RLIM suppresses hepatocellular carcinogenesis by up-regulating p15 and p21

Abstract

Hepatocellular carcinogenesis results from dysregulation of oncogenes and tumor suppressors that influence cellular proliferation, differentiation and apoptosis. p15 and p21 are cyclin-dependent kinase inhibitors, which arrest cell proliferation and serve as critical tumor suppressors. Here we report that the E3 ubiquitin ligase RLIM expression is downregulated in hepatocellular carcinoma patients, and correlated with p15 and p21 expression in clinical progression. In addition, we showed that RLIM overexpression suppresses the cell growth and arrests cell cycle progression of hepatocellular carcinoma. Mechanistically, we found that RLIM directly binds to MIZ1, disrupting the interaction between c-MYC and MIZ1, and enhancing p15 and p21 transcription. Our results demonstrate that RLIM is an important suppressor in hepatocellular carcinogenesis.

Keywords: MIZ1; RLIM; hepatocellular carcinogenesis; p15; p21.

Figure 1. The expressions of RLIM negatively correlate with the clinical progression of HCC and positively correlate with the expressions of p15 and p21

(A) The representative IHC staining of RLIM in human normal liver and HCC tissues. The expressions of RLIM were classified as absent/weak, moderate and strong. Upper images are lower magnification, and lower images are enlarged insets. Scale bars, 200 μm; 20 μm (insets). (B) Left panel: analysis showing the percentages of RLIM expressions in normal liver tissues and each HCC clinical stage, with the r and p values of the Spearman rank correlation test indicated. Right panel: the number of different expressions of RLIM in human normal liver and HCC tissues. (C) The expressions of RLIM, p15, p21 and c-MYC were categorized as high and low. IHC analyses of the representative cases are shown. Upper images are lower magnification, and lower images are enlarged insets. Scale bars, 200 μm; 20 μm (insets). (D) Left panel: stacked bar graphs showing the percentages of specimens with either low or high expression of p15, p21 and c-MYC relative to RLIM level. **, p< 0.01. Right panel: the number and percentage of high and low p15, p21 and c-MYC expressions relative to RLIM expressions, including χ² and p values from the Pearson’s chi-squared test.

Figure 2. RLIM enhances p15 and p21 expression through MIZ1 in vivo

(A-B) Overexpression of RLIM increases p15 and p21 gene expression in HCC cells. SK-Hep1 (A) and HepG2 (B) cells were infected with recombinant RLIM or RFP adenovirus for 48 h and then analyzed for real time-qPCR. (C-D) Silencing of RLIM decreases p15 and p21 gene expression in SK-Hep1 (C) and HepG2 (D) cells. (E-F) RLIM increases the expression of p15 and p21 luciferase reporter in a dose-dependent manner. HEK 293T cells were transfected with 0.2 μg of p15-Luc (E) or p21-Luc (F), and varying amounts of a RLIM construct (0, 0.1, 0.2, 0.3 μg). 48 h after transfection, the cells were examined for luciferase activity. (G-H) Silencing of RLIM reduces the expression of p15-Luc (G) and p21-Luc (H) in HEK 293T cells. (I-J) RLIM affects the expression of p15-Luc (I) and p21-Luc (J) through c-MYC and MIZ1. HEK 293T cells were co-transfected with p15-Luc and p21-Luc, with RLIM, c-MYC and MIZ1 constructs as indicated, and 48 h after transfection, the cells were examined for luciferase measurement. All the experiments were performed with co-transfection of Renilla-luciferase (20 ng) as an internal control. The data were derived from three independent experiments and expressed as mean + SEM, **, p< 0.01; *, p< 0.05.

Figure 3. RLIM interacts with MIZ1 and c-MYC

(A-B) HEK 293T cells were co-transfected with Myc-RLIM (MW: 72KDa) and Flag-MIZ1 (MW: 80KDa). 48 h post-transfection, cells were lysed and subjected to immunoprecipitation (IP) with Myc (A) or Flag (B) antibodies, respectively. The immunoprecipitates and whole cell lysate (WCL) were analyzed by immunoblotting. * indicates the position of IgG heavy chain. (C-D) HEK 293T cells were co-transfected with HA-RLIM (MW: 72KDa) and Myc-c-MYC (MW: 65KDa). 48 h post-transfection, cells were harvested and subjected to IP with HA (C) or Myc (D) antibodies, respectively. (E-F) IP of endogenous RLIM protein from SK-Hep1 (E) and HepG2 (F) cells. The associated c-MYC and MIZ1 proteins were analyzed by immunoblotting. (G) SK-Hep1 cells were fixed and immunostained with the fluorescently labeled antibodies against RLIM (red) and c-MYC or MIZ1 (green). The nuclei were counterstained with DAPI (blue). Scale bars: 50 μm. (H) SK-Hep1 cells were infected with RFP or RLIM recombinant adenovirus. Endogenous proteins were analyzed by immunoblotting with indicated antibodies. (I) SK-Hep1 cells were transfected with scramble siRNAs or siRNAs against RLIM. Endogenous proteins were analyzed by immunoblotting with indicated antibodies.

Figure 4. RLIM disrupts the c-MYC/MIZ1 interaction

(A) Left panel: Mapping the MIZ1 domains to interact with RLIM. HEK 293T cells were transfected with indicated constructs and their interaction was examined by immunoprecipitation (IP) and immunoblotting with indicated antibodies. Right panel: a schematic diagram of MIZ1 truncation constructs, which were taken from reference [9], and their interactions with RLIM. POZ and zinc fingers (ZF) 1-12 and 13 were indicated. (B) Left panel: Mapping the RLIM domains to interact with MIZ1. Right panel: a schematic diagram of RLIM truncation constructs and their interactions with MIZ1. LIM-binding domain (LIM-BD) and ring finger (RF) were indicated. (C-D) The differential effects of RLIM truncation constructs on the expression of p15-Luc (C) and p21-Luc (D). (E) The interaction between the exogenously expressed c-MYC and MIZ1 was reduced by the co-expression of HA-RLIM. HEK 293T cells were co-transfected with Flag-MIZ1 and Myc-c-MYC, with or without HA-RLIM (+: 1 μg; ++: 2 μg). (F-G) IP of the endogenous c-MYC protein in SK-Hep1 cells with RLIM overexpressed by recombinant adenovirus infection (F) or RLIM silenced with siRNAs (G). The co-precipitated MIZ1 was analyzed by immunoblotting and the quantitation was shown in the bottom panels. The data were derived from three independent experiments and expressed as mean + SEM, **, p< 0.01; *, p< 0.05.

Figure 5. RLIM suppresses HCC cell proliferation and cell cycle progression

(A) SK-Hep1 and HepG2 cells were infected with RLIM or RFP recombinant adenovirus. The cell growth were monitored by MTS assay at the indicated time points and expressed as mean ± SEM from triplicate experiments. (B) SK-Hep1 and HepG2 cells were infected with RLIM or RFP recombinant adenovirus for 18 h and then subjected to cell synchronization, followed by flow cytometry analysis. The data in the table were derived from three independent experiments, and represent the mean (SEM in bracket) from triplicate experiments. (C) A working model illustrating the mode of action for RLIM in the transcriptional regulation of p15 and p21, through interaction with the c-MYC/MIZ1 complex.