[Relationship between RAR-beta gene expression defect and its methylation].
OBJECTIVE
To evaluate the expression of RAR-beta gene in cervical carcinoma cell lines SiHa, HeLa, C33A and Caski and to analyze the relation between their gene expression and the promoter methylation of RAR-beta DNA.


METHODS
The expression of mRNA and protein of RAR-beta gene in the four cell lines were analyzed by RT-PCR, western blot and immunofluorescence, respectively. Methylation specific PCR (MSP) was used to check whether there was methylation in the promoter of RAR-beta gene. The demethylating agent 5-aza-2'-deoxycytidine (5-Aza-cdR) was used to treat methylated cell lines and the change of RAR-beta gene methylation and RAR-beta gene expression defects were observed. The cell proliferation was assayed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide method.


RESULTS
The mRNA and protein expression levels of RAR-beta in cell lines SiHa, HeLa, Caski and C33A were 0.25 +/- 0.08, 0, 0.60 +/- 0.19, 3.12 +/- 0.92 and 0.23 +/- 0.07, 0, 0.14 +/- 0.05, 0.68 +/- 0.21, respectively. The mRNA and protein expression of RAR-beta in SiHa, HeLa and Caski cell lines were decreased or silenced, whereas its expression increased in C33A cell line. MSP method showed that there were RAR-beta gene methylation in SiHa, HeLa and Caski cell lines, while there was no RAR-beta gene methylation in C33A cell line. After treated with 5-Aza-cdR, the mRNA and protein expression levels of RAR-beta in SiHa, HeLa, Caski and C33A cell lines were 1.82 +/- 0.59, 2.13 +/- 0.62, 1.67 +/- 0.43, 2.95 +/- 0.89 and 0.69 +/- 0.21, 0.83 +/- 0.29, 0.56 +/- 0.16, 0.64 +/- 0.20 respectively. The mRNA and protein levels of RAR-beta had a significant difference between before and after interference with 5-Aza-cdR in SiHa, HeLa, and Caski cell lines (P < 0.05). However, they had no significant difference between before and after interference with 5-Aza-cdR in C33A cell line (P > 0.05). The 5-Aza-cdR treatment could suppress cell proliferation.


CONCLUSIONS
The RAR-beta gene expression defects play an important role in the carcinogenesis of cervical cancer. Aberrant methylation in promotor region of RAR-beta gene may be an important mechanism for the loss of expression of RAR-beta gene.
Gao
Yan-ping
YP
Department of Obstetrics and Gynecology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Li
Min
M
Zhang
Ying-ying
YY
Wang
Han
H
He
Xiao-hong
XH
Wang
Ze-hua
ZH
D001374:Azacitidine / Q000494:pharmacology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019175:DNA Methylation; D005260:Female; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D018168:Receptors, Retinoic Acid / Q000096:biosynthesis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology
D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; C082925:retinoic acid receptor beta; D001374:Azacitidine