SIRT5-mediated PRKAA2 succinylation ameliorates apoptosis of human placental trophoblasts in hypertensive disorder complicating pregnancy.

PURPOSE: Hypertensive disorder complicating pregnancy (HDCP) is a serious clinical disorder syndrome during pregnancy. This study aims at finding novel targets for HDCP therapy. METHODS: HDCP-related mRNAs were firstly screened out and subjected to gene enrichment analysis. We chose protein kinase AMP-activated catalytic subunit alpha 2 (PRKAA2) as the research object. Thirty-nine HDCP patients at 32 to 40 weeks of gestation were selected as the HDCP group, and 39 normal controls who received cesarean section delivery at 37-42 weeks of pregnancy were enrolled in this study. Chorionic villi samples were collected within 30 min of delivery. The apoptosis of isolated placental trophoblasts was monitored to investigate the regulatory role of PRKAA2. RESULTS: PRKAA2 expression was further proven to be enhanced in the placental tissues of HDCP patients compared with that of normal puerpera. Subsequently, the results of flow cytometry analysis and western blot indicated that PRKAA2 overexpression accelerated primary placental cell apoptosis, while its knockdown attenuated cell apoptosis. Mechanistically, we determined that the level of PRKAA2 succinylation was elevated in the placental tissue of HDCP patients. Through in vitro succinylation assay and mutagenesis, we confirmed that sirtuin 5 (SIRT5) interacts with PRKAA2 at K69 and K260 to induce PRKAA2 desuccinylation. SIRT5 regulated primary HDCP cell apoptosis through PRKAA2. Finally, the animal study revealed that PRKAA2 elevates the systolic blood pressure of HDCP rat model. CONCLUSION: Our findings indicated that SIRT5-mediated PRKAA2 succinylation modulates placental cell apoptosis in HDCP, suggesting that PRKAA2 is a potential therapeutic target for HDCP treatment.

Molecular basis of bacterial DSR2 anti-phage defense and viral immune evasion.

Defense-associated sirtuin 2 (DSR2) systems are widely distributed across prokaryotic genomes, providing robust protection against phage infection. DSR2 recognizes phage tail tube proteins and induces abortive infection by depleting intracellular NAD+, a process that is counteracted by another phage-encoded protein, DSR Anti Defense 1 (DSAD1). Here, we present cryo-EM structures of Bacillus subtilis DSR2 in its apo, Tube-bound, and DSAD1-bound states. DSR2 assembles into an elongated tetramer, with four NADase catalytic modules clustered in the center and the regulatory-sensing modules distributed at four distal corners. Interestingly, monomeric Tube protein, rather than its oligomeric states, docks at each corner of the DSR2 tetramer to form a 4:4 DSR2-Tube assembly, which is essential for DSR2 NADase activity. DSAD1 competes with Tube for binding to DSR2 by occupying an overlapping region, thereby inhibiting DSR2 immunity. Thus, our results provide important insights into the assembly, activation and inhibition of the DSR2 anti-phage defense system.

MiR-34a-5p suppresses cutaneous squamous cell carcinoma progression by targeting SIRT6.

