[Transcription regulation of 5-Aza-2'-deoxycytidine on maspin gene demethylation in RKO human colorectal cell line].
OBJECTIVE
To detect the methylation status of 5'CpG island in the core promotor of maspin gene in RKO human colorectal cell line,and to explore the transcription regulation of DNA 5'CpG island demethylation on maspin tumor suppressor gene and its effect on the growth of cancer cell.


METHODS
The status of 5 'CpG island methylation of maspin gene in RKO human colorectal cell line was analyzed using methylation specific polymerase chain reaction (MSP). After treated with a specific demethylating agent, 5-Aza-2'-deoxycytidine, reverse transcription polymerase chain reaction (RT- PCR) was used to examine maspin gene expression. Cell proliferation was evaluated using MTT assay,distribution of cell cycle and rate of apoptosis were determined using flow cytometry.


RESULTS
The 5'CpG island methylation in the core promotor of maspin gene was detected in RKO human colorectal cell line. After treatment with three different concentration of 5-aza-2'-deoxycytidine, the expression of maspin mRNA increased 10.89, 16.91, 23.97 times respectively. MTT array showed the proliferation activity of RKO cell line was obviously reduced after 5-aza-2'-deoxycytidine treatment. The cells were arrested in G(0)/G(1) phase,and the apoptosis rates were 5.17%, 8.71% and 11.23% respectively compared with control group.


CONCLUSIONS
The 5'CpG island methylation is probably responsible for maspin expression silencing in RKO human colorectal cell line, 5-aza-2'-deoxycytidine may effectively cause demethylation and inhibit the growth of tumor cell by reactivating the gene transcription silenced by aberrant hypermethylation.
Tang
Bo
B
Department of General Surgery, Southwest Hospital, The Third Military Medical University, Chongqing, China. tangtbo@yahoo.com.cn
Peng
Zhi-hong
ZH
Yu
Pei-wu
PW
Zeng
Dong-zhu
DZ
Zhang
Chao
C
D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000187:drug effects; D019175:DNA Methylation; D000077209:Decitabine; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D015843:Serpins / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects*
C085344:SERPIN-B5; D015843:Serpins; D000077209:Decitabine; D001374:Azacitidine