Upregulated microRNA-125b-5p in patients with asthma-COPD overlap mediates oxidative stress and late apoptosis via targeting IL6R/TRIAP1 signaling.

BACKGROUND: Among patients with chronic obstructive pulmonary disease (COPD), some have features of both asthma and COPD-a condition categorized as asthma-COPD overlap (ACO). Our aim was to determine whether asthma- or COPD-related microRNAs (miRNAs) play a role in the pathogenesis of ACO. METHODS: A total of 22 healthy subjects and 27 patients with ACO were enrolled. We selected 6 miRNAs that were found to correlate with COPD and asthma. The expression of miRNAs and target genes was analyzed using quantitative reverse-transcriptase polymerase chain reaction. Cell apoptosis and intracellular reactive oxygen species production were evaluated using flow cytometry. In vitro human monocytic THP-1 cells and primary normal human bronchial epithelial (NHBE) cells under stimuli with cigarette smoke extract (CSE) or ovalbumin (OVA) allergen or both were used to verify the clinical findings. RESULTS: We identified the upregulation of miR-125b-5p in patients with ACO and in THP-1 cells stimulated with CSE plus OVA allergen. We selected 16 genes related to the miR-125b-5p pathway and found that IL6R and TRIAP1 were both downregulated in patients with ACO and in THP-1 cells stimulated with CSE plus OVA. The percentage of late apoptotic cells increased in the THP-1 cell culture model when stimulated with CSE plus OVA, and the effect was reversed by transfection with miR-125b-5p small interfering RNA (siRNA). The percentage of reactive oxygen species-producing cells increased in the NHBE cell culture model when stimulated with CSE plus OVA, and the effect was reversed by transfection with miR-125b-5p siRNA. In NHBE cells, siRNA transfection reversed the upregulation of STAT3 under CSE+OVA stimulation. CONCLUSIONS: Our study revealed that upregulation of miR-125b-5p in patients with ACO mediated late apoptosis in THP-1 cells and oxidative stress in NHBE cells via targeting IL6R and TRIAP1. STAT3 expression was also regulated by miR-125b-5p.

Long non-coding RNA FKSG29 regulates oxidative stress and endothelial dysfunction in obstructive sleep apnea.

Altered expressions of pro-/anti-oxidant genes are known to regulate the pathophysiology of obstructive sleep apnea (OSA).We aim to explore the role of a novel long non-coding (lnc) RNA FKSG29 in the development of intermittent hypoxia with re-oxygenation (IHR)-induced endothelial dysfunction in OSA. Gene expression levels of key pro-/anti-oxidant genes, vasoactive genes, and the FKSG29 were measured in peripheral blood mononuclear cells from 12 subjects with primary snoring (PS) and 36 OSA patients. Human monocytic THP-1 cells and human umbilical vein endothelial cells (HUVEC) were used for gene knockout and double luciferase under IHR exposure. Gene expression levels of the FKSG29 lncRNA, NOX2, NOX5, and VEGFA genes were increased in OSA patients versus PS subjects, while SOD2 and VEGFB gene expressions were decreased. Subgroup analysis showed that gene expression of the miR-23a-3p, an endogenous competitive microRNA of the FKSG29, was decreased in sleep-disordered breathing patients with hypertension versus those without hypertension. In vitro IHR experiments showed that knock-down of the FKSG29 reversed IHR-induced ROS overt production, early apoptosis, up-regulations of the HIF1A/HIF2A/NOX2/NOX4/NOX5/VEGFA/VEGFB genes, and down-regulations of the VEGFB/SOD2 genes, while the protective effects of FKSG29 knock-down were abolished by miR-23a-3p knock-down. Dual-luciferase reporter assays confirmed that FKSG29 was a sponge of miR-23a-3p, which regulated IL6R directly. Immunofluorescence stain further demonstrated that FKSGH29 knock-down decreased IHR-induced uptake of oxidized low density lipoprotein and reversed IHR-induced IL6R/STAT3/GATA6/ICAM1/VCAM1 up-regulations. The findings indicate that the combined RNA interference may be a novel therapy for OSA-related endothelial dysfunction via regulating pro-/anti-oxidant imbalance or targeting miR-23a-IL6R-ICAM1/VCAM1 signaling.

MicroRNA-23a-3p Down-Regulation in Active Pulmonary Tuberculosis Patients with High Bacterial Burden Inhibits Mononuclear Cell Function and Phagocytosis through TLR4/TNF-α/TGF-ß1/IL-10 Signaling via Targeting IRF1/SP1.