Cutaneous squamous cell carcinoma (cSCC) is a malignant tumor originating from epidermal or appendageal keratinocytes, with a rising incidence in recent years. Understanding the molecular mechanism driving its development is crucial. This study aims to investigate whether miR-34a-5p is involved in the pathogenesis of cSCC by targeting Sirtuin 6 (SIRT6).The expression levels of miR-34a-5p and SIRT6 were determined in 15 cSCC tissue specimens, 15 normal tissue specimens and cultured cells via real-time polymerase chain reaction (RT-qPCR). Pearson's correlation analysis was conducted to evaluate the relationship between miR-34a-5p and SIRT6 expression levels in cSCC tissues. A431 and SCL-1 cells were transfected with miR-34a-5p mimic, negative control or miR-34a-5p mimic together with recombinant plasmids containing SIRT6 gene. Cell counting kit-8, clone formation assay, wound healing assay, and flow cytometry were employed to assess the effects of these transfections on proliferation, migration, and apoptosis, respectively. The interaction between miR-34a-5p and SIRT6 was characterized using a dual-luciferase reporter assay.MiR-34a-5p expression was down-regulated in cSCC tissues significantly, while the SIRT6 expression was the opposite. A negative correlation was observed between the expression of miR-34a-5p and SIRT6 in cSCC tissues. Furthermore, overexpression of miR-34a-5p led to a significant reduction in the proliferation and migration abilities of A431 and SCL-1 cells, accompanied by an increase in apoptosis levels and a decrease in SIRT6 expression levels. MiR-34a-5p was identified as a direct target of SIRT6. Importantly, overexpression of SIRT6 effectively counteracted the inhibitory effect mediated by miR-34a-5p in cSCC cells.Our findings suggest that miR-34a-5p functions as a tumor suppressor in cSCC cells by targeting SIRT6.

The role of sirtuins and uncoupling proteins on vascular aging: The Northern Manhattan Study experience.

Aging affects all organs. Arteries, in particular, are among the most affected. Vascular aging (VA) is defined as age-associated changes in function and structure of vessels. Classical VA phenotypes are carotid intima-media thickness (IMT), carotid plaque (CP), and arterial stiffness (STIFF). Individuals have different predisposition to these VA phenotypes and their associated risk of cardiovascular events. Some develop an early vascular aging (EVA), and others are protected and identified as having supernormal vascular aging (SUPERNOVA). The mechanisms leading to these phenotypes are not well understood. In the Northern Manhattan Study (NOMAS), we found genetic variants in the 7 Sirtuins (SIRT) and 5 Uncoupling Proteins (UCP) to be differently associated with risk to developing VA phenotypes. In this article, we review the results of genetic-epidemiology studies to better understand which of the single nucleotide polymorphisms (SNPs) in SIRT and UCP are responsible for both EVA and SUPERNOVA.

Exosomes derived from adipose tissue-derived stem cells alleviated H2O2-induced oxidative stress and endothelial-to-mesenchymal transition in human umbilical vein endothelial cells by inhibition of the mir-486-3p/Sirt6/Smad signaling pathway.

Hypertrophic scar (HS) is characterized by excessive collagen deposition and myofibroblasts activation. Endothelial-to-mesenchymal transition (EndoMT) and oxidative stress were pivotal in skin fibrosis process. Exosomes derived from adipose tissue-derived stem cells (ADSC-Exo) have the potential to attenuate EndoMT and inhibit fibrosis. The study revealed reactive oxygen species (ROS) levels were increased during EndoMT occurrence of dermal vasculature of HS. The morphology of endothelial cells exposure to H2O2, serving as an in vitro model of oxidative stress damage, transitioned from a cobblestone-like appearance to a spindle-like shape. Additionally, the levels of endothelial markers decreased in H2O2-treated endothelial cell, while the expression of fibrotic markers increased. Furthermore, H2O2 facilitated the accumulation of ROS, inhibited cell proliferation, retarded its migration and suppressed tube formation in endothelial cell. However, ADSC-Exo counteracted the biological effects induced by H2O2. Subsequently, miRNAs sequencing analysis revealed the significance of mir-486-3p in endothelial cell exposed to H2O2 and ADSC-Exo. Mir-486-3p overexpression enhanced the acceleration of EndoMT, its inhibitors represented the attenuation of EndoMT. Meanwhile, the target regulatory relationship was observed between mir-486-3p and Sirt6, whereby Sirt6 exerted its anti-EndoMT effect through Smad2/3 signaling pathway. Besides, our research had successfully demonstrated the impact of ADSC-Exo and mir-486-3p on animal models. These findings of our study collectively elucidated that ADSC-Exo effectively alleviated H2O2-induced ROS and EndoMT by inhibiting the mir-486-3p/Sirt6/Smad axis.

