Integrative analysis of miRNA and mRNA profiles reveals that gga-miR-106-5p inhibits adipogenesis by targeting the KLF15 gene in chickens

Abstract

Background: Excessive abdominal fat deposition in commercial broilers presents an obstacle to profitable meat quality, feed utilization, and reproduction. Abdominal fat deposition depends on the proliferation of preadipocytes and their maturation into adipocytes, which involves a cascade of regulatory molecules. Accumulating evidence has shown that microRNAs (miRNAs) serve as post-transcriptional regulators of adipogenic differentiation in mammals. However, the miRNA-mediated molecular mechanisms underlying abdominal fat deposition in chickens are still poorly understood. This study aimed to investigate the biological functions and regulatory mechanism of miRNAs in chicken abdominal adipogenesis.

Results: We established a chicken model of abdominal adipocyte differentiation and analyzed miRNA and mRNA expression in abdominal adipocytes at different stages of differentiation (0, 12, 48, 72, and 120 h). A total of 217 differentially expressed miRNAs (DE-miRNAs) and 3520 differentially expressed genes were identified. Target prediction of DE-miRNAs and functional enrichment analysis revealed that the differentially expressed targets were significantly enriched in lipid metabolism-related signaling pathways, including the PPAR signaling and MAPK signaling pathways. A candidate miRNA, gga-miR-106-5p, exhibited decreased expression during the proliferation and differentiation of abdominal preadipocytes and was downregulated in the abdominal adipose tissues of fat chickens compared to that of lean chickens. gga-miR-106-5p was found to inhibit the proliferation and adipogenic differentiation of chicken abdominal preadipocytes. A dual-luciferase reporter assay suggested that the KLF15 gene, which encodes a transcriptional factor, is a direct target of gga-miR-106-5p. gga-miR-106-5p suppressed the post-transcriptional activity of KLF15, which is an activator of abdominal preadipocyte proliferation and differentiation, as determined with gain- and loss-of-function experiments.

Conclusions: gga-miR-106-5p functions as an inhibitor of abdominal adipogenesis by targeting the KLF15 gene in chickens. These findings not only improve our understanding of the specific functions of miRNAs in avian adipogenesis but also provide potential targets for the genetic improvement of excessive abdominal fat deposition in poultry.

Keywords: Abdominal fat; Adipogenesis; Chickens; KLF15; MiRNA; gga-miR-106-5p.

Fig. 1

Characteristics and expression profiles of miRNAs in chicken abdominal adipocytes at various stages of differentiation. A Length distribution of known miRNA sequences in chicken abdominal adipocytes; (B) Length distribution of novel miRNA sequences in chicken abdominal adipocytes; (C) PCA analysis of the 15 samples, based on the normalized expression levels of all the expressed miRNAs; (D) Histogram analysis of the number of differentially expressed (DE)-miRNAs in four comparisons (A0 vs. A12, A12 vs. A48, A48 vs. A72, and A72 vs. A120); (E) Clustering of short time series expression of all DE-miRNAs; (F) Significant profiles (profiles 8, 1, and 40) identified via STEM analysis

Fig. 2

Expression profiles of mRNAs in chicken abdominal adipocytes at various stages of differentiation. A Histogram of the number of differentially expressed genes (DEGs) in four comparisons (A0 vs. A12, A12 vs. A48, A48 vs. A72, A72 vs. A120); (B) Top 20 pathways identified via Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of all DEGs during adipogenic differentiation; (C) Heatmap of DEGs associated with lipid metabolism during adipogenesis; (D) Heatmap of differentially expressed transcriptional factors during adipogenesis

Fig. 3

Integrative analysis of differentially expressed (DE)-miRNA and differentially expressed genes (DEGs). A Top 20 signaling pathways identified via Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of differentially expressed targets of DE-miRNAs during adipogenic differentiation; (B) Regulatory networks involving DE-miRNAs and differentially expressed transcriptional factors; (C) Regulatory networks involving DE-miRNA, KLF15, DEGs related to lipid metabolism, and signaling pathways

Fig. 4

Expression patterns of adipocyte gga-miR-106-5p in vivo and in vitro. A Difference in volume between abdominal adipocytes in the abdominal fat tissues of chickens with high abdominal fat (HAbF) and low abdominal fat (LAbF), as determined via hematoxylin and eosin staining; (B) Difference in abdominal fat weight (AbFW) and abdominal fat percentage (AbFP) between HAbF and LAbF chickens; (C) Expression levels of gga-miR-106-5p in the abdominal fat tissues of HAbF and LAbF chickens; (D) Expression pattern of gga-miR-106-5p during abdominal preadipocyte proliferation in chickens; (E) Expression pattern of gga-miR-106-5p during adipogenic differentiation of chicken abdominal preadipocytes

Fig. 5

Effects of gga-miR-106-5p on abdominal preadipocyte proliferation in chickens. A CCK8 assay of chicken abdominal preadipocytes transfected with miR-106-5p agomir and the miR-106-5p agomir negative control (NC) at 12, 24, 48, 72, and 96 h post-transfection; (B) Detection of gga-miR-106-5p overexpression 48 h after transfecting miR-106-5p agomir in chicken abdominal preadipocytes; (C) Detection of the abundance of KLF15 mRNA in chicken abdominal preadipocytes treated with miR-106-5p agomir and miR-106-5p agomir NC; (D) Representative images from the EdU assay of chicken abdominal preadipocytes transfected with miR-106-5p agomir and miR-106-5p agomir NC for 48 h

