Yahong Wang

Pro-inflammatory response and oxidative stress induced by specific components in ambient particulate matter in human bronchial epithelial cells.

Previous studies have shown that biological effect of particulate matter (PM2.5) is involved in including chemical composition and mass concentration, but the precise components and biological action on human bronchial epithelial cell line (BEAS‐2B) are still unclear. The aim of this study was to evaluate the in vitro toxicity of PM2.5 collected at six urban sites in China, and to investigate how particle composition affects cytotoxicity. We used human bronchial epithelial (BEAS‐2B) cell lines as model in vitro to expose to PM2.5 from different source, and then reactive oxygen species (ROS), superoxide dismutase activity and total antioxidant capacity were analyzed. Furthermore, we estimated the polycyclic aromatic hydrocarbon (PAH) and transition metal and the endotoxin contents. The mRNA expression of IL‐1β and IL‐10 following exposure to PM2.5 was measured by QRT‐PCR. We also observed the mitochondrial membrane potential (MMP) using JC‐1 staining, and apoptosis of BEAS‐2B using flow cytometry. In addition, double‐stranded DNA breaks (DSBs) were assessed using γ‐H2AX immunofluorescence. Our results show that high concentrations of PAHs and elemental Ni were strongly associated with high apoptosis rates and high expression of IL‐1β, in addition, Fe element was associated with the ROS level, furthermore, Fe and Cr element were associated with DNA damage in BEAS‐2B cells. The cytotoxic effects of urban PM2.5 derived from six different cities in China appear dependent on the specific components in each. Our results indicate that air quality standards based on PM2.5 components may be more relevant than concentration–response functions (CRF).

Five miRNAs as Novel Diagnostic Biomarker Candidates for Primary Nasopharyngeal Carcinoma

MicroRNAs (miRNAs) play an essential role in the development and progression of nasopharyngeal carcinomas (NPC). Despite advances in the field of cancer molecular biology and biomarker discovery, the development of clinically validated biomarkers for primary NPC has remained elusive. In this study, we investigated the expression and clinical significance of miRNAs as novel primary NPC diagnostic biomarkers. We used an array containing 2, 500 miRNAs to identify 22 significant miRNAs, and these candidate miRNAs were validated using 67 fresh NPC and 25 normal control tissues via quantitative real-time PCR (qRT-PCR). Expression and correlation analyses were performed with various statistical approaches, in addition to logistic regression and receiver operating characteristic curve analyses to evaluate diagnostic efficacy. qRT-PCR revealed five differentially expressed miRNAs (miR-93-5p, miR-135b-5p, miR-205-5p and miR-183-5p) in NPC tissue samples relative to control samples (p<0.05), with miR-135b-5p and miR-205-5p being of significant diagnostic value (p<0.01). Moreover, comparison of NPC patient clinicopathologic data revealed a negative correlation between miR-93-5p and miR- 183-5p expression levels and lymph node status (p<0.05). These findings display an altered expression of many miRNAs in NPC tissues, thus providing information pertinent to pathophysiological and diagnostic research. Ultimately, miR-135b-5p and miR-205-5p may be implicated as novel NPC candidate biomarkers, while miR- 93-5p, miR-650 and miR-183-5p may find application as relevant clinical pathology and diagnostic candidate biomarkers.

AKT/mTOR and c-Jun N-terminal kinase signaling pathways are required for chrysotile asbestos-induced autophagy