The aim of this study is to explore the role of microRNAs (miR)-21/23a/146a/150/155 targeting the toll-like receptor pathway in active tuberculosis (TB) disease and latent TB infection (LTBI). Gene expression levels of the five miRs and predicted target genes were assessed in peripheral blood mononuclear cells from 46 patients with active pulmonary TB, 15 subjects with LTBI, and 17 non-infected healthy subjects (NIHS). THP-1 cell lines were transfected with miR-23a-3p mimics under stimuli with Mycobacterium TB-specific antigens. Both miR-155-5p and miR-150-5p gene expressions were decreased in the active TB group versus the NIHS group. Both miR-23a-3p and miR-146a-5p gene expressions were decreased in active TB patients with high bacterial burden versus those with low bacterial burden or control group (LTBI + NIHS). TLR2, TLR4, and interleukin (IL)10 gene expressions were all increased in active TB versus NIHS group. MiR-23a-3p mimic transfection reversed ESAT6-induced reduction of reactive oxygen species generation, and augmented ESAT6-induced late apoptosis and phagocytosis, in association with down-regulations of the predicted target genes, including tumor necrosis factor (TNF)-α, TLR4, TLR2, IL6, IL10, Notch1, IL6R, BCL2, TGF-ß1, SP1, and IRF1. In conclusion, the down-regulation of miR-23a-3p in active TB patients with high bacterial burden inhibited mononuclear cell function and phagocytosis through TLR4/TNF-α/TGF-ß1/IL-10 signaling via targeting IRF1/SP1.

miR-21-5p Under-Expression in Patients with Obstructive Sleep Apnea Modulates Intermittent Hypoxia with Re-Oxygenation-Induced-Cell Apoptosis and Cytotoxicity by Targeting Pro-Inflammatory TNF-α-TLR4 Signaling.

The purpose of this study is to explore the anti-inflammatory role of microRNAs (miR)-21 and miR-23 targeting the TLR/TNF-α pathway in response to chronic intermittent hypoxia with re-oxygenation (IHR) injury in patients with obstructive sleep apnea (OSA). Gene expression levels of the miR-21/23a, and their predicted target genes were assessed in peripheral blood mononuclear cells from 40 treatment-naive severe OSA patients, and 20 matched subjects with primary snoring (PS). Human monocytic THP-1 cell lines were induced to undergo apoptosis under IHR exposures, and transfected with miR-21-5p mimic. Both miR-21-5p and miR-23-3p gene expressions were decreased in OSA patients as compared with that in PS subjects, while TNF-α gene expression was increased. Both miR-21-5p and miR-23-3p gene expressions were negatively correlated with apnea hypopnea index and oxygen desaturation index, while TNF-α gene expression positively correlated with apnea hypopnea index. In vitro IHR treatment resulted in decreased miR-21-5p and miR-23-3p expressions. Apoptosis, cytotoxicity, and gene expressions of their predicted target genes-including TNF-α, ELF2, NFAT5, HIF-2α, IL6, IL6R, EDNRB, and TLR4-were all increased in response to IHR, while all were reversed with miR-21-5p mimic transfection under IHR condition. The findings provide biological insight into mechanisms by which IHR-suppressed miRs protect cell apoptosis via inhibit inflammation, and indicate that over-expression of the miR-21-5p may be a new therapy for OSA.

HOXA9 is a novel myopia risk gene.

PURPOSE: A recent meta-analysis revealed PAX6 as a risk gene for myopia. There is a link between PAX6 and HOXA9. Furthermore, HOXA9 has been reported to activate TGF-ß that is a risk factor for myopia. We speculate HOXA9 may participate in myopia development. METHODS: The Singapore GUSTO birth cohort provides data on children's cycloplegic refraction measured at age of 3 years and their methylation profile based on the umbilical cord DNA. The HOXA9 expression levels were measured in the eyes of mono-ocular form deprivation myopia in mice. The plasmid with the mouse HOXA9 cDNA was constructed and then transfected to mouse primary retinal pigment epithelial (RPE) cells. The expression levels of myopia-related genes and cell proliferation were measured in the HOXA9-overexpressed RPE cells. RESULTS: A total of 519 children had data on methylation profile and cycloplegic refraction. The mean spherical equivalent refraction (SE) was 0.90D. Among 8 SE outliers (worse than -2D), 7 children had HOXA9 hypomethylation. The HOXA9 levels in the retina of myopic eyes was 2.65-fold (p = 0.029; paired t-test) higher than the uncovered fellow eyes. When HOXA9 was over-expressed in the RPE cells, TGF-ß, MMP2, FGF2 and IGF1R expression levels were dose-dependently increased by HOXA9. However, over-expression of HOXA9 had no significant influence on IGF1 or HGF expression. In addition, HOXA9 also increased RPE proliferation. CONCLUSION: Based on the human, animal and cellular data, the transcription factor HOXA9 may promote the expression of pro-myopia genes and RPE proliferation, which eventually contribute to myopia development.

