Silencing of LLGL2 Suppresses the Estradiol-Induced BPH-1 Cell Proliferation through the Regulation of Autophagy

Abstract

Lethal giant larvae (Lgl) is an apical-basal polarity gene first identified in Drosophila. LLGL2 is one of the mammalian homologs of Lgl. However, little is known about its function in the prostate. In this study, to explore the new role of LLGL2 in the prostate, we examined the proliferative activity of a BPH-1 cell line, a well-established model for the human prostate biology of benign prostatic hyperplasia (BPH). The expression of LLGL2 was dose-dependently increased in BPH-1 cells after treatment with 17β-estradiol (E2). Additionally, E2 treatment increased the proliferation of the BPH-1 cells. However, the knockdown of LLGL2 with siRNA significantly suppressed the proliferation of the E2-treated BPH-1 cells. Moreover, si-llgl2 treatment up-regulated the expression of LC-3B, ATG7, and p-beclin, which are known to play a pivotal role in autophagosome formation in E2-treated BPH-1 cells. Overexpression of LLGL2 was able to further prove these findings by showing the opposite results from the knockdown of LLGL2 in E2-treated BPH-1 cells. Collectively, our results suggest that LLGL2 is closely involved in the proliferation of prostate cells by regulating autophagosome formation. These results provide a better understanding of the mechanism involved in the effect of LLGL2 on prostate cell proliferation. LLGL2 might serve as a potential target in the diagnosis and/or treatment of human BPH.

Keywords: LLGL2; autophagosome formation; benign prostatic hyperplasia; proliferation.

Figure 1

LLGL2 is highly expressed in testosterone-induced rat benign prostatic hyperplasia (BPH) in vivo. Representative photographs of immunostaining for LLGL2 (A–D). Prostate of control group; magnifications: ×100, scale bar = 100 μm (A) and magnifications: ×200, scale bar = 20 μm (B). Prostate of BPH group; magnifications: ×100, scale bar = 100 μm (C) and magnifications: ×200, scale bar = 20 μm (D). (E) LLGL2 protein expression in testosterone-induced rat BPH prostate tissue by Western blotting. (F) Graphical representation of the ratio of LLGL2 to β-actin. Values are expressed as the means ± standard deviation; *** p < 0.001 versus control group.

Figure 2

LLGL2 expression and BPH-1 cell proliferation in E2-treated BPH-1 cells. (A) E2 treatment (0, 10, and 100 nM) increased viability of BPH-1 cells. (B) Western blot data showing up-regulated protein expression of LLGL2 and cyclin D1 after treatment with E2 (0, 10, and 100 nM) for 24 h. Graphical representation of the ratio of LLGL2 and Cyclin D1 to β-actin. Values are expressed as the means ± standard deviation; ** p < 0.01 and *** p < 0.001 versus E0 group.

Figure 3

LLGL2 knockdown inhibits proliferation of E2-treated BPH-1 cells. BPH-1 cells were transiently transfected with a negative control siRNA (si-cont) or with LLGL2 siRNA (si-llgl2) against LLGL2 for 48 h. After that, cells were treated with E2 (0 and 100 nM) for 24 h. (A) Western blot data showing LLGL2 and Cyclin D1 protein expression levels in E2-treated BPH-1 cells after LLGL2 knockdown. (B) Cell morphology and confluence images of BPH-1 cells under a phase-contrast microscope (magnification 100×, scale bar = 100 μm). (C) Cell proliferation capacity of LLGL2 knockdown in E2-treated BPH-1 cells by CCK assay. (D) Cell cycle of LLGL2 knockdown in E2-treated BPH-1 cells detected by a flow cytometer. (E) Graphical representation of S-phase in LLGL2 knockdown in E2-treated BPH-1 cells. Values are expressed as the means ± standard deviation; *** p < 0.001 versus E0 + si-cont group. ## p < 0.01 versus E100 + si-cont group.

Figure 4

LLGL2 knockdown stimulates autophagosome formation in E2-treated BPH-1 cells. BPH-1 cells were transiently transfected with a negative control siRNA (si-cont) or with LLGL2 siRNA (si-llgl2) against LLGL2 for 48 h. After that, cells were treated with E2 (0 and 100 nM) for 24 h. (A) Western blot data of p-beclin, ATG7, LC3, and p62 protein expression levels in E2-treated BPH-1 cells after LLGL2 knockdown. (B) Representative images of endogenous LC3 puncta in E2-treated BPH-1 cells after LLGL2 knockdown. Cell nuclei were visualized by 6-diamino-2-phenylindole (DAPI; blue) staining and LC3-II was visualized with Alexa 488 conjugate (green) at 400× magnification. Scale bar = 10 μm. (C) Representative images of RFP-GFP-LC3 tandem fluorescent-tagged LC3 (RFP-GFP-LC3) in E2-treated BPH-1 cells after LLGL2 knockdown at 630× magnification. Scale bar = 50 μm.

Figure 5

LLGL2 overexpression in E2-treated BPH-1 cells. BPH-1 cells were transiently transfected with a negative control vector (Cont) or with LLGL2 overexpression plasmid (LLGL2 O/E, Strep-tag pEXPR-IBA 105-LLGL2) for 24 h. After that, cells were treated with E2 (0 and 100 nM) for 24 h. Western blot data of STrEP, LLGL2, cyclinD1, and LC3 in E2-treated BPH-1 cells after LLGL2 overexpression are shown.