Lysine demethylase KDM3A alleviates hyperoxia-induced bronchopulmonary dysplasia in mice by promoting ETS1 expression.

Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease among neonates, with increasing morbidity and mortality. This study aims to investigate the effect and mechanism of lysine demethylase 3A (KDM3A) on hyperoxia-induced BPD. Hyperoxia-induced BPD mouse and alveolar epithelial cell models were constructed. The effects of hyperoxia on lung development were evaluated by histological and morphological analysis. The levels of KDM3A, E26 transformation specific-1 (ETS1), H3 lysine 9 dimethylation (H3K9me2), and endoplasmic reticulum (ER) stress-related indexes were quantified by RT-qPCR, Western blot, and IF staining. Cell apoptosis was assessed by flow cytometry and TUNEL staining. Transfection of oe-ETS1, oe-KDM3A, and sh-ETS1 was applied in hyperoxia-induced alveolar epithelial cells to explore the mechanism of the KDM3A/ETS1 axis in hyperoxia-induced apoptosis. KDM3A inhibitor IOX1 was applied to validate the in vivo effect of KDM3A in hyperoxia-induced BPD mice. The results displayed that hyperoxia-induced BPD mice showed reduced body weight, severe destruction of alveolar structure, decreased radial alveolar count (RAC), and increased mean linear intercept (MLI) and mean alveolar diameter (MAD). Further, hyperoxia induction down-regulated ETS1 expression, raised ER stress levels, and increased apoptosis rate in BPD mice and alveolar epithelial cells. However, transfection of oe-ETS1 improved the above changes in hyperoxia-induced alveolar epithelial cells. Moreover, transfection of oe-KDM3A up-regulated ETS1 expression, down-regulated H3K9me2 expression, inhibited ER stress, and reduced apoptosis rate in hyperoxia-induced alveolar epithelial cells. In addition, transfection of sh-ETS1 reversed the inhibitory effect of KDM3A on hyperoxia-induced apoptosis by regulating ER stress. In vivo experiments, KDM3A inhibitor IOX1 intervention further aggravated BPD in newborn mice. In a word, KDM3A alleviated hyperoxia-induced BPD in mice by promoting ETS1 expression.

ETS1 Ameliorates Hyperoxia-Induced Bronchopulmonary Dysplasia in Mice by Activating Nrf2/HO-1 Mediated Ferroptosis.

PURPOSE: Bronchopulmonary dysplasia (BPD) is associated with hyperoxia-induced oxidative stress-associated ferroptosis. This study examined the effect of E26 oncogene homolog 1 (ETS1) on oxidative stress-associated ferroptosis in BPD. METHODS: Hyperoxia-induced A549 cells and neonatal mice were used to establish BPD models. The effects of ETS1 on hyperoxia-induced ferroptosis-like changes in A549 cells were investigated by overexpression of ETS1 plasmid transfection and erastin treatment. Glucose consumption, lactate production, and NADPH levels were assessed by the glucose, lactate, and NADP+/NADPH assay kits, respectively. The potential regulatory relationship between ETS1 and Nrf2/HO-1 was examined by treating hyperoxia-induced A549 cells with the Nrf2 inhibitor ML385. ETS1 effect on the Nrf2 promoter was explored by dual-luciferase reporter and chromatin immunoprecipitation assay. The effect of ETS1 on the symptoms of BPD mice was examined by injecting an adenovirus overexpressing ETS1. RESULTS: ETS1 overexpression increased hyperoxia-induced cell viability, glucose consumption, lactate production, and NADPH levels and reduced inflammation and apoptosis in A549 cells. In animal experiments, ETS1 overexpression prevented weight loss, airway enlargement, and reductions in radial alveolar counts in BPD mice, while reducing the mean linear intercept, mean alveolar diameter and inflammation. ETS1 overexpression suppressed PTGS2 and CHAC1 expression, reduced ROS, MDA and ferrous iron (Fe2+) production and increased GSH levels in hyperoxia-induced A549 cells and BPD mice. In addition, ETS1 can bind to the Nrf2 promoter region and thus promote Nrf2 transcription. ETS1 overexpression increased the mRNA and protein levels of Nrf2, HO-1, xCT, and GPX4 in hyperoxia-induced A549 cells and BPD mice. In hyperoxia-induced A549 cells, erastin and ML385 treatment abolished the effect of ETS1 overexpression. CONCLUSION: ETS1 is important in oxidative stress-related ferroptosis in a hyperoxia-induced BPD model, and the effect is partially mediated by the Nrf2/HO-1 axis.

