Analysis of HREV107 and RIG1 gene expression and the association with methylation in human cancer cells
| Autor: | Yu-Yen Hsu, 許玉燕 |
|---|---|
| Rok vydání: | 2005 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 93 HREV107 and RIG1 belong to the family of HREV107 type II tumor suppressor gene. Both genes are ubiquitously expressed in normal tissues. Expression of the genes are down-regulated in cancer cell lines and primary tumors. Ectopic H-rev107 or RIG1 expression leads to inhibition of cellular growth and induction of cellular apoptosis and differentiation. Aberrant DNA methylation of the promoter region is a key mechanism for inactivation of genes that suppress tumorigenesis. Most genes silenced by this mechanism contain CpG island in the promoter. Promoter of the murine H-rev107 gene contains CpG island, and DNA hypermethylation within island lesds to the gene silence. A CpG island is identified in human HREV107, whereas it is absent in the RIG1 gene. This study investigated role and mechanism of DNA methylation on expression of human HREV107 and RIG1 genes. Low levels of HREV107 expression was detected in CC-M2 and HL-60 cancer cells using the method of semi-quantitative reverse transcription polymerase chain reaction (PCR). The demethylation compound 5-AZA-2'-Deoxylcytidine (5-aza-dC) increased HREV107 mRNA levels in both cell lines in a dose dependent manner. The 5-aza-dC-mediated HREV107 induction was accompanied with a decrease in DNA methylation within the CpG island of HREV107, which was supported by methylation specific PCR, methylation specific DNA sequencing and combined bisulfite restriction analysis (COBRA). Also, treatment with the protein synthesis inhibitor cyclohexamide had no effect on 5-aza-dC induced HREV107 expression in HL-60 cells. Finally, the correlation between HREV107 expression and CpG methylation in cancer cells was analyzed using the COBRA method. CC-M1 and HT-29 cells that expressed the highest levels of HREV107 were the least methylated. Similar to the effect on HREV107, 5-aza-dC treatment for 3 to 5 days induced RIG1 mRNA levels in CC-M2 and HL-60 cells. However, 5-aza-dC had no effect on methylation of RIG1 promoter in CC-M2 cells determined by methylation specific PCR. The 5-aza-dC-mediated increased in RIG1 mRNA was not effected by treatment with CHX, in contrast to 2.6-fold induced in RIG1 expression by treatment with CHX (2 μM) in HL-60 cells. In conclusion, expression of HREV107 and RIG1 was regulated by 5-aza-dC at the epigenetic levels through different mechanism in CC-M2 and HL-60 cells. CpG island hypermethylation played a role in down-regulation of HREV107 mRNA levels. However, expression of RIG1 is not directly regulated by promoter CpG methylation. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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