Correction: Mitochondria-targeting nanozyme alleviating temporomandibular joint pain by inhibiting the TNFα/NF-κB/NEAT1 pathway.

Correction for 'Mitochondria-targeting nanozyme alleviating temporomandibular joint pain by inhibiting the TNFα/NF-κB/NEAT1 pathway' by Qian Bai et al., J. Mater. Chem. B, 2023, https://doi.org/10.1039/d3tb00929g.

Baicalin-modified polyethylenimine for miR-34a efficient and safe delivery.

The security and efficiency of gene delivery vectors are inseparable for the successful construction of a gene delivery vector. This work provides a practical method to construct a charge-regulated, hydrophobic-modified, and functionally modified polyethylenimine (PEI) with effective gene delivery and perfect transfection performance through a condensation reaction, named BA-PEI. The carrier was shown to possess a favorable compaction of miRNAs into positively charged nanoparticles with a hydrodynamic size of approximately 100 nm. Additionally, BA-PEI possesses perfect degradability, which benefits the release of miR-34a from the complexes. In A549 cells, the expression level of the miR-34a gene was checked by Western blotting, which reflects the transfection efficiency of BA-PEI/miR-34a. When miR-34a is delivered to the cell, the perfect anti-tumor ability of the BA-PEI/miR-34a complex was systematically evaluated with the suppressor tumor gene miR-34a system in vitro and in vivo. BA-PEI-mediated miR-34a gene transfection is more secure and effective than the commercial transfection reagent, thus providing a novel approach for miR-34a-based gene therapy.

DNMT1 determines osteosarcoma cell resistance to apoptosis by associatively modulating DNA and mRNA cytosine-5 methylation.

Cellular apoptosis is a central mechanism leveraged by chemotherapy to treat human cancers. 5-Methylcytosine (m5C) modifications installed on both DNA and mRNA are documented to regulate apoptosis independently. However, the interplay or crosstalk between them in cellular apoptosis has not yet been explored. Here, we reported that promoter methylation by DNMT1 coordinated with mRNA methylation by NSun2 to regulate osteosarcoma cell apoptosis. DNMT1 was induced during osteosarcoma cell apoptosis triggered by chemotherapeutic drugs, whereas NSun2 expression was suppressed. DNMT1 was found to repress NSun2 expression by methylating the NSun2 promoter. Moreover, DNMT1 and NSun2 regulate the anti-apoptotic genes AXL, NOTCH2, and YAP1 through DNA and mRNA methylation, respectively. Upon exposure to cisplatin or doxorubicin, DNMT1 elevation drastically reduced the expression of these anti-apoptotic genes via enhanced promoter methylation coupled with NSun2 ablation-mediated attenuation of mRNA methylation, thus rendering osteosarcoma cells to apoptosis. Collectively, our findings establish crosstalk of importance between DNA and RNA cytosine methylations in determining osteosarcoma resistance to apoptosis during chemotherapy, shedding new light on future treatment of osteosarcoma, and adding additional layers to the control of gene expression at different epigenetic levels.

Mitochondria-targeting nanozyme alleviating temporomandibular joint pain by inhibiting the TNFα/NF-κB/NEAT1 pathway.

Inflammatory cytokines that are secreted into the spinal trigeminal nucleus caudalis (Sp5C) may augment inflammation and cause pain associated with temporomandibular joint disorders (TMD). In a two-step process, we attached triphenylphosphonium (TPP) to the surface of a cubic liposome metal-organic framework (MOF) loaded with ruthenium (Ru) nanozyme. The design targeted mitochondria and was designated Mito-Ru MOF. This structure scavenges free radicals and reactive oxygen species (ROS) and alleviates oxidative stress. The present study aimed to investigate the effects and mechanisms by which Mito-Ru MOF ameliorates TMD pain. Intra-temporomandibular joint (TMJ) injections of complete Freund's adjuvant (CFA) induced inflammatory pain for ≥10 d in the skin areas innervated by the trigeminal nerve. Tumor necrosis factor-alpha (TNF-α), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), long non-coding RNA nuclear paraspeckle assembly transcript 1 (lncRNA NEAT1), and ROS also have been proved to be significantly upregulated in the Sp5C of TMD mice. Moreover, a single Mito-Ru MOF treatment alleviated TMD pain for 3 d and downregulated TNF-α, NF-κB, lncRNA NEAT1, and ROS. NF-κB knockdown downregulated NEAT1 in the TMD mice. Hence, Mito-Ru MOF inhibited the production of ROS and alleviated CFA-induced TMD pain via the TNF-α/NF-κB/NEAT1 pathway. Therefore, Mito-Ru MOF could effectively treat the pain related to TMD and other conditions associated with severe acute inflammatory activation.

