| Breast cancer(BC) is one of the most serious health threats to women worldwide. Our previous study selected a promising chemopreventive agent 3,6-dihydroxyflavone(3,6-DHF), and found 3,6-DHF significantly up-regulates miR-34 a and down-regulates mi R-21 in breast carcinogenesis, yet the upstream and downstream events of the anticancer mechanism remain unclear. Increasing evidence supports that the epigenetic variability at specific transcription regulation sites appears to be susceptible to modulation by diet and nutrition; some dietary components may suppress tumorigenesis and development by affecting the process of DNA methylation and histone modifications, which modulates the expression of certain key genes and the activation of crucial signaling pathways. Notably, the epigenetic silencing of miR-34 by aberrant Cp G methylation frequently occurs in various types of human cancer, including BC, up-regulation of mi R-21 expression has been shown to correlate with histone modifications. Thus, we hypothesized that 3,6-DHF may regulate the expressions of mi R-34 a and mi R-21 by modulating DNA methylation or histone modification. Mi R-34 a and mi R-21 play an important role in regulating the PI3K/Akt/m TOR signaling pathway, the tumor suppressor gene phosphatase and tensin homolog(PTEN) acts as the principal negative regulator of the PI3K/Akt/ mTOR signaling pathway and is a direct target of mi R-21, which negatively regulates its expression. Conversely, Notch-1, a direct target of miR-34 a, is also known to interact with the PI3K/Akt/mTOR signaling pathway. So we investigate the effects of 3,6-DHF on the PI3K/Akt/mTOR signaling pathway, and explored the roles of mi R-34 a and mi R-21.In the current study, with experimental models of carcinogenesis in rats, breast tissues(0, 2w) and tumors(18w) in MNU-treated rats were used to ananylze the level of p-Akt and TET(the ten-eleven translocation methylcytosine dioxygenase)1. With NNK and B[a]P-induced breast carcinogenic transformation in human breast epithelial MCF10 A cells, the cancer-associated properties were evaluated by reduced dependence on growth factors(RDGF) assay, anchorage-independent cell growth(AIG) assay and scratch/wound healing assay. With methylation-specific PCR(MSP), chromatin immunoprecipitation(Ch IP)-q PCR, siRNA, laser scanning confocal methods, we investigated the epigenetic effect of 3,6-DHF on mi R-34 a and miR-21; Furthermore, we used plasmid transfection, quantitative real-time PCR(qRT-PCR), western blot(WB), DNA dot blot to investigate the effect of 3,6-DHF on mi R-34 a through TET1 and DNA methyltransferase-1(DNMT1); With q RT-PCR, WB and si RNA, we investigated the effect of 3,6-DHF on PI3K/AKT/mTOR signaling pathway through modulating mi R-34 a and mi R-21.The main results and conclusions of this study are as follows:(1) 3,6-DHF administration(20 mg/kg, i.g.) significantly decreased the level of p-Akt(T308) and increased the level of TET1 in breast tissues(0, 2 w) and tumors(18 w) in MNU-treated rats. Using cancer-associated properties as the target endpoints, human breast epithelial MCF10 A cells treated with carcinogens(Car T) for 30 days showed aberrantly increased cell survival adapted to RDGF and AIG. Compared with CarT, cells co-treated with carcinogens and 3,6-DHF(C-DHF) for 30 days exhibited a significantly lower acquisition of RDGF and AIG. Similarly, the wound-healing assay showed that Car T cells required apparently increased mobility and proliferation to heal the wound, which can be suppressed by 3,6-DHF co-treatment. The qRT-PCR data revealed the significant down-regulation of mi R-34 a and up-regulation of miR-21 in cellular breast carcinogenesis, which could be mitigated by 3,6-DHF co-treatment.3,6-DHF treatment effectively modulated the expressions of mi R-34 a and miR-21 in BC cells MDA-MB-231(M231), MCF-7(M7) and MDA-MB-453(M453). A locked nucleic acid oligonucleotide complementary to the miR-34 a sequence and the pcDNA6.2-GW/ mi R-21 plasmids blocked the effects of 3,6-DHF on mi R-34 a and miR-21.(2) MSP detected the effect of 3,6-DHF on the methylation status of the mi R-34 a promoter in BC cells and in carcinogenesis, the data showed that the hypermethylation of mi R-34 a promoter was significantly inhibited in 30-d C-DHF and 3,6-DHF-treated BC cells compared with CarT and the control, respectively. ChIP-q PCR analysis for histone modifications associated with the active transcription state(H3K9-14 ac and H3K27ac) and the inactive transcription state(H3K27me3) on the mi R-21 promoter, the results showed that 3,6-DHF treatment significantly lowers the H3K9-14 ac on the mi R-21 promoter.