AIM: This study is aimed to investigate Runx2’s regulation by phytoestrogens resveratrol and genistein, screen for Runx2 mutation in Chinese cleidocranial dysplasia(CCD) patients, also determine the functional difference between Runx2’s two major isoforms-type I and type II. METHODS: The simian virus 40 large T antigens (SV40LT) gene was used to establish immortalized hBMSCs and the cell line were then identified by specific surface markers confirmation and pro-longed life span and renewable proliferative capacity illustration. Then we tested resveratrol and genistein’s regulation on the cell line by monitoring several osteoblastic markers and invested the possible involved pathway by western blotting. Subsequently, we determined the functional difference of Runx2’s two major isoforms-type I and type II-in osteoblastic differentiation by RNAi. Also for the first time we screened for the Runx2 mutations in Chinese CCD patients by DNA analysis. RESULTS: We successfully established a permanent hBMSCs by stable transfection of SV40LT. Based on this cell line platform, we illustrated that phytoestrogens resveratrol and genistein regulated Runx2’s expression in osteoblastic differentiation and might partly via MAPK pathway. We also found that type I and type II play a different role in osteoblastic differentiation process, during which type I major increased in the early phase and important for the cell proliferation while type II predominantly functioned in the late stage and necessary for the cell maturation. What’s more, we sequencing coding domains and promoter domains of Runx2 from 9 patients in 3 different CCD familial cases and found two nucleotide changes. CONCLUSION: In this paper, we set up an immortalized hBMSC line by stable transfection of SV40LT which brings convenience for the later studies, reveals that resveratrol and genistein are capable of promote osteoblastic differentiation and are probably via MAPK pathway, also finds that type I major functions in the early stage of osteoblastic differentiation while type II predominantly functions in the late stage, which for the first time distinguishes their effects and gave new hints for bone-related diseases therapies. Moreover, the investigation on RUNX2 mutations in CCD patients adds new records to the repertoire of RUNX2 mutations in China.
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