Abstract
Melanocortin receptor 4 (MC4R) is a key element in the mechanisms used to regulate both aspects of keeping the balance between energy uptake and energy expenditure. MC4R was knocked down by lentivirus-mediated shRNA expressing plasmids, which were controlled by the U6 promoter in bovine fibroblast cells, and the expression of MC4R was examined by the real time-PCR and Western blot analysis. Real time-PCR analysis was used to characterize the expression of Leptin, POMC, AGRP, MC3R and NPY gene. The relative genes [leptin, proopiomelanocortin (POMC), agouti-related peptide (AGRP), MC3R and neuropeptide Y (NPY)] expression level seemed to be closely associated with the MC4R gene in bovine fibroblast cell lines (BFCs). The levels of both MC4R mRNA and protein were significantly reduced by RNA interference (RNAi) mediated knockdown of MC4R in BFCs cells transfected with plasmid-based MC4R-specific shRNAs. The finding of this study demonstrated that vector based siRNA expression systems were an efficient approach to the knockdown of the MC4R gene expression in bovine fibroblast cells and they provided a new molecular basis for understanding the relationship of MC4R and other genes, which were responsible for the regulation of energy homeostasis by the melanocortin system. Key words: Melanocortin receptor 4 (MC4R), RNAi, bovine fibroblast cells, energy homeostasis.
Highlights
The melanocortin receptor 4 (MC4R), a G-protein coupled receptor, plays a pivotal role in controlling meal size and energy homeostasis in mammals (Govaerts et al, 2005; Adan et al, 2006)
The relative genes [leptin, proopiomelanocortin (POMC), agouti-related peptide (AGRP), MC3R and neuropeptide Y (NPY)] expression level seemed to be closely associated with the Melanocortin receptor 4 (MC4R) gene in bovine fibroblast cell lines (BFCs)
The levels of both MC4R mRNA and protein were significantly reduced by RNA interference (RNAi) mediated knockdown of MC4R in BFCs cells transfected with plasmid-based MC4R-specific short hairpin RNAs (shRNAs)
Summary
The melanocortin receptor 4 (MC4R), a G-protein coupled receptor, plays a pivotal role in controlling meal size and energy homeostasis in mammals (Govaerts et al, 2005; Adan et al, 2006). In spite of the evidence, presented about the role of several important factors in energy metabolism, the regulation and integration of the satiety pathway and the energy homeostasis have not been completely elucidated. These important factors are neuropeptide Y (NPY), leptin, proopiomelanocortin (POMC), Agouti-related peptide (AGRP) and their respective receptors (Pelleymounter et al, 1995; Stephens et al, 1995; Hu et al, 1996; Boston et al, 1997; Clement et al, 1998; Friedman and Halaas, 1998), whereas only few researches have been conducted on the relationship of MC4R with the expression of upstream regulatory genes in bovine. Synthetic siRNAs can be transferred into the cells to induce transient gene silencing, whereas short hairpin RNAs (shRNAs) could be driven by RNA polymerase III promoters
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