MiR-148a-3p/SIRT7 Axis Relieves Inflammatory-Induced Endothelial Dysfunction.

In endothelial cells, miR-148a-3p is involved in several pathological pathways, including chronic inflammatory conditions. However, the molecular mechanism of miR-148a-3p in endothelial inflammatory states is, to date, not fully elucidated. To this end, we investigated the involvement of miR-148a-3p in mitochondrial dysfunction and cell death pathways in human aortic endothelial cells (teloHAECs) treated with interleukin-6 (IL-6), a major driver of vascular dysfunction. The results showed that during IL6-activated inflammatory pathways, including increased protein levels of sirtuin 7 (SIRT7) (p < 0.01), mitochondrial stress (p < 0.001), and apoptosis (p < 0.01), a decreased expression of miR-148a-3p was observed (p < 0.01). The employment of a miR-148a mimic counteracted the IL-6-induced cytokine release (p < 0.01) and apoptotic cell death (p < 0.01), and ameliorated mitochondria redox homeostasis and respiration (p < 0.01). The targeted relationship between miR-148a-3p and SIRT7 was predicted by a bioinformatics database analysis and validated via the dual-luciferase reporter assay. Mechanistically, miR-148a-3p targets the 3' untranslated regions of SIRT7 mRNA, downregulating its expression (p < 0.01). Herein, these in vitro results support the role of the miR-148a-3p/SIRT7 axis in counteracting mitochondrial damage and apoptosis during endothelial inflammation, unveiling a novel target for future strategies to prevent endothelial dysfunction.

Leptin attenuates the osteogenic induction potential of BMP9 by increasing ß-catenin malonylation modification via Sirt5 down-regulation.

BMP9 has demonstrated significant osteogenic potential. In this study, we investigated the effect of Leptin on BMP9-induced osteogenic differentiation. Firstly, we found Leptin was decreased during BMP9-induced osteogenic differentiation and serum Leptin concentrations were increased in the ovariectomized (OVX) rats. Both in vitro and in vivo, exogenous expression of Leptin inhibited the process of osteogenic differentiation, whereas silencing Leptin enhanced. Exogenous Leptin could increase the malonylation of ß-catenin. However, BMP9 could increase the level of Sirt5 and subsequently decrease the malonylation of ß-catenin; the BMP9-induced osteogenic differentiation was inhibited by silencing Sirt5. These data suggested that Leptin can inhibit the BMP9-induced osteogenic differentiation, which may be mediated through reducing the activity of Wnt/ß-catenin signalling via down-regulating Sirt5 to increase the malonylation level of ß-catenin partly.

Rutaecarpine Alleviates Early Brain Injury-Induced Inflammatory Response Following Subarachnoid Hemorrhage via SIRT6/NF-[Formula: see text]B Pathway.

Subarachnoid hemorrhage (SAH), a specific subtype of cerebrovascular accident, is characterized by the extravasation of blood into the interstice between the brain and its enveloping delicate tissues. This pathophysiological phenomenon can precipitate an early brain injury (EBI), which is characterized by inflammation and neuronal death. Rutaecarpine (Rut), a flavonoid compound discovered in various plants, has been shown to have protective effects against SAH-induced cerebral insult in rodent models. In our study, we used a rodent SAH model to evaluate the effect of Rut on EBI and investigated the effect of Rut on the inflammatory response and its regulation of SIRT6 expression in vitro. We found that Rut exerts a protective effect on EBI in SAH rats, which is partly due to its ability to inhibit the inflammatory response. Notably, Rut up-regulated Sirtuin 6 (SIRT6) expression, leading to an increase in H3K9 deacetylation and inhibition of nuclear factor-kappa B (NF-[Formula: see text]B) transcriptional activation, thereby mediating the inflammatory response. In addition, further data showed that SIRT6 was proven to mediate the regulation of Rut on the microglial inflammatory response. These findings highlight the importance of SIRT6 in the regulation of inflammation and suggest a potential mechanism for the protective effect of Rut on EBI. In summary, Rut may have the potential to prevent and treat SAH-induced brain injury by interacting with SIRT6. Our findings may provide a new therapeutic strategy for the treatment of SAH-induced EBI.