Circulating cardiac MicroRNAs safeguard against dilated cardiomyopathy.

BACKGROUND: Cardiac-resident or -enriched microRNAs (miRNAs) could be released into the bloodstream becoming circulating cardiac miRNAs, which are increasingly recognized as non-invasive and accessible biomarkers of multiple heart diseases. However, dilated cardiomyopathy (DCM)-associated circulating miRNAs (DACMs) and their roles in DCM pathogenesis remain largely unexplored. METHODS: Two human cohorts, consisting of healthy individuals and DCM patients, were enrolled for serum miRNA sequencing (10 vs. 10) and quantitative polymerase chain reaction validation (46 vs. 54), respectively. Rigorous screening strategy was enacted to define DACMs and their potentials for diagnosis. DCM mouse model, different sources of cardiomyocytes, adeno-associated virus 9 (AAV9), gene knockout, RNAscope miRNA in situ hybridization, mRFP-GFP-LC3B reporter, echocardiography and transmission electron microscopy were adopted for mechanistic explorations. RESULTS: Serum miRNA sequencing revealed a unique expression pattern for DCM circulating miRNAs. DACMs miR-26a-5p, miR-30c-5p, miR-126-5p and miR-126-3p were found to be depleted in DCM circulation as well as heart tissues. Their expressions in circulation and heart tissues were proven to be correlated significantly, and a combination of these miRNAs was suggested potential values for DCM diagnosis. FOXO3, a predicted common target, was experimentally demonstrated to be co-repressed within cardiomyocytes by these DACMs except miR-26a-5p. Delivery of a combination of miR-30c-5p, miR-126-5p and miR-126-3p into the murine myocardium via AAV9 carrying an expression cassette driven by cTnT promoter, or cardiac-specific knockout of FOXO3 (Myh6-CreERT2 , FOXO3 flox+/+ ) dramatically attenuated cardiac apoptosis and autophagy involved in DCM progression. Moreover, competitively disrupting the interplay between DACMs and FOXO3 mRNA by specifically introducing their interacting regions into murine myocardium crippled the cardioprotection of DACMs against DCM. CONCLUSIONS: Circulating cardiac miRNA-FOXO3 axis plays a pivotal role in safeguarding against myocardial apoptosis and excessive autophagy in DCM development, which may provide serological cues for DCM non-invasive diagnosis and shed light on DCM pathogenesis and therapeutic targets.

Novel rare genetic variants of familial and sporadic pulmonary atresia identified by whole-exome sequencing.

Pulmonary atresia (PA) is a severe cyanotic congenital heart disease. Although some genetic mutations have been described to be associated with PA, the knowledge of pathogenesis is insufficient. The aim of this research was to use whole-exome sequencing (WES) to determine novel rare genetic variants in PA patients. We performed WES in 33 patients (27 patient-parent trios and 6 single probands) and 300 healthy control individuals. By applying an enhanced analytical framework to incorporate de novo and case-control rare variation, we identified 176 risk genes (100 de novo variants and 87 rare variants). Protein‒protein interaction (PPI) analysis and Genotype-Tissue Expression analysis revealed that 35 putative candidate genes had PPIs with known PA genes with high expression in the human heart. Expression quantitative trait loci analysis revealed that 27 genes that were identified as novel PA genes that could be affected by the surrounding single nucleotide polymorphism were screened. Furthermore, we screened rare damaging variants with a threshold of minor allele frequency at 0.5% in the ExAC_EAS and GnomAD_exome_EAS databases, and the deleteriousness was predicted by bioinformatics tools. For the first time, 18 rare variants in 11 new candidate genes have been identified that may play a role in the pathogenesis of PA. Our research provides new insights into the pathogenesis of PA and helps to identify the critical genes for PA.

CIRBP-OGFR axis safeguards against cardiomyocyte apoptosis and cardiotoxicity induced by chemotherapy.

