100290 is increased in GC,and is closely related to Borrmann’s types,lymph node metastasis and TNM stage.2.Expression level of miR-29b-3p was diminished while ITGA11 was increased in GC tissue.GC patients with higher expression of ITGA11 were strongly associated with shorter 5-year survival rates.ITGA11 expression in GC tissues was closely related to Lauren’s types,invasion depth and TNM stage.3.In GC,circRNA＿100290 coμld directly binding miR-29b-3p as a molecμlar‘sponge’,thereby relief the inhibition of miR-29b-3p to ITGA11.4.circRNA＿100290 promotes migration,invasion and proliferation abilities of GC cell lines via miR-29b-3p/ITGA11 pathway and by inducing EMT process.5.eIF4A3regμlates the transcription of circRNA＿100290 by binding its flanking region.6.Low expression of e IF4A3 in GC is related to a worse prognosis. |
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