Epigenetics: A Potential Mechanism Involved in the Pathogenesis of Various Adverse Consequences of Obstructive Sleep Apnea.

Epigenetics is defined as the heritable phenotypic changes which do not involve alterations in the DNA sequence, including histone modifications, non-coding RNAs, and DNA methylation. Recently, much attention has been paid to the role of hypoxia-mediated epigenetic regulation in cancer, pulmonary hypertension, adaptation to high altitude, and cardiorenal disease. In contrast to sustained hypoxia, chronic intermittent hypoxia with re-oxygenation (IHR) plays a major role in the pathogenesis of various adverse consequences of obstructive sleep apnea (OSA), resembling ischemia re-perfusion injury. Nevertheless, the role of epigenetics in the pathogenesis of OSA is currently underexplored. This review proposes that epigenetic processes are involved in the development of various adverse consequences of OSA by influencing adaptive potential and phenotypic variability under conditions of chronic IHR. Improved understanding of the interaction between genetic and environmental factors through epigenetic regulations holds great value to give deeper insight into the mechanisms underlying IHR-related low-grade inflammation, oxidative stress, and sympathetic hyperactivity, and clarify their implications for biomedical research.

MicroRNA let-7g possesses a therapeutic potential for peripheral artery disease.

Peripheral artery disease (PAD) is a manifestation of systemic atherosclerosis and conveys a significant health burden globally. Critical limb ischaemia encompasses the most severe consequence of PAD. Our previous studies indicate that microRNA let-7g prevents atherosclerosis and improves endothelial functions. This study aimed to investigate whether and how let-7g therapy may improve blood flow to ischaemic limbs. The present study shows that let-7g has multiple pro-angiogenic effects on mouse ischaemic limb model and could be a potential therapeutic agent for PAD. Mice receiving intramuscular injection of let-7g had more neovascularization, better local perfusion and increased recruitment of endothelial progenitor cells after hindlimb ischaemia. The therapeutic effects of let-7g's on angiogenesis are mediated by multiple regulatory machinery. First, let-7g increased expression of vascular endothelial growth factor-A (VEGF-A) and VEGF receptor-2 (VEGFR-2) through targeting their upstream regulators HIF-3α and TP53. In addition, let-7g affected the splicing factor SC35 which subsequently enhanced the alternative splicing of VEGF-A from the anti-angiogenic isoform VEGF-A165b towards the pro-angiogenic isoform VEGF-A164a . The pleiotropic effects of let-7g on angiogenesis imply that let-7g may possess a therapeutic potential in ischaemic diseases.

microRNA let-7g suppresses PDGF-induced conversion of vascular smooth muscle cell into the synthetic phenotype.

Platelet-derived growth factor (PDGF) can promote vascular smooth muscle cells (VSMCs) to switch from the quiescent contractile phenotype to synthetic phenotype, which contributes to atherosclerosis. We aimed to investigate the role of microRNA let-7g in phenotypic switching. Bioinformatics prediction was used to find let-7g target genes in the PDGF/mitogen-activated protein kinase kinase kinase 1 (MEKK1)/extracellular signal-regulated kinase (ERK)/Krüppel-like factor-4 (KLF4) signalling pathway that affects VSMC phenotypic switching. The luciferase reporter assay and let-7g transfection were used to confirm let-7g target genes. Two contractile proteins alpha-smooth muscle actin (α-SMA) and calponin were VSMC-specific genes and were measured as the indicators for VSMC phenotype. Lentivirus carrying the let-7g gene was injected to apolipoprotein E knockout (apoE-/- ) mice to confirm let-7g's effect on preventing atherosclerosis. Through the PDGF/MEKK1/ERK/KLF4 signalling pathway, PDGF-BB can inhibit α-SMA and calponin. The PDGFB and MEKK1 genes were predicted to harbour let-7g binding sites, which were confirmed by our reporter assays. Transfection of let-7g to VSMC also reduced PDGFB and MEKK1 levels. Moreover, we showed that let-7g decreased phosphorylated-ERK1/2 while had no effect on total ERK1/2. KLF4 can reduce VSMC-specific gene expression by preventing myocardin-serum response factor (SRF) complex from associating with these gene promoters. The immunoprecipitation assay showed that let-7g decreased the interaction between KLF4 and SRF. Further experiments demonstrated that let-7g can increase α-SMA and calponin levels to maintain VSMC in the contractile status. Injection of lentivirus carrying let-7g gene increased let-7g's levels in aorta and significantly decreased atherosclerotic plaques in the apoE-/- mice. We demonstrated that let-7g reduces the PDGF/MEKK1/ERK/KLF4 signalling to maintain VSMC in the contractile status, which further reduce VSMC atherosclerotic change.