Cold-inducible RNA-binding protein (CIRBP) is documented to be required for maintaining cardiac function, however, its role in chemotherapy-induced cardiotoxicity remains obscured. Herein, we report that CIRBP decreases cardiomyocyte apoptosis and attenuates cardiotoxicity through disrupting OGF-OGFR signal. CIRBP deficiency is involved in diverse chemotherapeutic agents induced cardiomyocyte apoptosis. Delivery of exogenous CIRBP to the mouse myocardium significantly mitigated doxorubicin-induced cardiac apoptosis and dysfunction. Specifically, OGFR was identified as a downstream core effector responsible for chemotherapy-induced cardiomyocyte apoptosis. CIRBP was shown to interact with OGFR mRNA and to repress OGFR expression by reducing mRNA stability. CIRBP-mediated cytoprotection against doxorubicin-induced cardiac apoptosis was demonstrated to largely involve OGFR repression by CIRBP. NTX as a potent antagonist of OGFR successfully rescued CIRBP ablation-rendered susceptibility to cardiac dyshomeostasis upon exposure to doxorubicin, whereas another antagonist ALV acting only on opioid receptors did not. Taken together, our results demonstrate that CIRBP confers myocardium resistance to chemotherapy-induced cardiac apoptosis and dysfunction by dampening OGF/OGFR axis, shedding new light on the mechanisms of chemo-induced cardiotoxicity and providing insights into the development of an efficacious cardioprotective strategy for cancer patients.

Suppression of Myocardial Hypoxia-Inducible Factor-1α Compromises Metabolic Adaptation and Impairs Cardiac Function in Patients With Cyanotic Congenital Heart Disease During Puberty.

BACKGROUND: Cyanotic congenital heart disease (CCHD) is a complex pathophysiological condition involving systemic chronic hypoxia (CH). Some patients with CCHD are unoperated for various reasons and remain chronically hypoxic throughout their lives, which heightens the risk of heart failure as they age. Hypoxia activates cellular metabolic adaptation to balance energy demands by accumulating hypoxia-inducible factor 1-α (HIF-1α). This study aims to determine the effect of CH on cardiac metabolism and function in patients with CCHD and its association with age. The role of HIF-1α in this process was investigated, and potential therapeutic targets were explored. METHODS: Patients with CCHD (n=25) were evaluated for cardiac metabolism and function with positron emission tomography/computed tomography and magnetic resonance imaging. Heart tissue samples were subjected to metabolomic and protein analyses. CH rodent models were generated to enable continuous observation of changes in cardiac metabolism and function. The role of HIF-1α in cardiac metabolic adaptation to CH was investigated with genetically modified animals and isotope-labeled metabolomic pathway tracing studies. RESULTS: Prepubertal patients with CCHD had glucose-dominant cardiac metabolism and normal cardiac function. In comparison, among patients who had entered puberty, the levels of myocardial glucose uptake and glycolytic intermediates were significantly decreased, but fatty acids were significantly increased, along with decreased left ventricular ejection fraction. These clinical phenotypes were replicated in CH rodent models. In patients with CCHD and animals exposed to CH, myocardial HIF-1α was upregulated before puberty but was significantly downregulated during puberty. In cardiomyocyte-specific Hif-1α-knockout mice, CH failed to initiate the switch of myocardial substrates from fatty acids to glucose, thereby inhibiting ATP production and impairing cardiac function. Increased insulin resistance during puberty suppressed myocardial HIF-1α and was responsible for cardiac metabolic maladaptation in animals exposed to CH. Pioglitazone significantly reduced myocardial insulin resistance, restored glucose metabolism, and improved cardiac function in pubertal CH animals. CONCLUSIONS: In patients with CCHD, maladaptation of cardiac metabolism occurred during puberty, along with impaired cardiac function. HIF-1α was identified as the key regulator of cardiac metabolic adaptation in animals exposed to CH, and pubertal insulin resistance could suppress its expression. Pioglitazone administration during puberty might help improve cardiac function in patients with CCHD.

Notch1 signaling mediated dysfunction of bone marrow mesenchymal stem cells derived from cyanotic congenital heart disease.

Bone marrow mesenchymal stem cells (BMSCs) derived from cyanotic congenital heart disease (CCHD) exhibit deficient multi-lineage differentiation potential due to the abnormal accumulation of D-galactose. However, the underlying mechanisms have not yet been explored. Here, the multi-lineage differentiation potential of the BMSCs from CCHD and non-CCHD (NCHD) patients were assessed. BMSCs from CCHD patients exhibited inferior multi-lineage differentiation potential with reduced Notch1 protein and mRNA level. Bisulfite sequencing PCR results showed the methylation level of Notch1 promoter was raised, which inhibited the binding of NF-Ya. Exposure BMSCs from NCHD patients with D-galactose under hypoxia (4% O2) decreased the expression of Notch1. And activating Notch1 partially restored the deficient BMSCs of CCHD patients. In conclusion, the impaired multi-lineage differentiation potential of BMSCs from CCHD patients is owing to the decreased Notch1 level with a remarkable hypermethylation in its promoter region. Activated Notch1 signaling pathway could partially restore the deficient BMSCs in the CCHD patients, which may provide a new method on cell therapy in patients with CCHD.

