Identification and characterization of microRNA candidates that are upregulated by 5-aza-2’-deoxycytidine treatment in hepatocellular carcinoma cells.

• Main Author: Leng, Charmaine Yong En.
• Other Authors: School of Biological Sciences
• Format: Final Year Project
• Language: English
• Published: 2009
• Subjects: DRNTU::Science::Biological sciences::Genetics
• Online Access: http://hdl.handle.net/10356/16345
• Institution: Nanyang Technological University

MicroRNAs are small non-coding RNA of 19-20 nucleotides, capable of negatively regulating gene expression by targeting specific mRNA gene sequences, which causes mRNA degradation or inhibits protein translation. This study aims to investigate whether DNA hypermethylation may contribute to microRNA suppression, thereby modulating their target mRNA expression during the development of hepatocellular carcinoma (HCC). Preliminary microarray data revealed 19 up-regulated microRNAs in Hep3B cells treated with a DNA demethylation agent, 5-aza-2’-deoxycytidine (5-aza-dC), with hsa-miR-886-5p showing the greatest increase in expression level. Using TaqMan RT-PCR, my finding confirmed that hsa-miR-886-5p expression was consistently up-regulated after 5-aza-dC treatment in 3 HCC cell lines – Hep3B, HepG2 and PLC/PRF/5. More than half of the cancerous liver tissues from 17 HCC patients were observed to have down-regulated hsa-miR-886-5p expression as compared with their surrounding non-cancerous liver tissues. To determine putative mRNA targets of hsa-miR-886-5p, HCC cells were overexpressed ectopically with hsa-miR-886-5p and screened for differentially expressed genes using microarrays. 102 and 53 probe sets were consistently down-regulated and up-regulated respectively, in all 3 HCC cell lines. Real time RT-PCR was performed to validate the microarray results. These findings might provide new insights to the mechanism regulating hsa-miR-886-5p, and the network of genes targeted by hsa-miR-886-5p.