Isolation And Identification Of Differentially Expressed Genes In Human Osteoblast-like MG-63 Cell Induced By 17Beta-Estradiol

Objective To obtain a series of differentially expressed cDNA fragments from human osteoblast-like osteosarcoma MG-63 cells induced by 17beta-estradiol, to identify some estrogen-responsive genes and to provide a new molecular basis for the protective effects of estrogens against the development of postmenopausal osteoporosis (PMOP) and the mechanisms of estrogen replacement therapy. Methods Modified cDNA representational difference analysis (RDA) was performed to isolate differentially expressed sequences between cDNA from MG-63 cell line treated with and without 17beta-estradiol. The sources of differentially expressed cDNA fragments were proved by Southern blot and "shotgun" Northern blot analysis. The fragments were then cloned into the pGEM-T easy vector and subtractive cDNA libraries were prepared in E.coli JM109 cells. The cDNA libraries were plated on LB/Amp+/X-gal/IPTG plates and white colonies were picked up randomly and individually grown in LB/amp+ medium in 96-well plates. After PCR, colonies were individually blotted onto a Hybond N membrane. Membranes were hybridized with -32P-labeled subtracted or unsubtracted cDNA. Clones showing a strong hybridization signal with the forward-subtracted probes compared with the reverse-subtracted ones and vice-versa were selected for DNA sequencing and homologue analysis. Northern blot analysis was performed after release of the pGEM-T easy insert with EcoR I.Results Through cDNA RDA, three upregulated and two downregulated expressed fragments were isolated in the fourth subtraction hybridization using cDNA from MG-63 cells induced by 17beta-estradiol and cDNA from untreated MG-63 cells as"tester" amplicon, respectively. These fragments were proved to really come from the "tester" amplicons by Southern blotting and proved to be really upregulated or downregulated expressed after E2-treated through "shotgun" Northern blotting analysis. We obtained nearly 600 cDNA clones with positive insert from MG-63 cells induced by E2 and 120 differentially expressed clones through dot blotting. Forty clones including 20 upregulated and 20 downregulated were sequenced and 36 sequences (17 upregulated and 19 downregulated ones) obtained, some of them highly homologous to known genes, with three of them proved to be upregulated expressed, one downregulated and one's expression without obvious change through Northern blotting.Conclusion cDNA RDA is one of the most effective methods to isolate differentially expressed genes and we have screened differentially expressed genes rapidly through cDNA RDA combined with cDNA arrays. Some of the genes of human osteoblast-like osteosarcoma MG-63 cells were differentially expressed induced by 17beta-estradiol. The results show that 17 -E2 regulates the expression of genes associated with bone matrix, supplying energy, steroid hormone metabolism, gene transcription, inflammatory reaction and tumorigenesis. These data might provide a new molecular basis for the protective effects of estrogens against the development of PMOP.