METTL3 aggravates cell damage induced by Streptococcus pneumoniae via the NEAT1/CTCF/MUC19 axis.

Disruption of the alveolar barrier can trigger acute lung injury. This study elucidated the association of methyltransferase-like 3 (METTL3) with Streptococcus pneumoniae (SP)-induced apoptosis and inflammatory injury of alveolar epithelial cells (AECs). AECs were cultured and then infected with SP. Furthermore, the expression of METTL3, interleukin (IL)-10, IL-6, tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1), long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1), mucin 19 (MUC19), N6-methyladenosine (m6A), and NEAT1 after m6A modification were detected by qRT-PCR, Western blot, and enzyme-linked immunosorbent, m6A quantification, and methylated RNA immunoprecipitation-qPCR analyses, respectively. Moreover, the subcellular localization of NEAT1 was analyzed by nuclear/cytosol fractionation assay, and the binding between NEAT1 and CCCTC-binding factor (CTCF) was also analyzed. The results of this investigation revealed that SP-induced apoptosis and inflammatory injury in AECs and upregulated METTL3 expression. In addition, the downregulation of METTL3 alleviated apoptosis and inflammatory injury in AECs. METTL3-mediated m6A modification increased NEAT1 and promoted its binding with CTCF to facilitate MUC19 transcription. NEAT1 or MUC19 overexpression disrupted their protective role of silencing METTL3 in AECs, thereby increasing apoptosis and inflammatory injury. In conclusion, this is the first study to suggest that METTL3 aggravates SP-induced cell damage via the NEAT1/CTCF/MUC19 axis.

Real-world tobacco cessation practice in China: findings from the prospective, nationwide multicenter China National Tobacco Cessation Cohort Study (CNTCCS).

Background: Tobacco cessation is proven to be the most effective and cost-effective strategy for smokers to reduce their risk of smoking-related disease and premature death. Providing effective, efficient, safe, and patient-centred tobacco cessation treatment to reach those who need them is a significant challenge. To date, only a few nationwide studies in China have assessed the overall clinical care practice and treatment outcome of tobacco cessation. Methods: This a prospective, nationwide, multicenter, cohort study covering all Eastern China, Northwest China, Central China, North China, Southwest China, Northeast China, and South China. Participants who were current smokers aged 18-85 years attending clinic for smoking cessation were included. All the participants were treated with 3-month cessation treatment and followed up for 3 months. Data were collected prospectively using online system. The primary outcome was 7-day point abstinence rate at 24 weeks, validated biochemically by an expired carbon monoxide level of less than 10 ppm. The participants lost to follow-up or not providing validation were included as non-abstainers. Findings: A representative sample of 3557 participants were recruited and 2943 participants were included into this analysis. These participants had mean age of 53.05 years, and 94.8% were males, with 75.8% showing symptoms of tobacco dependence. A total of 965 (32.8%) participants were treated with Bupropion + behavioural counselling, followed by 935 (31.8%) with behavioural counselling, 778 (26.4%) with Varenicline + behavioural counselling, 135 (4.6%) with alternative treatments + behavioural counselling, and 130 (4.4%) with nicotine replacement therapy (NRT) + behavioural counselling. After 3-month treatment and 3-month follow-up, 21.74% of the participants quit smoking at 24 weeks. In the multivariable-adjusted analyses, quitting smoking was significantly associated with female, higher socioeconomic status, poor health condition, different treatment received, and less smoking intensity. The tobacco cessation treatment varied widely across different areas of China. In particular, the areas with higher usage of cessation medication were associated with better cessation treatment outcome. Interpretation: The CNTCCS is the first large-scale nationwide cohort study of smoking cessation in China. Rich data collected from this prospective cohort study provided the opportunity to evaluate the clinical practice of tobacco cessation treatment in China. Funding: Chinese Academy of Medical Sciences (CAMS) Initiative for Innovative Medicine (CAMS 2021-I2M-1-010), Heilongjiang Provincial Science and Technology Key Program (2022ZXJ03C02), and National Key R&D Program of China (grant no. 2017YFC1309400).

