ObjectiveTo 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). MethodsHey1 lentivirus and Hey1 short hairpin 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-shHey1 group); the cells of control groups (C3H10T1/2 group and C3H10T1/2-Blank group) were cultured with normal medium. The mRNA 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. ResultsC3H10T1/2 cell lines with different Hey1 expression levels were successfully established. In osteogenesis compared with BMP-9+C3H10T1/2 group, overexpression of Hey1 enhanced the mRNA 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). ConclusionExpression 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.