Fig. 6

Effects of gga-miR-106-5p on the adipogenic differentiation of chicken abdominal preadipocytes. A Detection of miR-106-5p overexpression after transfecting miR-106-5p agomir into the differentiated abdominal adipocytes of chicken; (B) Detection of the abundance of KLF15 mRNA in the differentiated abdominal adipocytes of chicken treated with miR-106-5p agomir and miR-106-5p agomir negative control (NC); (C) Spectrophotometric analysis of lipid droplet content via Oil red O staining of the differentiated chicken abdominal adipocytes transfected with miR-106-5p agomir and miR-106-5p agomir NC; (D) Oil red O staining of the differentiated chicken abdominal adipocytes transfected with miR-106-5p agomir and miR-106-5p agomir NC; (E) Representative images of Nile red fluorescent staining of the differentiated chicken abdominal adipocytes transfected with miR-106-5p agomir and miR-106-5p agomir NC

Fig. 7

Validation of the KLF15 gene as a direct target of gga-miR-106-5p. A Potential gga-miR-106-5p binding sites in the 3′ UTR of the KLF15 gene; (B) Construction and validation of dual-luciferase reporter vectors for the validation of gga-miR-106-5p targeting the KLF15 gene. The sequences with or without the binding sites of gga-miR-106-5p and the 3′ UTR of the KLF15 gene were cloned into the psiCHECK-2 vector. WT: wild-type vector; mut: mutant vector; hRluc: Renilla luciferase; hluc+: firefly luciferase; (C, D) Validation of the interaction between gga-miR-106-5p and the 3′ UTR of the KLF15 gene via a dual-luciferase reporter assay in 293 T cells and DF1 cells. Data are presented as mean ± SD (n = 3)

Fig. 8

Expression patterns of the KLF15 gene in adipocytes in vivo and in vitro. A mRNA expression levels of KLF15 in the abdominal fat tissues of chickens with high (HAbF) and low (LAbF) abdominal fat; (B) mRNA expression pattern of KLF15 during the proliferation of chicken abdominal preadipocytes; (C) mRNA expression pattern of KLF15 during the adipogenic differentiation of chicken abdominal preadipocytes

Fig. 9

Effects of KLF15 on the proliferation of chicken abdominal preadipocytes. A CCK8 assay of chicken abdominal preadipocytes transfected with pcDNA3.1-KLF15-EGFP and pcDNA3.1-EGFP at 12, 24, 48, 72, and 96 h post-transfection; (B) CCK8 assay of chicken abdominal preadipocytes transfected with siKLF15 and siNC at 12, 24, 48, 72, and 96 h post-transfection; (C) Representative images from the EdU assay of chicken abdominal preadipocytes transfected with pcDNA3.1-EGFP and pcDNA3.1-KLF15-EGFP at 48 h post-transfection; (D) Representative images from the EdU assay of chicken abdominal preadipocytes transfected with siNC and siKLF15 at 48 h post-transfection; (E) mRNA expression levels of KLF15, PCNA, MKI67, and CDK1 after KLF15 overexpression; (F) mRNA expression levels of KLF15, PCNA, MKI67, and CDK1 after KLF15 knockdown

Fig. 10

Effects of KLF15 on the adipogenic differentiation of chicken abdominal preadipocytes. A Oil red O staining of the differentiated chicken abdominal adipocytes transfected with pcDNA3.1-EGFP and pcDNA3.1-KLF15-EGFP; (B) Representative images of Nile red fluorescent staining of the differentiated chicken abdominal adipocytes transfected with pcDNA3.1-EGFP and pcDNA3.1-KLF15-EGFP; (C) Spectrophotometric analysis of lipid droplet content via Oil red O staining of the differentiated chicken abdominal adipocytes transfected with pcDNA3.1-EGFP and pcDNA3.1-KLF15-EGFP; (D) mRNA expression levels of KLF15, PPARγ, CEBPα, SLC27A1, ACSL1, ACACA, FASN, and AGPAT2 in the differentiated chicken abdominal adipocytes transfected with pcDNA3.1-EGFP and pcDNA3.1-KLF15-EGFP; (E) Oil red O staining of the differentiated chicken abdominal adipocytes transfected with siNC and siKLF15; (F) Representative images of Nile red fluorescent staining of the differentiated chicken abdominal adipocytes transfected with siNC and siKLF15; (G) Spectrophotometric analysis of lipid droplet content via Oil red O staining of the differentiated chicken abdominal adipocytes transfected with siNC and siKLF15; (H) mRNA expression levels of KLF15, PPARγ, CEBPα, SLC27A1, ACSL1, ACACA, FASN, and AGPAT2 in the differentiated chicken abdominal adipocytes transfected with siNC and siKLF15

Fig. 11

Proposed model of gga-miR-106-5p regulation of abdominal adipogenesis in chicken