The Role Of CircMAN1A2 In Promoting The Progression Of Gastric Cancer Induced By Helicobacter Pylori

BackgroundGastric cancer is the third most common malignant tumor in the world,and China is one of the countries with high incidence of gastric cancer.According to statistics in 2018,the incidence and mortality of gastric cancer in East Asia still ranks first in the world,so the pathogenesis of gastric cancer is crucial for research.The occurrence of gastric cancer is a multi-factor,multi-step process in which Helicobacter pylori infection is generally considered to be one of the important initiating factors in the development and progression of gastric cancer.In 1994,the World Health Organization listed Helicobacter pylori as a class I carcinogen.Helicobacter pylori is a Gram-negative bacterium mainly colonized in the gastric mucosa.Helicobacter pylori infection is not only the main cause of chronic gastritis and peptic ulcer,but also contribute to the process of malignant transformation of gastric mucosa from chronic inflammation.Studies have shown that Helicobacter pylori can induce abnormal changes in the proliferation and apoptosis of gastric mucosal epithelial cells by regulating the expression of non-coding RNA.CircRNA is one kind of non-coding RNA with a circular structure and it has a high content and high stability in eukaryotic cells.Recent studies have found that the novel RNA molecule circRNA is involved in the development of gastric cancer.However,the specific mechanism of circRNA in the development of gastric cancer is still unclear.In view of the fact that Helicobacter pylori infection is an important factor in the malignant transformation of gastric mucosa,it is important to investigate the role of circRNA in Helicobacter pylori-mediated malignant transformation of gastric mucosa.In this study,the expression profile of circRNA in gastric cancer cells after Helicobacter pylori infection was obtained by circRNA sequencing analysis,and the circular RNA circMAN1A2 which was up-regulated after Helicobacter pylori infection was screened.The role and molecular mechanism of circMAN1A2 in gastric cancer was investigated by a series of experiments.PurposeTo investigate the regulatory role of circular RNA circMAN1A2 in the development and progression of Helicobacter pylori-mediated gastric carcinogenesis,and further elucidate the molecular mechanism of Helicobacter pylori-mediated gastric carcinogenesis.Method1.CircRNA expression profiling of Helicobacter pylori infected gastric cancer cell linesHelicobacter pylori-infected gastric cancer cells AGS and PBS-treated control cells were detected by circular RNA sequencing,and the sequencing results were analyzed by bioinformatics.2.CircMAN1A2 expression analysis in gastric cancer cell lines and tissuesThe expression of circMAN1A2 was detected by qRT-PCR in gastric cancer cell lines AGS,Hp-negative and Hp-positive human gastritis tissues,and the results were compared with the sequencing results.At the same time,the localization of circMAN1A2 in cells was detected by qualitative nuclear separation experiments.3.The expression and mechanism of Helicobacter pylorn infection regulation circMAN1A2AGS cells were transfected with wild-type Helicobacter pylori,CagA-deficient Helicobacter pylori,CagA plasmid and control plasmid,respectively,and expression of circMAN1A2 by different treatments were detected by qRT-PCR.4.Cell function study of CircMAN1A2Transfection of specific siRNA in gastric cancer cell lines AGS and BGC-823 to interfere with the expression of circMAN1A2,followed by cell scratch assay to detect the migration ability of AGS and BGC-823.Transwell assay was used to detect the migration and invasion ability of cells after interference with circMAN1A2.The proliferation ability of AGS and BGC-823 after interference with circMAN1A2 was detected by CCK-8 assay and cell colony formation assay.5.CircMAN1A2 regulation mechanism in gastric cancer cellsRNAhybrid and miRanda analysis were used to find miRNAs binding to circMAN1A2 and their binding sequences,and luciferase reporter assay was used to detect whether circMAN1A2 can bind to miRNA.Correlation between circMAN1A2 and miRNA target gene MTA2 expression was detected by qRT-PCR.6.Expression of CircMAN1A2 in gastric cancer