MicroRNA-34a and microRNA-21 play roles in the chemopreventive effects of 3,6-dihydroxyflavone on 1-methyl-1-nitrosourea-induced breast carcinogenesis

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 mu g/ml in plasma of rats within 2 hours after administration, and was 21.7 +/- 3.8 mu g/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.