The homeobox a5 (Hoxa5) plays considerable roles in the differentiation and lipid metabolism of adipocytes. However, the current knowledge about the mechanistic roles and functions of Hoxa5 in goat subcutaneous preadipocyte remains unclear. Therefore, Hoxa5 loss-of-function and gain-of-function was performed to reveal its functions in adipogenesis. For differentiation, overexpression of Hoxa5 notably increased the expression of adipogenic genes (PPARγ, CEBP/α, CEBP/β, AP2, and SREBP1), as well as promoted goat subcutaneous preadipocyte lipid accumulation. Knockdown of Hoxa5 mediated by siRNA technique significantly inhibited its differentiation and suppressed the accumulation of lipid droplets. Regarding proliferation, overexpression of Hoxa5 reduced the number of cells stained with crystal violet, and inhibited mRNA expression of the marker genes including CCNE1, PCNA, CCND1, and CDK2, and also significantly reduced EdU-positive rates. Consistently, knockdown of Hoxa5 demonstrated the opposite tendency. In conclusion, these data demonstrated that Hoxa5 promotes adipogenic differentiation of goat subcutaneous preadipocyte and inhibits its proliferation in vitro.
The presence or absence of subcutaneous adipose accumulation will affect the energy storage, insulation resistance and metabolism of animals. Proliferation and differentiation of preadipocytes play a significant role in lipid deposition. The objective of this study was to clone the goat CXCL17 gene and investigate its potential functions on goat subcutaneous preadipocytes’ proliferation by gaining or losing function in vitro. The goat CXCL17 gene was cloned by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and bioinformatics analysis was performed. The expression of the CXCL17 gene in the different goat tissues and adipocytes at different differentiation stages was detected by real-time fluorescence quantitative PCR (qPCR). The results showed that the cloned sequence of goat CXCL17 gene is 728 bp and the CDS region is 357 bp, encoding 118 amino acids. CXCL17 protein is located in nucleus, cytoplasm, mitochondria and extracellular matrix. Tissue-expression profiles revealed that CXCL17 expressed in all of the examined tissues. In visceral tissues, the highest expression level was found in lung (p < 0.01); in muscle tissues, the highest CXCL17 expression level was found in the longissimus dorsi (p < 0.01) and in adipose tissues, the highest expression level was found in subcutaneous adipose (p <0.01). Compared with those cells before differentiation, CXCL17 expression levels upregulated at 48 h (p < 0.01), 72 h (p < 0.01), 120 h (p < 0.01) and downregulated at 96 h (p < 0.01). Furthermore, the results of crystal violet staining and semi-quantitative assay showed that transfection with 1 μg CXCL17 expression plasmid reduced the cell numbers in vitro. Meanwhile, the expression of CCND1 was significantly decreased. A similar consequence happened after interfering with CXCL17 expression. However, plasmid transfected with 2 μg pEGFPN1-CXCL17 increased the number of cells in vitro. These results suggest that CXCL17 is involved in the proliferation of goat subcutaneous preadipocytes.
PDZK1IP1 is highly expressed in tumor tissue and has been identified as a tumor biomarker. However, the role of PDZK1IP1 in goat subcutaneous preadipocyte differentiation remains largely unknown. The molecular mechanism of autophagy in regulating the differentiation of goat subcutaneous preadipocytes has not been clarified yet. In our study, PDZK1IP1 gain of function and loss of function were performed to reveal its functions in preadipocyte differentiation and autophagy. Our results showed that the overexpression of PDZK1IP1 inhibited the differentiation of goat subcutaneous preadipocytes, whereas it promoted autophagy. Consistently, the knockdown of PDZK1IP1 demonstrated the opposite tendency. Next, we investigated whether PDZK1IP1 inhibited the differentiation of goat preadipocytes by regulating autophagy. We found that inhibiting autophagy can rescue the PDZK1IP1-induced differentiation restraint in goat subcutaneous preadipocytes. In conclusion, PDZK1IP1 acts as a regulator of adipogenesis, and inhibits goat subcutaneous preadipocyte differentiation through promoting autophagy. Our results will contribute to further understanding the role and mechanism of PDZK1IP1 in controlling adipogenesis.
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