Re-expression of RASSF1A by 5-Aza-CdR induced demethylation of the promoter region in human biliary tract carcinoma cells.
Hypermethylation of the promoter region is an important mean for the transcriptional repression of a number of cancer-associated genes, and over-expression and/or increased activity of DNA methyltransferase are considered to be the main cause of promoter hypermethylation. In order to further explore the epigenetic mechanism of tumor suppressor gene RASSF1A inactivation, 5-aza-2'-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor, was used to treat the human biliary tract carcinoma cell line QBC-939 at the concentration of 5 micromol/L for 24 h in this study. After the chemical intervention with 5-Aza-CdR, the methylation status in the promoter region of RASSF1A gene was detected by methylation specific PCR (MS-PCR), and the expression alteration of RASSF1A mRNA and protein were observed by RT-PCR and Western Blot respectively. Following the treatment with 5-Aza-CdR, methylation status in the promoter region of RASSF1A gene was reversed from methylation to unmethylation. A 280 bp DNA band which represented RASS1FA expression at transcriptional level and a 40 kDa (1 kDa=0.9921 ku) protein band which represented RASSF1A expression at protein level were detected by RT-PCR and Western Blot respectively in the experimental group cells and there were no corresponding bands in the control group cells. The experimental results suggest that 5-Aza-CdR can induce demethylation in the promoter region of RASSF1A. It can also reverse epigenetic transcriptional silencing caused by DNA methylation and induce the re-expression of RASSF1A in QBC-939. This study also suggest that the mechanism of RASSF1A inactivation is very closely related to the methylation of the promoter region, which may provide a new epigenetic understanding for tumor related gene inactivation and the pathogenesis of biliary tract carcinoma.
Zuo
Shi
S
Department of General Survery, Tongji Hospital, Tongji Medical College, Huazhong Science & Technology University, Wuhan 430030, China. zuoshi1991@yahoo.com.cn
Chen
Yongjun
Y
Xu
Lining
L
Tang
Qibin
Q
Zou
Shengquan
S
D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001661:Biliary Tract Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000187:drug effects*; D000077209:Decitabine; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
C411762:RASSF1 protein, human; D012333:RNA, Messenger; D025521:Tumor Suppressor Proteins; D000077209:Decitabine; D001374:Azacitidine