Abstract:
ObjectiveRNA interference (RNAi) is used to inhibit Notch intracellular domain (NICD) expression in MC3T3-E1 cells. The aim of RNAi is to observe the effect of the inhibition of the NICD expression on cell proliferation and function-related gene expression in MC3T3-E1 cells exposed to 2 Gy radiation.
MethodsThe MC3T3-E1 cells were established to inhibit the NICD. The NICD expression of cells was detected by using qRT-PCR and Western blot. MC3T3-E1 and inhibited NICD MC3T3-E1 cells were irradiated with 2 Gy. Then, the proliferation and function-related gene expression were detected through BrdU incorporation and qRT-PCR.
ResultsNICD expression in MC3T3-E1 cells could be inhibited by the RNAi technology. The inhibition of NICD expression could interfere with the proliferation of precursor osteoblasts and osteoblasts. The proliferation of precursor osteoblasts, osteoblasts, and NICD RNAi osteoblasts significantly decreased after 2 Gy irradiation. The function-related gene expression of each target cell is as follows. ① The expression of Runt-related transcription factor 2 (Runx2) was upregulated in precursor osteoblasts (t=2.353, P < 0.05) and downregulated in the NICD RNAi precursor osteoblasts (t=2.353, P < 0.05) after 2 Gy irradiation. ② The expression of alkaline phosphatase (ALP) was upregulated in precursor osteoblasts and osteoblasts and the NICD RNAi precursor osteoblasts (t=3.182, 3.345, 3.555, all P < 0.05) and was downregulated in the NICD RNAi osteoblasts (t=5.045, P < 0.01) after 2 Gy irradiation. ③ The expression of receptor activator of nuclear factor κB ligand (RANKL) was downregulated in precursor osteoblasts (t=2.541, P < 0.05) and was upregulated in osteoblasts and NICD RNAi precursors osteoblast (t=3.299, P < 0.05; t=10.212, P < 0.01) after 2 Gy irradiation. However, the inhibition of the NICD expression could cause an opposite change in other cells (t=0.765, P > 0.05). ④ The expression of osteoprotegerin (OPG) was downregulated in precursor osteoblasts and osteoblasts (t=2.994 and 2.782, P < 0.05) after 2 Gy irradiation. However, the inhibition of the NICD expression could cause expression upregulation in precursor osteoblasts and expression downregulation in osteoblasts (t=5.841, P < 0.01). ⑤ The expression of macrophage-colony stimulating factor (M-CSF) in the target cells exhibited the same trend as the expression of RANKL after 2 Gy irradiation.
ConclusionsThe inhibition of the NICD expression exerts different effects on the differentiation of irradiated osteoblasts. The inhibition of the NICD expression could cause a series of changes including:①It may decrease the proliferation of precursor osteoblasts and osteoblasts and protect the proliferation of differentiating precursor osteoblasts after irradiation. ②It can significantly inhibit the differentiation of precursors osteoblasts after irradiation and reduce bone loss through regulating the expression of Runx2. ③The osteoblasts did not show the regulated function of the osteoclasts through the RANKL/OPG/RANK system. ④The osteoblasts can exhibit the inhibited function of the osteoclasts through the expression of M-CSF.