The Role Of M6A Modification And LncRNA NBR2 In Pancreatic Cancer

BackgroundPancreatic cancer is one of the most deadly cancers in humans and its 5-year survival rate is less than 10%.With the development of sequencing technology and biotechnology,m6A modification and lncRNA has attracted a lot of attention in cancer research.Due to the tumor specificity and stability of lncRNA,it may be the potential tumor markers.It’s of great clinical significance to find a novel lncRNA for diagnosis and treatment of pancreatic cancer.AimBy analyzing the data sets of pancreatic cancer from the International Cancer Genome Consortium(ICGC)and the Cancer Genome Atlas Program(TCGA),and using the next generation sequencing(NGS)and MeRIP sequencing technology,lncRNA NBR2 has been screened out with higher expression level and m6A modification in pancreatic cancer patients.Further studies have been performed to investigate the biological function and mechanisms of lneRNA NBR2 in pancreatic cancer.MethodFirstly,by using the data sets of ICGC and TCGA,the relationship between m6A RNA methylation regulatory factors and m6A RNA methylation related genes has been systematically analyzed as well as their relationship with survival time,including univariate Cox regression analysis,LASSO Cox regression analysis,risk prognosis model,STRING,Spearman and consistent cluster analysis.Secondly,NGS and MeRIP sequencing technology was used to detect the expression level of lncRNAin pancreatic cancer patientsand then lncRNA NBR2 with m6A modification has been screened out with higher expression level.Thirdly,biological function has been done in SW1990 cells with overexpressing lncRNA NBR2 and CAPAN2 cells with silencing lncRNA NBR2,including subcellular cloning,apoptosis,cell cycle arrest and cell migration in vitro as well as tumor forming capacity in vivo.Fourthly,Immunohistochemistry was used to detect the expression level of lncRNA NBR2 in pancreatic cancer tissues and normal tissues.The correlation between lncRNA NBR2 and clinocopahtological features,overall and disease-free survival were analyzed.Finally,by using online bioinformatics website(Targetscan)for prediction and TCGA data analysis,the target miRNA of lncRNA NBR2 and the target of miRNA were screened out and then verified by the luciferase reporter assay.ResultFirstly,by the data analysis of pancreatic cancer from ICGC and TCGA,it has revealed that 4 m6A methylation regulatory factors were significantly correlated with AJCC staging,including RBM15,METTL14,FTO and ALKBH5,while m6A methylation related genes EGFR,PLEC,BLM and PLK1 were closely associated with others genes as the "Hub" genes in the network.AJCC stage,T and N stage,KRAS mutation status and x8q23.3 CNV fragment mutation were significantly different between the low risk group and the high risk group.The expression of the 4 m6A RNA methylation related genes were significantly among the 7 subgroups,including PLK1,ZNF596,BCAT1 and COL7A1.Secondly,lncRNA NBR2 with m6A modification has been screened out with higher expression level in pancreatic cancer patients by using NGS and MeRIP sequencing technology.lncreased expression level of lncRNA NBR2 could enhance cell proliferation and migration but inhibit cell apoptosis in vitro and promote tumorigenesis in vivo,while decreased expression level of lncRNA NBR2 inhibited cell proliferation and migration but enhance cell apoptosis in vitro and suppress tumorigenesis in vivo.Thirdly,Higher expression of lncRNA NBR2 was observed in pancreatic cancer than normal tissues and IncRNA NBR2 was significantly correlated with clinicopathological features.Poor overall and disease-free survival was observed in higher expression group of lncRNA NBR2.Finally,by the analysis of Targetsacan and TCGA data and the identification of luciferase reporter assay,miR-193b-3p has been screened out to be the target of lncRNA NBR2 and E2F6 has been the target of miR-193b-3p.ConclusionLncRNA NBR2 with m6A modification may serve as an effective indicator for diagnossi in pancreatic cancer and the lncRNA NBR2/miR-193b-3p/E2F6 axis is likely to be an important pathway regulating cell cycle arrest,proliferation,apoptosis and migration in pancreatic cancer.