Screening of down-regulated expression genes in human osteoblast-like MG-63 cells induced by 17beta-estradiol and its preliminary analysis

Objective To obtain a series of downregulated expression of cDNA fragments from human osteoblast-like MG-63 cells induced by 17beta-estradiol (E2), to identify E2-related genes, and to try to elucidate the probable mechanisms of bone protective effects of estrogen.Methods Modified cDNA representational difference analysis (RDA) was performed to isolate differentially expressed cDNA sequences from MG-63 cells treated with and without E2. The differentially expressed cDNA fragments were reconfirmed by Southern blot and “shotgun” Northern blot analysis, then cloned into pGEM-T easy vector. After blue/white selection, the white clones were picked up individually to sequence and compare the homology. Northern blotting was introduced to confirm the expression of the differentially expressed cDNA clones before and after E2 treatment.Results Two downregualted cDNA fragments expressions were isolated and verified by RDA. Twenty downregulated clones were then sequenced to obtain 19 sequences with high homologous to the known genes representing 14 different genes, which were associated with bone matrix formation, gene transcription and signalling transduction, and regulation of cell differentiation and proliferation. One gene was confirmed downregulated expression after E2 treatment by Northern blotting.Conclusions cDNA RDA is one of the most effective techniques to identify differentially expressed genes. The downregulated expressed genes induced by E2 from MG-63 might participate in bone reconstruction induced by E2 to play protective effect on bone.