The Effect Of Methylation Inhibitor On MicroRNAs Expression In Pancreatic Cancer Cell Line BxPC-3

Objective：To explore the effect of the DNA methylation inhibitor5-Azacytidine onmicroRNAs expression by using LNATM microarray in pancreatic cancer cell lineBxPc-3, and obtain the profile of microRNA regulated by DNA methylation.Methods：Pancreatic cancer cell line BxPc-3were cultured and divided into theexperimental group treated with5-aza-zdeoxycytidine for72hours and the controlgroup treated with the same volume of FBS. RNAs were extracted from organic cellwith lysis buffer by utilizing filter separator. Then we measured their concentrationand purified them determined by ultraviolet spectrophotometer. The integrity ofRNAs was tested with electrophoresis. Capture probes were set for microarray plateand miRNAs were labeled in dual color mix (cy3/cy5).Through hybridization，stringency washing and performing microRNA array, we clustered scanner image,processed data and analyzed result. Total RNAs were isolated and purified in theprotocol Trizol way，and we used1%agarose electrophoresis to text nucleotidesintegrity. Based on the microarray result，we selected3aberrant-expressed miRNAsto manipulate RT-PCR for further reliability of the result that miRNAs dsregulatedexpression.Result：1. On basis of analysis of miRNA biochip，63miRNAs with increasingexpression and21miRNAs with decrease expression were screened in accordancewith taking RATIO>2as over-expressed standard and RATIO<0.5asdown-expressed criterion, including6miRNAs (miR-605、miR-720、miR-197-4、miR-34b、miR-148a and miR-205)up-regulated distinctly while3miRNA(smiR-569、miR-1207-5p and miR-1268)deregulated significantly，and miRNA expressionprofile was obtained. 2.3of9notably dysregulated-expressed miRNAs(miR-34b、miRNA-148a andmiR-205)were optimized for RT-PCR to validate the array revealed result, and hadhigh expression levels comparing with the controls, as same change trend ofmicroarray.Conclusion：Through respective performing miRNA array and RT-PCR with pancreaticcancer cell line BxPC-3of the demethylation group and the control group, somemiRNAs were identified to have ectopic expression due to methylation, and obtainedthe profile of miRNAs performed with demethylation. It is indicated that DNAmethylation had an important role in some miRNA dysregulated expression andmiRNA made a difference to early diagnosis and therapeutic method of pancreaticcancer.