Neutrophilic asthma is a steroid-resistant condition linked to immunometabolic dysregulation. While the NAD+-dependent deacetylase Sirtuin 6 (SIRT6) regulates immune responses, its role in neutrophilic asthma remains unknown. Utilizing multiple human samples and neutrophilic asthma murine model, we identify macrophage SIRT6 as a key regulator that governs airway neutrophil infiltration in severe asthma. Myeloid-specific Sirt6 deletion attenuates allergen-induced airway neutrophil infiltration by suppressing lactate dehydrogenase A (LDHA)-mediated lactate production and neutrophil-recruiting chemokines secretion. Mechanistically, SIRT6 directly interacts with LDHA and deacetylates LDHA at lysine 261 (K261) via SIRT6-N-terminal domain. Lactate accumulation promotes histone H4 lysine 12 (H4K12) lactylation, up-regulating Cxcl1 and Cxcl2 transcription to drive airway neutrophil infiltration. Importantly, we screen flavonoid astragalin as a specific SIRT6 inhibitor that attenuates airway neutrophil infiltration in severe asthmatic mice. Collectively, our findings reveal a critical role of the SIRT6-mediated metabolic reprogramming in neutrophilic asthma and establish SIRT6 as a promising therapeutic target.

Severe asthma frequently manifests as a neutrophilic phenotype associated with dysregulated Th17 cell response, yet the molecular mechanism regulating Th17 cell-driven pathology remain poorly defined. Here, we identified histone deacetylase (HDAC) 10 as a critical regulator of Th17 cell differentiation in severe asthma. HDAC10 expression in CD4+ T cells was upregulated in asthmatic mice and patients with asthma. CD4+ T cell-specific deletion of Hdac10 attenuated neutrophilic airway inflammation by dampening Th17 cell differentiation and subsequent IL-17A secretion. Mechanistically, HDAC10 directly bound to signal transducer and activator of transcription 3 (STAT3) and deacetylated it at lysine 631 (K631) in Th17 cells, a post-translational modification essential for Il-17a transcription and subsequent neutrophilic airway inflammation. Importantly, pharmacological inhibition of HDAC10 abrogated Th17 cell response and neutrophilic airway inflammation. Together, our findings reveal an unrecognized role for HDAC10 in governing Th17 cell pathogenicity, highlighting HDAC10 as a promising therapeutic target for severe asthma.

Dysregulated macrophage polarization is a pivotal driver of airway inflammation in asthma, yet the underlying molecular mechanisms remain incompletely understood. Here, we demonstrate that histone acetyltransferase KAT8 exacerbates allergic airway inflammation by promoting M2 macrophage polarization in asthma. KAT8 expression was significantly upregulated in lung macrophages of asthmatic mice and in bone marrow-derived macrophages (BMDMs) stimulated with house dust mite (HDM). Macrophage-specific KAT8 deficiency attenuated allergic airway inflammation and inhibited M2 macrophage polarization by suppressing signal transducer and activator of transcription 3 (STAT3) signaling. Mechanistically, KAT8 directly interacted with STAT3 and targeted it for acetylation, thereby driving M2 macrophage polarization. Importantly, pharmacological inhibition of KAT8 reduced M2 macrophage polarization and attenuated allergic airway inflammation. These findings establish KAT8 as a critical regulator of macrophage-driven allergic inflammation via STAT3 acetylation, highlighting its potential as a therapeutic target for asthma.

Macrophages play central role in driving airway inflammation during asthma pathogenesis, though the molecular mechanism governing their function remains incompletely understood. Here, we demonstrate that lysine acetyltransferase 8 (KAT8) plays a critical role in papain-induced neutrophilic airway inflammation through post-translational modification of poly(C)-binding protein 1 (PCBP1). We show that papain exposure significantly upregulates KAT8 expression in lung macrophages both in vivo and in vitro. Myeloid-specific Kat8 deficiency conferred protection against papain-induced airway inflammation, reducing CXCL1 and CXCL2 production and consequent neutrophil recruitment. Mechanistically, KAT8 interacted with PCBP1 via its C2HC zinc finger domain and acetylated it at lysine 119 (K119), an essential modification for CXCL1 and CXCL2 secretion. Moreover, KAT8 enhanced M1 macrophage polarization in papain-exposed mice. Importantly, pharmacological inhibition of KAT8 significantly attenuated neutrophil infiltration and allergic airway inflammation in vivo. Our findings establish KAT8 as a pivotal regulator of neutrophilic airway inflammation through PCBP1 acetylation and highlight the KAT8-PCBP1 axis as a promising therapeutic target for severe asthma.