Chrysotile asbestos is closely associated with excess mortality from pulmonary diseases such as lung cancer, mesothelioma, and asbestosis. Although multiple mechanisms in which chrysotile asbestos fibers induce pulmonary disease have been identified, the role of autophagy in human lung epithelial cells has not been examined. In this study, we evaluated whether chrysotile asbestos induces autophagy in A549 human lung epithelial cells and then analyzed the possible underlying molecular mechanism. Chrysotile asbestos induced autophagy in A549 cells based on a series of biochemical and microscopic autophagy markers. We observed that asbestos increased expression of A549 cell microtubule-associated protein 1 light chain 3 (LC3-II), an autophagy marker, in conjunction with dephosphorylation of phospho-AKT, phospho-mTOR, and phospho-p70S6K. Notably, AKT1/AKT2 double-knockout murine embryonic fibroblasts (MEFs) had negligible asbestos-induced LC3-II expression, supporting a crucial role for AKT signaling. Chrysotile asbestos also led to the phosphorylation/activation of Jun N-terminal kinase (JNK) and p38 MAPK. Pharmacologic inhibition of JNK, but not p38 MAPK, dramatically inhibited the protein expression of LC3-II. Moreover, JNK2(-/-) MEFs but not JNK1(-/-) MEFs blocked LC3-II levels induced by chrysotile asbestos. In addition, N-acetylcysteine, an antioxidant, attenuated chrysotile asbestos-induced dephosphorylation of P-AKT and completely abolished phosphorylation/activation of JNK. Finally, we demonstrated that chrysotile asbestos-induced apoptosis was not affected by the presence of the autophagy inhibitor 3-methyladenine or autophagy-related gene 5 siRNA, indicating that the chrysotile asbestos-induced autophagy may be adaptive rather than prosurvival. Our findings demonstrate that AKT/mTOR and JNK2 signaling pathways are required for chrysotile asbestos-induced autophagy. These data provide a mechanistic basis for possible future clinical applications targeting these signaling pathways in the management of asbestos-induced lung disease.

Particulate matter 2.5 induces autophagy via inhibition of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin kinase signaling pathway in human bronchial epithelial cells.

Particulate matter 2.5 (PM2.5) is a significant risk factor for asthma. A recent study revealed that autophagy was associated with asthma pathogenesis. However, the specific mechanisms underlying PM2.5-induced autophagy in asthma have remained elusive. In the present study, PM2.5-induced autophagy was evaluated in Beas-2B human bronchial epithelial cells and the potential molecular mechanisms were investigated. Using electron microscopy, immunofluorescence staining and immunoblot studies, it was confirmed that PM2.5 induced autophagy in Beas-2B cells as a result of PM2.5-mediated inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway in Beas-2B cells. LY294002, a PI3K inhibitor, reduced the accumulation of microtubule-associated protein 1 light chain 3 II and attenuated the effect of PM2.5. Phosphorylated (p-)p38, p-extracellular signal-regulated kinase and p-c-Jun N-terminal kinase were dephosphorylated following exposure to PM2.5. The roles of p53, reactive oxygen species scavenger tetramethylthiourea and autophagy inhibitor 3-methyladenine in PM2.5-induced autophagy in Beas-2B cells were also investigated. The results suggested that the PI3K/Akt/mTOR signaling pathway may be a key contributor to PM2.5-induced autophagy in Beas-2B cells. The results of the present study therefore provided an a insight into potential future clinical applications targeting these signaling pathways, for the prevention and/or treatment of PM2.5-induced lung diseases.

miR-363-3p inhibits tumor growth by targeting PCNA in lung adenocarcinoma

Increasing evidence suggests that microRNAs play key roles in lung cancer. Our previous study demonstrated that microRNA 363-3p (miR-363-3p) is downregulated in lung cancer tissues. In this study, we demonstrated that overexpression of miR-363-3p inhibits the proliferation and colony formation of A549 and H441 cells, while silencing of miR-363-3p has the converse effects. The anti-oncogenic function of miR-363-3p was verified in a mouse tumor xenograft model. Furthermore, cell cycle analysis showed miR-363-3p can induce S phase arrest by downregulating Cyclin-D1 and upregulating Cyclin-dependent kinase-2 in lung adenocarcinoma cells. Additionally, miR-363-3p enhances cell apoptosis, whereas miR-363-3p inhibitor prevents apoptosis and leads to downregulation of Bax and Bak expression. The anti-proliferative function of miR-363-3p toward lung cancer cells may be explained by its ability to inhibit the activation of the mTOR and ERK signaling pathways. Using target prediction software and luciferase reporter assays, we identified PCNA as a specific target of miR-363-3p. miR-363-3p can decreased the accumulation of endogenous PCNA in lung adenocarcinoma cells. Moreover, exogenous expression of PCNA relieve the inhibition of miR-363-3p on cell proliferation, colony formation and mTOR and ERK signaling pathways. Taken together, our data indicate that miR-363-3p suppresses tumor growth by targeting PCNA in lung adenocarcinoma.

