Folliculin deficiency effect on Raw264.7 cells

Osteoclast differentiation is a dynamic differentiation process, which is accompanied by dramatic changes in metabolic status as well as in gene expression. Recent findings have revealed an essential connection between metabolic reprogramming and dynamic gene expression changes during osteoclast differentiation. However, the upstream regulatory mechanisms that drive these metabolic changes in osteoclastogenesis remain to be elucidated. We demonstrate that induced deletion of a tumor suppressor gene, Folliculin (Flcn), in mouse osteoclast precursors causes severe osteoporosis in 3 weeks through excess osteoclastogenesis. Flcn deficient osteoclast precursors (Raw264.7 cells) reveal cell autonomous accelerated osteoclastogenesis. For the purpose of elucidating the molecular mechanism of accelerated osteoclastogenesis in Flcn deficient Raw264.7 cells, we performed DNA microarray analysis. Raw264.7 cells were transfected with scramble or Flcn targeting siRNA, followed by a medium change at 24hr after transfection. Cells were cultured after an additional 48hr and total RNA was collected and purified using the RNeasy Micro kit. cDNA preparation and hybridization of the probe arrays were performed according to the manufacturer's instructions. Affymetrix GeneChip Mouse Genome 430 2.0 arrays were applied.