Genistein inhibits prostate cancer cell growth by targeting miR-34a and oncogenic HOTAIR

Abstract

Objective: Genistein is a soy isoflavone that has antitumor activity both in vitro and in vivo. It has been shown that genistein inhibits many type of cancers including prostate cancer (PCa) by regulating several cell signaling pathways and microRNAs (miRNAs). Recent studies suggest that the long non-coding RNAs (lncRNAs) are also involved in many cellular processes. At present there are no reports about the relationship between gensitein, miRNAs and lncRNAs. In this study, we focused on miRNAs, lncRNA that are regulated by genistein and investigated their functional role in PCa.

Method: Microarray (SurePrint G3 Human GE 8×60K) was used for expression profiling of genistein treated and control PCa cells (PC3 and DU145). Functional assay (cell proliferation, migration, invasion, apoptosis and cell cycle assays) were performed with the PCa cell lines, PC3 and DU145. Both in vitro and in vivo (nude mouse) models were used for growth assays. Luciferase reporter assays were used for binding of miR-34a to HOTAIR.

Results: LncRNA profiling showed that HOTAIR was highly regulated by genistein and its expression was higher in castration-resistant PCa cell lines than in normal prostate cells. Knockdown (siRNA) of HOTAIR decreased PCa cell proliferation, migration and invasion and induced apoptosis and cell cycle arrest. miR-34a was also up-regulated by genistein and may directly target HOTAIR in both PC3 and DU145 PCa cells.

Conclusions: Our results indicated that genistein inhibited PCa cell growth through down-regulation of oncogenic HOTAIR that is also targeted by tumor suppressor miR-34a. These findings enhance understanding of how genistein regulates lncRNA HOTAIR and miR-34a in PCa.

Figure 1. Effect of genistein treatment on PCa cells (PC3 and DU145).

(A) Genistein significantly inhibits cell viability. Cell viability was analyzed by the MTS cell proliferation assay after 4 days treatment (25 µM). **, P<0.0001. *, P<0.01. Data are presented as the mean ± SE. (B) Apoptosis assay using flow cytometry. Representative quadrant figures of control and genistein (25 µM) treated cells in PC3 (left) and DU145 (right) cells. P<0.05. (C) The typical figures of cell cycle analysis of control, or genistein (25 µM) treated cells are shown. The bar chart shown on the right of each figures represents the percentage of the cells in G0/G1, S, or G2/M phase as indicated. *P<0.05.

Figure 2. Effect of genistein treatment and expression of HOTAIR and miR-34a in PCa cells.

(A) Expression of HOTAIR in PCa cell lines (LNCaP, PC3 and DU145) and normal prostate epithelial cells (RWPE-1). Real-time PCR showed that the expression levels of HOTAIR were up-regulated in castration-resistant PCa cell lines (PC3 and DU145). Data are presented as mean ± SE. *, P<0.05. (B) Expression levels of HOTAIR after treatment with genistein (25 µM). HOTAIR expression decreased by 30–40% in genistein treated cells compared with controls. HOTAIR expression was normalized to GAPDH. *, P<0.05. (C) Expression of miR-34a in PCa cell lines (PC3 and DU145) and normal prostate epithelial cells (RWPE-1). Real-time PCR showed that the expression levels of miR-34a were down-regulated in castration-resistant PCa cell lines (PC3 and DU145). miR-34a expression was normalized to RNU48. (D) Expression levels of miR-34a after treatment with genistein (25 µM) in DU145 cell. P<0.05.

Figure 3. miR-34a regulates HOTAIR expression.

(A) Putative miR-34a binding and mutated sites in HOTAIR. (B) Luciferase reporter assays using vectors encoding putative binding sites. PC3 and DU145 cells were transiently transfected with Pre-miR miRNA precursor or negative control, followed by transient transfection with basic vector or wild-type reporter plasmids or mutated plasmids for 24 hours. Reporter activity was measured by luciferase assay and normalized to the activity of Renilla luciferase. Data are presented as the mean ± SE. *, P<0.05. (C) The expression level of HOTAIR was determined by quantitative real-time PCR analyses after transfection with miR-34a mimics and negative control in PCa cell lines (PC3 and DU145). HOTAIR expression was normalized to GAPDH. *, P<0.05.

Figure 4. siRNA knockdown of HOTAIR and effect on PCa cell viability.

(A) HOTAIR expression levels in PCa cell lines (PC3 and DU145) were determined by real-time PCR at 96 hours after transient transfection of siRNA. Gene expression was normalized to GAPDH. Data are presented as the mean ± SE. **, P<0.0001. *, P<0.0005. (B) Knockdown of HOTAIR significantly inhibits cell viability. Cell viability was analyzed by the MTS cell proliferation assay 96 hours after transient transfection. (C) Knockdown of HOTAIR significantly inhibits cell migration. After transfection (48 hours), a wound was formed by scraping and measured after 24 hours. Representative images of wound healing assay are shown at 200× magnification. **, P<0.0001. (D) Knockdown of HOTAIR significantly decreased cell invasion. Representative images of invasion assay are shown at 200× magnification. **, P<0.0001. (E) Representative images of tumors in nude mice 5 weeks after subcutaneous injection of transfected HOTAIR siRNA DU145 cell lines or control cell lines and time course of tumor growth.

Figure 5. siRNA knockdown of HOTAIR and effect on PCa apoptosis and cell cycle.

(A) Apoptosis assay using flow cytometry. Representative quadrant figures of control and si-HOTAIR treated cells in PC3 (upper) and DU145 (lower) cells. **, P<0.005. *, P<0.05. (B) Typical figures of cell cycle analysis of control, or si-HOTAIR treated cells are shown. The bar chart shown on the right of each figure represents the percentage of the cells in G0/G1, S, or G2/M phase as indicated. **, P<0.001. *, P<0.005.