Chronic hypoxia-induced Cirbp hypermethylation attenuates hypothermic cardioprotection via down-regulation of ubiquinone biosynthesis.

Therapeutic hypothermia is commonly used during cardiopulmonary bypass (CPB) to protect the heart against myocardial injury in cardiac surgery. Patients who suffer from chronic hypoxia (CH), such as those with certain heart or lung conditions, are at high risk of severe myocardial injury after cardiac surgery, but the underlying mechanisms are unknown. This study tested whether CH attenuates hypothermic cardioprotection during CPB. Using a rat model of CPB, we found that hypothermic cardioprotection was impaired in CH rats but was preserved in normoxic rats. Cardiac proteomes showed that cold-inducible RNA binding protein (CIRBP) was significantly (P = 0.03) decreased in CH rats during CPB. Methylation analysis of neonatal rat cardiomyocytes under CH and myocardium specimens from patients with CH showed that CH induced hypermethylation of the Cirbp promoter region, resulting in its depression and failure to respond to cold stress. Cirbp-knockout rats showed attenuated hypothermic cardioprotection, whereas Cirbp-transgenic rats showed an enhanced response. Proteomics analysis revealed that the cardiac ubiquinone biosynthesis pathway was down-regulated during CPB in Cirbp-knockout rats, resulting in a significantly (P = 0.01) decreased concentration of ubiquinone (CoQ10). Consequently, cardiac oxidative stress was aggravated and adenosine 5'-triphosphate production was impaired, leading to increased myocardial injury during CPB. CoQ10-supplemented cardioplegic solution improved cardioprotection in rats exposed to CH, but its effect was limited in normoxic rats. Our study suggests that an individualized cardioprotection strategy should be used to fully compensate for the consequences of epigenetic modification of Cirbp in patients with CH who require therapeutic hypothermia.

Author Correction: HuR regulates telomerase activity through TERC methylation.

In the original version of this Article, the affiliation details for Fan Yang were incorrectly given as 'Key Laboratory of Regenerative Medicine of Ministry of Education, Institute of Aging and Regenerative Medicine, Jinan University, Guangzhou, 510632, China' and 'Leibniz Institute for Age Research - Fritz Lipmann Institute, Friedrich-Schiller University of Jena, Jena, 07745, Germany'. This has now been corrected in both the PDF and HTML versions of the Article.

HuR regulates telomerase activity through TERC methylation.

Telomerase consists of the catalytic protein TERT and the RNA TERC. Mutations in TERC are linked to human diseases, but the underlying mechanisms are poorly understood. Here we report that the RNA-binding protein HuR associates with TERC and promotes the assembly of the TERC/TERT complex by facilitating TERC C106 methylation. Dyskeratosis congenita (DC)-related TERC U100A mutation impair the association of HuR with TERC, thereby reducing C106 methylation. Two other TERC mutations linked to aplastic anemia and autosomal dominant DC, G107U, and GC107/108AG, likewise disrupt methylation at C106. Loss-of-HuR binding and hence lower TERC methylation leads to decreased telomerase activity and telomere shortening. Furthermore, HuR deficiency or mutation of mTERC HuR binding or methylation sites impair the renewal of mouse hematopoietic stem cells, recapitulating the bone marrow failure seen in DC. Collectively, our findings reveal a novel function of HuR, linking HuR to telomerase function and TERC-associated DC.

Hypoxia induces senescence of bone marrow mesenchymal stem cells via altered gut microbiota.

Systemic chronic hypoxia is a feature of many diseases and may influence the communication between bone marrow (BM) and gut microbiota. Here we analyse patients with cyanotic congenital heart disease (CCHD) who are experiencing chronic hypoxia and characterize the association between bone marrow mesenchymal stem cells (BMSCs) and gut microbiome under systemic hypoxia. We observe premature senescence of BMSCs and abnormal D-galactose accumulation in patients with CCHD. The hypoxia that these patients experience results in an altered diversity of gut microbial communities, with a remarkable decrease in the number of Lactobacilli and a noticeable reduction in the amount of enzyme-degraded D-galactose. Replenishing chronic hypoxic rats with Lactobacillus reduced the accumulation of D-galactose and restored the deficient BMSCs. Together, our findings show that chronic hypoxia predisposes BMSCs to premature senescence, which may be due to gut dysbiosis and thus induced D-galactose accumulation.

RNA methyltransferase NSUN2 promotes stress-induced HUVEC senescence.