(3) q RT-PCR, WB, DNA dot blot analysis results showed that 3,6-DHF treatment significantly up-regulates the expression of TET1 and 5hm C in M231 cells. Immunostaining results showed that 3,6-DHF administration up-regulates TET1 and 5hm C in xenografted breast tumor of M231 cells;WB data showed that the levels of TET1 significantly decreased in Car T cells, Co-treatment with 3,6-DHF could effectively counteracted the silencing of TET1. Immunostaining results showed that TET1 level significantly decreased in breast tissues and tumors in MNU-treated rats, while 3,6-DHF administration(20mg/kg,i.g.) could effectively up-regulate the expression of TET1. MSP detections showed that 3,6-DHF treatment could effectively decrease the methylation level of TET1 promoter in BC cells.(4) DNMT1 inhibitor testing showed 3,6-DHF decreased the DNMT activity in BC cells. Using Autodock4, we blind-docked 3,6-DHF to the DNMT1 target by setting grid sizes that included the entire DNMT1 molecule, the results showed that 3,6-DHF docked to the putative cytosine pocket with the lowest binding energies of-7.1 kcal/mol. WB results showed that transfecting DNMT1 plasmids over-expressed DNMT1 in M231 cells and significantly down-regulated TET1. DNMT1 over-expression significantly reduced the promotional effect of 3,6-DHF on TET1. MSP detections showed that DNMT1 over-expression blocked the effect of 3,6-DHF on the methylation of TET1 promoter. We blocked TET1 expression by siRNA in M231 cells, TET1 siRNA abrogated the promotional effect of 3,6-DHF on TET1 level. qRT-PCR analysis showed that TET1 silencing down-regulated the mi R-34 a level and significantly reduced 3,6-DHF-induced up-regulation of mi R-34 a. Furthermore, the MSP detections showed that 3,6-DHF decreased the methylation level of miR-34 a promoter, and TET1 inhibition blocked the demethylation effect of 3,6-DHF.(5) WB detections showed that the levels of PI3K(p85, p110), p-Akt(Thr308, Ser473) and p-mTOR(S2448, S2481) in CarT cells significantly increased in a time-dependent manner, co-treatment with 3,6-DHF could effectively suppress the cancer-promoting signaling pathway. An ELISA-based kinase activity assay results showed that the activity of Akt and m TOR in Car T cells significantly increased in a time-dependent manner, which could be counteracted by 3,6-DHF co-treatment. Immunostaining results showed that p-Akt level significantly increased in breast tissues and tumors in MNU-treated rats, which could be effectively inhibited by 3,6-DHF administration(20mg/kg, i.g.). 3,6-DHF treatment could significantly down-regulate the levels of PI3 K, p-Akt and p-mTOR and consequently reduce the activity of Akt and mTOR in BC cells. The immunohistochemistry for p-Akt(T308) in xenografted breast tumors of M231 cells showed that 3,6-DHF administration(20mg/kg, i.g.) could significantly suppress the activation of Akt in vivo. We further investigated the effects of 3,6-DHF on the downstream targets of the PI3K/Akt/m TOR signaling pathway, the results showed that 3,6-DHF treatment could significantly suppress the activation of the downstream targets, including ribosomal p70-S6 kinase(S6K70), ribosomal protein S6(rp S6), eukaryotic translation initiation factor(eIF)4B and eukaryotic translation initiation factor 4E-binding protein(4EBP)1.(6) WB results showed that 3,6-DHF treatment significantly up-regulated PTEN and down-regulated Notch-1, Hes-1 in carcinogenesis and in BC cells. By transfecting the cells with a locked nucleic acid oligonucleotide complementary to the mi R-34 a sequence or pcDNA6.2-GW/miR-21 plasmids, we blocked the effects of 3,6-DHF on the two mi RNAs. Results showed that the nucleic acid oligonucleotide blocked miR-34 a function, while the pcDNA6.2-GW/miR-21 plasmids led to the substantial production of mi R-21 in M231 cells. Inhibition of mi R-34 a or over-expression of mi R-21 significantly reduced the effects of 3,6-DHF on Notch-1 and PTEN, and consequently weakened the suppression of 3,6-DHF on the activation of Akt and mTOR.Conclusion(1) 3,6-DHF inhibited carcinogen-induced breast carcinogenic transformation. 3,6-DHF co-treatment could mitigate significant down-regulation of miR-34 a and up-regulation of miR-21 in cellular breast carcinogenesis. 3,6-DHF treatment effectively modulates the expressions of mi R-34 a and miR-21 in BC cells.(2) MSP and ChIP-qPCR data indicated that 3,6-DHF up-regulated mi R-34 a expression via demethylation, while down-regulated mi R-21 expression by modulating histone modification. Further study indicated 3,6-DHF improveed TET1 level by repression of DNMT1 and demethylation of TET1 promoter, and reactivated mi R-34 a via TET1- mediated demethylation.(3) By transfecting with a locked nucleic acid oligonucleotide complementary to the mi R-34 a sequence or pc DNA6.2-GW/mi R-21 plasmids, results showed that 3,6-DHF treatment significantly up-regulated PTEN and down-regulated Notch-1, Hes-1 through mi R-34 a and miR-21, indicated 3,6-DHF suppressed the PI3K/Akt/ mTOR signaling pathway through mi R-34 a and mi R-21, consequently inhibited breast carcinogenesis. |
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