Purification and characterization of a phospholipase by Photobacterium damselae subsp. piscicida from cobia Rachycentron canadum.

Toxicity of the extracellular products (ECPs) and the lethal attributes of phospholipase secreted by pathogenic Photobacterium damselae subsp. piscicida from cobia Rachycentron canadum was studied. An extracellular lethal toxin in the ECPs was partially purified by using Fast Protein Liquid Chromatography system. A protein band (27 kDa) exhibited phospholipase activity on Native-PAGE (by 0.3% egg yolk agar-overlay), was excised and eluted. The pI value of the purified phospholipase was determined as 3.65 and was determined as a phospholipase C by using the Amplex™ Red phosphatidylcholine -Specific phospholipase C Assay kit. The phospholipase showed maximum activity at temperature around 4-40 °C and maximal activity at pH between 8 and 9. The enzyme was inhibited by ethylenediamine-tetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS); but was activated by Ca(2+) and Mg(2+) and inactivated by Zn(2+) and Cu(2+) . Both the ECPs and phospholipase were hemolytic against erythrocytes of cobia and lethal to the fish with LD50 values of 3.25 and 0.91 µg protein g(-1) fish, respectively. In toxicity neutralization test, the rabbit antisera against the phospholipase could neutralize the toxicity of ECPs, indicating that the phospholipase is a major extracellular toxin produced by the bacterium.

Sex Differences in the Expression of Cardiac Remodeling and Inflammatory Cytokines in Patients with Obstructive Sleep Apnea and Atrial Fibrillation.

Although there is a link between obstructive sleep apnea (OSA) and atrial fibrillation (AF) and numerous investigations have examined the mechanism of AF development in OSA patients, which includes cardiac remodeling, inflammation, and gap junction-related conduction disorder, there is limited information regarding the differences between the sexes. This study analyzes the impact of sex differences on the expression of cardiac remodeling, inflammatory cytokines, and gap junctions in patients with OSA and AF. A total of 154 individuals diagnosed with sleep-related breathing disorders (SRBDs) were enrolled in the study and underwent polysomnography and echocardiography. Significant OSA was defined as an apnea-hypopnea index (AHI) of ≥15 per hour. Exosomes were purified from the plasma of all SRBD patients and incubated in HL-1 cells to investigate their effects on inflammatory cytokines and GJA1 expression. The differences in cardiac remodeling and expression of these biomarkers in both sexes were analyzed. Of the 154 enrolled patients, 110 patients were male and 44 patients were female. The LA sizes and E/e' ratios of male OSA patients with concomitant AF were greater than those of control participants and those without AF (all p < 0.05). Meanwhile, female OSA patients with AF had a lower left ventricular ejection fraction than those OSA patients without AF and control subjects (p < 0.05). Regarding the expression of inflammatory cytokines and GJA1, the mRNA expression levels of GJA1 were lower and those of IL-1ß were higher in those male OSA patients with AF than in those male OSA patients without AF and control subjects (p < 0.05). By contrast, mRNA expression levels of HIF-1α were higher in those female OSA patients with and without AF than in control subjects (p < 0.05). In conclusion, our study revealed sex-specific differences in the risk factors and biomarkers associated with AF development in patients with OSA.

Biochemical characterization of a phospholipase A2 from Photobacterium damselae subsp. piscicida.

