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. 2014 Mar 18;110(6):1645-54.
doi: 10.1038/bjc.2014.48. Epub 2014 Feb 6.

Genistein Downregulates onco-miR-1260b and Upregulates sFRP1 and Smad4 via Demethylation and Histone Modification in Prostate Cancer Cells

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Free PMC article

Genistein Downregulates onco-miR-1260b and Upregulates sFRP1 and Smad4 via Demethylation and Histone Modification in Prostate Cancer Cells

H Hirata et al. Br J Cancer. .
Free PMC article

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Abstract

Background: Recently several microRNAs (miRNAs) have been found to be regulated by genistein in cancer cells. In this study, we focused on the gene regulatory effect of genistein on microRNA and its target genes in prostate cancer (PC).

Methods: Initially, we investigated the effect of genistein on prostate cancer cells and identified that the expression of miRNA-1260b was decreased by genistein. We performed functional analyses and investigated the relationship between miRNA-1260b expression and prostate cancer patient outcomes. Two target genes (sFRP1 and Smad4) of miR-1260b were identified based on computer algorithm and 3'UTR luciferase assay was carried out to determine direct miRNA regulation of the genes.

Results: Genistein promoted apoptosis while inhibiting prostate cancer cell proliferation, invasion and TCF reporter activity in PC cells. MiR-1260b was highly expressed in prostate cancer tissues and significantly downregulated by genistein in PC cells. After knocking down miR-1260b, cell proliferation, invasion, migration and TCF reporter activity were decreased in PC cells. Western analysis and 3'UTR luciferase assay showed that the two target genes (sFRP1 and Smad4) were directly regulated by miR-1260b. The expression of sFRP1 and Smad4 was significantly decreased in prostate cancer tissues. Genistein also increased expression of these two genes via DNA demethylation and histone modifications.

Conclusions: Our data suggest that genistein exerts its anti-tumour effect via downregulation of miR-1260b that targeted sRRP1 and Smad4 genes in prostate cancer cells. The expression of sFRP1 and Smad4 was also modulated by genistein via DNA methylation or histone modifications in PC cell lines.

Figures

Figure 1
Figure 1
Effect of genistein on prostate cancer cell lines (PC-3 and DU-145). Two cell lines were treated with DMSO (control) or genistein (25μM) for 4 days. (A) Cell viability assay, (B) invasion assay, (C) wound healing assay, (D) apoptosis assay-FACS based, (E) cell cycle analysis, (F) TOPflash luciferase assay. Error bars represent±s.d.
Figure 2
Figure 2
miR-1260b expression and association with clinical parameters in prostate cancer tissues. (A) miR-1260b expression in cell lines (RWPE-1, PC-3, DU-145), (B) Effect of genistein on miR-1260b expression in prostate cancer cell lines. (C) miR-1260b expression in human clinical samples (PC and BPH).
Figure 3
Figure 3
Effect of miR-1260b overexpression in normal prostate cells (RWPE-1). A normal prostate cell line (RWPE-1) was transiently transfected with either miR-1260b precursor or control (miR-NC). (A) Relative miR-1260b expression, (B) cell viability assay, (C) wound healing assay.
Figure 4
Figure 4
Effect of miR-1260b knockdown on prostate cancer cells (PC-3, DU-145). Two prostate cancer cell lines (PC-3 and DU-145) were transiently transfected with either miR-1260b inhibitor or miR-negative control (miR-NC inhibitor). (A) Relative miR-1260b expression (miR-NC inhibitor or miR-1260b inhibitor transfected PC cells), (B) cell viability assay (miR-NC inhibitor or miR-1260b inhibitor transfected PC cells), (C) invasion assay, (D) wound healing assay (16–17 h). (E) Apoptosis assay, (F) cell cycle analysis, (G) TCF reporter assay. Error bars represent ±s.d.
Figure 5
Figure 5
Expression of miR-1260b target genes (sFRP1 and Smad4) in BPH and PC tissues and relationship with miR-1260b in prostate cancer cell lines. (A) sFRP1 expression level in BPH and PC tissues, (B) Smad4 expression level in BPH and PC tissues, (C) 3′UTR Luciferase assay (miR-NC and miR-1260b precursor), (D) expression of sFRP1, Smad4 and beta-tubulin protein in miR-NC inhibitor or miR-1260b inhibitor transfected prostate cancer cells (PC-3, DU-145). Error bars represent±s.d.
Figure 6
Figure 6
Effect of genistein on sFRP1 and Smad4 genes in prostate cancer cells. (A) sFRP1 and Smad4 expression in DMSO (control) and genistein 25 μM (G25) treated prostate cancer cells. (B) Representative bisulphite modified genomic DNA sequencing of the sFRP1 promoter region from control and genistein-treated PC-3 cells. (C) Representative bisulphite modified genomic DNA sequencing of the Smad4 promoter region from control and genistein-treated PC-3 cells. (D) Quantification of ChIP real-time PCR analysis in PC-3 cells in sFRP1 and Smad4 genes promoter region in control and genistein-treated 786-O cells.

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