Gene expression patterns of Sirtuin family members (SIRT1 TO SIRT7): Insights into pathogenesis and prognostic of Myelodysplastic neoplasm.

To assess and validate the gene expression profile of SIRTs (SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7) in relation to the pathogenesis and prognostic progression of Myelodysplastic neoplasm (MDS). Eighty bone marrow samples of patients with de novo MDS were diagnosed according to WHO 2022 and IPSS-R criteria. Ten bone marrow samples were obtained from elderly healthy volunteers and used as control samples. Gene expression levels of all SIRTs were assessed using RT-qPCR assays. Downregulation of SIRT2 (p = 0.009), SIRT3 (p = 0.048), SIRT4 (p = 0.049), SIRT5 (p = 0.046), SIRT6 (p = 0.043), and SIRT7 (p = 0.047) was identified in MDS patients compared to control individuals. Also, we identified that while SIRT2-7 genes are typically down-regulated in MDS patients compared to normal controls, there are relative expression variations among MDS patient subgroups. Specifically, SIRT4 (p = 0.029) showed increased expression in patients aged 60 or above, and both SIRT2 (p = 0.016) and SIRT3 (p = 0.036) were upregulated in patients with hemoglobin levels below 8 g/dL. SIRT2 (p = 0.045) and SIRT3 (p = 0.033) were highly expressed in patients with chromosomal abnormalities. Different SIRTs exhibited altered expression patterns concerning specific MDS clinical and prognostic characteristics. The downregulation in SIRTs genes (e.g., SIRT2 to SIRT7) expression in Brazilian MDS patients highlights their role in the disease's development. The upregulation of SIRT2 and SIRT3 in severe anemia patients suggests a potential link to manage iron overload-related complications in transfusion-dependent patients. Moreover, the association of SIRT2/SIRT3 with genomic instability and their role in MDS progression signify promising areas for future research and therapeutic targets. These findings underscore the importance of SIRT family in understanding and addressing MDS, offering novel clinical, prognostic, and therapeutic insights for patients with this condition.

Targeting NAD+ Metabolism: Preclinical Insights into Potential Cancer Therapy Strategies.

NAD+ is one of the most important metabolites for cellular activities, and its biosynthesis mainly occurs through the salvage pathway using the nicotinamide phosphoribosyl transferase (NAMPT) enzyme. The main nicotinamide adenine dinucleotide (NAD) consumers, poly-ADP-ribose-polymerases and sirtuins enzymes, are heavily involved in DNA repair and chromatin remodeling. Since cancer cells shift their energy production pathway, NAD levels are significantly affected. NAD's roles in cell survival led to the use of NAD depletion in cancer therapies. NAMPT inhibition (alone or in combination with other cancer therapies, including endocrine therapy and chemotherapy) results in decreased cell viability and tumor burden for many cancer types. Many NAMPT inhibitors (NAMPTi) tested before were discontinued due to toxicity; however, a novel NAMPTi, KPT-9274, is a promising, low-toxicity option currently in clinical trials.

Hepatic Sirt6 activation abrogates acute liver failure.