Photobacterium damselae subsp. piscicida (Phdp) is the causative agent of fish photobacteriosis (pasteurellosis) in cultured cobia (Rachycentron canadum) in Taiwan. A component was purified from the extracellular products (ECP) of the bacterium strain 9205 by fast protein liquid chromatography (FPLC) and identified as a phospholipase. An N-terminal sequence of 10 amino acid residues, QDQPNLDPGK, was determined by mass spectroscopy (MS) and found to be identical with that of another Phdp phospholipase (GenBank accession no. BAB85814) at positions 21 to 30. The corresponding gene sequence of the phospholipase (GenBank accession no. AB071137) was employed to design primers for amplification of the sequence by the polymerase chain reaction (PCR). The PCR products were transformed into Escherichia coli, and a recombinant protein product was obtained which was purified as a His-tag fusion protein by Ni-metal affinity chromatography. A single 43-kDa band was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Phosphatidylcholine was degraded by this protein to lysophosphatidylcholine and a fatty acid. These products were characterized by thin-layer (TLC) and gas chromatography (GC), respectively, allowing the identification of the protein as a phospholipase A2. The recombinant protein had maximum enzymatic activity between pH 4 and 7, and at 40 degrees C. The activity was inhibited by Zn(2+) and Cu(2+), activated by Ca(2+) and Mg(2+), and completely inactivated by dexamethasone and p-bromophenacyl bromide. A rabbit antiserum against the recombinant protein neutralized the phospholipase A2 activity in the ECP of Phdp strain 9205 and the recombinant protein itself. The recombinant protein was toxic to cobia of about 5 g weight with an LD50 value between 2 and 4 microg protein/g fish. The results revealed phospholipase A2 as a fish toxin in the ECP of Phdp strain 9205.

Autophagy impairment in patients with obstructive sleep apnea modulates intermittent hypoxia-induced oxidative stress and cell apoptosis via hypermethylation of the ATG5 gene promoter region.

BACKGROUND: Autophagy is a catabolic process that recycles damaged organelles and acts as a pro-survival mechanism, but little is known about autophagy dysfunction and epigenetic regulation in patients with obstructive sleep apnea (OSA). METHODS: Protein/gene expressions and DNA methylation levels of the autophagy-related genes (ATG) were examined in blood leukocytes from 64 patients with treatment-naïve OSA and 24 subjects with primary snoring (PS). RESULTS: LC3B protein expression of blood monocytes, and ATG5 protein expression of blood neutrophils were decreased in OSA patients versus PS subjects, while p62 protein expression of cytotoxic T cell was increased, particularly in those with nocturia. ATG5, ULK1, and BECN1 gene expressions of peripheral blood mononuclear cells were decreased in OSA patients versus PS subjects. LC3B gene promoter regions were hypermethylated in OSA patients, particularly in those with excessive daytime sleepiness, while ATG5 gene promoter regions were hypermethylated in those with morning headache or memory impairment. LC3B protein expression of blood monocytes and DNA methylation levels of the LC3B gene promoter region were negatively and positively correlated with apnea hyponea index, respectively. In vitro intermittent hypoxia with re-oxygenation exposure to human THP-1/HUVEC cell lines resulted in LC3B/ATG5/ULK1/BECN1 down-regulations and p62 up-regulation along with increased apoptosis and oxidative stress, while rapamycin and umbilical cord-mesenchymal stem cell treatment reversed these abnormalities through de-methylation of the ATG5 gene promoter. CONCLUSIONS: Impaired autophagy activity in OSA patients was regulated by aberrant DNA methylation, correlated with clinical phenotypes, and contributed to increased cell apoptosis and oxidative stress. Autophagy enhancers may be novel therapeutics for OSA-related neurocognitive dysfunction.

Unraveling the Pathogenesis of Asthma and Chronic Obstructive Pulmonary Disease Overlap: Focusing on Epigenetic Mechanisms.

Asthma and COPD overlap (ACO) is characterized by patients presenting with persistent airflow limitation and features of both asthma and COPD. It is associated with a higher frequency and severity of exacerbations, a faster lung function decline, and a higher healthcare cost. Systemic inflammation in COPD and asthma is driven by type 1 T helper (Th1) and Th2 immune responses, respectively, both of which may contribute to airway remodeling in ACO. ACO-related biomarkers can be classified into four categories: neutrophil-mediated inflammation, Th2 cell responses, arachidonic acid-eicosanoids pathway, and metabolites. Gene-environment interactions are key contributors to the complexity of ACO and are regulated by epigenetic mechanisms, including DNA methylation, histone modifications, and non-coding RNAs. Thus, this review focuses on the link between epigenetics and ACO, and outlines the following: (I) inheriting epigenotypes without change with environmental stimuli, or epigenetic changes in response to long-term exposure to inhaled particles plus intermittent exposure to specific allergens; (II) epigenetic markers distinguishing ACO from COPD and asthma; (III) potential epigenetic drugs that can reverse oxidative stress, glucocorticoid insensitivity, and cell injury. Improved understanding of the epigenetic regulations holds great value to give deeper insight into the mechanisms, and clarify their implications for biomedical research in ACO.

