MiR-449a/SIRT1/PGC-1? Is Necessary For Mitochondrial Biogenesis Induced By T-2 Toxin

T-2 toxin is one of the type A trichothecenes produced mainly by the Fusarium genus.Due to its broad distribution and highly toxic nature,it causes great concern as a threat to human health and animal breeding.In addition to its ribotoxic effects,T-2 toxin exposure leads to mitochondrial dysfunction,reactive oxygen species(ROS)accumulation and eventually cell apoptosis.Involved in the mechanisms of T-2 toxin toxicity,we observed that mitochondrial biogenesis and function was highly activated in animal cells exposed to low dosages of T-2 toxin.Then we screened the up-regulated genes related to mitochondrial biogenesis in animal cells exposed to T-2 toxin,and knockdown or overexpression the selected genes to further verify whether they were involved in the induction of T-2 toxin.Furthermore,we detected the promoter areas and miRNAs related to the genes for explaining how T-2 toxin regulated the expression of the key genes.Finally,we found out the signal pathway involved in mitochondrial biogenesis induced by T-2 toxin.Low dosages of T-2 toxin significantly increased the levels of mitochondrial biogenesis,mtDNA copy number,mitochondrial mass,ROS production and ATP content in HEK293T,HepG2 and HeLa cells.The genes related to mitochondrial biogenesis were assessed in the cells treated by T-2 toxin,the mRNA levels of SIRT1 and TFAM were significantly up-regulated.Western blot showed that SIRT1 and TFAM protein levels were significantly up-regulated only in HEK293T and HepG2 cells.PGC-1?is a downstream target gene of SIRT1.The protein activity of PGC-1?is related to its posttranslational modification status.We detected that T-2 toxin did not change the protein level of PGC-1?in treated HEK293T,HepG2 and HeLa cells.The post-translational modification of PGC-1?mainly included phosphorylation and acetylation.SIRT1 is an NAD~+-dependent deacetylase.In subsequent experiments,the acetylation level of PGC-1?was mainly detected.The immunoprecipitation(IP)analysis revealed that T-2toxin significantly down-regulated the acetylation level of PGC-1?,whereas,this phenomenon could not be observed in the SIRT1 knockdown cells,regardless of whether they were exposed to T-2 toxin or not.Further analysis revealed that the overexpression of SIRT1 and knockdown of SIRT1could respectively increase and reduce the mtDNA copy number in HEK293T and HepG2cells.After treatment with T-2 toxin,we observed a significant increase in mitochondrial biogenesis.However,the knockdown of SIRT1 reversed the increase in mtDNA copy number in both cells treated by T-2 toxin,suggesting that SIRT1 is involved in the induction of mitochondrial biogenesis by T-2 toxin.So we proposed that whether the presence of SIRT1 would reduce cell death under T-2 toxin treatment.Next,we examined whether knockdown of SIRT1 could affect cell viability in both cells under T-2 toxin treatment.The MTT assay showed that the cell death increased in the SIRT1 knockdown cells under T-2 toxin treatment,which suggested that the self-protection of the cells in T-2toxin exposure mainly depend on the T-2 toxin-mediated overexpression of SIRT1.Our data have confirmed that the overproduction of SIRT1 in response to T-2 toxin exposure is the main reason for the upregulation of mitochondrial biogenesis.However,the molecular mechanisms of T-2 toxin-induced up-regulation of SIRT1 remain unclear.To unveil the mechanisms of the T-2-induced overexpression of SIRT1,we assessed SIRT1expression at the transcription level and the post-transcription level in T-2 treated cells.We first excluded the transcriptional activation of SIRT1 expression by the construction of SIRT1 promoter-luciferase reporters in T-2 toxin-treated cells.Promoter analysis by the dual-luciferase assay showed that T-2 toxin induction in the promoter is limited,as no significant induction was observed.Next,we addressed whether miRNAs were involved in the regulation of SIRT1 expression.The miRNA target prediction program miRanda was used to identify miRNAs that target the 3'UTR of SIRT1.The expression levels of the putative SIRT1-targeting miRNAs after in T-2 toxin treatment were determined by RT-qPCR,miR-449a was selected for further analysis because it was significantly downregulated.The overexpression of miR-449a reversed the heightened expression of SIRT1 induced by T-2 toxin and led to no apparent increase in mtDNA copy number.Transfection of miR-449a mimics significantly inhibited the deacetylation of PGC-1?in both cells under T-2 toxin treatment.The MTT assay showed that the cell death increased in the cells transfected with miR-449a mimics than in control cells exposed to T-2 toxin.These data suggested that miR-449a plays a key role in the upregulation of mitochondrial biogenesis in the T-2 toxin-treated cells.In this study,we proved that an early stage of T-2 toxin exposure exerted positive effects on mitochondrial biogenesis in human cells.This study is the first to report that SIRT1 is necessary for the induction of mitochondrial biogenesis by T-2 toxin in human cells.In addition,T-2 toxin suppresses miR-449a expression;the downregulation of miR-449a upregulates SIRT1 expression;the overexpression of SIRT1 leads to the increased accumulation of deacetylated PGC-1?;and the high accumulation of deacetylated PGC-1?activates mitochondrial biogenesis and function.We conclude that the pathway of miR-449a/SIRT1/PGC-1?regulated mitochondrial biogenesis in animal cells exposed to T-2 toxin.This study provides a reliable reference for revealing the molecular mechanisms of T-2 toxin in animal cells that produce different effects at different concentrations.