The Role Of SIRT6 In Silica Nanoparticle-Induced Stress

As high-tech ultrafine inorganic materials develop,silica nanoparticles(SiO2 NP)are widely applied in various fields.Its potential safety issues for human health have drawn much attentions.Studies have shown that SiO2 NP can enter the body through the respiratoty system,digestive system,skin,blood system and other means,contributes to organ injury.SiO2 NP exposure could induce the generation of reactive oxygen species(ROS),which leads to celluar damage.However,the current understanding of the cellular processes that respond to oxidative stress induced by SiO2 NP remains to be identified.Our previous study demonstrated SiO2 NP induced the expression of FST in mouse lung tissue as well as in human lung epithelial cells.The elevated FST inhibited SiO2 NP-induced ROS production and cell apoptosis.Interestingly,the levels of Ac-H3(K9/18),an active gene marker,at FST promoter region was significantly increased during SiO2 NP treatment,indicating an activation of FST transcription.However,the detailed mechanism underlying SiO2 NP-induced FST transcription remains elusive.Sirtuins are a class of proteins that possess either mono-ADP-ribosyltransferase,or NAD+-dependent deacylase activity.Sirtuins have been implicated in influencing a wide range of cellular processes like aging,transcription,apoptosis,inflammation and stress resistance,as well as energy efficiency and alertness during low-calorie situations.Many of the family members are implicated in cellular stress response.To address which of sirtuins regulates FST transcription,we transfected cells with siRNA targeting SIRT1-7 and examined FST mRNA level.Data showed that down-regulation of SIRT1 and SIRT6 induced a significant increase of FST mRNA.Both SIRT1 and SIRT6 are nuclear localized and act as transcriptional co-factors.To determine whether SIRT1 and SIRT6 regulated FST expression by binding to its promoter,chromatin immunoprecipitation was carried out.Data shows that SIRT6 directly regulates FST transcription while SIRT1 regulates FST expression indirectly.Knockdown of SIRT6 significantly increased both the mRNA and protein level of FST.Cells over-expressing SIRT6 displayed a marked decrease in FST expression compared with null vector-transfected cells.These data indicated that FST transcription was indeed repressed by SIRT6 under normal growth condition.To determine whether SIRT6 deacetylates histone H3 at FST promoter,we performed ChIP assay to analyze Ac-H3K9 and Ac-H3K56 levels at FST promoter region after silencing SIRT6.Data showed that knockdown of SIRT6 indeed significantly increased both Ac-H3K9 level and Ac-H3K56 level at FST promoter region.To address whether SIRT6 responds to SiO2 NP stress,A549 cells were treated with SiO2 NP and SIRT6 expression was detected.The mRNA level of SIRT6 started to decrease 1 hour after SiO2 NP treatment,while the protein level started to decrease at 3 hours of SiO2 NP treatment.Furthermore,we intratracheally instilled mice with SiO2 NP and detected SIRT6 expression.Data showed that both the mRNA level and the protein level of SIRT6 decreased after 1 day of SiO2 NP treatment.Altogether,our results revealed that SiO2 NP treatment decreases SIRT6 expression.The reduced level of SIRT6 mRNA may result from either transcriptional depression or post-transcriptional regulation.We therefore first detected changes in SIRT6 gene promoter activity using a luciferase reporter.Data showed that the transcriptional activity of SIRT6 promoter was not significantly induced under SiO2 NP treatment.Next,we examined the stability of SIRT6 mRNA.Actinomycin D was used to block de novo RNA synthesis,and then the persistence of the existing SIRT6 mRNA was measured.Our results revealed that SiO2 NP treatment led to substantial stabilization of the SIRT6 mRNA.The half-life of total SIRT6 mRNA is longer in control cells than SiO2 NP-treated cells.These results suggested that SiO2 NP-triggered SIRT6 mRNA down-regulation is due to mRNA destabilization.To identify whether SIRT6 functions in cellular damage,SIRT6-expressing A549 cells were treated with SiO2 NP.Our results revealed that SiO2 NP treatment for 2 hours induced more ROS production in SIRT6-expressing group,meanwhile SiO2-NP treatment for 12 hours induced more cell apoptosis,indicating that SIRT6 promotes SiO2 NP-induced ROS production and cell apoptosis.In this study,we identified SIRT6 as a negative regulator of FST transcription SIRT6 de-acetylates H3K9 and H3K56 level at FST promoter region,which induces FST gene silencing under normal condition.SiO2 NP exposure decreases SIRT6 expression,which loosens FST chromatin and activates FST gene transcription.