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. 2023 Jun 15;18(1):32.
doi: 10.1186/s13062-023-00388-4.

H19X-encoded microRNAs induced by IL-4 in adipocyte precursors regulate proliferation to facilitate differentiation

Choijamts Munkhzul #  1   2 , Ji-Min Lee #  1 , Boseon Kim  1   2 , Thi Thanh My Nguyen  1 , Rehna Paula Ginting  1   2 , Dahee Jeong  1   2 , Young-Kook Kim  3 , Min-Woo Lee  4   5 , Mihye Lee  6   7
Affiliations

H19X-encoded microRNAs induced by IL-4 in adipocyte precursors regulate proliferation to facilitate differentiation

Choijamts Munkhzul et al. Biol Direct. .

Abstract

Adipose tissue, an organ critical for systemic energy homeostasis, is influenced by type 2 immunity in its development and function. The type 2 cytokine interleukin (IL)-4 induces the proliferation of bipotential adipocyte precursors (APs) in white fat tissue and primes these cells for differentiation into beige adipocytes, which are specialized for thermogenesis. However, the underlying mechanisms have not yet been comprehensively examined. Here, we identified six microRNA (miRNA) genes upregulated upon IL-4 stimulation in APs, miR-322, miR-503, miR-351, miR-542, miR-450a, and miR-450b; these are encoded in the H19X locus of the genome. Their expression is positively regulated by the transcription factor Klf4, whose expression also increases upon IL-4 stimulation. These miRNAs shared a large set of target genes, of which 381 genes were downregulated in mRNA expression upon IL-4 stimulation and enriched in Wnt signaling pathways. Two genes with downregulated expression, Ccnd1 and Fzd6, were repressed by H19X-encoded miRNAs. Additionally, the Wnt signaling activator LiCl downregulated the expression of this group of miRNAs in APs, indicating that Wnt signaling-related genes and these miRNAs form a double-negative feedback regulatory loop. This miRNA/Wnt feedback regulation modulated the elevated proliferation of APs induced by IL-4 stimulation and contributed to priming them for beige adipocyte differentiation. Moreover, the aberrant expression of these miRNAs attenuates the differentiation of APs into beige adipocytes. Collectively, our results suggest that H19X-encoded miRNAs facilitate the transition of APs from proliferation to differentiation in the IL-4-mediated regulation.

