Lack of PPAR β/ δ-Inactivated SGK-1 Is Implicated in Liver Carcinogenesis

Abstract

Objective: The present study examined the role of PPARβ/δ in hepatocellular carcinoma (HCC).

Methods: The effect of PPARβ/δ on HCC development was analyzed using PPARβ/δ-overexpressed liver cancer cells and PPARβ/δ-knockout mouse models.

Results: PPARβ/δ ^(-/-) mice were susceptible to diethylnitrosamine- (DEN-) induced HCC (87.5% vs. 37.5%, p < 0.05). In addition, PPARβ/δ-overexpressed HepG2 cells had reduced proliferation, migration, and invasion capabilities accompanied by increased apoptosis and cell cycle arrest at the G0/G1 phase. Moreover, differential gene expression profiling uncovered that the levels of serine/threonine-protein kinase (SGK-1) mRNA and its encoded protein were reduced in PPARβ/δ-overexpressed HepG2 cells. Consistently, elevated SGK-1 levels were found in PPARβ/δ ^(-/-) mouse livers as well as PPARβ/δ-knockdown human SMMC-7721 HCC cells. Chromatin immunoprecipitation (ChIP) assays followed by real-time quantitative polymerase chain reaction (qPCR) assays further revealed the binding of PPARβ/δ to the SGK-1 regulatory region in HepG2 cells.

Conclusions: Due to the known tumor-promoting effect of SGK1, the present data suggest that PPARβ/δ-deactivated SGK1 is a novel pathway for inhibiting liver carcinogenesis.

Figure 1

Role of PPARβ/δ in the upregulation of HCC. Mouse livers were excised after eight months of DEN treatment. (a) The photograph shows reduced tumor growth in the PPARβ/δ^(+/+) mice compared to the PPARβ/δ^(-/-) mice. (b) Hematoxylin-eosin-stained liver tissue sections of mice. (c) Representative histological results from HCC tissues showing HCC in hematoxylin-eosin-stained liver tissue sections of mice (magnification, 100x and 400x). Arrows indicate microscopic HCC. (d) Incidence of HCC development in PPARβ/δ^(+/+) and PPARβ/δ^(-/-) mice, which were kept under observation for eight months after the administration of DEN.

Figure 2

Effect of PPARβ/δ overexpression on cell growth, apoptosis, and cell cycle regulation. HepG2 cells were stably transfected with pEGFP-PPARβ/δ or pEGFP vector. (a) PPARβ/δ expression was analyzed in five different cell lines using western blot. (b) The relative mRNA expression levels for PPARβ/δ were evaluated by qPCR. The PPARβ/δ mRNA expression level was significantly higher in the PPARβ/δ-overexpressed cells than in the control cells (p < 0.001). (c) Western blotting analysis to evaluate the PPARβ/δ expression levels in HepG2 cells transfected with pEGFP-PPARβ/δ or the control vector. (d) Cell proliferation was assessed by the CCK-8 assay at the indicated time points. (e) The effect of PPARβ/δ on cancer cell growth was confirmed by a colony formation assay. Colonies were stained with 0.1% crystal violet and counted. (f) Representative histogram plots of the flow cytometry analysis. The numbers of cells in the G0/G1 and S+G2 phases were determined by flow cytometry. (g) The effect of PPARβ/δ on apoptosis was determined by FACS using an annexin V apoptosis assay. Annexin V-positive apoptotic cells were significantly increased in pEGFP-PPARβ/δ-transfected cells compared with pEGFP vector-transfected cells. Values are the mean of ± standard deviation from three replicate experiments. ^∗p < 0.05, ^∗∗p < 0.01.

Figure 3

Effect of PPARβ/δ on liver cancer cell motility and invasion capability, as assessed by wound healing and Matrigel invasion assays. HepG2 cells stably transfected with pEGFP-PPARβ/δ or the pEGFP vector (control) were subjected to (a) a wound healing assay and (b) a cell migration assay. Representative pictures were taken under an inverted microscope at the indicated time points. (c) Cell motility was quantified by counting the cells that migrated through the Matrigel membrane under a light microscope (×100). The relative cell number ratio was expressed as the mean ± standard deviation. ∗∗p < 0.001, compared to the control. (d) Representative images of the cell invasion ability of HepG2 cells transfected with pEGFP-PPARβ/δ or the control vector after 48 h. (e) Quantification of cell invasion was estimated by counting the cells that invaded through the Matrigel membrane under a light microscope (×100). The data are expressed as the mean ± standard deviation. ^∗∗p < 0.01, compared to the control.

Figure 4

PPARβ/δ regulates the expression of SGK-1. (a) Western blot analysis of SGK-1 in PPARβ/δ-overexpressed HepG2 cells. (b, c) The mRNA levels of PPARβ/δ and SGK-1 were determined in shPPARβ/δ cells using qPCR, respectively. (d) Representative images of immunohistochemical staining from PPARβ/δ^(-/-) mice and control mice, with a higher expression of SGK-1 in PPARβ/δ^(-/-) mice. (e) ChIP-qPCR assays confirmed that the transcription factor PPARβ/δ can specifically bind to the regulatory region of SGK1 in HepG2 cells. Bars correspond to the mean ± standard deviation. ^∗p < 0.05, compared to the isotype-matched IgG control (IgG). (f) Whole-genome microarray analysis of gene expression in HepG2 cells transfected with PPARβ/δ_pEGFP-N1 or empty vector. Functional annotation was carried out in tabulation.