[Effects of Akt2-siRNA on chemotherapeutic sensitivity and drug resistance in human lung cancer cells].

OBJECTIVE: To explore the effects of oncogene protein v-akt-siRNA on the sensitivity of human lung cancer cell line NCI-H446 to cisplatin and drug resistance proteins in human lung cancer cells. METHODS: The small interfering siRNA expression vector targeting Akt2 gene (siAkt2) was constructed. And the NCI-H446 cells were transfected with negative control vector or siRNA vector. The expressions of Akt2-mRNA and lung resistance-related protein (LRP) and P-glycoprotein (P-gp) were detected by reverse transcription-polymerase chain reaction and immunocytochemistry respectively. NCI-H446 and transfected cells were treated by cisplatin for 24 h. The cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay and cell apoptotic rate detected by flow cytometry. RESULTS: Akt2-mRNA decreased significantly in the transfected NCI-H446 cells versus the non-transfection group. And the expressions of LRP and P-gp proteins decreased significantly in the transfection group versus the control group (P < 0.01). The cell proliferation rate decreased from (60.2 ± 2.8)% to (34.7 ± 2.6)% (P < 0.01). The cell apoptotic rate increased from (19.3 ± 1.6)% to (38.8 ± 1.2)% after a therapy of cisplatin (P < 0.01). CONCLUSION: The siRNA targeting Akt2 can decrease the Akt2 expression, increase the chemotherapeutic sensitivity to cisplatin and partially reverse the cisplatin resistance of NCI-H446. The mechanism may be through the lowered expressions of LRP and P-gp.

[Changes of HMGB1 and RAGE in induced sputum from patients with bronchial asthma].

OBJECTIVE: To explore the relationship between HMGB1 (high mobility group box-1) protein and receptor for advanced glycosylation end products (RAGE) and the nosogenesis and severity of bronchial asthma. METHODS: Based on the criteria, the asthma group included 64 acute-onset asthma patients while the control group had 20 healthy cases. The asthma group received a 4-week combination inhalation therapy of budesonide and formoterol. Lung functions and induced sputum examinations were conducted before and after treatment. The percentage of a second forced expiratory volume in the predicted value (FEV(1)%) was recorded. A differential count of neutrophilic leukocyte in reduced sputum was performed. And the sputum levels of HMGB1 and RAGE were detected by ELISA (enzyme linked immunosorbent assay). RESULTS: Prior to treatment, the neutrophilic leukocyte percentage and the levels of HMGB1 and RAGE were all higher than those of control group (P < 0.01). The induced sputum of severe asthma patients had higher levels of neutrophilic leukocyte percentage and HMGB1 than those of mild cases (P < 0.01). But the level of RAGE showed no statistical significance among mild, moderate or severe asthma cases (P > 0.05). The post-treatment levels of neutrophilic leukocyte percentage, HMGB1 and RAGE were lower as compared with the pre-treatment ones (P < 0.01). These three parameters in uncontrolled cases were higher than those in completely controlled cases (P < 0.05); in asthma group, both HMGB1 and RAGE had a negative correlation with FEV(1)% (r = -0.830, r = -0.632, P < 0.01); in induced sputum, both HMGB1 and RAGE had a positive correlation with FEV(1)% (r = 0.820, r = 0.623, P < 0.01). The levels of HMGB1 and RAGE were positively correlated (r = 0.929, P < 0.01). CONCLUSION: Both HMGB1 and RAGE participate in the inflammatory process of asthmatic airway. HMGB1 is correlated with the severity of asthma. And the levels of HMGB1 and RAGE in induced sputum may be employed as reference indices for the observation of therapeutic effects.

[Correlation between the airway inflammations and levels of stromal cell-derived factor 1 in induced sputum of asthmatic patients].