MiR-4673 Modulates Paclitaxel-Induced Oxidative Stress and Loss of Mitochondrial Membrane Potential by Targeting 8-Oxoguanine-DNA Glycosylase-1

Background: Our previous study identified a novel microRNA, miR-4673, which is upregulated in A549 cells exposed to paclitaxel (PTX). In this study, we investigated the role of miR-4673 in PTX-induced cytotoxicity. Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, apoptosis assay, 5,5’,6,6’-Tetrachloro-1,1’,3,3’-tetraethyl-imidacarbocyanine iodide (JC-1) staining and 2’,7’-Dichlorofluorescein (DCFH) staining were used to evaluate cell viability, apoptosis, mitochondrial membrane potential (MMP) loss and reactive oxygen species (ROS) generation in A549 and H1299 cells. Bioinformatics analysis and Luciferase reporter assay were used to explore whether 8-oxoguanine-DNA glycosylase-1 (OGG1) is a target gene of miR-4673. Results: Enforced expression of miR-4673 decreased cell viability and increased PTX-induced apoptosis, MMP loss and reactive oxygen species (ROS) generation in A549 and H1299 cells. Bioinformatics analysis, which was used to identify potential target of miR-4673, revealed a binding site of miR-4673 in 3’UTR of OGG1. Luciferase reporters assays showed that miR-4673 specifically binds to ‘CUGUUGA’ in 3’UTR of OGG1. Enforced expression of miR-4673 decreased accumulation of OGG1. In addition, silencing OGG1 enhanced inhibitory effects of PTX on apoptosis, MMP loss and ROS generation, which is similar to effects of miR-4673. Moreover, enforced expression of OGG1 compromised promoting effects of miR-4673 on PTX-induced apoptosis, MMP loss and ROS generation. Conclusion: miR-4673 modulates PTX-induced apoptosis, MMP loss and ROS generation by targeting OGG1.

Reduced apurinic/apyrimidinic endonuclease activity enhances the antitumor activity of oxymatrine in lung cancer cells

Lung cancer is the leading cause of cancer-related deaths worldwide and is associated with a very poor outcome. Oxymatrine exerts antitumor effects by inducing apoptosis and inhibiting the proliferation of different cancer cells; however, the anticancer effects and mechanism of action of oxymatrine have not been evaluated sufficiently in human lung cancer cells. Thus, the present study aimed to investigate the anticancer effects of oxymatrine in human lung cancer cells and identify the molecular mechanisms underlying these effects. MTT assays demonstrated that oxymatrine significantly inhibited the proliferation of A549 and H1299 cells in a time- and dose-dependent manner. In addition, flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assays suggested that oxymatrine treatment may induce lung cancer cell apoptosis in a dose-dependent manner. Furthermore, we detected that oxymatrine induced a significant increase in DNA damage and the expression of PARP and phosphorylated H2AX, and a significant decrease in that of nuclear APE1 and AP endonuclease activity in A549 cells. APE1 knockdown cells (APE1shRNA) plus oxymatrine treatment reduced cells proliferation and induced apoptosis more seriously than control shRNA cells. This appeared to be a consequence of an increase in the number of apurinic/apyrimidinic (AP) sites, DNA damage, PARP and H2AX phosphorylation, which together resulted in the induction of apoptosis. In contrast, the sensitizing effects of APE1 overexpression plus oxymatrine treatment did not occur in APEOE cells. These findings reveal a potential mechanism of action for oxymatrine-induced apoptosis and suggest that oxymatrine is a promising potential therapeutic agent for the treatment of lung cancer.