Exposure to fine particulate matter (PM2.5) represents a leading environmental cause of pulmonary inflammation and diseases, yet the underlying cellular mechanisms remain incompletely understood. Here, we identify histone deacetylase 10 (HDAC10) in macrophages as a critical regulator of PM2.5-induced airway inflammation by governing autophagic flux. PM2.5 exposure upregulated HDAC10 expression specifically in lung macrophages both in vivo and in vitro. Myeloid-specific Hdac10 deletion markedly attenuated PM2.5-induced airway inflammation and inflammatory cytokine production by inhibiting macrophage autophagy. Mechanistically, HDAC10 interacted with Beclin1 and deacetylated it at lysine 5 (K5), a modification critical for autophagic flux and subsequent inflammatory responses. Pharmacological inhibition of HDAC10 with salvianolic acid B reduced Beclin1 deacetylation, suppressed macrophage autophagy, and ameliorated PM2.5-induced lung inflammation. Clinically, elevated HDAC10 expression and reduced Beclin1 acetylation were observed in lung tissues from chronic obstructive pulmonary disease (COPD) patients, where HDAC10 mRNA levels correlated positively with the heightened lung inflammation. Our findings reveal a previously unrecognized HDAC10-Beclin1 axis that links PM2.5 exposure to macrophage autophagy and pulmonary inflammation, providing potential therapeutic targets for PM2.5-related respiratory diseases.

Background Aberrant activation of macrophages is associated with pathogenesis of acute lung injury (ALI). However, the potential pathogenesis has not been explored. Objectives We aimed to identify whether histone deacetylase (HDAC) 10 is involved in lipopolysaccharide (LPS)-exposed ALI and reveal the underlying pathogenesis by which it promotes lung inflammation in LPS-exposed ALI via modifying P62 with deacetylation. Methods We constructed an ALI mice model stimulated with LPS to determine the positive effect of Hdac10 deficiency. Moreover, we cultured murine alveolar macrophage cell line (MH-S cells) and primary bone marrow-derived macrophages (BMDMs) to explore the pro-inflammatory activity and mechanism of HDAC10 after LPS challenge. Results HDAC10 expression was increased both in mice lung tissues and macrophage cell lines and promoted inflammatory cytokines production exposed to LPS. Hdac10 deficiency inhibited autophagy and inflammatory response after LPS stimulation. In vivo, Hdac10(fl/fl)-LysMCre mice considerably attenuated lung inflammation and inflammatory cytokines release exposed to LPS. Mechanistically, HDAC10 interacts with P62 and mediates P62 deacetylation at lysine 165 (K165), by which it promotes P62 expression and increases inflammatory cytokines production. Importantly, we identified that Salvianolic acid B (SAB), an HDAC10 inhibitor, reduces lung inflammatory response in LPS-stimulated ALI. Conclusion These results uncover a previously unknown role for HDAC10 in regulating P62 deacetylation and aggravating lung inflammation in LPS-induced ALI, implicating that targeting HDAC10 is an effective therapy for LPS-exposed ALI.