Lef1 is transcriptionally activated by Klf4 and suppresses hyperoxia-induced alveolar epithelial cell injury.

PURPOSE: Bronchopulmonary dysplasia (BPD) is a long-term respiratory condition. More than a quarter of extremely premature newborns are harmed by BPD. At present, there are no apparent effective drugs or treatments for the condition. In this study, we aimed to investigate the functional role and mechanism of lymphoid enhancer-binding factor 1 (Lef1) in BPD in vitro. MATERIALS AND METHODS: Blood samples from BPD patients and healthy volunteers were gathered, and an in vitro model of BPD was developed in alveolar epithelial cells (AECs) MLE-12 induced by hyperoxia. Then expression of krüppel-like factor 4 (KLF4/Klf4) and LEF1/Lef1 were evaluated. After Lef1 overexpressing plasmid and the vector were transfected into hyperoxia-induced MLE-12 cells, cell proliferation assays were carried out. Cell apoptosis was investigated by a flow cytometry assay, and apoptosis related proteins Bcl-2, cleaved-caspase 3 and 9 were analyzed by a western blot assay. The binding between Klf4 and Lef1 promoter predicted on the JASPAR website was verified using luciferase and ChIP assays. For further study of the mechanism of Klf4 and Lef1 in BPD, gain-of-function experiments were performed. RESULTS: The mRNA levels of KLF4/Klf4 and LEF1/Lef1 were diminished in clinical BPD serum samples and hyperoxia-induced MLE-12 cells. Overexpression of Lef1 stimulated AEC proliferation and suppressed AEC apoptosis induced by hyperoxia. Mechanically, Klf4 bound to Lef1's promoter region and aids transcription. Moreover, the results of gain-of-function experiments supported that Klf4 could impede AEC damage induced by hyperoxia via stimulating Lef1. CONCLUSION: Klf4 and Lef1 expression levels were declined in hyperoxia-induced AECs, and Lef1 could be transcriptionally activated by Klf4 and protect against hyperoxia-induced AEC injury in BPD. As a result, Lef1 might become a prospective therapeutic target for BPD.

LncRNA SNHG6 accelerates hyperoxia-induced lung cell injury via regulating miR-335 to activate KLF5/NF-κB pathway.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is a common chronic lung disease in premature infants, and its pathogenesis has not been clarified. Long non-coding RNAs (lncRNA) have important functions in cell bioactivity. However, their role in developmental lung disease remains unclear. OBJECTIVE: The aim of this study was to demonstrate the role of lncRNA SNHG6 (SNHG6) in BPD and its underlying mechanisms. METHODS: The blood of patients with BPD were collected, and BPD model of BEAS-2B cells was established by hyperoxia method. SNHG6, miR-335 and KLF5 mRNA expression were detected by RT-qPCR. Western blot was conducted to measure the levels of apoptosis-related proteins' expression and NF-κB pathway related proteins. BEAS-2B cell viability and apoptosis were assessed by CCK-8 and flow cytometry, respectively. Assay Kit was applied to detect ROS, MDA and SOD levels, respectively. ELISA was performed to assess the levels of inflammatory factors. The binding site of miR-335 with SNHG6 or KLF5 were predicted by using DIANA or TargetScan, and which was verified by double luciferase reporter assay. RESULTS: Firstly, SNHG6 was highly expressed and miR-335 was lowly expressed in BPD model, SNHG6 knockdown and miR-335 mimics both alleviated hyperoxia-induced lung cell injury, and SNHG6 targeted miR-335. Subsequently, KLF5 was targeted by miR-335, and KLF5 promoted lung cell injury via activating NF-κB pathway. Furthermore, SNHG6 mediated lung cell injury via regulating the miR-335/KLF5/NF-κB pathway. CONCLUSION: Our research confirmed that SNHG6 mediated hyperoxia-induced lung cell injury via regulating the miR-335/KLF5/NF-κB pathway. These findings suggest that SNHG6 serves as promising targets for the treatment of newborns with BPD.

