Effect of gene CRTC2 on the differentiation of subcutaneous precursor adipocytes in goats
Effect of gene CRTC2 on the differentiation of subcutaneous precursor adipocytes in goats
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Objective: The aim of this study was to obtain goat <i>CRTC2</i> gene sequence and elucidate its biological properties, and further study the impact of overexpression and interference of <i>CRTC2</i> on the cell differentiation of goat subcutaneous precursor adipocytes.Methods: The sequence of goat <i>CRTC2</i> was cloned by reverse transcription (RT)-polymerase chain reaction (PCR) and its molecular characterization was analyzed. The expression of <i>CRTC2</i> 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 <i>CRTC2</i> 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 <i>CRTC2</i> gene was 2363 bp long (coding sequence [CDS] 2082 bp), encoding 693 amino acids. The relative expression levels of <i>CRTC2</i> gene were highest in liver and then in kidney (p<0.05). During differentiation, the highest expression of <i>CRTC2</i> in subcutaneous precursor adipocytes was observed at 120 of differentiating (p<0.01). In addition, we found that overexpression of <i>CRTC2</i> significantly increased the expression of lipid metabolism-related genes (<i>C/EBPα</i>, <i>C/EBPβ</i>, <i>PPARγ</i>, <i>DGAT1</i>, <i>DGAT2</i>, <i>ACC</i>, <i>FASN</i>, <i>SREBP1</i>, <i>AP2</i>, <i>LPL</i>, <i>ATGL</i>) and promoted lipid accumulation. We then chemically synthesized goat <i>CRTC2</i> small interfering RNA and transfected it into goat subcutaneous precursor adipocytes. The results revealed that SiRNA-mediated interference with <i>CRTC2</i> significantly inhibited its differentiation and suppressed lipid droplet aggregation.Conclusion: So, this study indicates that <i>CRTC2</i> 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 <i>CRTC2</i> in lipid deposition 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 indepth study of the role of CRTC2 in lipid deposition in goats.