Differential Modulation of miR-122 Transcription by TGFβ1/BMP6: Implications for Nonresolving Inflammation and Hepatocarcinogenesis

Abstract

Chronic inflammation is widely recognized as a significant factor that promotes and worsens the development of malignancies, including hepatocellular carcinoma. This study aimed to explore the potential role of microRNAs in inflammation-associated nonresolving hepatocarcinogenesis. By conducting a comprehensive analysis of altered microRNAs in animal models with liver cancer of various etiologies, we identified miR-122 as the most significantly downregulated microRNA in the liver of animals with inflammation-associated liver cancer. Although previous research has indicated the importance of miR-122 in maintaining hepatocyte function, its specific role as either the trigger or the consequence of underlying diseases remains unclear. Through extensive analysis of animals and in vitro models, we have successfully demonstrated that miR-122 transcription is differentially regulated by the immunoregulatory cytokines, by the transforming growth factor-beta 1 (TGFβ1), and the bone morphogenetic protein-6 (BMP6). Furthermore, we presented convincing evidence directly linking reduced miR-122 transcription to inflammation and in chronic liver diseases. The results of this study strongly suggest that prolonged activation of pro-inflammatory signaling pathways, leading to disruption of cytokine-mediated regulation of miR-122, may significantly contribute to the onset and exacerbation of chronic liver disease.

Keywords: BMP6; Smad4; TGFβ1; inflammation; liver cancer; miR-122.

Figure 1

miR-122 is downregulated in inflammatory hepatocellular carcinoma. miR−122 is significantly downregulated in the tumor tissue isolated from the livers of lymphotoxin (LT, Purple) c-Myc (blue) over-expressing animals but not in the tissue isolated from (DEN, orange) animals. (A) Heat map depicting unsupervised hierarchical clustering of HCC models with respect to controls. The color scale illustrates the fold change of miRNAs across the samples. (B) Visualization of miRNAs downregulated in the tumor tissue of LT and c-myc models but not in the DEN model. (C) miR−122 expression as box plots (n = 5). (D) Representative image on the livers of wild type (ET) and LPC^{ΔTraf2, Casp8} mice at 6 and 52 weeks. (E) Representative HE staining of the liver of control (WT) and LPC^{ΔTraf2, Casp8} mice at 6 and 52 weeks of age. (F) Expression of miR−122 in the liver of WT and LPC^{ΔTraf2, Casp8} mice at 6 and 52 weeks (normalized to miR-15a, n = 4). Results are represented as mean ± SD, significant differences were evaluated by using the one-way ANOVA or the t-test as appropriate (ns = Not significant; *, p ≤ 0.05; and ***, p ≤ 0.0001).

Figure 2

miR−122 is differentially regulated in the liver of Hjv and Tmprss6 Ko animals. (A) Model of potential regulation of miR−122 associated with signaling downstream to Hfe. (B) qPCR analysis of miR−122 expression in the liver of Hjv KO (n = 5) and Tmprss6 KO animals (n = 5). (C) qPCR analysis of hepcidin and pri−miR−122 expression in primary mouse hepatocytes stimulated with either BMP6 (50 ng/mL; n = 4) or TGFβ1 (5 ng/mL; n = 4). (D) Proposed model of Smad signaling upstream to miR−122 transcription. Results are represented as mean ± SD. Differences were evaluated by using the t-test (ns = Not significant; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; and ****, p ≤ 0.0001).

Figure 3

Mouse miR−122 promoter regions respond to BMP6 and TGFβ stimulation. (A) Different lengths of upstream sequences of the mouse miR−122 promoter region were isolated and cloned in the luciferase reporter vector. Hepa1−6 cells were transfected with constructs and treated with BMP6 or TGFβ1, or left untreated (n = 5). Cell were lysed 48 h after the transfection and Luciferase activity was measured. Data represent average luciferase activity in % of control (n = 5). (B) Mouse primary hepatocytes were transfected with anti−Smad4 siRNA or scramble oligos (CTRL). Levels of pri−miR−122 were measured 18 h after TGFβ1 administration (n = 4). Results are represented as mean ± SD, significant differences were evaluated by using the one-way ANOVA or the t-test as appropriate (ns = Not significant; *, p ≤ 0.05; and **, p ≤ 0.01).

