Identification of Reduced Fibronectin 1 Gene Expression in Osteoblasts following Hydrogen Peroxide Treatment using Subtractive Gene Cloning

Abstract

Bone formation steadily declines with age resulting in a significant loss of bone mass, while reactive oxygen species (ROS) are thought to be major contributors to the aging process. In the present study, we attempted to identify which gene had an altered transcription level when osteoblasts were treated with ROS, H₂O₂, using a subtraction gene cloning technique. A gene clone was obtained from cDNA of cells from the osteoblastic cell line MC3T3-E1 after subtraction of the cDNA of H₂O₂-treated cells and designated as MC-ROS-7. To confirm the gene product, the DNA sequence of MC-ROS-7 was determined and homology was assessed using a nucleotide-nucleotide BLAST (BLASTN) search of the NCBI database, which revealed a 99.7% homology with fibronectin 1 (FN) gene. A significant lower level of FN mRNA was found in H₂O₂-treated cells when compared to that in non-treated cells using endpoint RT-PCR and real-time PCR assays. Our results suggest that a reduction of FN gene expression by ROS is involved in the decline of bone formation in osteoblasts during the ageing process.