Background: Perturbation of macrophage homeostasis is one of the key mechanisms of airway inflammation in asthma. However, the exact mechanisms remain poorly understood. Objectives: We sought to examine the role of histone deacetylase (HDAC) 10 as an epigenetic regulator that governs macrophage M2 program and promotes airway inflammation in asthma, and to elucidate the underlying mechanisms. Methods: Peripheral blood and airway biopsies were obtained from healthy individuals and asthmatic patients. Asthma was induced by exposure to allergen in mice with myeloid-specific deletion of Hdac10 (Hdac10fl/fl-LysMCre) mice. HDAC10 inhibitor Salvianolic acid B (SAB), STAT3 selective agonist Colivelin, and the specific PI3K/Akt activator 1,3-Dicaffeoylquinic acid (DA) were also used in asthmatic mice. For cell studies, THP1 cells, primary mouse bone marrow derived macrophage (BMDMs) were used and related signaling pathways was investigated. Results: HDAC10 expression was highly expressed by macrophages and promoted M2 macrophage activation and airway inflammation in asthmatic patients and mice. Hdac10fl/fl-LysMCre mice were protected from airway inflammation in experimental asthma model. Hdac10 deficiency significantly attenuated STAT3 expression and decreased M2 macrophage polarization following allergen exposure. Mechanistically, HDAC10 directly binds STAT3 for deacetylation in macrophages, by which it promotes STAT3 expression and activates the macrophage M2 program. Importantly, we identified SAB as a HDAC10 inhibitor that had protective effects against airway inflammation in mice. Conclusions: Our results revealed that HDAC10-STAT3 interaction governs macrophage polarization to promote airway inflammation in asthma, implicating HDAC10 as a therapeutic target.

Dysregulation of IL-17A is closely associated with airway inflammation and remodeling in severe asthma. However, the molecular mechanisms by which IL-17A is regulated remain unclear. Here we identify epithelial sirtuin 6 (SIRT6) as an epigenetic regulator that governs IL-17A pathogenicity in severe asthma. Mice with airway epithelial cell-specific deletion of Sirt6 are protected against allergen-induced airway inflammation and remodeling via inhibiting IL-17A-mediated inflammatory chemokines and mesenchymal reprogramming. Mechanistically, SIRT6 directly interacts with RORγt and mediates RORγt deacetylation at lysine 192 via its PPXY motifs. SIRT6 promotes RORγt recruitment to the IL-17A gene promoter and enhances its transcription. In severe asthma patients, high expression of SIRT6 positively correlates with airway remodeling and disease severity. SIRT6 inhibitor (OSS_128167) treatment significantly attenuates airway inflammation and remodeling in mice. Collectively, these results uncover a function for SIRT6 in regulating IL-17A pathogenicity in severe asthma, implicating SIRT6 as a potential therapeutic target for severe asthma.

Background: Excessive salt intake is considered as an important risk factor for cognitive impairment, which might be the consequence of imbalanced intestinal homeostasis. Objective: To investigate the effects of dietary salt on the gut microbiota and cognitive performance and the underlying mechanisms. Methods: Adult female C57BL/6 mice were maintained on either normal chow (control group, CON) or sodium-rich chow containing 8% NaCl (high-salt diet, HSD) for 8 weeks. Spatial learning and memory ability, short-chain fatty acids (SCFAs) concentrations, gut bacterial flora composition, blood-brain barrier permeability, and proinflammatory cytokine levels and apoptosis in the brain were evaluated. Results: The mice fed a HSD for 8 weeks displayed impaired learning and memory abilities. HSD significantly reduced the proportions of Bacteroidetes (S24-7 and Alloprevotella) and Proteobacteria and increased that of Firmicutes (Lachnospiraceae and Ruminococcaceae). SCFA concentrations decreased in the absolute concentrations of acetate, propionate, and butyrate in the fecal samples from the HSD-fed mice. The HSD induced both BBB dysfunction and microglial activation in the mouse brain, and increased the IL-1β, IL-6, and TNF-α expression levels in the cortex. More importantly, the degree of apoptosis was higher in the cortex and hippocampus region of mice fed the HSD, and this effect was accompanied by significantly higher expression of cleaved caspase-3, caspase-3, and caspase-1. Conclusion: The HSD directly causes cognitive dysfunction in mice by eliciting an inflammatory environment and triggering apoptosis in the brain, and these effects are accompanied by gut dysbiosis, particularly reduced SCFA production.