Acute liver failure (ALF) is a deadly illness due to insufficient detoxification in liver induced by drugs, toxins, and other etiologies, and the effective treatment for ALF is very limited. Among the drug-induced ALF, acetaminophen (APAP) overdose is the most common cause. However, the molecular mechanisms underlying APAP hepatoxicity remain incompletely understood. Sirtuin 6 (Sirt6) is a stress responsive protein deacetylase and plays an important role in regulation of DNA repair, genomic stability, oxidative stress, and inflammation. Here, we report that genetic and pharmacological activation of Sirt6 protects against ALF in mice. We first observed that Sirt6 expression was significantly reduced in the liver tissues of human patients with ALF and mice treated with an overdose of APAP. Then we developed an inducible Sirt6 transgenic mice for Cre-mediated overexpression of the human Sirt6 gene in systemic (Sirt6-Tg) and hepatic-specific (Sirt6-HepTg) manners. Both Sirt6-Tg mice and Sirt6-HepTg mice exhibited the significant protection against APAP hepatoxicity. In contrast, hepatic-specific Sirt6 knockout mice exaggerated APAP-induced liver damages. Mechanistically, Sirt6 attenuated APAP-induced hepatocyte necrosis and apoptosis through downregulation of oxidative stress, inflammation, the stress-activated kinase JNK activation, and apoptotic caspase activation. Moreover, Sirt6 negatively modulated the level and activity of poly (ADP-ribose) polymerase 1 (PARP1) in APAP-treated mouse liver tissues. Importantly, the specific Sirt6 activator MDL-800 exhibited better therapeutic potential for APAP hepatoxicity than the current drug acetylcysteine. Furthermore, in the model of bile duct ligation induced ALF, hepatic Sirt6-KO exacerbated, but Sirt6-HepTg mitigated liver damage. Collectively, our results demonstrate that Sirt6 protects against ALF and suggest that targeting Sirt6 activation could be a new therapeutic strategy to alleviate ALF.

Sirt7/HIC1 complex participates in hyperglycaemia-mediated EndMT via modulation of SDC1 expression in diabetic kidney disease and metabolic memory.

Diabetic kidney disease (DKD), a primary microvascular complication arising from diabetes, may result in end-stage renal disease. Epigenetic regulation of endothelial mesenchymal transition (EndMT) has been recently reported to exert function in metabolic memory and DKD. Here, we investigated the mechanism which Sirt7 modulated EndMT in human glomerular endothelial cells (HGECs) in the occurrence of metabolic memory in DKD. Lower levels of SDC1 and Sirt7 were noted in the glomeruli of both DKD patients and diabetes-induced renal injury rats, as well as in human glomerular endothelial cells (HGECs) with high blood sugar. Endothelial-to-mesenchymal transition (EndMT) was sustained despite the normalization of glycaemic control. We also found that Sirt7 overexpression associated with glucose normalization promoted the SDC1 expression and reversed EndMT in HGECs. Furthermore, the sh-Sirt7-mediated EndMT could be reversed by SDC1 overexpression. The ChIP assay revealed enrichment of Sirt7 and H3K18ac in the SDC1 promoter region. Furthermore, hypermethylated in cancer 1 (HIC1) was found to be associated with Sirt7. Overexpression of HIC1 with normoglycaemia reversed high glucose-mediated EndMT in HGECs. The knockdown of HIC1-mediated EndMT was reversed by SDC1 upregulation. In addition, the enrichment of HIC1 and Sirt7 was observed in the same promoter region of SDC1. The overexpressed Sirt7 reversed EndMT and improved renal function in insulin-treated diabetic models. This study demonstrated that the hyperglycaemia-mediated interaction between Sirt7 and HIC1 exerts a role in the metabolic memory in DKD by inactivating SDC1 transcription and mediating EndMT despite glucose normalization in HGECs.

Dual modifying of MAVS at lysine 7 by SIRT3-catalyzed deacetylation and SIRT5-catalyzed desuccinylation orchestrates antiviral innate immunity.

