Influence of silencing the MC4R gene by lentivirusmediated RNA interference in bovine fibroblast cells
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.