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  • br The p c kit

    2020-03-24


    3.3. The p-c-kit/MAPK-p38/E2F1 cascade is involved in ASC/SCF-induced miR20b downregulation in BC cells
    To elucidate the signaling pathways involved in ASC-induced miR20b downregulation in BC cells, we examined whether the MAPK pathway was activated in 4T1 cells treated with SCF and found that the activation of SCF/c-Kit triggers phosphorylation of ERK (p-ERK) and the upregulation of p38, which could inhibit the expression of E2F1 in the nucleus (Fig. 3A). In consideration of the ERK1/2 is the only downstream protein target of MEK1/2, and to further confirm the contribution of the MAPK p38 pathway to E2F1 expression and ASC-induced migration and invasion of the BC cells, we treated 4T1 cells with a MEK-specific siRNA and a p38-specific siRNA and analyzed the changes in E2F1 levels by western blot. We found that the E2F1 ex-pression levels in the nuclei were significantly enhanced by p38-specific siRNA following the phospho-P38 decrease (Fig. 3B, Supplementary Fig. S2A–C). Interestingly, the MEK-specific siRNA did not significantly enhance the expression of E2F1 in the nuclei (Fig. 3B, Supplementary Fig. S2C). In addition, the ASC-induced migration and invasion of cancer cells were abrogated by the p38-specific siRNA (Supplementary Fig. S2D–E). 
    Considering that the promoter region of miR20b contains E2F1 Dorsomorphin according to the MATCH software (Fig. 3C), we in-vestigated whether E2F1 regulates miR20b at the transcriptional level. The results showed that the expression levels of miR106a-363, primary precursors and mature miR20b-5p were significantly changed when 4T1 cells were transfected with an E2F1-specific siRNA (Supplementary Fig. S2F). To further explore whether E2F1 can regulate the expression of miR106a-363 clusters directly, 4T1 cells with stable expression of E2F1 were cotransfected with the miR106a-363-promoter-driven luci-ferase plasmid. We found that the overexpression of Dorsomorphin E2F1 markedly increased miR106a-363 promoter activity in 4T1 cells (Fig. 3D). A ChIP assay demonstrated that ASCs induced the binding of E2F1 to the promoter region of miR106a-363 in 4T1 cells through the SCF/MAPK p38 cascade (Fig. 3E–F). These results indicate that ASCs induce miR20b downregulation in BC cells at the transcriptional level through the production of SCF and the subsequent SCF-dependent induction of the MAPK-p38/E2F1 cascade in BC cells.
    3.4. ASC induction of miR20b downregulation activates HIF-1α and VEGFA
    To further investigate the mechanism of miR20b regulation of the migration and invasion of breast cancer cells, we hypothesized that HIF-1α and VEGFA may be the target genes of miR20b according to the predictions of the PicTar, miRWalk and TargetScan programs. Then, the luciferase reporter plasmids containing HIF-1α-3′UTR or VEGFA-3′UTR with a wild type miR20b-bingding site or a mutated miR20b-bingding site were cotransfected with a miR20b agomir in 4T1 breast cancer cells (Fig. 4A–B). As shown in Fig. 4A and Fig. 4B, the overexpression of miR20b significantly inhibited the luciferase reporter activity of the wild-type HIF-1α-3′UTR and the VEGFA-3′UTR but not the activity of the mutant HIF-1α-3′UTR and the VEGFA-3′UTR constructs. A previous study showed that HIF-1α can bind to the VEGFA promoter and re-activate its activity in BC cells. Therefore, a ChIP assay was performed to detect the proteins bound to the VEGFA promoter in 4T1 cells. We found that the binding of HIF-1α to the VEGFA promoter was sig-nificantly decreased in the 4T1 cells transfected with miR20b and co-cultured with ASCs compared to that in the untreated cells (Fig. 4C, Supplementary Fig. S3A). In agreement with a report of Sandra Cascio et al. on miR20b [14], our results showed that HIF-1α levels were considerably reduced in the nuclei of 4T1 cells transfected with miR20b and stimulated with ASCs compared to the levels in the 4T1 cells treated with ASCs, but which was higher than the levels in untreated 4T1 cells (Fig. 4D, Supplementary Fig. S3B). In addition, we found that the overexpression of HIF-1α markedly increased the ASC-induced breast cancer cell migration and invasion, whereas miR20b upregula-tion markedly attenuated HIF-1α promoter activity (Fig. 4E–F). These results indicate that ASCs can induce BC cell migration and invasion through the SCF/miR20b/HIF-1α/VEGFA signaling cascade.
    3.5. SCF released from c-kit+ ASCs induces BC cell metastasis through inhibition of miR20b in vivo
    To confirm that ASC-stimulated BC cells exhibit the EMT phenotype in vitro, we detected the mRNA levels of the EMT-related genes in untreated 4T1 cells, 4T1 cells overexpressing miR20b, and 4T1 cells