To effectively protect the host from viral infection while avoiding excessive immunopathology, the innate immune response must be tightly controlled. However, the precise regulation of antiviral innate immunity and the underlying mechanisms remain unclear. Here, we find that sirtuin3 (SIRT3) interacts with mitochondrial antiviral signaling protein (MAVS) to catalyze MAVS deacetylation at lysine residue 7 (K7), which promotes MAVS aggregation, as well as TANK-binding kinase I and IRF3 phosphorylation, resulting in increased MAVS activation and enhanced type I interferon signaling. Consistent with these findings, loss of Sirt3 in mice and zebrafish renders them more susceptible to viral infection compared to their wild-type (WT) siblings. However, Sirt3 and Sirt5 double-deficient mice exhibit the same viral susceptibility as their WT littermates, suggesting that loss of Sirt5 in Sirt3-deficient mice may counteract the increased viral susceptibility displayed in Sirt3-deficient mice. Thus, we not only demonstrate that SIRT3 positively regulates antiviral immunity in vitro and in vivo, likely via MAVS, but also uncover a previously unrecognized mechanism by which SIRT3 acts as an accelerator and SIRT5 as a brake to orchestrate antiviral innate immunity.

[The Effect of SIRT5 Deletion on Recovery of Hematopoietic Stem Cells after Injury in Mouse].

OBJECTIVE: To investigate the effect of deacylase Sirtuin 5 in the recovery of hematopoietic stem cells (HSCs) after treated by 5-FU in mouse. METHODS: Flow cytometry was used to analyze the effect of SIRT5 deletion on the proportion of hematopoietic stem/progenitor cells (HSPCs) in bone marrow (BM), the proportion of T cells, B cells and myeloid cells (TBM) in peripheral blood (PB) and spleen, and the development of T cells in thymus. Mouse were treated with 5-FU to study the effect of SIRT5 deletion on the cell cycle, apoptosis and the proportion of HSPCs in BM. The effect of SIRT5 deletion on the proliferation of HSCs was analyzed by flow sorting in vitro. RESULTS: SIRT5 deletion did not affect the development of T cells in thymus and the proportion of TBM cells in PB and spleen compared with wild type mice. SIRT5 deletion increased proportion of HSPCs in BM. After 5-FU treatment, the proportion of HSCs in SIRT5 deletion mice was significant decreased (P < 0.05), the HSPC in SIRT5 deletion mice was activated from G0 to G1 phase (P < 0.05), and the proportion of early apoptosis increased (P < 0.05). By monoclonal culture in vitro, the ability of HSCs to form clones in SIRT5 deletion mice was decreased significantly (P < 0.05). CONCLUSION: SIRT5 deletion lead to a decreased the ability of HSCs to clone in vitro. SIRT5 deletion is not conducive to the recovery of HSPCs injury in mice under hematopoietic stress.

Epigenetic mechanism of SET7/9-mediated histone methylation modification in high glucose-induced ferroptosis in retinal pigment epithelial cells.

Ferroptosis of the retinal pigment epithelial (RPE) cells leads to retinal neuron injury and even visual loss. Our study aims to investigate the role of the SET domain with lysine methyltransferase 7/9 (SET7/9) in regulating high glucose (HG)-induced ferroptosis in RPE cells. The cell model was established by HG treatment. The levels of SET7/9 and Sirtuin 6 (SIRT6) were inhibited and Runt-related transcription factor 1 (RUNX1) was overexpressed through cell transfection, and then their levels in ARPE-19 cells were detected. Cell viability and apoptosis was detected. The levels of reactive oxygen species, malondialdehyde, glutathione, ferrous ion, glutathione peroxidase 4, and acyl-CoA synthetase long-chain family member 4 were detected. SET7/9 and trimethylation of histone H3 at lysine 4 (H3K4me3) levels in the RUNX1 promoter region and RUNX1 level in the SIRT6 promoter region were measured. The relationship between RUNX1 and SIRT6 was verified. SET7/9 and RUNX1 were highly expressed while SIRT6 was poorly expressed in HG-induced ARPE-19 cells. SET7/9 inhibition increased cell viability and inhibited cell apoptosis and ferroptosis. Mechanistically, SET7/9 increased H3K4me3 on the RUNX1 promoter to promote RUNX1, and RUNX1 repressed SIRT6 expression. Overexpression of RUNX1 or silencing SIRT6 partially reversed the inhibitory effect of SET7/9 silencing on HG-induced ferroptosis. In conclusion, SET7/9 promoted ferroptosis of RPE cells through the SIRT6/RUNX1 pathway.