The Impact of Intermittent Hypoxemia on Left Atrial Remodeling in Patients with Obstructive Sleep Apnea Syndrome.

Obstructive sleep apnea syndrome (OSAS) is a significant risk factor for left atrial (LA) remodeling. Intermittent hypoxemia occurs during the sleep cycle in patients with OSAS and plays a crucial role in cardiovascular pathologies such as stroke, arrhythmia, and coronary artery disease. However, there is very little information about the role of intermittent hypoxemia in LA remodeling in patients with OSAS. In total, 154 patients with sleep-related breathing disorders (SRBD) were prospectively recruited for this study. All enrolled SRBD patients underwent polysomnography and echocardiography. Significant OSAS was defined as an oxygen desaturation index (ODI) of ≥10 per hour. Intermittent hypoxia/reoxygenation (IHR) stimulation was used to test the effect of hypoxia on the viability, reactive oxygen species, apoptosis, and inflammation-associated cytokine expression in the HL-1 cell line. To investigate the effect of patients' exosomes on HIF-1 and inflammation-associated cytokine expression, as well as the relationship between ODI and their expression, exosomes were purified from the plasma of 95 patients with SRBD and incubated in HL-1 cells. The LA size was larger in patients with significant OSAS than in those without. There was a significant association between ODI, lowest SpO2, mean SpO2, and LA size (all p < 0.05) but not between the apnea-hypopnea index and LA size. IHR condition caused increased LDH activity, reactive oxygen species (ROS) levels, and apoptosis in HL-1 cells and decreased cellular viability (all p < 0.05). The expression of HIF-1α, TNF-α, IL-6, and TGF-ß increased in the IHR condition compared with the control (all p < 0.05). The expression of HIF-1α, IL-1ß, and IL-6 increased in the HL-1 cells incubated with exosomes from those patients with significant OSAS than those without (all p < 0.05). There was a significantly positive correlation between ODI and the expression of HIF-1α, TNF-α, IL-1ß, IL-6, and TGF-ß; a significantly negative correlation between mean SpO2 and IL-6 and TGF-ß; and a significantly negative correlation between the lowest SpO2 and HIF-1α (all p < 0.05). In conclusion, intermittent hypoxemia was strongly associated with LA remodeling, which might be through increased ROS levels, LDH activity, apoptosis, and the expression of HIF-1α and inflammation-associated cytokines.

Next generation sequencing reveals miR-431-3p/miR-1303 as immune-regulating microRNAs for active tuberculosis.

OBJECTIVES: RNA therapeutics is an emerging field that widens the range of treatable targets and would improve disease outcome through bypassing the antibiotic bactericidal targets to kill Mycobacterium tuberculosis (M.tb). METHODS: We screened for microRNA with immune-regulatory functions against M.tb by next generation sequencing of peripheral blood mononuclear cells, followed by validation in an independent cohort. RESULTS: Twenty three differentially expressed microRNAs were identified between 12 active pulmonary TB patients and 4 healthy subjects, and 35 microRNAs before and after 6-month anti-TB therapy. Enriched predicted target pathways included proteoglycan, HIF-1 signaling, longevity-regulating, central carbon metabolism, and autophagy. We validated miR-431-3p down-regulation and miR-1303 up-regulation accompanied with corresponding changes in their predicted target genes in an independent validation cohort of 46 active TB patients, 30 latent TB infection subjects, and 24 non-infected healthy subjects. In vitro experiments of transfections with miR-431-3p mimic/miR-1303 short interfering RNA in THP-1 cells under ESAT-6 stimuli showed that miR-431-3p and miR-1303 were capable to augment and suppress autophagy/apoptosis/phagocytosis of macrophage via targeting MDR1/MMP16/RIPOR2 and ATG5, respectively. CONCLUSIONS: This study provides a proof of concept for microRNA-based host-directed immunotherapy for active TB disease. The combined miR-431-3p over-expression and miR-1303 knock-down revealed new vulnerabilities of treatment-refractory TB disease.

GJA1 Expression and Left Atrial Remodeling in the Incidence of Atrial Fibrillation in Patients with Obstructive Sleep Apnea Syndrome.

