LncRNA IMFNCR Promotes Intramuscular Adipocyte Differentiation by Sponging miR-128-3p and miR-27b-3p

Abstract

Poultry meat quality is affected by many factors, among which intramuscular fat (IMF) is predominant. IMF content affects the tenderness, juiciness, and flavor of chicken. An increasing number of studies are focusing on the functions of lncRNAs in adipocyte differentiation. However, little is known about lncRNAs associated with intramuscular adipocyte differentiation. In the present study, we focused on an up-regulated lncRNA during intramuscular adipogenetic differentiation, which we named intramuscular fat-associated long non-coding RNA (IMFNCR). IMFNCR promotes intramuscular adipocyte differentiation. In-depth analyses showed that IMFNCR acts as a molecular sponge for miR-128-3p and miR-27b-3p and that PPARG is a direct target of miR-128-3p and miR-27b-3p in chicken. High-fat and high-protein diet inhibited chicken IMFNCR level in vivo. Moreover, IMFNCR level was positively correlated with PPARG mRNA level in chicken breast muscle tissues, a vital corollary to ceRNA function. Altogether, our research showed that IMFNCR acts as a ceRNA to sequester miR-128-3p and miR-27b-3p, leading to heightened PPARG expression, and thus promotes intramuscular adipocyte differentiation. Taken together, our findings may contribute to a more thorough understanding of chicken IMF deposition and the improvement of poultry meat quality.

Keywords: ceRNA; chicken; intramuscular adipocyte differentiation; long non-coding RNA; miRNA.

FIGURE 1

Induction of differentiation in intramuscular and abdominal preadipocyte. The basic medium consisted of DMEM/F-12 and 10% FBS. The induction differentiation medium consisted of basic medium, insulin, dexamethasone, 3-isobutyl-1-methylxanthine, and oleate. The maintenance medium consisted of basic medium, insulin, and oleate. The induction differentiation medium was replaced with the maintenance medium at 48 h, whereas the maintenance medium was replaced with basic medium at 96 h.

FIGURE 2

Characteristics of chicken IMFNCR in different tissues and cells. (A) Tissue expression profile of IMFNCR by qRT-PCR. (B) Tissue expression profile of IMFNCR by semi-quantitative PCR. (C) The expression dynamics of IMFNCR in breast muscle tissues at different physiological periods. (D) The expression level of IMFNCR in abdominal and intramuscular preadipocyte. Data represent means ± SEM (n = 3). ^∗p ≤ 0.05; ^∗∗p ≤ 0.01.

FIGURE 3

Subcellular localization and encoding ability prediction of IMFNCR. (A) Intramuscular preadipocyte were stained with FITC-labeled IMFNCR probes and visualized by fluorescence. (B) The RNA sequences of IMFNCR, GHR-AS, and GAPDH were put into the Coding Potential Calculator (CPC) program, and both IMFNCR and GHR-AS were predicted to be non-coding RNAs, while GAPDH was identified to code for protein. (C) IMFNCR is mainly localized in the cytoplasm of intramuscular preadipocyte. RNA isolated from cytoplasm (Cyto) and nuclear (Nuc) fractions of preadipocyte and adipocyte was used to analyze the expression level of IMFNCR by semi-quantitative PCR. GAPDH mRNA was used as control.

FIGURE 4

Induction of chicken intramuscular preadipocyte differentiation. Oil Red O staining (A) and qRT-PCR (B) analysis of IMFNCR and adipogenic markers, PPARG, and CEBPA confirm the identity of chicken intramuscular preadipocyte. Gene expression is plotted as fold-change relative to day 0 (mean ± SEM, n = 3, ^∗p ≤ 0.05, ^∗∗p ≤ 0.01).

FIGURE 5

lncRNA IMFNCR was a potential target gene of miR-128-3p and miR-27b-3p. (A) RNA hybrid predicted miR-128-3p and miR-27b-3p binding sites in IMFNCR. Mfe, minimal free energy (kcal/mol). Nucleotides of the miR-128-3p and miR-27b-3p seed region (positions 2–8) are marked in red. Mutated nucleotides are in blue. (B) Expression pattern of miR-128-3p and miR-27b-3p during chicken intramuscular preadipocyte differentiation. (C) KEGG pathway analysis of intersection target genes of miR-128-3p and miR-27b-3p. (mean ± SEM, n = 6, ^∗p ≤ 0.05, ^∗∗p ≤ 0.01).

