Study The Abnormal Methylation Of MiRNA In Pancreatic Cancer Cell Line By Methylation Chip + TA Clone Sequencing Technology

ObjectiveTo study the methylation of the promoter region in miRNA in pancreatic cancer cell line PANC-1 and normal pancreatic tissue,find the miRNA of abnormal methylation associated with pancreatic cancer.MethodSelect the miRNA with promoter hypermethylation in pancreatic cancer cell lines or normal pancreatic tissue by the DNA methylation chip, Use the approach of BSP(bisulfite genomic sequencing PCR) and TA clone sequencing to further validation. Extraction the genomic DNA of pancreatic cancer cell line BXPC-3, CFPAC-1, PANC-1, SW1990, use the approach of COBRA (combined bisulfite restriction analysis,) to detect of miRNA promoter in those cell lines.ResultWe screened 8 miRNAs from the DNA methylation chips ,then selected five of them for sequencing,as the result show that: in the PANC-1, the methylation status of miRNA-615, 663, 663b was significantly higher than in normal tissues (60.6%: 7.6%, 88.8%: 22.2%, 94.4%: 13.0%),; the methylation status of miRNA-675 was no significant difference between PANC-1 and normal pancreatic tissue (76.0%: 100%). The results of COBRA showed the promoter of those miRNAs have different levels of methylation in pancreatic cancer cell lines.Conclusion "DNA methylation chip + TA clone sequencing" can be used as an accurate and effective manner to screening the aberrant methylation of miRNA promoter. In BXPC-3, CFPAC-1,PANC-1 and SW1990,the promoter of miRNA-615,663, 663b,675 exist in varying degrees of methylation.