Obstructive sleep apnea syndrome (OSAS) is an important risk factor for atrial fibrillation (AF). GJA1 gene encoding connexin43, a major protein in cardiac gap junctions, plays a crucial role in the synchronized contraction of the heart and in cardiac arrhythmia. However, little is known regarding the role of GJA1 expression in the incidence of AF in patients with OSAS. All prospectively enrolled OSAS patients underwent polysomnography, electrocardiography, a 24-h Holter test, and echocardiography. Moderate-to-severe OSAS was defined as an apnea-hypopnea index (AHI) ≥ 15. Exosomes were purified from the plasma of all OSAS patients and incubated in HL-1 cells to investigate the effect of exosomes from patients with and without AF on GJA1 expression. A total of 129 patients were recruited for this study; 26 were excluded due to an AHI < 15. Of the 103 enrolled patients, 21 had AF, and 82 did not. Multivariate analysis showed diabetes mellitus, lower sleep efficiency, lower left ventricular ejection fraction, and larger left atrial (LA) size were independent predictors of AF occurrence in OSAS patients. The area under the receiver operating characteristic curve for LA with a size ≥38.5 mm for predicting AF occurrence in OSAS patients was 0.795 (95% confidence interval [0.666, 0.925]); p < 0.001). GJA1 expression in HL-1 cells incubated with exosomes from OSAS patients with AF was lower than that with exosomes from patients without AF after controlling for age and sex and was negatively correlated with the AHI and oxygen desaturation index (ODI), especially during the non-rapid eye movement period (NREM) of OSAS patients with AF (all p < 0.05). LA size was an independent predictor of AF occurrence in OSAS patients. The AHI and ODI in the NREM period of OSAS patients with AF were negatively correlated with GJA1 expression in HL-1 cells, which offers a hint that GJA1 may play a crucial role in the development of AF in patients with OSAS.

H3K23/H3K36 hypoacetylation and HDAC1 up-regulation are associated with adverse consequences in obstructive sleep apnea patients.

The aim of this study is to determine the roles of global histone acetylation (Ac)/methylation (me), their modifying enzymes, and gene-specific histone enrichment in obstructive sleep apnea (OSA). Global histone modifications, and their modifying enzyme expressions were assessed in peripheral blood mononuclear cells from 56 patients with OSA and 16 matched subjects with primary snoring (PS). HIF-1α gene promoter-specific H3K36Ac enrichment was assessed in another cohort (28 OSA, 8 PS). Both global histone H3K23Ac and H3K36Ac expressions were decreased in OSA patients versus PS subjects. H3K23Ac expressions were further decreased in OSA patients with prevalent hypertension. HDAC1 expressions were higher in OSA patients, especially in those with excessive daytime sleepiness, and reduced after more than 6 months of continuous positive airway pressure treatment. H3K79me3 expression was increased in those with high C-reactive protein levels. Decreased KDM6B protein expressions were noted in those with a high hypoxic load, and associated with a higher risk for incident cardiovascular events or hypertension. HIF-1α gene promoter-specific H3K36Ac enrichment was decreased in OSA patients versus PS subjects. In vitro intermittent hypoxia with re-oxygenation stimuli resulted in HDAC1 over-expression and HIF-1α gene promoter-specific H3K36Ac under-expression, while HDAC1 inhibitor, SAHA, reversed oxidative stress through inhibiting NOX1. In conclusions, H3K23/H3K36 hypoacetylation is associated with the development of hypertension and disease severity in sleep-disordered breathing patients, probably through up-regulation of HDAC1, while H3K79 hypermethylation is associated with higher risk of cardiovascular diseases, probably through down-regulation of KDM6B.

MicroRNA Sequencing Analysis in Obstructive Sleep Apnea and Depression: Anti-Oxidant and MAOA-Inhibiting Effects of miR-15b-5p and miR-92b-3p through Targeting PTGS1-NF-κB-SP1 Signaling.