FIGURE 6

lncRNA IMFNCR functions as a miRNA sponge. (A) IMFNCR knockdown decreased PPARG and FABP4 expression. (B) IMFNCR knockdown inhibited intramuscular adipogenesis, as indicated by Oil Red O staining and quantified by microplate reader at 510 nm. (C) IMFNCR knockdown inhibited the protein expression of PPARG. (D) IMFNCR knockdown increased the expression of miR-128-3p and miR-27b-3p. (E) miR-128-3p and miR-27b-3p inhibited the expression of IMFNCR. (F) miR-128-3p and miRNA mixture overexpression inhibited Rluc expression of the Rluc activity of psiCH2-IMFNCR-WT, while psiCH2-IMFNCR-WT-Mut no longer responded to miRNA mixture (miR-128-3p and miR-27b-3p). MiR-128-3p and miR-27b-3p mimics were transfected into DF1 cells, together with psiCH2-IMFNCR-WT, or psiCH2-IMFNCR-WT-Mut. The data represent means ± SEM (n = 3). ^∗p ≤ 0.05; ^∗∗p ≤ 0.01.

FIGURE 7

IMFNCR promotes intramuscular adipocyte differentiation by sponging miR-128-3p and miR-27b-3p. (A) The putative miR-128-3p and miR-27b-3p-binding sites at PPARG 3′UTR (green) are evolutionarily conserved across species. (B) miR-128-3p and miR-27b-3p suppresses PPARG translation. miR-128-3p and miR-27b-3p were transfected into DF1 cells, along with PPARG-UTR-WT but not PPARG-UTR-Mut. The luciferase activity was analyzed 48 h later. (C) miR-128-3p suppresses the expression of PPARG and IRS1. Intramuscular preadipocyte was infected with miR-128-3p mimics or mimics Control at 37°C, followed by the addition of fresh growth medium. RNA and protein were extracted 48 h later, and levels were accessed by qRT-PCR and (D) western blot. (E) IMFNCR siRNA and inhibitor (miR-128-3p inhibitor, mixture inhibitor) or mimics (miR-128-3p mimics, mixture mimics) were transfected into chicken intramuscular preadipocyte. qRT-PCR assays were performed to determine the expression levels of PPARG and IMFNCR. (F) Negative control inhibitor (NC inhibitor) or miR-128-3p or mixture (miR-128-3p and miR-27b-3p) inhibitor was transfected into chicken intramuscular preadipocyte. Forty-eight hours later, qRT-PCR assays were performed to determine the expression levels of miR-128-3p, miR-27b-3p, PPARG, and FABP4. (G) IMFNCR siRNA and miR-27b-3p were transfected into miRNA mimics NC or miR-128-3p mimics. At 48 h intracellular triglyceride content was measured by Oil Red O staining and (H) quantified by microplate reader at 510 nm. The data represent means ± SEM (n = 3). ^∗p ≤ 0.05; ^∗∗p ≤ 0.01.

FIGURE 8

Effects of dietary nutrient level on IMFNCR expression. (A) HFD inhibits IMFNCR expression in breast muscle tissues. The breast muscle tissues of chicken were collected after 3-weeks feeding (C, control diet group; L, low-fat diet group; H, high-fat diet group). (B) HPD inhibits IMFNCR expression in breast muscle tissues. The breast muscle tissues of chicken were collected after 3-weeks feeding (C, control diet group; L, low-protein diet group; H, high-protein diet group). Data are presented as means ± SEM, n = 6, ^∗p ≤ 0.05, ^∗∗p ≤ 0.01. ^#No significant difference.

FIGURE 9

Proposed model of IMFNCR regulation on intramuscular adipocyte differentiation. High-protein and High-fat diet inhibit the expression of IMFNCR; IMFNCR promotes intramuscular adipogenesis differentiation by functioning as a ceRNA, which sequesters miR-128-3p and miR-27b-3p, thereby protecting PPARG transcripts from miRNA-mediated suppression in chicken.