The identification of new biomarkers (e.g., metabolic biomarkers) will facilitate not only the diagnosis of stroke but also the differentiation of stroke subtypes, especially the discrimination of ischaemic stroke from intracerebral hemorrhage. Herein, we develop for the first time an ultra-high-pressure liquid chromatography tandem mass spectrometry (UHPLC-MS)-based targeted metabolomic method to screen the metabolic biomarkers of stroke and identify the fatty acid metabolite 20-hydroxy-leukotriene B4 (20-OH-LTB4) and its key enzyme cytochrome P450 family 4 subfamily F member 2 (CYP4F2) as the potential biomarkers for differentiating healthy persons, acute ischemic stroke (AIS) patients, and intracerebral hemorrhage stroke (ICH) patients. We evaluated 158 fatty acids and their metabolites in 177 serum samples obtained from 65 healthy volunteers, 70 AIS patients and 42 ICH patients, and identified the potential biomarkers associated with ICH by using multivariate statistical analysis. We found that 20-OH-LTB4 and arachidonic acid can be used to discriminate ICH patients from healthy individuals, and 20-OH-LTB4 and 17, 18-epoxy-eicosatetraenoic acid (7,18-EpETE) can be used to differentiate the subtypes of ICH and AIS. Especially, 20-OH-LTB4 may function as a potential biomarker for ICH diagnosis and risk assessment, and it can discriminate ICH patients from healthy individuals and AIS patients. Moreover, we identified CYP4F2 protein as a potential biomarker of ICH for prevention and treatment assessment. This method may provide a powerful platform for ICH diagnosis, prevention, and treatment assessment.

目的 探讨大气细颗粒物(PM)增强香烟暴露小鼠炎症反应的机制.方法 用野生型(WT)及IL-17A基因敲除(IL-17A-/-)小鼠,按随机数字表法随机分为对照组、熏烟组、PM组、熏烟+PM组,每组6～8只.采用香烟烟雾暴露装置烟熏,气道滴注方法吸入PM,观察气道炎症反应,连续干预3个月后取检.用HE检测肺组织炎症浸润、用ELISA及RT-PCR检测肺组织炎症因子表达、免疫组化检测平滑肌表达、Masson染色观察胶原沉积及细胞流式检测分泌IL-17A的淋巴细胞类型.体外实验,用不同浓度的香烟提取物(CSE)和/或大气细微颗粒(PM)刺激人气道上皮(HBE)细胞,用IL-17A siRNA转染在HBE细胞中敲除IL-17A基因,用RT-PCR检测炎症因子表达.结果 与对照组、熏烟组及PM组比较,熏烟联合PM组小鼠肺组织炎症因子(CXCL1、TFG-β1、IL-6及IL-17A)、胶原沉积及平滑肌表达明显增高.相反,IL-17A-/-小鼠能缓解上述指标.流式细胞检测发现PM主要通过调控CD4+细胞促进IL-17A表达,增强熏烟诱导的炎症反应.在体外,CSE、PM分别干预HBE细胞均能诱导IL-6、IL-8表达,而CSE联合PM干预HBE细胞能进一步增加IL-6、IL-8表达.敲除HBE细胞IL-17A基因后,能缓解IL-6、IL-8表达.结论 PM能诱导IL-17A表达,加剧熏烟小鼠肺组织的炎症反应、胶原沉积及平滑肌增生,提示针对IL-17A信号通路靶向治疗可能对缓解PM导致的慢性阻塞性肺疾病急性加重有效.

Background We have reported that heparin-binding epidermal growth factor (HB-EGF) is increased in patients with chronic obstructive pulmonary disease (COPD) and associated with collagen deposition, but the mechanisms remain unclear. In the present study, we aimed to investigated the inflammatory cytokines secreted by bronchial epithelial cells following exposure to HB-EGF that promoted proliferation and migration of human lung fibroblast. Methods HB-EGF-induced inflammatory cytokines were assayed in two airway epithelial cells (primary human bronchial epithelial cells [HBECs] and BEAS-2B cells). Moreover, the culture supernatants derived from HB-EGF-treated HBECs and BEAS-2B cells were added to human primary lung fibroblasts. The effect of culture supernatants on proliferation and migration of fibroblasts was assessed. Results IL-8 expression was significantly increased in bronchial epithelial cells treated with HB-EGF, which was at least partially dependent on NF-kB pathways activation. HB-EGF-induced IL-8 was found to further promote lung fibroblasts proliferation and migration, and the effects were attenuated after neutralizing IL-8. Conclusions These findings suggest that HB-EGF may be involved in the pathology of airway fibrosis by induction of IL-8 from airway epithelium, subsequently causing lung fibroblasts proliferation and migration. Thus, inhibition of HBEGF and/or IL-8 production could prevent the development of airway fibrosis by modulating fibroblast activation.