The aim of this study was to identify novel microRNAs related to obstructive sleep apnea (OSA) characterized by intermittent hypoxia with re-oxygenation (IHR) injury. Illumina MiSeq was used to identify OSA-associated microRNAs, which were validated in an independent cohort. The interaction between candidate microRNA and target genes was detected in the human THP-1, HUVEC, and SH-SY5Y cell lines. Next-generation sequencing analysis identified 22 differentially expressed miRs (12 up-regulated and 10 down-regulated) in OSA patients. Enriched predicted target pathways included senescence, adherens junction, and AGE-RAGE/TNF-α/HIF-1α signaling. In the validation cohort, miR-92b-3p and miR-15b-5p gene expressions were decreased in OSA patients, and negatively correlated with an apnea hypopnea index. PTGS1 (COX1) gene expression was increased in OSA patients, especially in those with depression. Transfection with miR-15b-5p/miR-92b-3p mimic in vitro reversed IHR-induced early apoptosis, reactive oxygen species production, MAOA hyperactivity, and up-regulations of their predicted target genes, including PTGS1, ADRB1, GABRB2, GARG1, LEP, TNFSF13B, VEGFA, and CXCL5. The luciferase assay revealed the suppressed PTGS1 expression by miR-92b-3p. Down-regulated miR-15b-5p/miR-92b-3p in OSA patients could contribute to IHR-induced oxidative stress and MAOA hyperactivity through the eicosanoid inflammatory pathway via directly targeting PTGS1-NF-κB-SP1 signaling. Over-expression of the miR-15b-5p/miR-92b-3p may be a new therapeutic strategy for OSA-related depression.

Epigenome-wide association study on asthma and chronic obstructive pulmonary disease overlap reveals aberrant DNA methylations related to clinical phenotypes.

We hypothesized that epigenetics is a link between smoking/allergen exposures and the development of Asthma and chronic obstructive pulmonary disease (ACO). A total of 75 of 228 COPD patients were identified as ACO, which was independently associated with increased exacerbations. Microarray analysis identified 404 differentially methylated loci (DML) in ACO patients, and 6575 DML in those with rapid lung function decline in a discovery cohort. In the validation cohort, ACO patients had hypermethylated PDE9A (+ 30,088)/ZNF323 (- 296), and hypomethylated SEPT8 (- 47) genes as compared with either pure COPD patients or healthy non-smokers. Hypermethylated TIGIT (- 173) gene and hypomethylated CYSLTR1 (+ 348)/CCDC88C (+ 125,722)/ADORA2B (+ 1339) were associated with severe airflow limitation, while hypomethylated IFRD1 (- 515) gene with frequent exacerbation in all the COPD patients. Hypermethylated ZNF323 (- 296) / MPV17L (+ 194) and hypomethylated PTPRN2 (+ 10,000) genes were associated with rapid lung function decline. In vitro cigarette smoke extract and ovalbumin concurrent exposure resulted in specific DNA methylation changes of the MPV17L / ZNF323 genes, while 5-aza-2'-deoxycytidine treatment reversed promoter hypermethylation-mediated MPV17L under-expression accompanied with reduced apoptosis and decreased generation of reactive oxygen species. Aberrant DNA methylations may constitute a determinant for ACO, and provide a biomarker of airflow limitation, exacerbation, and lung function decline.

Aberrant DNA methylation levels of the formyl peptide receptor 1/2/3 genes are associated with obstructive sleep apnea and its clinical phenotypes.

BACKGROUND: FPR1 over-expression and insufficiency of FPR2 and FPR3 are associated with disease severity of obstructive sleep apnea (OSA). We hypothesized that epigenetic modification of the FPR1/2/3 genes may underlie intermittent hypoxia with re-oxygenation (IHR) injury in OSA. METHODS: DNA methylation levels over 17 CpG sites of the FPR1/2/3 genes and their gene expression levels in the peripheral blood mononuclear cells were determined in 40 treatment-naïve OSA patients, 12 severe OSA patients under long-term continuous positive airway pressure treatment, 16 primary snoring (PS) subjects, and 10 healthy non-snorers (HS). RESULTS: Both -524 and -264 CpG sites of the FPR1 gene were hypomethylated in treatment-naïve OSA versus HS, while -264 CpG site methylation level was negatively correlated with FPR1/FPR3 gene expression ratio and associated with prevalent diabetes mellitus. Both +8802 and +8845 CpG sites of the FPR2 gene were hypermethylated in treatment-naive OSA versus HS, while hypermethylated +9132 and +9150 CpG sites were both associated with prevalent hypertension. FPR3 gene expression and DNA methylation levels over -842/-516 CpG sites of the FPR3 gene were both decreased in treatment-naive OSA versus HS, while hypermethylated -429 CpG site was associated with elevated serum C-reactive protein level. In vitro IHR stimuli in human monocytic THP-1 cells resulted in gene promoter hypomethylation-mediated FPR1 over-expression, increased production of reactive oxygen species, and increased cell apoptosis, which could be reversed with re-methylation agent, folic acid, treatment. CONCLUSIONS: Aberrant DNA methylation patterns of the FPR1/2/3 gene promoters contribute to disease severity and diabetes mellitus or cardiovascular disease in OSA patients, probably through regulating FPR1/2/3 gene expressions.