Establishment And Functional Study Of Regulable Osterix Expression Based On Tet-on System

Objetives:In an attempt to establish the functional expression of Osterix with doxycycline (DOX) induced Tet-on regulating system in mouse myogenic stem cell line C2C12, an ideal experimental platform was provided for investigation of biological function and further studies of Osterix.Methods:The open reading frame encoding Osterix was amplified by Polymerse Chain Reaction.Then insert the PCR product into the reponse plasmid pTRE. The pTet-on regulating plasmid was transfected into C2C12, and stable expression of Tet-on was established in C2C12 through G418 select. Then the response plasmid pTRE and the recombinant plasmid pTRE-Osx was steadily transfected into positive C2C12-pTet cells with hygromycin B(Hyg B) screen. DOX was used to induce the expression of Osterix and a cell clone sensitive to Dox was selected. The best-induced concentration was determined with different concentration of Dox induction. The response time was also detected by quantitive PCR. Growth curves and cell cycle distribution were detected after Osterix gene up-regulated expression with DOX induction by MTT and flow cytometry methods. To study the potential of osteogenic differentiation, alkaline phosphatase(ALP) was strained by Calcium-Cobalt and alizarin red staining methods. The early bone makers and other related gene were investigated by quantetive PCR.Results:The recombinant plasmid pTRE-Osx and stable cell lines were successfully constructed. The expression of Osterix reached the highest level in the presence of DOX concentration reached 14μg/mL. At that concentration, the cell line was begin to responsed to overexpress Osterix after 1h induced by DOX, and the highest express level presented after DOX induced the C2C12-pTet-pTRE-Osx cell line for 18h. The growth capacity of C2C12-pTet-pTRE-Osx was significantly suppressed, compared with the controls. FCM analysis indicated that S phrase cell rate of C2C12-pTet-pTRE-Osx was markedly higher than that of C2C12-pTet-pTRE cells as control. The results of ALP and alizarin red staining showed that the ALP levels in C2C12 induced by BMP-2 were significantly higher than that of other two groups. While, little difference showed in C2C12-pTet-pTRE-Osx compared with C2C12-pTet-pTRE as negative control after induced by DOX. Early bone marker was up-regulated by overexpression of Osterix. The Wnt antagonist Dkkl was also up-regulated while Pitx2 (paired-like homeodomain transcription factor) was down-regulated after Osterix gene overexpres-sion with DOX induction.Conclusions:In this study, functional expression of Osterix under DOX induced Tet-on regulation system was successfully established in C2C12 cell line. The osteogensis-related transcription factor Osterix was detected to inhibit cell proliferation and promote osteoblast differentiation in C2C12 cell line.