OBJECTIVE: To evaluate concentrations of stromal cell-derived factor 1 (SDF-1) and IL-17 in induced sputum supernatants from asthmatic patients before and after treatment with glucocorticosteroids. METHODS: Induced sputum was collected from 30 healthy controls and 99 patients with chronic persistent asthma from 2009-2010. Sputum samples were obtained before and after 4 week treatment with inhaled glucocorticosteroids. The sputum concentrations of SDF-1 and IL-17 were measured by ELISA. RESULTS: The FEV(1)% and the asthma control score of patients with severe asthma were decreased as compared with patients with moderate persistent and mild persistent asthma (F = 457.448 and 79.271, all P < 0.01). The concentrations of SDF-1, IL-17 and the percentage of eosinophils were increased in asthma group compared with control subjects (all P < 0.01), but the percentage of sputum neutrophils was lower than that in the healthy controls (P < 0.01). The percentage of sputum neutrophils and eosinophils and the level of SDF-1 and IL-17 in patients with severe persistent asthma were significantly higher than those in patients with mild persistent asthma (all P < 0.05). The percentage of sputum neutrophils and eosinophils were negatively correlated with FEV(1)% (r = -0.409 and -0.316, all P < 0.05). The levels of IL-17 and SDF-1 were positively correlated with the percentage of sputum neutrophils and eosinophils (all P < 0.01). The levels of IL-17 were positively correlated with the levels of SDF-1 (r = 0.872, P < 0.01). After glucocorticosteroid therapy, the percentage of eosinophils and neutrophils, the levels of IL-17 and SDF-1 decreased significantly in all patients (all P < 0.01), while the percentage of sputum neutrophils and the levels of IL-17 and SDF-1 in uncontrolled patients increased significantly compared with the controlled and partly controlled groups (all P < 0.05). CONCLUSIONS: SDF-1 and IL-17 may contribute to airway inflammation in asthma by chemotactic activity towards neutrophils. The concentration of SDF-1 may be used to evaluate the inflammation and the therapeutic effects.

[Levels of HMGB1 in induced sputum from patients with asthma and chronic obstructive pulmonary disease].

OBJECTIVE: To explore the relationship between the sputum levels of high mobility group protein B1 (HMGB1) and airway inflammation in bronchial asthma and chronic obstructive pulmonary disease (COPD) patients. METHODS: A total of 57 patients with persistent asthma [per Global Initiative for Asthma (GINA) guidelines], 30 patients with stable COPD [stratified by Global Initiative for COPD (GOLD) status] and 20 control subjects were recruited. After completing an asthma control questionnaire, spirometry was performed before sputum induction. The ratio of forced expiratory volume in the first second (FEV(1))/predictive value (FEV(1)%Pre) and neutrophil differential count in induced sputum were recorded. The concentrations of HMGB1 in the supernatant of sputum were measured by ELISA (enzyme-linked immunosorbent assay). RESULTS: The sputum concentrations of HMGB1 in the asthmatics and COPD patients were significantly higher than those of the control subjects [(291 ± 55) and (511 ± 39) vs (61 ± 5) ng/L, all P < 0.01]. And they were significantly negatively correlated with FEV(1)%Pre in all subjects. The levels of HMGB1 in induced sputum of COPD patients were significantly higher than those of asthmatics and healthy controls (P < 0.01). No significant difference existed in the levels of HMGB1 between patients with eosinophilic and noneosinophilic asthma [(290 ± 55) vs (292 ± 54) ng/L, P > 0.05]. The HMGB1 levels with COPD stage II and stage III were significantly higher than those with stage I [(526 ± 29) and (541 ± 29) vs (471 ± 18) ng/L]. The differences of sputum neutrophil percentage were statistically significant in mild, moderate and severe asthma [(27 ± 2)%, (36 ± 4)%, (49 ± 4)%]. And the sputum levels of HMGB1 were significantly higher in the patients with moderate and severe asthma [(312 ± 14) vs (347 ± 11) ng/L]. And the levels of HMGB1 in asthmatic and COPD patients were positively correlated with neutrophil percentage. According to the multivariate analysis, neutrophil percentage and FEV(1)%Pre were independent predictors of sputum HMGB1, but not smoking, age, gender and eosinophilic percentage. CONCLUSION: HMGB1 may contribute to airway inflammation through its higher expression in bronchial asthma and COPD patients.