Effect Of Hey1 Of Notch Signaling Pathway On Osteogenic Differentiation And Proliferation Of C3H10T1/2 Cells Induced By Bone Morphogenetic Protein 9

Objective : The mouse fibroblast cells C3H10T1/2 were cultured in vitro to simulate the stem cells, lentivirus were used to change the expression level of Hey1 in C3H10T1/2 cells, then the cells with different Hey1 expression were induced by BMP-9 conditioned medium to investigate the effect of Notch signaling pathway important target Hey1 expression on the differentiation and proliferation of C3H10T1/2 cells induced by bone morphogenetic protein 9(BMP-9).Methods : Hey1 lentivirus and Hey1 sh RNA lentivirus were constructed and used to infect C3H10T1/2 cells to change the expression level of Hey1 in C3H10T1/2 cells. C3H10T1/2 cells infected with LV-Blank(empty plasmid) as control. The Hey1 expression levels of different groups were detected by fluorescence microscope, real-time fluorescence quantitative PCR, and Western blot. The C3H10T1/2 cells with different Hey1 expression level were induced by BMP-9 conditioned medium(BMP-9+C3H10T1/2 group, BMP-9+C3H10T1/2-Hey1 group, and BMP-9+C3H10T1/2-sh Hey1 group); the cells of control groups(C3H10T1/2 group and C3H10T1/2-Blank group) were cultured with normal medium. The m RNA and protein expression levels of osteogenesis related transcription factors(Runx2, osteopontin, and osteocalcin) were detected at 48 hours by real-time fluorescence quantitative PCR and Western blot assay. The cells proliferation and cycles were detected by MTT assay at 4, 5, 6, and 7 days and flow cytometry at 4, 5, and 10 days. The alkaline phosphatase(ALP) activity was analyzed by ELISA and observed by ALP staining at 4 and 7 days.Results : C3H10T1/2 cell lines with different Hey1 expression levels were successfully established. In the osteogenesis, compared with BMP-9+C3H10T1/2 group, overexpression of Hey1 enhanced the m RNA and protein expressions of transcription factors(Runx2, osteopontin, and osteocalcin), and the expression of osteogenic differentiation marker(ALP)(P<0.05); however, inhibition of Hey1 expression significantly decreased the above indexes(P<0.05). In cell proliferation activity compared with BMP-9+C3H10T1/2 group, overexpression of Hey1 increased absorbance(A) value in MTT assay and pecentage of G2+S cells in cytometry assay, but inhibition of Hey1 expression significantly decreased the indexes(P<0.05).Conclusion : Expression of Hey1 is the important link in the osteogenic differentiation process of C3H10T1/2 cells induced by BMP-9, and plays an important role in the regulation of early cell proliferation. Overexpression of Hey1 presents the osteogenetic differentiation synergies, and promote cell proliferation, inhibition of Hey1 expression appears strong antagonism effect on osteogenetic differentiation while inhibiting cell proliferation in early stage.