Mitochondrial and Nuclear DNA Variants in Amyotrophic Lateral Sclerosis: Enrichment in the Mitochondrial Control Region and Sirtuin Pathway Genes in Spinal Cord Tissue.

Amyotrophic Lateral Sclerosis (ALS) is a progressive disease with prevalent mitochondrial dysfunctions affecting both upper and lower motor neurons in the motor cortex, brainstem, and spinal cord. Despite mitochondria having their own genome (mtDNA), in humans, most mitochondrial genes are encoded by the nuclear genome (nDNA). Our study aimed to simultaneously screen for nDNA and mtDNA genomes to assess for specific variant enrichment in ALS compared to control tissues. Here, we analysed whole exome (WES) and whole genome (WGS) sequencing data from spinal cord tissues, respectively, of 6 and 12 human donors. A total of 31,257 and 301,241 variants in nuclear-encoded mitochondrial genes were identified from WES and WGS, respectively, while mtDNA reads accounted for 73 and 332 variants. Despite technical differences, both datasets consistently revealed a specific enrichment of variants in the mitochondrial Control Region (CR) and in several of these genes directly associated with mitochondrial dynamics or with Sirtuin pathway genes within ALS tissues. Overall, our data support the hypothesis of a variant burden in specific genes, highlighting potential actionable targets for therapeutic interventions in ALS.

Silencing KLF6 Alleviates Cigarette Smoke Extract-Induced Mitochondrial Dysfunction in Bronchial Epithelial Cells by SIRT4 Upregulation.

Background: The incidence of chronic obstructive pulmonary disease (COPD) is increasing year by year. Kruppel-like factor 6 (KLF6) plays an important role in inflammatory diseases. However, the regulatory role of KLF6 in COPD has not been reported so far. Methods: The viability of human bronchial epithelial cells BEAS-2B induced by cigarette smoke extract (CSE) was detected by CCK-8 assay. The protein expression of KLF6 and sirtuin 4 (SIRT4) was appraised with Western blot. RT-qPCR and Western blot were applied to examine the transfection efficacy of sh-KLF6 and Oe-KLF6. Cell apoptosis was detected using flow cytometry. The levels of inflammatory factors IL-6, TNF-α and IL-1ß were assessed with ELISA assay. DCFH-DA staining was employed for the detection of ROS activity and the levels of oxidative stress markers SOD, CAT and MDA were estimated with corresponding assay kits. The mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) content and Complex I activity were evaluated with JC-1 staining, ATP colorimetric/fluorometric assay kit and Complex I enzyme activity microplate assay kit. With the application of mitochondrial permeability transition pore detection kit, mPTP opening was measured. Luciferase report assay was employed to evaluate the activity of SIRT4 promoter and chromatin immunoprecipitation (ChIP) to verify the binding ability of KLF6 and SIRT4 promoter. Results: KLF6 expression was significantly elevated in CSE-induced cells. KLF6 was confirmed to suppress SIRT4 transcription. Interference with KLF6 expression significantly inhibited cell viability damage, cell apoptosis, inflammatory response, oxidative stress and mitochondrial dysfunction in CSE-induced BEAS-2B cells, which were all reversed by SIRT4 overexpression. Conclusion: Silencing KLF6 alleviated CSE-induced mitochondrial dysfunction in bronchial epithelial cells by SIRT4 upregulation.

SIRT5 promote malignant advancement of chordoma by regulating the desuccinylation of c-myc.

