去甲基化剂对胰腺癌细胞BxPC3中microRNAs表达影响.pdfVIP

摘要 关键词：胰腺癌；DNA去甲基化；MicroRNA。 III Abstract ABSTRACT objective： To explore the effect of the DNA methylation inhibitor 5-Azacytidine on microRNAs expression by using LNATM microarray in pancreatic cancer cell line BxPc一3，and obtain the profile ofmicroRNA regulated by DNA methylation． Methods： Pancreatic cancer cell line BxPc-3 were cultured and divided into the experimental group treated with 5-aza—zdeoxycytidine for 72 hours and the control group treated with the same volume of FBS．RNAs were extracted from organic cell with lysis buffer by utilizing filter separator．Then we measured their concentration and purified them determined by ultraviolet spectrophotometer．The integrity of RNAs was tested with electrophoresis．Capture probes were set for microarray plate and 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 the protocol Trizol way，and we used 1％agarose electrophoresis to text nucleotides integrity．Based on the microarray result，we selected 3 aberrant-expressed miRNAs to manipulate RT—PCR for further reliability of the result that miRNAs dsregulated expression． Result： 1．On basis of analysis of miRNA biochip，63 miRNAs with increasing expression and 21 miRNAs with decrease expression were screened in accordance with taking RATIO2 as over-expressed standard and RATIO0．5 as down-expressed criterion．including 6 miRNAs(miR-605、miR-720、miR-1 97-4、 miR-34b、miR-1 48aand miR-205)up-regulated distinctly while 3 miRNAs(miR-569、 mil0 l 207—5p and miR-1268)deregulated significantly，and miRNA expression profile was obtained． IV Abstract 2．3 of 9 notably dysregulated-expressed miRNAs(miR·34b、miRNA-148a and miR一205)were optimized for RT-PCR to validate the array revealed result，and had high expression levels comparing with the controls，as same change trend of microarray． Conclusion： Through respective perfor