Global Transcriptome Analysis During Adipogenic Differentiation and Involvement of Transthyretin Gene in Adipogenesis in Cattle

Abstract

Adipose tissue plays central role in determining the gustatory quality of beef, but traditional Chinese beef cattle have low levels of fat content. We applied RNA-seq to study the molecular mechanisms underlying adipocyte differentiation in Qinchuan cattle. A total of 18,283 genes were found to be expressed in preadipocytes and mature adipocytes, respectively. 470 of which were significantly differentially expressed genes (DEGs) [false discovery rate (FDR) values < 0.05 and fold change ≥ 2]. In addition, 4534 alternative splicing (AS) events and 5153 AS events were detected in preadipocytes and adipocytes, respectively. We constructed a protein interaction network, which suggested that collagen plays an important role during bovine adipogenic differentiation. We characterized the function of the most down-regulated DEG (P < 0.001) among genes we have detected by qPCR, namely, the transthyretin (TTR) gene. Overexpression of TTR appears to promote the expression of the peroxisome proliferator activated receptor γ (PPARγ) (P < 0.05) and fatty acid binding Protein 4 (FABP4) (P < 0.05). Hence, TTR appears to be involved in the regulation of bovine adipogenic differentiation. Our study represents the comprehensive approach to explore bovine adipocyte differentiation using transcriptomic data and reports an involvement of TTR during bovine adipogenic differentiation. Our results provide novel insights into the molecular mechanisms underlying bovine adipogenic differentiation.

Keywords: adipocyte differentiation; alternative splicing; cattle; differentially expressed genes; transcriptome analysis; transthyretin.

FIGURE 1

Venn diagram of global genes (A), transcript isoforms (B), and potential novel genes (C).

FIGURE 2

Comparison of AS patterns in preadipocytes and adipocytes. (A) Number of AS events in preadiopocytes and adipocytes. (B) Overlap of five types of AS genes in preadipocytes and adipocytes.

FIGURE 3

DEGs detection. (A) Volcano plot of bovine adipogenesis related DEGs expression profiling. Red plots represent genes up-regulated significantly and green plots represent genes down-regulated significantly. (B) Heat map of DEGs. Hierarchical cluster and cluster tree analysis was used to create the dendrogram. The heat map compares fold change patterns of the most highly DEGs in preadipocytes and adipocytes. Each line represents one gene. Left two panels: adipocyte; Right two panels: preadipocytes. Red indicates high expression; blue indicates low expression.

FIGURE 4

GO annotation (A) and KEGG analysis (B) of DEGs.

FIGURE 5

Protein interaction networks encoded by DEGs enriched in the top 3 KEGG pathways. Red and green points represent genes up-regulated and down-regulated, respectively.

FIGURE 6

Relative expression level of 16 DEGs selected with high or low expression levels in RNA-seq data and qPCR detection. The expression level in preadipocytes is defined as “1.” In qPCR, all samples were measured in triplicate, and 4 preadipocyte samples and 4 adipocyte samples were used. The data represents mean ± SEM (Standard Error).

FIGURE 7

TTR is involved in bovine adipogenic differentiation. (A) The expression of TTR during bovine adipocyte differentiation. (B) On 9th day, overexpression of TTR increased mRNA expression of PPARγ and FABP4. (C) On 11th day, overexpression of TTR increased mRNA expression of PPARγ and FABP4. (D) TNFα treatment down regulate the expression of TTR. (E) TTR overexpression also promote protein expression of PPARγ and FABP4. Each treatment is in triplicate. OV-TTR: overexpression vector of bovine TTR gene. OV-NC: empty pcDNA3.1 (+) plasmid without any insertion fragment. ^∗P < 0.05. ^∗∗P < 0.01. ^∗∗∗P < 0.001.