Mechanism Of MiR-375 Regulating H.pylori-induced Epithelial–Mesenclymal Transition In Gastric Cancer

Gastric cancer(Gastric cancer)ranks fourth in global incidence and second in mortality.It is one of the most common malignant tumors.Helicobacter pylori(H.pylori)infection has become a high-risk factor for gastric cancer.The Health Organization(WHO)designated H.pylori as a Class I carcinogen in 1994.The incidence of gastric cancer in my country is very high,and the annual morbidity and mortality are more than twice the world level.Most patients with gastric cancer are already in the middle and advanced stages when they are diagnosed,with various metastases.An important reason for its late discovery,refractory treatment,and high mortality is its easy invasion and metastasis,and epithelial-mesenchymal transition(EMT)is the key mechanism of gastric cancer invasion and metastasis.Micro RNAs(miRNAs)are small single-stranded RNAs with a size of about 18-26 bases.They are produced from a single-stranded RNA precursor with a hairpin structure and processed by Dicer enzyme.It can regulate target genes at the post-transcriptional level.expression.This article mainly discusses the role and mechanism of miR-375 in H.pylori-infected gastric cancer EMT.ObjectiveTo study the mechanism of miR-375 regulating H.pylori-mediated gastric cancer epithelial-mesenchymal transition through targeted binding to JAK2Methods1.Screening of differential miRNAs and m RNA in H.pylori infection gastric mucosal cell model(1)Construct H.pylori-infected gastric normal mucosal cells and gastric adenocarcinoma models,use whole transcriptome sequencing analysis,use volcano graphs to infer the overall distribution of differential miRNAs and m RNAs,and then aggregate the differentially expressed miRNAs and m RNAs Class analysis to screen out the miRNAs and m RNAs that are differentially expressed in the H.pylori-infected gastric mucosal cell model.(2)Immunohistochemistry(IHC),Immunofluorescence technique(IF)and RT-PCR to detect the expression levels of miR-375,AEG-1,JAK2 and EMT-related factors and markers(E-cadherin,Vimentin,Snail)in clinical gastric cancer tissues.2.The targeting relationship of miR-375 to AEG-1 and JAK2 genes(1)Use miRWalk,miRanda,Targetscan and other bioinformatics software to predict the binding sites of miR-375 to AEG-1 and JAK2 genes.(2)Construct the p MIR-GLO-AEG-1-3’UTR wt/mut and p MIR-GLO-JAK2-3’UTR wt/mut luciferase reporter vector,and verify the correctness of the recombinant plasmid by gene sequencing.(3)Confirm the targeting relationship of miR-375 to AEG-1 and JAK2 genes by luciferase reporter method,Western Blot and immunofluorescence techniques.3.Study on the mechanism of miR-375 regulating "H.pylori-induced gastric cancer EMT"(1)Construct a gastric cancer cell model with low expression and overexpression of miR-375,and analyze the correlation between miR-375 and "H.pylori-induced gastric cancer EMT" using scratch-repair,Transwell and immunofluorescence techniques.(2)Design JAK2 si RNA according to the relevant principles of small interfering RNA(si RNA),verify the interference efficiency of the designed JAK2 si RNA,and screen JAK2 si RNA with higher interference efficiency;target JAK2 gene silence by JAK2 si RNA Use scratch repair experiments,Transwell and other experiments to detect whether miR-375 affects Helicobacter pylori-induced gastric cancer migration through JAK2.Result1.Screening of differential miRNAs and m RNA in H.pylori infection gastric mucosal cell model(1)Full transcription sequencing analysis of H.pylori infected gastric mucosal cell model found that in the normal gastric mucosal cell model infected with H.pylori,21 miRNAs were up-regulated and 11 were down-regulated;m RNAs had 3107 up-regulated expressions.3156 were down-regulated;in the H.pylori-infected gastric cancer cell model,41 miRNAs were up-regulated and 32 were down-regulated;m RNAs had 4042 up-regulated expression and2851 down-regulated expression.In addition,it was found that miR-375 showed low expression in normal gastric mucosa and gastric cancer cell models infected by H.pylori,while AEG-1 and JAK2 showed high expression.(2)The expression of miR-375 in clinical gastric cancer tissues was significantly reduced by RT-PCR,immunohistochemistry,and immunohistochemical techniques,and the expression of AEG-1 and JAK2 were significantly increased in clinical gastric cancer tissues.E-cadherin,a marker of EMT epithelial cells,is low in clinical gastric cancer tissues,and the related transcription factor Snail1 and Vimentin,a marker of mesenchymal cells,are both highly expressed.2.The targeting relationship of miR-375 to AEG-1 and JAK2 genes(1)Using miRanda,miRWalk,Targetscan and other bioinformatics software to predict the binding sites of miR-375 to AEG-1 and JAK2 genes,it was found that miR-375 could theoretically interact with the 3’-UTR region of AEG-1 and JAK2 genes.Binding at multiple sites.(2)Gene sequencing confirmed that the recombinant plasmids p MIR-GLO-AEG-1-3’UTR wt/mut and p MIR-GLO-JAK2-3’UTR wt/mut had no frameshift and other mutations,and the sequences were completely correct.(3)It is confirmed that miR-375 can target AEG-1 and JAK2 genes by luciferase reporter method,Western Blot and immunofluorescence techniques.3.Study on the mechanism of miR-375 regulating "H.pylori-induced gastric cancer EMT"(1)Compared with mimic nc,miR-375 mimics can significantly down-regulate the expression of JAK2 in H.pylori-infected gastric cancer cells;compared with inhibitor nc,miR-375 inhibitors can significantly up-regulate the expression of JAK2.(2)Techniques such as scratch-repair and Transwell confirm that miR-375 can affect the invasion and migration ability of gastric adenocarcinoma cell line BGC-823.(3)RT-PCR and Western Blot confirmed that JAK2 si RNA can effectively down-regulate the protein expression of JAK2;and JAK2 si RNA + miR-375 mimics can more effectively inhibit the protein expression of JAK2.(4)It was confirmed by scratch repair and Transwell experiment that the migration and invasion ability of gastric cancer cells was significantly reduced after JAK2 si RNA interfered with JAK2 expression;and JAK2 si RNA + miR-375 mimics could effectively inhibit the migration and invasion ability of gastric cancer cells.Conclusion1.In H.pylori-infected gastric mucosal cell models and clinical gastric cancer tissues,miRNAs such as miR-375 showed low expression,while genes such as AEG-1 and JAK2 showed high expression.2.In gastric cancer cells,miR-375 can target the AEG-1 and JAK2 genes,and regulate the H.pylori-induced EMT of gastric cancer by inhibiting the expression of JAK2.