Summary
SIRT6 plays a protective role in hypoxia-induced apoptosis in MC3T3-E1 cells, and that strategies might be beneficial for the treatment of ischemic bone disease.
Abstract
Introduction
Hypoxic–ischemia injury of the bone causes consequential osteonecrosis. Angiogenesis is considered essential for proper bone regeneration in ischemic necrosis of the bone. SIRT6, a nicotinamide adenosine dinucleotide–dependent deacetylase and a class III histone deacetylase, plays a critical role in angiogenesis. However, the effects of SIRT6 overexpression or activation on osteoblastic differentiation remain unclear. In the present study, we investigated the role of SIRT6 in the survival of osteoblast cells against hypoxia stimulus as well as the underlying mechanism.
Methods
MC3T3-E1 osteoblast cells were used. Apoptosis was induced by hypoxic treatment. MC3T3-E1 cells were transfected with adenovirus SIRT6. For SIRT6 silencing experiment, the transfection of cells were done using three siRNA duplexes directed at different regions of SIRT6 or scrambled siRNA pool. The relative levels of SIRT6 mRNA were quantified by using real-time PCR. Western blot experiments were done after the specific treatment and sample collection. MTT assay was used to estimate cell viability. Caspase 3/7 activity was assayed.
Results
The expression of SIRT6 mRNA appeared to be markedly down-regulated in the hypoxia-treated group compared to the matched normal group. Meanwhile, western blot analysis revealed that the expression of SIRT6 level was markedly down-regulated in hypoxia-treated group at protein level. SIRT6 level was effectively down-regulated by the transfection of SIRT6 siRNA, and significantly enhanced by SIRT6 overexpression. Compared with the control group, SIRT6 overexpression could significantly increase cell viability, and significantly decrease the percentage of apoptotic cells and the activity of caspase-3/7 in response to hypoxia treatment. By contrast, SIRT6 knockdown via treatment with SIRT6 siRNA exhibited the opposite phenotype.
Conclusions
These results suggest that SIRT6 plays a protective role in hypoxia-induced apoptosis in MC3T3-E1 cells, and that strategies might be beneficial for the treatment of ischemic bone disease.
Keywords: SIRT6, Hypoxia, Apoptosis, Osteoblast
Acknowledgments:This work was supported by grants from Shandong Provincial Natural Science Foundation, China (ZR2014CQ037) and Chinese National Natural Science Foundation (81400182)