INTRODUCTION: miRNAs are very important regulators in biological processes such 
as development, cellular differentiation, and carcinogenesis. Given the 
important role of miRNAs in tumorigenesis and development, it is worth 
investigating whether some miRNAs play roles in the anticancer mechanism of 
flavonoids. However, such a role has not yet been reported. We previously 
selected the promising anticancer agent 3,6-dihydroxyflavone (3,6-DHF) in 
pharmacodynamic experiments, which may serve as a leading compound for 
developing more potent anticancer drugs or chemopreventive supplements. The 
present study aims to investigate the chemopreventive activities of 3,6-DHF 
against mammary carcinogenesis.
METHODS: The experimental model of breast carcinogenesis was developed by 
intraperitoneal injection of 1-methyl-1-nitrosourea (MNU). The bioavailability 
of 3,6-DHF in rats was detected by HPLC. The expression of microRNA-34a 
(miR-34a) and microRNA-21 (miR-21) was evaluated by real-time quantitative 
RT-PCR. Cell apoptosis was analyzed by flow cytometry or terminal 
deoxynucleotidyl transferase dUTP nick end-labeling assay. The mitochondrial 
membrane potential was assayed using 
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide dye by 
confocal laser scanning microscopy. The level of cytochrome C in cytosol was 
evaluated by western blotting.
RESULTS: Our study showed that oral administration of 3,6-DHF effectively 
suppressed MNU-induced breast carcinogenesis in rats, decreasing the cancer 
incidence by 35.7%. The detection of bioavailability indicated that the 
concentration of 3,6-DHF was 2.5 ± 0.4 μg/ml in plasma of rats within 2 hours 
after administration, and was 21.7 ± 3.8 μg/ml in urine within 24 hours. Oral 
administration of 3,6-DHF to BALB/c nude mice bearing breast cancer cell 
xenografts also significantly suppressed tumor growth in vivo. Furthermore, our 
study revealed that the global upregulation of miR-21 and downregulation of 
miR-34a in breast carcinogenesis could be reversed by 3,6-DHF, which 
significantly upregulated miR-34a expression and decreased miR-21 expression - 
inducing apoptosis of breast cancer cells in vitro and in vivo. Overexpression 
of miR-34a induced by plasmid transfection or inhibition of miR-21 by 
oligonucleotides markedly promoted the pro-apoptotic effect of 3,6-DHF. 
Inactivation of miR-34a or overproduction of miR-21 compromised the anticancer 
effects of 3,6-DHF.
CONCLUSION: These findings indicate that 3,6-DHF is a potent natural 
chemopreventive agent, and that miR-34a and miR-21 play roles in MNU-induced 
breast carcinogenesis and the anticancer mechanism of flavonoids.