miR-133b Regulation of Connective Tissue Growth Factor: A Novel Mechanism in Liver Pathology

Abstract

miRNAs are involved in liver regeneration, and their expression is dysregulated in hepatocellular carcinoma (HCC). Connective tissue growth factor (CTGF), a direct target of miR-133b, is crucial in the ductular reaction (DR)/oval cell (OC) response for generating new hepatocyte lineages during liver injury in the context of hepatotoxin-inhibited hepatocyte proliferation. Herein, we investigate whether miR-133b regulation of CTGF influences HCC cell proliferation and migration, and DR/OC response. We analyzed miR-133b expression and found it to be down-regulated in HCC patient samples and induced in the rat DR/OC activation model of 2-acetylaminofluorene with partial hepatectomy. Furthermore, overexpression of miR-133b via adenoviral system in vitro led to decreased CTGF expression and reduced proliferation and Transwell migration of both HepG2 HCC cells and WBF-344 rat OCs. In vivo, overexpression of miR-133b in DR/OC activation models of 2-acetylaminofluorene with partial hepatectomy in rats, and 3,5-diethoxycarbonyl-1,4-dihydrocollidine in mice, led to down-regulation of CTGF expression and OC proliferation. Collectively, these results show that miR-133b regulation of CTGF is a novel mechanism critical for the proliferation and migration of HCC cells and OC response.

Figure 1

Tumor suppressor effects of microRNA 133b. A: Patient samples [hepatocellular carcinoma (HCC) and normal adjacent liver tissue] and human HCC HepG2 and Huh-7 cell lines plated in 12-well plates were collected and analyzed by TaqMan miRNA RT-PCR for miR-133b expression levels. B: Phase-contrast and fluorescence images of HepG2 cells transduced for 72 hours with adenovirus vectors expressing either green fluorescent protein (GFP) only (Ad-control) or GFP-miR-133b (Ad-miR-133b). C and D: Semiquantitative PCR assay showing miR-133b expression (C) and down-regulation of connective tissue growth factor (CTGF) mRNA (D) after adenovirus transduction of HepG2 cells. Graphs represent quantitation of the results. E: Immunoblotting analysis and quantitation of CTGF protein expression in transduced HepG2 cells. F and G: Decreased CTGF-induced proliferation (F) and migration (G) of miR-133b–transduced HepG2 cells. Data are shown as means ± SEM (A and C–F). n = 8 (A, HCC and normal adjacent liver tissues); n = 6 (A, human HCC HepG2 and Huh-7 cell lines). ^∗P ≤ 0.05 (t-test). Scale bar = 80 μm (B).

Figure 2

Induction of miR-133b expression after 2-acetylaminofluorene (2-AAF)/partial hepatectomy (PHx) and in vitro effects of miR-133b overexpression in WBF-344 rat oval cells. A: Time course of the miR-133b expression in the rat liver tissues collected from normal and 2-AAF/PHx–treated animals from day (d) 1 to day 21. B: Induction of miR-133b expression and connective tissue growth factor (CTGF) mRNA by 2-AAF/PHx was assayed by RT-PCR in situ hybridization (ISH) and Ctgf ISH, respectively. C: Endogenous Ctgf and Gapdh mRNA levels in transduced WBF-344 for 72 hours were assayed by semiquantitative PCR. D: Transduced WBF-344 cells were transfected with CTGF 3′ untranslated region containing plasmid, and luciferase reporter assay was performed 48 hours after transfection. E and F: Reduction of the CTGF-induced proliferation (E) and migration of WBF-344 cells overexpressing miR-133b (F). Data are shown as the means ± SEM (A and D–F). n = 6 (D); n = 3 (A, rats per time point, E, and F). ^∗P ≤ 0.05 (t-test). Scale bar = 50 μm (B).

Figure 3

Overexpression of miR-133b via adenoviral transduction system in vivo leads to down-regulation of connective tissue growth factor (CTGF) expression and oval cell response. Adenovirus vectors containing either green fluorescent protein (GFP)-control or GFP-miR-133b were delivered via intraportal injection at the time of 70% partial hepatectomy (PHx). Animals were sacrificed at day 9 after PHx. A: Paraformaldehyde-fixed and paraffin-embedded liver tissue sections were stained with immunofluorescence anti-GFP (green) antibody, anti-Ctgf (red); and costained for Ck19 and Ki-67 or anti-Sox9. For the GFP and Ctgf staining, slides were counterstained with DAPI (blue) for nuclear visualization. B: Semiquantitative PCR showing GFP and miR-133b transduction of rat livers. C: Graphs represent quantitation performed by ImageJ software version 1.48v (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Six to 12 images obtained from stained slides of three different rat livers were used for quantitation. Data are presented as means ± SEM (C). ^∗P ≤ 0.05 (t-test). Scale bar = 50 μm (A). 2-AAF, 2-acetylaminofluorene.

Figure 4

In vivo delivery of miR-133b in 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)–treated mice reduces ductular reactions and oval cell proliferation. Mice that were given the DDC diet for 1 day were injected on the tail vein with adenovirus expressing green fluorescent protein (GFP) only (Ad-control) or GFP-miR-133b (Ad-miR-133b) and were fed a DDC diet for an additional 8 days. Animals were sacrificed, and liver tissues were analyzed. A: The immunofluorescence staining for GFP shows localization of transduced adenovirus in DDC-damaged livers. Red represents autofluorescence obtained from porphyrin plugs. B: TaqMan real-time PCR analysis detects high levels of miR-133b in infected livers. C and D: Lower levels of connective tissue growth factor (CTGF) mRNA and protein are detected by RT-PCR (C) and immunohistochemistry (D). Three different animals from each group were analyzed. E: Dual immunofluorescence staining for Ki-67 and A6 was performed to detect proliferating biliary epithelial cells. F: Biliary cells positive for both A6 and Ki-67 were counted from eight slides from three different animals on the basis of dual staining for Ki-67 and A6. Nuclei were counterstained with DAPI (blue; A and E) or nuclear fast red (D). G: Diagram illustrates how miR-133b down-regulation of CTGF expression leads to the decrease of hepatocellular carcinoma (HCC) cell proliferation and migration and to the reduction of ductular reactions (DRs)/oval cell (OC) response during liver injury (ie, 2-acetylaminofluorene/partial hepatectomy in rats or DDC in mice). Data are shown as means ± SEM (B and F). ^∗P ≤ 0.05 (t-test). Scale bar = 75 μm (A, D, and E). PV, portal vein.