The tRNA methyltransferase NSUN2 delays replicative senescence by regulating the translation of CDK1 and CDKN1B mRNAs. However, whether NSUN2 influences premature cellular senescence remains untested. Here we show that NSUN2 methylates SHC mRNA in vitro and in cells, thereby enhancing the translation of the three SHC proteins, p66SHC, p52SHC, and p46SHC. Our results further show that the elevation of SHC expression by NSUN2-mediated mRNA methylation increased the levels of ROS, activated p38MAPK, thereby accelerating oxidative stress- and high-glucose-induced senescence of human vascular endothelial cells (HUVEC). Our findings highlight the critical impact of NSUN2-mediated mRNA methylation in promoting premature senescence.

NSun2 delays replicative senescence by repressing p27 (KIP1) translation and elevating CDK1 translation.

A rise in the levels of the cyclin-dependent kinase (CDK) inhibitor p27KIP1 is important for the growth arrest of senescent cells, but the mechanisms responsible for this increase are poorly understood. Here, we show that the tRNA methyltransferase NSun2 represses the expression of p27 in replicative senescence. NSun2 methylated the 5'-untranslated region (UTR) of p27 mRNA at cytosine C64 in vitro and in cells, thereby repressing the translation of p27. During replicative senescence, increased p27 protein levels were accompanied by decreased NSun2 protein levels. Knockdown of NSun2 in human diploid fibroblasts (HDFs) elevated p27 levels and reduced the expression of CDK1 (encoded by CDK1 mRNA, a previously reported target of NSun2), which in turn further repressed cell proliferation and accelerated replicative senescence, while overexpression of NSun2 exerted the opposite effect. Ectopic overexpression of the p27 5'UTR fragment rescued the effect of NSun2 overexpression in lowering p27, increasing CDK1, promoting cell proliferation, and delaying replicative senescence. Our findings indicate that NSun2-mediated mRNA methylation regulates p27 and CDK1 levels during replicative senescence.

NSun2 Promotes Cell Growth via Elevating Cyclin-Dependent Kinase 1 Translation.

The tRNA methytransferase NSun2 promotes cell proliferation, but the molecular mechanism has not been elucidated. Here, we report that NSun2 regulates cyclin-dependent kinase 1 (CDK1) expression in a cell cycle-dependent manner. Knockdown of NSun2 decreased the CDK1 protein level, while overexpression of NSun2 elevated it without altering CDK1 mRNA levels. Further studies revealed that NSun2 methylated CDK1 mRNA in vitro and in cells and that methylation by NSun2 enhanced CDK1 translation. Importantly, NSun2-mediated regulation of CDK1 expression had an impact on the cell division cycle. These results provide new insight into the regulation of CDK1 during the cell division cycle.

Methylation by NSun2 represses the levels and function of microRNA 125b.

Methylation is a prevalent posttranscriptional modification of RNAs. However, whether mammalian microRNAs are methylated is unknown. Here, we show that the tRNA methyltransferase NSun2 methylates primary (pri-miR-125b), precursor (pre-miR-125b), and mature microRNA 125b (miR-125b) in vitro and in vivo. Methylation by NSun2 inhibits the processing of pri-miR-125b2 into pre-miR-125b2, decreases the cleavage of pre-miR-125b2 into miR-125, and attenuates the recruitment of RISC by miR-125, thereby repressing the function of miR-125b in silencing gene expression. Our results highlight the impact of miR-125b function via methylation by NSun2.

The tRNA methyltransferase NSun2 stabilizes p16INK4 mRNA by methylating the 3'-untranslated region of p16.

The impact of methylation of the 3'-untranslated region (UTR) of a messenger RNA (mRNA) remains largely unknown. Here we show that NSun2, a transfer RNA methyltransferase, inhibits the turnover of p16(INK4) mRNA. Knockdown of NSun2 reduces p16 expression by shortening the half-life of the p16 mRNA, while overexpression of NSun2 stabilizes the p16 mRNA. In vitro methylation assays show that NSun2 methylates the p16 3'UTR at A988. Knockdown of NSun2 reduces the stability of the EGFP-p16 chimeric reporter transcripts bearing wild-type p16 3'UTR, but not p16 3'UTR with a mutant methylation site. Methylation by NSun2 prevents the association of p16 3'UTR with HuR, AUF1 and Ago2/RISC, and prevents the recruitment of EGFP-p16 3'UTR chimeric transcripts to processing bodies. In response to oxidative stress, NSun2 is essential for elevating p16 expression levels. We conclude that NSun2-mediated methylation of the p16 3'UTR is a novel mechanism to stabilize p16 mRNA.