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, 117 (1), 454-463

Identification of Ppar γ-modulated miRNA Hubs That Target the Fibrotic Tumor Microenvironment

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Identification of Ppar γ-modulated miRNA Hubs That Target the Fibrotic Tumor Microenvironment

Ivana Winkler et al. Proc Natl Acad Sci U S A.

Abstract

Liver fibrosis interferes with normal liver function and facilitates hepatocellular carcinoma (HCC) development, representing a major threat to human health. Here, we present a comprehensive perspective of microRNA (miRNA) function on targeting the fibrotic microenvironment. Starting from a murine HCC model, we identify a miRNA network composed of 8 miRNA hubs and 54 target genes. We show that let-7, miR-30, miR-29c, miR-335, and miR-338 (collectively termed antifibrotic microRNAs [AF-miRNAs]) down-regulate key structural, signaling, and remodeling components of the extracellular matrix. During fibrogenic transition, these miRNAs are transcriptionally regulated by the transcription factor Pparγ and thus we identify a role of Pparγ as regulator of a functionally related class of AF-miRNAs. The miRNA network is active in human HCC, breast, and lung carcinomas, as well as in 2 independent mouse liver fibrosis models. Therefore, we identify a miRNA:mRNA network that contributes to formation of fibrosis in tumorous and nontumorous organs of mice and humans.

Keywords: PPARγ; fibrosis; hepatocellular carcinoma; microRNAs.

Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
A subset of miRNAs predicted to target ECM-linked and fibrosis-associated genes in mHCC. (A) Sirius Red staining of control, nodular, and tumor liver samples isolated from SRF-VP16iHep mice. (Scale bar, 50 μm.) (B) Quantification of Sirius Red signal shown in A. (C) Network of miRNA:mRNA pairs, which encompasses predicted miRNA targeting of genes contributing to the ECM-related pathways highlighted in dark gray in SI Appendix, Fig. S1C. Genes are grouped in structural (upper right), signaling (upper left), and remodeling (middle right) components of the ECM, as well as genes related to integrin signaling (middle left) and Rho signaling (bottom). miRNAs mmu-miR-30e-5p, mmu-miR-30d-5p, mmu-miR-338-3p, mmu-miR-335-3p, mmu-miR-29c-3p, mmu-let-7a-5p, mmu-let-7c-5p, and mmu-let-7g-5p, which are predicted to target all ECM-related proteins of the network, were further experimentally characterized in the remainder of this study. Additionally, we chose to further characterize gene expression and predicted miRNA-mediated targeting of a subset of genes, which represent key structural, remodeling, and signaling components of the ECM. We highlighted these genes using red circles. Rims of gene nodes: red, ECM-related genes characterized further in this study and predicted to be targeted by the here-characterized miRNAs; blue, genes not characterized in this study but predicted to be targeted by the here-characterized miRNAs; and gray, genes not characterized in this study and predicted to be targeted by the here-noncharacterized miRNAs. Data are shown as median, first, and third quartiles (“box”) and 95% confidence interval of median (“whiskers”). ***p value 0.001.
Fig. 2.
Fig. 2.
AF-miRNAs are down-regulated and fibrosis-associated genes are up-regulated in murine HCC. (A) Volcano plot of genes identified in RNA-seq. Fibrosis-related genes characterized in this study are shown in red and significantly dysregulated genes (threshold 2-fold) in violet. (B) Volcano plot of miRNAs identified in sRNA-seq. AF-miRNAs characterized in this study are depicted in red (also listed in C), and significantly dysregulated miRNAs (threshold 1.5-fold) are depicted in violet. (C) sRNA-seq–derived, normalized read counts (log2-transformed) of AF-miRNAs in control (blue bars) and tumor (red bars) samples of SRF-VP16iHep mice. Data are shown as mean and SEM. **padj value 0.01, ***padj value 0.001.
Fig. 3.
Fig. 3.
AF-miRNAs are down-regulated and fibrosis-associated genes are up-regulated in the pHSC fibrosis model. (A) Relative expression of mature miRNAs in inactive (freshly isolated) and activated (prolonged in vitro culturing) pHSCs. (B–D) Relative expression of fibrosis-associated structural (B), remodeling (C), and signaling (D) genes of the ECM in inactive and activated pHSCs. All samples are normalized to a randomly chosen control sample. Data are shown as mean and SEM. *p value 0.05, **p value 0.01, ***p value 0.001.
Fig. 4.
Fig. 4.
AF-miRNAs target structural, signaling, and remodeling components of the ECM. (A and B) Activities of wild-type and mutant (mutated miRNA site) 3’-UTR–luciferase constructs derived from (A) Pdgfa in NIH/3T3 cells transfected with miR-29c and scrambled miRNA mimic and (B) Tgfbr1 in NIH/3T3 cells transfected with let-7g and scrambled miRNA mimic. Let-7g– and miR-29c–transfected samples are colored in the plots according to the luciferase construct schematic. Samples transfected with scrambled miRNA mimic are shown in white (Neg. ctrl). (C and D) Relative expression of putative let-7 target genes associated with fibrosis in stable Lin28a-overexpressing NIH/3T3 cells (C) and putative miR-29c target genes associated with fibrosis in NIH/3T3 cells transfected with miR-29c mimics (D). (A and B) Data are shown as median, first, and third quartiles (“box”) and 95% confidence interval of median (“whiskers”). (C and D) Data are shown as mean and SEM. *p value 0.05, **p value 0.01, ***p value 0.001.
Fig. 5.
Fig. 5.
Transcription factor Pparγ regulates expression of AF-miRNAs. (A) Relative expression of Pparg in the pHSC in vitro culture model. (B) Relative expression of AF-pri-miRNAs in the pHSC in vitro culture model. (C) Schematic representation of predicted Pparγ binding to Mus musculus (Left) and Homo sapiens (Right) miRNA-encoding gene promoters. miRNA-encoding gene promoters are shown in red, whereas mature miRNAs are indicated in blue. miRNAs located in exons or introns of protein-coding genes share the promoter of the respective protein-coding genes. In the cases of miRNAs located in intergenic regions of the genome, the nearest neighboring genes are shown. Pparγ is depicted on the individual miRNA-encoding gene promoter if it is predicted to bind to the respective promoter. (D) Relative expression of AF-miRNAs in a stable Pparγ-overexpressing GRX hepatic stellate cell line. (E) ChIP analysis of Pparγ binding to the promoters of AF-miRNAs in a Pparγ-overexpressing GRX (red bars) and control GRX (blue bars) hepatic stellate cell line. (A–E) Data are shown as mean and SEM. *p value 0.05, **p value 0.01, ***p value 0.001.
Fig. 6.
Fig. 6.
Schematic displays summarizing the regulation of AF-miRNAs and their target genes. (A) Circos plot summarizing the regulation of AF-miRNAs and their target genes. PPARG (black bar) is shown to up-regulate the here-validated miRNAs let-7g-5p, let-7c-5p, let-7a-5p, miR-338-3p, and miR-29c-3p. (B) Graphical model summarizing the transcriptional regulation of AF-miRNA–encoding genes by PPARG in HSCs. Reduced expression of PPARG upon activation of HSCs causes down-regulation of AF-miRNA expression. This permits elevated levels of profibrotic mRNAs, leading to the formation of a fibrotic ECM.

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