The c-Jun N-terminal kinase signaling pathway mediates chrysotile asbestos-induced alveolar epithelial cell apoptosis

Exposure to chrysotile asbestos exposure is associated with an increased risk of mortality in combination with pulmonary diseases including lung cancer, mesothelioma and asbestosis. Multiple mechanisms by which chrysotile asbestos fibers induce pulmonary disease have been identified, however the role of apoptosis in human lung alveolar epithelial cells (AEC) has not yet been fully explored. Accumulating evidence implicates AEC apoptosis as a crucial event in the development of both idiopathic pulmonary fibrosis and asbestosis. The aim of the present study was to determine whether chrysotile asbestos induces mitochondria-regulated (intrinsic) AEC apoptosis and, if so, whether this induction occurs via the activation of mitogen-activated protein kinases (MAPK). Human A549 bronchoalveolar carcinoma-derived cells with alveolar epithelial type II-like features were used. The present study showed that chrysotile asbestos induced a dose- and time-dependent decrease in A549 cell viability, which was accompanied by the activation of the MAPK c-Jun N-terminal kinases (JNK), but not the MAPKs extracellular signal-regulated kinase 1/2 and p38. Chrysotile asbestos was also shown to induce intrinsic AEC apoptosis, as evidenced by the upregulation of the pro-apoptotic genes Bax and Bak, alongside the activation of caspase-9, poly (ADP-ribose) polymerase (PARP), and the release of cytochrome c. Furthermore, the specific JNK inhibitor SP600125 blocked chrysotile asbestos-induced JNK activation and subsequent apoptosis, as assessed by both caspase-9 cleavage and PARP activation. The results of the present study demonstrated that chrysotile asbestos induces intrinsic AEC apoptosis by a JNK-dependent mechanism, and suggests a potential novel target for the modulation of chrysotile asbestos-associated lung diseases.

Nickle(II) ions exacerbate bleomycin-induced pulmonary inflammation and fibrosis by activating the ROS/Akt signaling pathway

Nickle (Ni) is a heavy metal found in particulate matter. We previously reported that Ni ions are strongly associated with high apoptosis rates and high expression of IL-1β in human bronchial epithelial cells following exposure to PM2.5; however, the effects of Ni ions on pulmonary fibrosis have not been fully elucidated. In the current study, we evaluated whether Ni ions can exacerbate bleomycin (BLM)-induced pulmonary fibrosis in a mouse model and illustrated the potential mechanism. Ni ions inhibited cell proliferation and induced apoptosis in A549 and MRC-5 cells. BLM-induced lung injury and fibrosis in mice were significantly enhanced by nickel treatment, and these findings were also supported by inflammatory cell accumulation in bronchoalveolar lavage fluid and elevated levels of pro-inflammatory cytokines in lung tissues. Ni ions also increased extracellular matrix protein levels, including those of type I collagen and MMP9 in mouse lung tissues and cell lines. Moreover, Ni ions promoted the phosphorylation of AKT in this mouse model. The effect of increased collagen levels and MMP9 expression was inhibited by blocking the AKT phosphorylation. Together, these findings suggest AKT activation as a critical contributor to this Ni-exacerbated pulmonary fibrotic process.

TFPI2AS1, a novel lncRNA that inhibits cell proliferation and migration in lung cancer

ABSTRACT Accumulating evidence demonstrates that a series of differentially expressed lncRNAs is important in tumorigenesis. However, the function of many of the lncRNAs in lung cancer remains elusive. In the present study, we used microarray analysis to identify lncRNAs that are dysregulated in non-small-cell lung cancer (NSCLC) as compared with normal tissues. Among the dysregulated lncRNAs, we identified TFPI2AS1, an antisense transcript of the tumor suppressor TFPI2 (tissue factor pathway inhibitor 2). TFPI2AS1 was shown to be markedly upregulated in NSCLC patient tumors as compared to paired non-tumor samples. TFPI2AS1 knockdown increased NSCLC cell proliferation and migration, which was associated with enhanced G1/S transition and downregulation of cyclin D1 and cyclin-dependent kinases 2 (CDK2), while TFPI2AS1 overexpression had the opposite effect. Knockdown and overexpression experiments also suggested that TFPI2AS1 regulates NSCLC cell migration and AKT activation. Moreover, TFPI2AS1 is a positive regulator of TFPI2. Our findings bring new insights for understanding the role of TFPI2AS1 in mediating the proliferation and migration of NSCLC cells by regulating TFPI2 expression.