Shikonin, a natural naphthoquinone extracted from the roots of Lithospermumery throrhizon, possesses multiple pharmacological properties, including antioxidant, anti-inflammatory and antitumor effects. It has been hypothesized that the properties of shikonin are associated with its oxygen free radical scavenging abilities. However, the mechanism underlying the antioxidant activity of shikonin is not completely understood. The aim of the present study was to investigate the effect of shikonin against H2O2-induced oxidative injury in HT29 cells and to explore the underlying molecular mechanism. The concentration and duration of H2O2 treatment to cause maximal damage, and the effects of shikonin (2.5, 5 or 10 µg/ml) on the activity of H2O2-induced HT29 cells were determined by MTT assay. The apoptotic rate in HT29 cells was determined by annexin V/propidium iodide staining. HT29 cell cycle alteration was also analyzed by propidium iodide staining. Reactive oxygen species (ROS) production was assessed by monitoring 2',7'-dichlorofluorescin in diacetate fluorescence. Mitochondrial membrane potentials were determined by JC-1 staining. The activities of malondialdehyde, superoxide dismutase, caspase-9 and caspase-3 were measured using spectrophotometric assays. The expression levels of Bcl-2, Bax and cytochrome c were determined by western blotting. The results suggested that shikonin increased cell viability, reduced cell apoptosis and increased the proliferation index in H2O2-treated HT29 cells. Shikonin also significantly inhibited increases in intracellular reactive oxygen species (ROS), restored the mitochondrial membrane potential, prevented the release of lactic dehydrogenase and decreased the levels of superoxide dismutase and malondialdehyde in H2O2-induced HT29 cells. Furthermore, shikonin significantly decreased caspase-9 and caspase-3 activities, increased Bcl-2 expression and decreased Bax and cytochrome c expression levels in H2O2-induced HT29 cells. The results indicated that shikonin protected against H2O2-induced oxidative injury by removing ROS, ameliorating mitochondrial dysfunction, attenuating DNA oxidative damage and inhibiting mitochondrial pathway-mediated apoptosis.

MafF is a member of the basic leucine zipper (bZIP) transcription factor Maf family and is commonly downregulated in multiple cancers. But the expression and function of MafF in hepatocellular carcinoma (HCC) remain unclear. In this study, we investigated the relationship between endogenous MafF expression and HCC progression and explored the regulatory mechanism of MafF expression in HCC. We found that MafF decreased in HCC tissues and cells. Lentivirus-mediated MafF overexpression inhibited HCC cell proliferation and induced cell apoptosis. Bioinformatics analysis and luciferase assay identified MafF as a direct target of miR-224-5p. RNA pull-down assay demonstrated that circular RNA circ-ITCH could sponge miR-224-5p specifically in HCC. The rescue experiments further elucidated that the expression and antitumor effects of MafF could be regulated via the circ-ITCH/miR-224-5p axis. This study verified that MafF acted as a tumor suppressor in HCC and revealed the upstream regulation mechanism of MafF, which provided a new perspective for potential therapeutic targets of HCC.