Chordoma is a relatively rare and locally aggressive malignant tumor. Sirtuin (SIRT)5 plays pivotal roles in various tumors, but the role of SIRT5 in chordoma has not been found. This study was performed to investigate the regulatory effects of SIRT5 on cell proliferation, migration, and invasion and the underlying mechanism in chordoma. A xenograft tumor mouse model was established to assess tumor growth. Reverse transcription-quantitative polymerase chain reaction was used to analyze the mRNA levels of SIRT5 and c-myc. The effects of SIRT5 and c-myc on cell proliferation, migration, and invasion of chordoma cells were detected by cell counting kit-8, colony formation, and Transwell assays. The interaction between SIRT5 and c-myc was evaluated by co-immunoprecipitation (IP) assay. The succinylation of c-myc was analyzed by IP and Western blot. The results showed that SIRT5 expression was upregulated in chordoma tissues and cells. SIRT5 interacted with c-myc to inhibit the succinylation of c-myc at K369 site in human embryonic kidney (HEK)-293T cells. Silencing of SIRT5 suppressed the cell proliferation, migration, and invasion of chordoma cells, while the results were reversed after c-myc overexpression. Moreover, silencing SIRT5 suppressed tumor growth in mice. These findings suggested that SIRT5 promoted the malignant advancement of chordoma by regulating the desuccinylation of c-myc.

The sirtuin-associated human senescence program converges on the activation of placenta-specific gene PAPPA.

Sirtuins are pro-longevity genes with chromatin modulation potential, but how these properties are connected is not well understood. Here, we generated a panel of isogeneic human stem cell lines with SIRT1-SIRT7 knockouts and found that any sirtuin deficiency leads to accelerated cellular senescence. Through large-scale epigenomic analyses, we show how sirtuin deficiency alters genome organization and that genomic regions sensitive to sirtuin deficiency are preferentially enriched in active enhancers, thereby promoting interactions within topologically associated domains and the formation of de novo enhancer-promoter loops. In all sirtuin-deficient human stem cell lines, we found that chromatin contacts are rewired to promote aberrant activation of the placenta-specific gene PAPPA, which controls the pro-senescence effects associated with sirtuin deficiency and serves as a potential aging biomarker. Based on our survey of the 3D chromatin architecture, we established connections between sirtuins and potential target genes, thereby informing the development of strategies for aging interventions.

SIRT3 and SIRT4 double-genes remodeled the mitochondrial network to induce hepatocellular carcinoma cell line differentiation and suppress malignant phenotypes.

Tumor cells with damaged mitochondria undergo metabolic reprogramming, but gene therapy targeting mitochondria has not been comprehensively reported. In this study, plasmids targeting the normal hepatocyte cell line (L-O2) and hepatocellular carcinoma cell line were generated using three genes SIRT3, SIRT4, and SIRT5. These deacetylases play a variety of regulatory roles in cancer and are related to mitochondrial function. Compared with L-O2, SIRT3 and SIRT4 significantly ameliorated mitochondrial damage in HCCLM3, Hep3B and HepG2 cell lines and regulated mitochondrial biogenesis and mitophagy, respectively. We constructed double-gene plasmid for co-express SIRT3 and SIRT4 using the internal ribosome entry site (IRES). The results indicated that the double-gene plasmid effectively expressed SIRT3 and SIRT4, significantly improved mitochondrial quality and function, and reduced mtDNA level and oxidative stress in HCC cells. MitoTracker analysis revealed that the mitochondrial network was restored. The proliferation, migration capabilities of HCC cells were reduced, whereas their differentiation abilities were enhanced. This study demonstrated that the use of IRES-linked SIRT3 and SIRT4 double-gene vectors induced the differentiation of HCC cells and inhibited their development by ameliorating mitochondrial dysfunction. This intervention helped reverse metabolic reprogramming, and may provide a groundbreaking new framework for HCC treatment.