Keywords: Adipocyte precursor; Beige adipocyte; Interleukin-4; Proliferation; Wnt signaling; microRNA.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
IL-4 promotes cellular expansion and beige commitment of adipocyte precursors (APs). A Schematic of IL-4 induced priming of APs. APs isolated from scWAT were cultured in vitro. IL-4 was added into the growth medium one day after cell seeding and cells were harvested for analysis on the indicated days B The cell proliferation rate of APs cultured in IL-4 containing media (n = 3). C The beige adipogenic markers (Pgc1-α, Pparg, Ear2, and Tmem26) mRNA levels were examined by qPCR in APs (n = 3). Longer incubation of APs with IL-4 induces higher expression levels of markers. D mRNA expression levels of the white adipogenic markers (Zfp423 and Asc1) and the brown adipogenic markers (Ebf2 and Pdk4) were analyzed by qPCR in APs (n = 3). E The mRNA levels of Ucp1, Ap2, and Pgc1-α genes were measured by quantitative PCR (qPCR) in differentiated adipocytes (n = 3). When APs were pre-incubated with IL-4 for four days before the induction of differentiation, the expressions of beige and adipogenic markers in differentiated adipocytes increased to a significantly greater extent than those in control cells. F The protein levels of UCP1 were examined by western blotting in differentiated adipocytes. Pre-incubation of APs with IL-4 leads to higher levels of UCP1 expression in differentiated adipocytes. Biological triplicates for each condition were examined. The quantified signal intensities in the western blotting are presented as a bar graph (n = 3). MDI refers to the induction of differentiation by an adipogenic cocktail. Data are presented as mean ± SEM. *** p < 0.005, **p < 0.01, *p < 0.05
Fig. 2
Fig. 2
Differentially expressed genes in IL-4–treated APs. A Transcriptomic analysis of control and IL-4–treated APs (four-day sample). The mRNA-seq results are presented as a volcano plot (Total 18,345 genes). The genes of log2 fold change > 1 (red) or < -1 (blue) with p.adj < 0.05 are highlighted. Pgc1-α, Ear2, and Tmem26 are labeled as the beige adipogenic markers. B KEGG pathway analysis of upregulated (log2 fold change > 1, p.adj < 0.05, upper panel, red) and downregulated (log2 fold change < -1, p.adj < 0.05, lower panel, blue) genes. The number of genes that belongs to each pathway is indicated by circle size and significance (p-value) is presented by color
Fig. 3
Fig. 3
Differentially expressed miRNAs in IL-4–treated Aps. A The analysis of differentially expressed miRNAs using the small RNA sequencing data of control and IL-4–treated APs (two-day sample). Differentially expressed miRNAs in IL-4–treated APs are marked on the volcano plot as follows, fold change > 1.5 (red) or < -1.5 (blue), p < 0.05. The vertical dashed lines indicate the fold change of 1.5 (black) and 2.0 (blue). B The genomic structure of the H19X-encoded locus. miRNA genes are indicated by pink bars. The genomic region used for transcription factor enrichment analysis is marked with a red line. C The primary transcripts of miR-322, miR-351, and miR-542 were examined by qPCR for the expression levels in APs incubated with IL-4 for 0, 2, and 4 days (n = 3). The let-7b primary transcript was used as a control. D The abundance of mature miRNAs, miR-322-5p, miR-351-5p, and miR-542-3p were examined by qPCR in APs incubated with IL-4 for 0, 2, and 4 days (n = 3). E Transcription factor enrichment analysis for the promoter region of H19X-encoded miRNAs. Top-ranked transcription factors are presented in the table. F Klf4 mRNA levels were measured by qPCR in APs incubated with IL-4 for four days (n = 3). G Klf4 mRNA and primary transcripts of miR-322, miR-503, miR-351, miR-542, and miR-450b were analyzed by qPCR for the expression levels after APs were treated with Klf4 siRNA (n = 3). Data are presented as mean ± SEM. ***p < 0.005, **p < 0.01, *p < 0.05
Fig. 4
Fig. 4
H19X-encoded miRNAs share target genes enriched in Wnt signaling pathways. A Analysis of shared target genes of H19X-encoded miRNAs using TargetScan. The numbers of overlapping target genes are presented in the table. B KEGG pathway analysis of genes that are downregulated by IL-4 and targeted by two or more miRNAs. C A functional module analysis for genes that are downregulated by IL-4 and targeted by two or more miRNAs. Among several cluster modules, a regulatory network of Wnt signaling–related genes is shown
Fig. 5
Fig. 5
Ccnd1 and Fzd6 are repressed by H19X-encoded miRNAs. A The gene structures of Ccnd1 and Fzd6 are shown with binding sites for H19X-encoded miRNAs in the 3′ UTR. Ccnd1 mRNA expression was significantly repressed by mimic treatment of miR-322-5p or miR-503-5p, and not miR-450b-5p. Fzd6 mRNA levels were reduced by approximately 40% upon mimic treatment of miR-450b or miR-542-3p (n = 3). B The CCND1 protein levels decreased with miR-322-5p and miR-503-5p mimic treatment. The quantified signal intensities in the western blotting are presented as a bar graph (n = 2). C The positions and sequences of miRNA binding sites in the 3′ UTR of Ccnd1 and Fzd6 are presented (upper panel). The seed region of miRNA is highlighted in red and is base paired with the matched sequence of each target mRNA. The binding activity of miRNAs to the predicted sites in 3′ UTR was measured using a dual-luciferase reporter assay (n = 3). The luciferase activity was reduced by transfection of the corresponding miRNA when the reporter contained the 3′ UTR fragment of Ccnd1 or Fzd6. Data are presented as mean ± SEM. ***p < 0.005, **p < 0.01, *p < 0.05
Fig. 6
Fig. 6
The negative feedback loop of miRNA/Wnt affects the proliferation and beige commitment in IL4-treated Aps. A LiCl treatment reduced the abundance of mature miRNAs to less than half that observed in control APs. miR-322-5p, miR-351-5p, and miR-542-3p were examined by qPCR as representatives of H19X-encoded miRNAs (n = 3). B The mRNA expression levels of Wnt signaling–related genes were measured by qPCR (n = 3). LiCl treatment increased the mRNA levels of Ccnd1 and Fzd6. The transfection of miRNA mimics together with LiCl treatment alleviated the effect of the Wnt activator, LiCl. C, D The proliferation of APs was analyzed by MTT assay (n = 3). C LiCl treatment enhanced the cell proliferation, and transfection of miR-322-5p/miR-503-5p and miR-450b-5p/miR-542-3p mimics lowered the LiCl-induced proliferation. D IL-4-induced cell proliferation was suppressed by the transfection of miR-322-5p/miR-503-5p and miR-450b-5p/miR-542-3p mimics. E, F The proliferation of APs was analyzed by flow cytometry (E) and immunocytochemistry (F). The percentage of Ki67-positive cells increased after six days of incubation in IL-4 containing medium but decreased upon additional transfection of miR-322-5p/miR-503-5p mimics. Scale: 100 µm G The mRNA expression levels of beige adipogenic markers were measured by qPCR (n = 3). IL-4-induced upregulation of Pparg and Pgc1-α expressions was compromised by LiCl treatment. Data are presented as mean ± SEM. ***p < 0.005, **p < 0.01, *p < 0.05.
Fig. 7
Fig. 7
Dysregulation of H19X-encoded miRNAs in APs affects their differentiation into beige adipocytes. A, B Cells were cultured in a differentiation medium for beige fat lineage and harvested for analysis at several time points (n = 3). A Mature miRNAs, miR-322-5p and miR-542-3p, were downregulated in differentiated beige adipocytes. B The primary transcripts of miR-503 and miR-542 were downregulated immediately upon the differentiation of adipocytes. The let-7b primary transcript was used as a control. C-E APs were transiently transfected with miRNA mimic mix (miR-322-5p/miR-503-5p/miR-450b-5p/miR-542-3p) for 24 h and stimulated with an adipogenic cocktail for the differentiation. Cells were harvested for analysis at several time points after the induction of differentiation (day 0, 2, 4, 6, and 8) (C) The representative images of Oil Red O staining in differentiated beige adipocytes at day 8 (left panel). The quantified intensities of Oil Red O staining are shown (right panel). The pretreatment of miRNA mimic mix reduced the number of differentiated adipocytes. Scale bar, 100 µm (D) The mRNA levels of Ucp1 and Ap2 were analyzed during beige adipocyte differentiation (n = 2). The pretreatment of miRNA mimic mix (miR-322-5p/miR-503-5p/miR-450b-5p/miR-542-3p) attenuated the expression of Ucp1 and Ap2. E The protein levels of CCND1 and PLIN1 were examined by western blotting during beige adipocyte differentiation. miRNA mimic mix downregulated CCND1 protein levels and delayed the expression of PLIN1 protein. Data are presented as mean ± SEM. ***p < 0.005, **p < 0.01, *p < 0.05
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