Background: Omega-3 polyunsaturated fatty acids (omega-3 PUFAs) have significant multiple antitumor roles. However, whether epigenetic DNA hydroxymethylation enrolls in the anticancer process of omega- 3 PUFAs is still not clear yet. Objective: To expound the interaction between the anti-tumor role of omega-3 PUFAs and the DNA demethylation pathway and thus provide a firm foundation for deepening our understanding on anticancer mechanism of omega-3 PUFAs. Methods: Colorectal Cancer (CRC) model rats were induced to generate tumor by N-methyl-N-nitrosourea and their counterparts treated with omega-3 PUFAs during the induction. The blood samples from different treatment groups of rats [Normal Control group (NC), colorectal cancer model group (CRC) and omega-3 PUFAs Medication Group (MG)] were used as experimental materials. Genomic 5-hydroxymethylocytosine (5hmC) content was quantified using LC-MS/MS, and the expression of ten-eleven translocation dioxygenase 1 (TET1), catalyzing the generation of 5hmC, was also evaluated by quantitative real-time PCR. Results: We observed lower tumor incidence and small tumor size in MG group when compared with CRC group, supporting the effective anticancer role of omega-3 PUFAs. Due to the formation of CRC, 5hmC level was dramatically dropped in CRC group when compared with the NC group. Notably, 5hmC percentage in MG group remarkably increased close to NC group and was significantly higher than that in the CRC group. Consistent alteration pattern of TET1 expressions in mRNA was also observed in the tested groups of rats. Conclusion: The anticancer effect of omega-3 PUFAs was positively correlated with global 5hmC accumulation and TET1 expression, suggesting DNA hydroxymethylation pathway was factually involved in the anticancer process of omega-3 PUFAs.

Context: Shikonins, a series of natural occurring naphthoquinones extracted from Arnebia euchroma (Royle) Jonst. (Boraginaceae), have antitumor activities and low toxicity. Objective: To illuminate potential activity and mechanism of shikonins against colorectal cancer (CRC). Materials and methods: Five shikonins were isolated from A. euchroma, and elucidated by extensive spectroscopic analysis. Anti-proliferative activities of shikonins (0-100 μg/mL) on human colorectal cells were evaluated by MTT and CCK-8 for 24 or 48 h. Cell apoptosis and cycle distribution were examined by FCM analysis. The expression of PI3K/Akt/mTOR pathway mRNAs and proteins was analysed by RT-PCR and Western blot, respectively. Cell viability, cell apoptosis, cell cycle and protein expression were measured, when co-treated with PI3K/Akt/mTOR pathway inhibitors. The in vivo activity of deoxyshikonin was evaluated using xenograft tumour model. Results: Deoxyshikonin and another four shikonins were isolated and identified. Deoxyshikonin exhibited anti-proliferative activity with IC50 of 10.97 μM against HT29 cells. Moreover, the percentage of early apoptotic cells and G0/G1 cells increased from 1 to 29% and 44 to 67% with 0-50 μg/mL deoxyshikonin, respectively. Deoxyshikonin also down-regulated the expression of PI3K, p-PI3K, Akt, p-Akt308 and mTOR proteins in HT29 and DLD-1 cells. Moreover, LY294002, NVP-BEZ235 and MK-2206 can make deoxyshikonin more cell proliferation inhibited, cell cycle arrested at G0/G1 and apoptosis promoted. In vivo study, the weight of tumour tissues at deoxyshikonin groups was significantly reduced compared with the control group, and PI3K, p-PI3K, Akt, p-Akt308 and mTOR expression was decreased. Discussion and conclusions: We can conclude that deoxyshikonin isolated from Arnebia euchroma inhibited CRC through the PI3K/Akt/mTOR pathway.

目的:探讨湿热对溃疡性结肠炎(UC)大鼠模型的影响.方法:采用内因(高脂高糖饮食)和外因(高湿高温气候)因素刺激,再辅以DNCB/乙酸灌肠建立湿热UC大鼠模型,探讨湿热对UC大鼠证候学、病理学及免疫学指标的变化.结果:湿热UC比单纯UC更早出现与大肠湿热UC患者相似的临床症状,DAI、病理学评分均升高;湿热UC血清SOD、MPO、MIF和组织TXB水平明显高于单纯UC(P ＜0.05);湿热UC血清和组织MPO、P选择素、组织MIF、TXB2与单纯UC表达水平一致,均比正常组升高.结论:血清SOD、MPO、MIF和组织TXB2水平可作为判断湿热病因所致UC的免疫标识物;湿热能促进UC的发生和进一步加重炎症程度,是UC的主要病因.