Figure 4

pri−miR−122 expression is differentially regulated in human hepatoma cells in response to TGFβ and BMP6 stimulation. (A) Effect of TGFβ1 and BMP6 stimulation on Hamp, Smad7, and pri−miR−122 expression in Huh−7 human−derived hepatoma cells, 3 and 6 h after stimulation (Normalized to GAPDH, n = 8). (B) Response of the human miR−122 promoter to TGFβ1 and BMP6. Four promoter constructs encompassing different lengths of the human miR−122 promoter were transfected into Huh−7 cells. After transfection, cells were starved for 24 h and then treated with either TGFβ or BMP6. Data represent average luciferase activity in % of serum−free control (n = 5). (C) Serum−starved Huh−7 cells were first incubated with TGFβ receptor type 1 inhibitor SB431542 (5 µM or 10 µM) for 3 h before stimulation with TGFβ1 or vehicle control for another 3 h. The levels of selected genes were measured by qPCR (Normalized to ACTB). Results are represented as mean ± SD, significant differences were evaluated by using the one-way ANOVA or the t-test as appropriate (ns = Not significant; *, p ≤ 0.05; and ***, p ≤ 0.001).

Figure 5

miR-122 expression is reduced in the liver of LCMV WE-infected mice and in the livers of CCl4-treated animals. (A) Wild-type C57-Bl6 mice were infected with 2 × 10⁶ PFUs of LCMV WE and either euthanized before the infection (naïve) or at 4, 6, 8, 19, 12, and 15 days after infection (n = 3/time point). (B) qPCR analysis of miR-122 expression in RNA extracted from the liver and in the sera of animals included in the experiment shown in A; blood parameters for Bilirubin and ALT levels measure in the infected mice. (C) Representative macroscopical alterations and visualization of fibrosis by Sirius red staining mice that received CCl4 or vehicle (oil; CTRL) treatment for 8 weeks. (D) Blood parameters for Aspartate aminotransferase (AST), alanine aminotransferase (ALT), Glutamate dehydrogenase (GLDH), and (E) qPCR analysis of miR-122 levels measured in CCl4 and control mice. Results are represented as mean ± SD, significant differences were evaluated by using the one-way ANOVA or the t-test as appropriate (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; and ****, p ≤ 0.0001).

Figure 6

Analysis of gene expression in the livers of LT and c-myc mice. (A) Unsupervised hierarchical clustering of the genes regulated in the livers of LT α/β and c-Myc overexpressing mice. The color scale illustrates the fold change of mRNAs across the samples (Generated by Altanalyze). (B) Overlap between significantly downregulated genes and miRWalk predicted miR−122 targets. (C) ShinyGO enrichment analysis of potential miR−122 targets. (D) qPCR analysis of the expression of highly enriched genes in the liver of LPC^{ΔTraf2, Casp8} animals at 6 and 52 weeks (n = 4). (E) Representative livers and HE staining of camiN-mirE and matching control (CTRL) untreated mice. (F) qPCR analysis of miR−122, Anln, Hmmr, Tpx2, Ccnb1, and Ccnb2, in the RNAs extracted from the livers of camiN-mirE (n = 7) and CTRL (n = 4) mice. Results are represented as mean ± SD, significant differences were evaluated by using the t-test (ns = Not significant; *, p ≤ 0.05; **, p ≤ 0.01; and ***, p ≤ 0.001).

Figure 7

Analysis of gene expression in the livers of CCl4-injected mice and in activated HSCs. (A) qPCR expression of Anln, Hmmr, Tpx2, Ccnb1, and Ccnb2, in the livers of CCl4-treated (n = 5) and oil-injected control (CTRL) mice (n = 5). (B) Relative expression of GOIs in naïve and activated mouse HSCs and in the RNA extracted from the livers of WT animals (n = 3). Results are represented as mean ± SD, significant differences were evaluated by the using t-test (ns = Not significant; *, p ≤ 0.05).

Figure 8

ANLN, HMMR, TPX2 CCNB1, and CCNB2, are significantly upregulated in human liver cancer. (A) Kaplan–Meier survival curves for ANLN, HMMR, TPX2, CCNB1, and CCNB2, in TCGA LIHC cohorts. (B) qPCR expression for ANLN, HMMR, TPX2, CCNB1, and CCNB2 in RNAs extracted from Huh−7 hepatoma cells transfected with either control scrambled oligo or miR−122 antagomiR (n = 5). Results are represented as mean ± SD, significant differences were evaluated by using the t-test (*, p ≤ 0.05; and ****, p ≤ 0.0001).

Figure 9

Proposed models for regulatory networks up-stream to miR−122. (A) Model 1; positive feedback loop during normal control of iron homeostasis. LSEC release BMP6 in response to high iron storage, BMP6-activated Smad signaling leads to both the activation of HAMP and miR−122 transcription. In this model the role of miR−122 is to fine-tune HAMP transcription via inhibiting HJV and HFE translation. (B) Model 2; negative feedback loop in response to liver injury (e.g., HSCs activation) and inflammation. Activated HSCs or immune cells release TGFβ1. Following TGFβ1 binding to its receptor, canonical Smad signaling is activated leading to (i) downregulation of miR−122 transcription; and (ii) increase of TGF-signaling through the reduction of miR−122-mediated inhibition. In this model, the role of miR−122 downregulation is an activate inflammatory response via the release of miR−122 inhibitions on its target genes.