Sphingosine Kinase-1 (SPHK1) promotes inflammation in infantile pneumonia by regulating NLRP3 inflammasome and SIRT1 expression.

BACKGROUND: Infantile pneumonia is an acute inflammatory disorder of the lung caused by mycoplasma pneumonia. SPHK1 (sphingosine kinase-1) signaling pathway is involved in the process of inflammatory diseases. However, whether SphK1 regulates inflammatory responses in infantile pneumonia remains unclear. In this study, we investigated the role of SPHK1 in infantile pneumonia and its underlying mechanisms. METHODS: Serum samples of 12 patients with infantile pneumonia and healthy controls were obtained from Hunan Children's Hospital. To induce pneumonia, mice were administrated with LPS (lipopolysaccharide) into the lung. RAW264.7 cells were used as an in vitro macrophage model stimulated with LPS or PBS for 4 h. RESULTS: SPHK1 mRNA level and protein level in the LPS-treated mice and patients with infantile pneumonia were significantly increased. SPHK1 promoted inflammation and lung injury in mice with infantile pneumonia. The knockdown of SPHK1 expression inhibited inflammation and restrained lung injury in mice with infantile pneumonia. SPHK1 overexpression also exacerbated inflammation in RAW264.7 cells stimulated by LPS, and SPHK1 silencing reduced inflammatory responses. We further showed that SPHK1 induced NLRP3 (NLR Family Pyrin Domain Containing 3) activity by inhibiting SIRT1 expression. CONCLUSION: Our study demonstrated that SPHK1 promotes inflammation of infantile pneumonia by modulating NLRP3 inflammasome via the regulation of SIRT1 expression and mitochondrial permeability transition.

ETS1 Ameliorates Hyperoxia-Induced Alveolar Epithelial Cell Injury by Regulating the TGM2-Mediated Wnt/ß-Catenin Pathway.

PURPOSE: Bronchopulmonary dysplasia (BPD) is a chronic lung disease that affects newborns who need oxygen therapy, and high-concentration oxygen therapy may cause neonatal morbidity and mortality in newborns. E26 oncogene homologue 1 (ETS1) and transglutaminase 2 (TGM2) have been reported to be associated with lung cell injury. However, the mechanism of ETS1 in regulating BPD is still unclear. METHODS: Hyperoxia-induced A549 cells to simulate hyperoxia-induced alveolar epithelial cell injury. MTT assays and colony formation assays were performed to investigate the proliferation of A549 cells. Flow cytometry was carried out to quantify the apoptosis of A549 cells. The expression levels of ETS1 and TGM2 were quantified by qRT-PCR. The protein expression levels of ETS1, TGM2, ß-catenin, c-Jun and MET were measured by western blot. Overexpression of ETS1, overexpression of TGM2, overexpression of ETS1 with downregulation of TGM2 and overexpression of TGM2 with inhibition of Wnt/ß-catenin pathway were performed to investigate the role of ETS1, TGM2 and Wnt/ß-catenin pathways in hyperoxia-induced alveolar epithelial cell injury. RESULTS: Hyperoxia decreased the proliferation and promoted the apoptosis of cells in a time-dependent manner. Moreover, overexpression of ETS1 rescued the effect of hyperoxia on proliferation and apoptosis. In addition, overexpression of TGM2 participated in the regulation of hyperoxia-induced proliferation and apoptosis. ETS1 regulated hyperoxia-induced alveolar epithelial cell injury through the Wnt/ß-catenin pathway via TGM2. CONCLUSION: ETS1 ameliorates hyperoxia-induced alveolar epithelial cell injury through the TGM2-mediated Wnt/ß-catenin pathway.

