Effect of gene CRTC2 on the differentiation of subcutaneous precursor adipocytes in goats

Abstract

Objective: The aim of this study was to obtain goat CRTC2 gene sequence and elucidate its biological properties, and further study the impact of overexpression and interference of CRTC2 on the cell differentiation of goat subcutaneous precursor adipocytes.

Methods: The sequence of goat CRTC2 was cloned by reverse transcription (RT)-polymerase chain reaction (PCR) and its molecular characterization was analyzed. The expression of CRTC2 gene in goat tissues and subcutaneous precursor adipocytes differentiated from 0 to 120 h was examined by quantitative real-time PCR (qRT-PCR). The effects of CRTC2 on the subcutaneous precursor adipocyte differentiation were investigated by using liposome transfection, Bodipy, Oil Red O staining and qPCR.

Results: The results showed that the cloned goat CRTC2 gene was 2363 bp long (coding sequence [CDS] 2082 bp), encoding 693 amino acids. The relative expression levels of CRTC2 gene were highest in liver and then in kidney (p<0.05). During differentiation, the highest expression of CRTC2 in subcutaneous precursor adipocytes was observed at 120 of differentiating (p<0.01). In addition, we found that overexpression of CRTC2 significantly increased the expression of lipid metabolism-related genes (C/EBPα, C/EBPβ, PPARγ, DGAT1, DGAT2, ACC, FASN, SREBP1, AP2, LPL, ATGL) and promoted lipid accumulation. We then chemically synthesized goat CRTC2 small interfering RNA and transfected it into goat subcutaneous precursor adipocytes. The results revealed that SiRNA-mediated interference with CRTC2 significantly inhibited its differentiation and suppressed lipid droplet aggregation.

Conclusion: So, this study indicates that CRTC2 is a positive regulator that promoting cell differentiation of subcutaneous adipocyte in goats, which lays the foundation for an in-depth study of the role of CRTC2 in lipid deposition in goats.

Keywords: Adipogenesis; Biological Property; CRTC2; Goat; Subcutaneous Precursor Adipocytes.

Figure 1

Sequence analysis of the goat CRTC2 gene. (A) Goat CRTC2 gene amplification results. (B) CRTC2 gene sequence alignment with predicted sequence. (C) Amino acid composition of CRTC2 protein.

Figure 2

Bioinformatics analysis of the CRTC2 gene in goats. (A) Secondary-structure prediction. (B) Functional structure domain prediction. (C) Tertiary-structure prediction. (D) CRTC2 protein phosphorylation site prediction. (E) Subcellular localization. (F) Interaction protein prediction of goat CRTC2.

Figure 3

Tissue and temporal expression analysis of goat CRTC2 gene. (A) Distribution of CRTC2 in different tissues of goats. (B) Temporal expression profile of goat CRTC2 gene in subcutaneous precursor adipocytes. ^a–c p<0.05, ^A–I p<0.01.

Figure 4

Effect of overexpression of goat CRTC2 on adipocyte differentiation in subcutaneous precursor adipocytes. (A) Effect of overexpression of pEGFP-CRTC2 double enzyme digest product. (B) qRT-PCR and protein level image of overexpressing CRTC2 efficiency. (C) Oil red O staining and Semi-quantitative evaluation of absorbance assay of Oil Red O content performed at 490 nm. (D) Bodipy staining. (E) Effects of overexpression of CRTC2 on expression of lipid metabolism marker genes. * p<0.05, ** p<0.01. DAPI, 4′,6-diamidino-2-phenylindole; TG, triglycerides; qRT-PCR, quantitative reverse transcription polymerase chain reaction.

Figure 5

Effect of Si-CRTC2 on adipocyte differentiation in goat subcutaneous precursor adipocytes. (A) Transfection efficiency of Si-CRTC2 by qRT-PCR and WB. (B) Oil Red O staining. C: Semi-quantitative evaluation of absorbance assay for Oil Red O content at 490 nm. (D) BODIPY staining. (E) Effects of Si-CRTC2 on expression of marker genes of lipid metabolism. * p<0.05, ** p<0.01. DAPI, 4′,6-diamidino-2-phenylindole; TG, triglycerides; qRT-PCR, quantitative reverse transcription polymerase chain reaction; WB, Western blot.