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, 108 (10), 2070-8

Genistein Downregulates onco-miR-1260b and Inhibits Wnt-signalling in Renal Cancer Cells

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Genistein Downregulates onco-miR-1260b and Inhibits Wnt-signalling in Renal Cancer Cells

H Hirata et al. Br J Cancer.

Abstract

Background: Wnt-signalling has an important role in renal cancer and it is modulated by genistein in other cancers. Recently, microRNAs (miRNAs) have emerged as new regulators of gene expression. Thus, we focused on miRNAs to examine the regulatory mechanism of genistein on the Wnt-signalling pathway in renal cell carcinoma (RCC).

Methods: Initially, we investigated the effect of genistein on Wnt-signalling (TOPflash reporter assay (TCF reporter assays)) in renal cancer cells, and using microarray identified candidate miRNAs whose expression was decreased by genistein. We performed functional analyses and investigated the relationship between miRNA expression and renal cancer patient outcomes. We also did 3'UTR luciferase assays to look at direct miRNA regulation of Wnt-signalling-related genes.

Results: Genistein promoted apoptosis while inhibiting RCC cell proliferation and invasion. Genistein also decreased TCF reporter activity in RCC cells. We found that miR-1260b was highly expressed and significantly downregulated by genistein in RCC cells. The expression of miR-1260b was significantly higher in renal cancer tissues compared with normal, and significantly related to overall shorter survival. In addition, miR-1260b promoted renal cancer cell proliferation and invasion in RCC cells. The 3'UTR luciferase activity of target genes (sFRP1, Dkk2, Smad4) was significantly decreased and their protein expression significantly upregulated in miR-1260b inhibitor-transfected renal cancer cells.

Conclusion: Our data suggest that genistein inhibited Wnt-signalling by regulating miR-1260b expression in renal cancer cells.

Figures

Figure 1
Figure 1
Genistein effect on renal cancer cell lines (786-O, A-498 and Caki-2). Three cell lines were treated with DMSO or genistein (25 μℳ) for 4 days. (A) Cell viability assay. (B) Invasion assay. (C) Apoptosis assay-FACS based. (D) TCF/LEF reporter assays. Error bars represent ±s.d.
Figure 2
Figure 2
miR-1260b expression and association with clinical parameters in renal cancer tissues. (A) miR-1260b expression in human clinical samples and renal cancer cell lines (786-O, A-498 and Caki-2). (B) Association of miR-1260b with clinic-pathological parameters. (C) Kaplan-Meier plots of overall survival.
Figure 3
Figure 3
Effect of miR-1260b overexpression on renal cancer cell function (786-O and A-498).Two renal cancer cell lines (786-O and A-498) were transiently transfected with either miR-1260b precursor or control (miR-NC). (A) Cell viability assay. (B) Invasion assay. (C) Flow cytometric analysis of apoptosis in miR-NC or miR-1260b-transfected renal cancer cells.
Figure 4
Figure 4
(A) Effect of genistein on miR-1260b expression in renal cancer cell lines, (B1) Overexpression of miR-1260b after miR-NC or miR-1260b precursor transfection. (B2) TCF/LEF reporter assays. (C1) Relative miR-1260b expression after NC-inhibitor or miR-1260b inhibitor transfection. (C2) TCF/LEF reporter assays.Genistein, miRNA-1260b and TCF reporter assay.
Figure 5
Figure 5
miR-1260b binds to the 3′UTR of Dkk2, sFRP1 and Smad4 mRNAs and downregulates expression. (A) Dkk2, sFRP1 and Smad4 3′UTR sequence and complementary miR-1260b binding site. (B) 3′UTR luciferase assay (miR-NC and miR-1260b precursor), error bars represent ±s.d.. (C) Protein expression of Dkk2, sFRP1, Smad4 and β-tubulin in miR-NC inhibitor or miR-1260b inhibitor-transfected renal cancer cells (786-O, A-498).
Figure 6
Figure 6
Effect of sFRP1, Dkk2 and Smad4 overexpression on renal cancer cell (A-498) function. At 24 h after transfection of either pCMV6-empty, pCMV6-sFRP1, pCMV6-Dkk2 or pCMV6-Smad4 into renal cancer cell (A-498), sFRP1, Dkk2 and Smad4 expression levels were verified by real-time RT-PCR (A) and western analysis (B). (C) Cell viability assay. (D) Invasion assay. (E) Apoptosis assay. Error bars represent ±s.d.

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