Overexpression of xeroderma pigmentosum group C decreases the chemotherapeutic sensitivity of colorectal carcinoma cells to cisplatin.

Xeroderma pigmentosum group C (XPC) is a DNA-damage-recognition gene active at the early stage of DNA repair. XPC also participates in regulation of cell-cycle checkpoint and DNA-damage-induced apoptosis. In the present study, the expression levels of genes involved in nucleotide excision repair (NER) were assessed in human colorectal cancer (CRC) tissue. This analysis revealed that expression of XPC mRNA significantly increased in colorectal carcinoma tissues compared with matched normal controls. Expression of XPC gradually increased along with the degree of progression of CRC. In vitro, an XTT assay demonstrated that small interfering RNA (siRNA) targeting XPC significantly increased the sensitivity of CRC SW480 cells to cisplatin, whereas cells transfected with a XPC-overexpression plasmid became more resistant to cisplatin. Furthermore, flow cytometry revealed that the proportion of apoptotic cells significantly increased in XPC-knockdown cells upon cisplatin treatment. However, the overexpression XPC significantly increased the resistance of cells to cisplatin. In vivo, tumor growth was significantly reduced in tumor-bearing mice when the XPC gene was knocked down. Upregulation of the expression of pro-apoptotic Bcl-associated X and downregulation of the anti-apoptotic B-cell lymphoma 2 proteins was observed in the implanted tumor tissue. In conclusion, XPC serves a key role in chemotherapeutic sensitivity of CRC to cisplatin, meaning that it may be a potential target for chemotherapy of CRC.

Angiogenesis-related genes may be a more important factor than matrix metalloproteinases in bronchopulmonary dysplasia development.

We characterized the expression profile of angiogenesis-related genes (ARG) and matrix metalloproteinase (MMP) genes in preterm infants, with and without bronchopulmonary dysplasia (BPD). We reanalyzed a gene expression dataset for preterm infants from the Gene Expression Omnibus database using the Gene-Cloud of Biotechnology Information platform. A total of 1,652 genes were differentially (1.2-fold change) expressed: 811 were highly expressed in infants with BPD, and 841 were highly expressed in those without BPD. Twenty-eight and 11 ARGs were upregulated in infants with and without BPD, respectively. Among 27 detected MMPs and TIMPs, MMP8, MMP9, MMP25, TIMP2 and TIMP3 were differently expressed. Levels of THBS1, MMP8, MMP9, MMP25, TIMP2 and TIMP3 increased as severity of BPD and retinopathy of prematurity (ROP) increased, whereas ETS1, LEF1 and SPOCK2 exhibited the opposite trend. Expression of ETS1 and LEF1 had a fitting rate of R2 = 0.849 and P < 0.001. ELISAs showed a positive correlation between THBS1 and CD36 (receptor of THBS1) levels in serum samples from preterm infants. Our study indicates that the upregulation of THBS1 and downregulation of ETS1, LEF1 promotes BPD in preterm infants by disrupting blood vessel formation rather than by dysregulation of MMPs and TIMPs.

Susceptibility and transmissibility of pigeons to Asian lineage highly pathogenic avian influenza virus subtype H5N1.

To determine whether or not pigeons are susceptible to infection with Asian lineage highly pathogenic (HP) avian influenza virus (AIV) subtype H5N1 and can serve as a transmission host for H5N1 HPAIV, we experimentally infected 187 young and adult pigeons with five different isolates of H5N1 HPAIV and co-habited some experimentally infected pigeons with susceptible specific pathogen free chickens. Results showed that all infected pigeons remained clinically healthy during the observation period. No gross lesions or histopathological changes were observed in the infected pigeons, and haemagglutination inhibition antibodies were not detected in serum samples of the infected pigeons. Additionally, all chickens placed in contact with AIV H5N1 infected pigeons remained healthy, and no virus or haemagglutination inhibition antibodies were detected in samples from the chickens. Our data suggest that pigeons are not susceptible to Asian lineage H5N1 HPAIV and do not transmit the virus to chickens.