CDC20 Regulates the Osteogenic Differentiation of hASCs through Proteasome-dependent Degradation of p65

doi:10.13701/j.cnki.kqyxyj.2022.06.015

Abstract

Abstract: Objective: To investigate the role of cell division cycle 20 (CDC20) in the osteogenesis of human adipose-derived stem cells (hASCs) and its underlying mechanisms. Methods: After 0, 7, and 14 days of osteogenic induction, RT-qPCR and Western blot assays were used to determine CDC20 level. In CDC20 stable knockdown hASCs, ALP staining and Western blot assays were performed to examine the expressions of related markers of osteogenic differentiation. The CDC20 stable knockdown hASCs mixed with β-tricalcium phosphate (β-TCP) were implanted under the skin of nude mice. Eight weeks later, the mice were sacrificed and ectopic bone were measured. In CDC20 overexpressed hASCs, ALP staining and quantification were performed to examine the osteogenic differentiation of hASCs. Co-immunoprecipitation and western blot assays were conducted to explore the relationship of CDC20 and p65 in hASCs. The CDC20 and p65 double knockdown hASCs were constructed to evaluate the effect of p65 in osteogenesis manipulated by CDC20. Results: CDC20 was significantly induced in hASCs during osteogenic differentiation (P＜0.05). After the osteogenic induction, ALP quantification of CDC20 stable knockdown hASCs [(7.31±0.25)、(11.01±0.49) U/gprot] was significantly lower than that of control [(16.00±0.35) U/gprot] (P＜0.05). Western blot results showed the decreased RUNX2 protein level in CDC20 knockdown group. The CDC20 stable knockdown hASCs mixed with β-TCP and implanted under the skin of nude mice represented less bone formation compared to control cells. After the osteogenic induction, ALP quantification of CDC20 overexpression in hASCs [(20.74±0.53) U/gprot] was significantly higher than that of control [(12.58±0.42) U/gprot](P＜0.05). CDC20 interacted with p65. The expression of p65 increased after the knockdown of CDC20. Overexpression of CDC20 led to decreased p65, and the treatment of MG132 (the proteasome inhibitor) reversed the trend. After osteogenic differentiation of CDC20 and p65 double knockdown hASCs,the ALP staining and quantification results illustrated that CDC20 regulated osteogenic differentiation of hASCs in a p65-dependent manner. Conclusion: CDC20 promoted the osteogenic differentiation of hASCs through proteasome-dependent degradation of p65,thus providing a potential target for bone regeneration.

Key words: hASCs, CDC20, osteogenic differentiation, p65

DU Yangge, CHENG Yawen, GUO Qian, ZHANG Ping, ZHOU Yongsheng. CDC20 Regulates the Osteogenic Differentiation of hASCs through Proteasome-dependent Degradation of p65[J]. Journal of Oral Science Research, 2022, 38(6): 565-571.

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