tissues and plasma and its feasibility as a diagnostic marker for gastric cancerDetection of circMAN1A2 in human gastric cancer tissues and adjacent gastric mucosa tissues and plasma of healthy people and preoperative plasma of gastric cancer patients was done by qRT-PCR.The correlation between the expression of circMAN1A2 and the pathological parameters of gastric cancer patients were analyzed by SPSS,and the feasibility of circMAN1A2 as a diagnostic marker for gastric cancer was evaluated.Result1.Analysis of circRNA expression profiles of Helicobacter pylori infected gastric cancer cell linesThe sequencing results showed that a total of 18308 circRNAs were detected in the two cell lines,of which 101 were differentially expressed circRNAs.GO analysis and KEGG analysis showed that these circRNAs may be involved in tumor proliferation and metastasis.CircMAN1A2 was significantly up-regulated in Hp-transfected AGS cells compared to PBS-treated AGS cells,and bioinformatic analysis revealed that circMAN1A2 has a binding site for miR-1236-3p.2.CircMAN1A2 is mainly located in the cytoplasm and is highly expressed in the Hp-infected gastric cancer cell line and Hp-positive gastritis.CircMAN1A2 is a circRNA derived from an autosomal exon.Hp infection can up-regulate the expression of circMAN1A2 in the gastric cancer cell line AGS.And circMAN1A2 is highly expressed in Hp-positive gastritis tissue compared to Hp-negative gastritis tissue.In addition,circMAN1A2 is primarily localized in the cytoplasm.3.Helicobacter pylori induced up-regulation of circMAN1A2 expressionHelicobacter pylori-infected gastric cancer cell lines AGS and BGC-823 can up-regulate the expression of circMAN1A2,and this up-regulation is time-dependent,but does not depend on the Helicobacter pylori virulence factor CagA,while the contact of live bacteria is necessary for the up-regulation of circMAN1A2 expression ·4.CircMAN1A2 promotes migration,invasion and proliferation of gastric cancer cell lines AGS and BGC-823Compared with other gastric cancer cells,circMAN1A2 is most abundantly expressed in the gastric cancer cell line BGC-823.CircMAN1A2 specific siRNA was transfected into gastric cancer cells.Cell migration assay,Transwell assay,CCK-8 proliferation assay and colony formation assay showed that the migration,invasion and proliferation of gastric cancer cell lines AGS and BGC-823 decreased after interference with circMAN1A2.Furthermore,Helicobacter pylori partially reversed the down-regulation of AGS and BGC-823 cell proliferation induced by inhibition of circMAN1A2 expression.5.CircMAN1A2 binds to miR-1236-3p via sponge mechanism to regulate the expression of MTA2CircMAN1A2 can bind to miR-1236-3p through a sponge mechanism.After interfering with circMAN1A2,the expression of miR-1236-3p target gene MTA2 is down-regulated.The double luciferase reporter gene assay confirmed the interaction between circMAN1A2 and miR-1236-3p.qRT-PCR results showed that the expression of circMAN1A2 was positively correlated with the expression of MTA2 in gastric cancer tissue samples,r2=0.619.6.The expression of CircMAN1A2 is related to TNM staging of gastric tumors.Compared with normal gastric mucosa and healthy human plasma samples,circMAN1A2 was up-regulated in gastric cancer tissues and preoperative plasma samples from gastric cancer patients.Data analysis showed that the expression of circMAN1A2 was associated with TNM staging of gastric cancer.ConclusionHelicobacter pylori infection induced up-regulation of circMAN1A2 in gastric cancer cell lines,and the induction was independent of virulence factor CagA.CircMAN1A2 can bind to miR-1236-3p through the sponge mechanism to indirectly regulate the expression of target gene MTA2,promote the migration,invasion and proliferation of gastric cancer cells,and play a crucial role in the regulation and development of gastric cancer.In this study,we investigated the role of circMAN1A2 in the development and progression of Helicobacter pylori-mediated gastric cancer and its related molecular mechanisms from three aspects of cell level,molecular level and human tissue level.It revealed the carcinogenic mechanism of Helicobacter pylori,suggesting that circMAN1A2 may be used as diagnostic marker and intervention target for gastric cancer.