M6A Modification Of LncRNA-KCNK15-AS Regμlates EPAB Mediated Pancreatic Cancer Proliferation,Migration And Invasion

Background:Pancreatic cancer is one of the most malignant tumors,with low ealy diagnosis rate and low surgicai removal rate,and the incidence and mortality of pancreatic cancer are increasing year by year.Despite the continuous progress of medical diagnosis and treatment technology improved the survival rate of most cancer patients,the prognosis of pancreatic cancer has not improved significantly.Genome and transcriptional research data shows that protein encoding genes are a small proportion of the genome(1-2%),while most of the genome encodes noncoded RNAs.Long noncoding RNA(lncRNA)is a class of RNA molecules with a length of more than 200 nucleotides and without coding ability;but they participates in many important biological processes in the form of RNA.LncRNA mutation and abnormal expression are closely related to human diseases,including tumors.It is found that some lncRNAs are dysregulated in multiple human tumors.Although some of the differences in lncRNA expression might be secondary,most lncRNAs play important roles as tumor promoting or supressing genes in cell transformation.m6 A modification,as a novel level in regulating RNA,plays an important role in post transcriptional regulation of RNA,especially influcing the structural stability and expression of lncRNAs.This indicates that lncRNA may present m6 A modification and participate in the regulation of pancreatic cancer migration and invasion.Objective: To investigate the effect of m6 A modified long noncodingRNA-KCNK15-AS(KCNK15-AS)on the migration and invasion of pancreatic cancer,and to explore the molecular mechanism involving EPAB.Method:Three pair of pancreatic cancer tissues and adjacent normal tissues are applied for lncRNA microarray analysis.The key KCNK15-AS,was selected as the research object.The pancreatic cancer tissues and pancreatic cancer cells were collected to verify the expression of KCNK15-AS,followed by a clinical correlation analysis.Pancreatic cancer cells stably expressing KCNK15-AS was constructed,and measured for the effect of KCNK15-AS overexpression on proliferation,migration and invasion.In order to identify the m6 A modification ofKCNK15-AS,the ultraviolet cross linked RIP,together with RT-qPCR were performed using pancreatic cancer cells and normal pancreatic ductal epithelial cells.The demethylation transferase ALKBH5 overexpressed and knocked-down pancreatic cancer cells were constructed and applied for migration and invasion experiments,as well as measured for m6 A modification.RT-qPCR was performed to verify the variation of the KCNK15-AS adjacent genes.EPAB was selected as the target gene and its protein expression was measure in pancreatic cancer cells,using normal pancreatic duct epithelial cells as control.The pancreatic cancer cells expressing EPAB were constructed,and tested for migration and invasion ability.Then we knocked down EPAB in pancreatic cancer cells stably overexpressing KCNK15-AS,and the cells were tested for migration and invasion ability.The promoter plasmids of EAPB and luciferase reporter gene experiments were constructed to verify the effect of KCNK15-AS on promoter activity of EPAB.Result:The expression of KCNK15-AS was lower in pancreatic cancer tissues than normal cells and shows a correlation with the lymph node metastasis of pancreatic cancer.Overexpression of KCNK15-AS inhibited the proliferation,migration and invasion of pancreatic cancer cells.The UV cross linked RIP assayshowed that KCNK15-AS presented more m6 A modified RNA in pancreatic cancer cells than that of normal pancreatic ductal epithelial cells.The RT-qPCR and Westem blot experiments showed that ALKBH5 was downregulated in pancreatic cancer cells.Migration and invasion assay indicates that ALKBH5 acted as the tumor suppressor in pancreatic cancer.Meanwhile,the expression of KCNK15-AS was up-regulated after the expression of ALKBH5,and the rate of KCNK15-AS methylation was reduced.Knocking down ALKBH5 on pancreatic cancer cells showed the opposite results.The RT-qPCR showed that EPAB expression presented with the most change among KCNK15-AS adjacent genes,thus it was selected as the target gene.The RT-qPCR and westem blot experiments showed that EAPB was deregulated.Overexpressing EPAB in pancreatic cancer cells resulted in compromised proliferation,migration and invasion ability.Knocking down EPAB in pancreatic cancer cell overexpressing KCNK15-AS resulted in the opposite result.At last,luciferase reporter gene experiments showed that KCNK15-AS activates the promoter of EPAB and promotes its transcription.Conclusions:m6A methylation of KCNK15-AS in pancreatic cancer cell results in its compromised expression.KCNK15-AS regulates EPAB expression and inhibits the proliferation,migration and invasion of pancreatic cancer cells.