Promoter hypermethylation of FANCF plays an important role in the occurrence of ovarian cancer through disrupting Fanconi anemia-BRCA pathway

Objective: Individuals with Fanconi anemia ( FA) are predisposed to develop ovarian cancer than those without FA, this is largely contributed to promoter methylation of the FANCF gene and subsequent disruption of the FA-BRCA pathway. In an attempt to understand more molecular genetic bases of ovarian cancer, we examined the expression of the FANCF and the status of the promoter methylation of the FANCF gene in ovarian cancer. Methods: Seven ovarian cancer cell lines and eighteen ovarian cancer specimens were selected for this study. Both genomic DNA and total RNA were extracted from fresh tissues and cell lines. The DNA was treated with bisulfite and then analyzed with methylation-specific PCR (MSP) to detect FANCF methylation. The expression of FANCF mRNA was detected with Reverse transcription-polymerase chain reaction (RT-PCR). Additionally, the proliferation of cell lines before and after the treatment with demethylating agent 5-Aza-2'-deoxycytidine(5-ADC) was examined with 3-(4,5-dimethylthiazol-2-yl)- 2, 5-diphenyltetrazolium bromide(MTT) assay. Results: The expression of FANCF mRNA decreased in most ovarian cancers as compared with those in normal ovarian tissues. Similarly, the level of FANCF protein decreased in ovarian cancers. The decrease of FANCF was due, in part, to FANCF methylation ( five of 12 specimens). The methylation rates were 27.8% ( five of 18 specimens) in primary tumors and 14.3% ( one of seven cell lines) in established ovarian cancer cell lines respectively. The treatment of ovarian cancer cells with 5-ADC contributed to the following results: the inhibition of DNA promoter methylation, the reactivation of FANCF mRNA expression and protein, and the subsequent reduction in the proliferation of tumor cells both in vitro and in vivo. Conclusions: The results showed that FANCF methylation regulates the expression of FANCF at both mRNA and protein levels. Methylation-induced inactivation of FANCF plays an important role in the occurrence of ovarian cancers via disrupting the FA-BRCA pathway.