Identification and Characterization of microRNAs Responsible for Regulating GNMT Gene Expression

• Main Authors: Pin-Chen Huang, 黃品程
• Other Authors: Yin-Min Chen
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/30249098615590034522

碩士 === 國立陽明大學 === 微生物及免疫學研究所 === 102 === Glycine N-methyltransferase (GNMT) is involved in one-carbon metabolism and regulates the ratio of S-adenosylmethionine and S-adenosylhomocystine. GNMT expresses abundantly in normal liver but is down-regulated in hepatocellular carcinoma (HCC). Our previous finding suggests that GNMT is a tumor suppressor for HCC. However, the mechanism of GNMT down-regulation is not clear. Many studies showed that microRNA (miRNA) deregulation is involved in tumorigenesis. Therefore, the objective of this thesis is to study the role of miRNA in GNMT down-regulation. We identified 4 candidate miRNAs, miR-17, miR-93-5p, miR-224 and miR-491-5p, by literature review and using MiRWalk predicting tool. Results from qRT-PCR analysis demonstrated a significant negative correlation between miR-224 and GNMT expression in 10 HCC specimens. We next further investigated whether miR-224 regulates GNMT expression and found that GNMT expression was significantly lower in miR-224 mimic transfected cells than in control cells, which can be reversed by addition of miR-224 inhibitor. The predicted target site of miR-224 is in the coding region (nt601~609) of GNMT. We cloned mutant GNMT gene fragment (GNMTMT601-9-Flag) into pCMV5a to compare existed wild type plasmid, PCMV5aGNMTWT-Flag. Results from targeting assay showed that GNMTWT-Flag expression was down-regulated by miR-224, while GNMTMT601-9-Flag expression was not affected by miR-224. In consistent with this finding, results from luciferase reporter assay showed that the luciferase activity of GNMTWT was decreased upon transfection of miR-224 mimics compared with GNMTMT601-9. Furthermore, we found that overexpression of GNMT reduced cell proliferation and this effect can be partially reversed by overexpression of miR-224. We further found similar results using cologenic assay. Finally, we used qRT-PCR to detect the expression of miR-224 and GNMT in tumorous and tumor adjacent from 78 HCC specimens. The results showed that among HBsAg positive HCC patients, miR-224 expression in the tumor tissues was significantly higher than that in the tumor adjacent tissues and significant negative correlation was found between miR-224 and GNMT expression. miR-224 expression in non-HBV/HCV associated HCC patients’ tumor tissues was also significantly higher than that in the tumor adjacent tissues. In summary, miR-224 is involved in the down-regulation of GNMT expression via targeting at its coding region nucleotide residues 601-609. This finding is important for the understanding of the mechanism of down-regulation of GNMT and may shed light to the pathogenesis of HCC.