BackgroundTumor suppressor gene p16 promoter hypermethylation has been widely studied in colorectal cancer (CRC), yet its clinicopathological significance remains controversial. The methylation alterations of other regions within p16 gene are still rarely researched. The present study aimed to explore the methylation changes of p16 gene body in CRC and to find whether they were associated with clinicopathological staging of CRC.MethodsPaired colorectal cancer tissues and corresponding adjacent normal tissues from 30 CRC patients were collected. The methylation levels of two CpG islands within p16 gene body, exon 1 and exon 2, were accurately assessed simultaneously by a LC-MS/MS method. The p16 protein expressions were assessed by immunohistochemistry assay. Statistical analyses were carried out using SPSS 17.0 software. Heat-map analysis was carried out by HemI 1.0 software.ResultsIn the present study, CRC tissues showed more highly methylated than adjacent normal tissues at both CpG islands of p16 gene. And exon 2 hypermethylation was higher and more frequent than exon 1. The ROC curve analysis showed that the simultaneous use of both indicators had excellent sensitivity and specificity for distinguishing CRC tissues and adjacent normal tissues. Following, the methylation level of p16 exon 1/2 was negatively related to p16 protein expression. Further correlation analysis revealed that p16 exon 1 hypermethylation was associated with N/Dukes staging (p=0.033), and p16 exon 2 hypermethylaiton was associated with T staging (p=0.035).ConclusionsThe p16 gene body was remarkably hyper-methylated in CRC tissues and associated with p16 protein expression and cancer clinicopathological staging. The combination of p16 exon 1 and exon 2 could better reflect the overall methylation status of p16 gene body and provide potential biomarkers of CRC.

Ambiguous alteration patterns of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) involved in Alzheimer's disease (AD) obstructed the mechanism investigation of this neurological disorder from epigenetic view. Here, we applied a fully quantitative and validated LC-MS/MS method to determine genomic 5mC and 5hmC in the brain cortex of 3 month-aged (12, 15, and 18 month) AD model mouse and found significant increases of 5mC and 5hmC levels in different months of AD mouse when compared with age-matched wild-type control and exhibited rising trend from 12-month to 18-month AD mouse, thereby supporting genomic DNA methylation and hydroxymethylation were positively correlated with developing AD.

Tumor suppressor genes (TSGs) with DNA methylation has been suggested as effective biomarkers for cancer identification and promoter methylation in TSGs has been widely discussed. However, the feasibility of TSG exon methylation used for discriminating cancers was not fully clarified yet. In this study, genomic DNA was isolated from colorectal tissues and responding cancer-adjacent counterparts, and then bisulfite-converted. The exon 1 region of four popular TSGs including ALX4, FBN1, MLH1 and BCL2 was amplified from bisulfite-treated DNA, using nest- polymerase chain reaction (PCR) technique. The purified amplicon was hydrolyzed and then used to detect the methylation level using a robust and convenient LC-MS/MS method. The methodological validation revealed the favorable sensitivity and accuracy of established LC-MS/MS approach. The LC-MS/MS result showed that the methylation level of ALX4 and FBN1 exon in colorectal cancerous tissues (23.70 +/- 10.85 and 33.23 +/- 6.64) was significant higher than that in cancer-adjacent tissues (12.15 +/- 7.08 and 22.08 +/- 4.46) with statistic difference, and their value of area under curve (AUC) in receiver operator characteristic curve (ROC) analysis were higher than 0.8. On the other hand, the methylation level of MLH1 and BCL2 was 13.95 +/- 6.93 and 15.46 +/- 2.41 in CRC tissues and 13.87 +/- 3.47 and 12.84 +/- 1.91 in cancer-adjacent tissues, respectively. It is demonstrated that the methylation alteration of MLH1 and BCL2 exhibited a non-differential association between two groups. The finding indicated exon methylation of ALX4 and FBN1 could effectively distinguish CRC from non-CRC tissue and exon-based methylation should be used as potential biomarkers for cancer identification. [Graphics] .