A novel microRNA miR-4433a-3p as a potential diagnostic biomarker for lung adenocarcinoma.

Background: Lung adenocarcinoma is one of the leading causes of cancer-related deaths because of the lack of early specific clinical indicators. MicroRNAs (miRNAs) have become the focus in lung cancer diagnosis. Further studies are required to explore miRNA expression in the serum of lung adenocarcinoma patients and their correlation with therapy and analyse specific messenger RNA targets to improve the specificity and sensitivity of early diagnosis. Methods: The Toray 3D-Gene miRNA array was used to compare the expression levels of various miRNAs in the sera of patients with lung adenocarcinoma and healthy volunteers. Highly expressed miRNAs were selected for further analysis. To verify the screening results, serum and pleural fluid samples were analysed using qRT-PCR. Serum levels of the miRNAs and their correlation with the clinical information of patients with lung adenocarcinoma were analysed. The functions of miRNAs were further analysed using the Kyoto Encyclopedia of Gene and Genomes and Gene Ontology databases. Results: Microarray analysis identified 60 and 50 miRNAs with upregulated and downregulated expressions, respectively, in the serum of patients with lung adenocarcinoma compared to those in healthy individuals. Using qRT-qPCR to detection of miRNAs expression in the serum or pleural effusion of patients with early and advanced lung adenocarcinoma, we found that miR-4433a-3p could be used as a diagnostic marker and therapeutic evaluation indicator for lung adenocarcinoma. Serum of miR-4433a-3p levels significantly correlated with the clinical stage. miR-4433a-3p may be more suitable than other tumour markers for the early diagnosis and evaluation of therapeutic effects in lung adenocarcinoma. miR-4433a-3p may affect tumour growth and metastasis by acting on target genes (PIK3CD, UBE2J2, ICMT, PRDM16 and others) and regulating tumour-related signalling pathways (MAPK signal pathway, Ras signalling pathway and others). Conclusion: miR-4433a-3p may serve as a biomarker for the early diagnosis of lung adenocarcinoma and monitoring of therapeutic effects.

Development and validation of a prospective study to predict the risk of readmission within 365 days of respiratory failure: based on a random survival forest algorithm combined with COX regression modeling.

BACKGROUND: There is a need to develop and validate a widely applicable nomogram for predicting readmission of respiratory failure patients within 365 days. METHODS: We recruited patients with respiratory failure at the First People's Hospital of Yancheng and the People's Hospital of Jiangsu. We used the least absolute shrinkage and selection operator regression to select significant features for multivariate Cox proportional hazard analysis. The Random Survival Forest algorithm was employed to construct a model for the variables that obtained a coefficient of 0 following LASSO regression, and subsequently determine the prediction score. Independent risk factors and the score were used to develop a multivariate COX regression for creating the line graph. We used the Harrell concordance index to quantify the predictive accuracy and the receiver operating characteristic curve to evaluate model performance. Additionally, we used decision curve analysiso assess clinical usefulness. RESULTS: The LASSO regression and multivariate Cox regression were used to screen hemoglobin, diabetes and pneumonia as risk variables combined with Score to develop a column chart model. The C index is 0.927 in the development queue, 0.924 in the internal validation queue, and 0.922 in the external validation queue. At the same time, the predictive model also showed excellent calibration and higher clinical value. CONCLUSIONS: A nomogram predicting readmission of patients with respiratory failure within 365 days based on three independent risk factors and a jointly developed random survival forest algorithm has been developed and validated. This improves the accuracy of predicting patient readmission and provides practical information for individualized treatment decisions.

LincR-PPP2R5C Promotes Th2 Cell Differentiation Through PPP2R5C/PP2A by Forming an RNA-DNA Triplex in Allergic Asthma.

PURPOSE: The roles and mechanisms of long noncoding RNAs (lncRNAs) in T helper 2 (Th2) differentiation from allergic asthma are poorly understood. We aimed to explore a novel lncRNA, LincR-protein phosphatase 2 regulatory subunit B' gamma (PPP2R5C), in Th2 differentiation in a mouse model of asthma. METHODS: LincR-PPP2R5C from RNA-seq data of CD4+ T cells of asthma-like mice were validated and confirmed by quantitative reverse transcription polymerase chain reaction, northern blotting, nuclear and cytoplasmic separation, and fluorescence in situ hybridization (FISH). Lentiviruses encoding LincR-PPP2R5C or shRNA were used to overexpress or silence LincR-PPP2R5C in CD4+ T cells. The interactions between LincR-PPP2R5C and PPP2R5C were explored with western blotting, chromatin isolation by RNA purification assay, and fluorescence resonance energy transfer. An ovalbumin-induced acute asthma model in knockout (KO) mice (LincR-PPP2R5C KO, CD4 conditional LincR-PPP2R5C KO) was established to explore the roles of LincR-PPP2R5C in Th2 differentiation. RESULTS: LncR-PPP2R5C was significantly higher in CD4+ T cells from asthmatic mice ex vivo and Th2 cells in vitro. The lentivirus encoding LincR-PPP2R5C suppressed Th1 differentiation; in contrast, the short hairpin RNA (shRNA) lentivirus decreased LincR-PPP2R5C and Th2 differentiation. Mechanistically, LincR-PPP2R5C deficiency suppressed the phosphatase activity of the protein phosphatase 2A (PP2A) holocomplex, resulting in a decline in Th2 differentiation. The formation of an RNA-DNA triplex between LincR-PPP2R5C and the PPP2R5C promoter enhanced PPP2R5C expression and activated PP2A. LincR-PPP2R5C KO and CD4 conditional KO decreased Th2 differentiation, airway hyperresponsiveness and inflammatory responses. CONCLUSIONS: LincR-PPP2R5C regulated PPP2R5C expression and PP2A activity by forming an RNA-DNA triplex with the PPP2R5C promoter, leading to Th2 polarization in a mouse model of acute asthma. Our data presented the first definitive evidence of lncRNAs in the regulation of Th2 cells in asthma.

IL-33/ST2 axis deficiency exacerbates neutrophil-dominant allergic airway inflammation.

OBJECTIVE: The IL-33/ST2 axis has been extensively investigated in type 2 eosinophilic inflammation. Here, we aimed to investigate the role of the IL-33/ST2 axis in neutrophil-dominant allergic airway inflammation. METHODS: House-dust mite (HDM) extract and lipopolysaccharide (LPS) were administered to establish a murine model of neutrophil-dominant allergic airway inflammation. The formation of neutrophilic extracellular traps (NETs) in the lung tissues was demonstrated by immunofluorescence imaging. Mature IL-33 in bronchoalveolar lavage fluid (BALF) was detected by Western blotting. The neutrophilic chemokine KC produced by bone marrow-derived macrophages (BMDMs) or primary alveolar epithelial cells was measured with a commercial ELISA kit. RESULTS: In the present study, we observed neutrophilic inflammation and tight junction damage in the lungs of mice sensitised with HDM and LPS. Furthermore, sensitisation with HDM and LPS resulted in the formation of NETs, accompanied by increased levels of mature IL-33 in the BALF. Moreover, LPS damaged the epithelial tight junction protein occludin directly or indirectly by inducing NET formation. Surprisingly, IL-33 deficiency augmented neutrophilia and epithelial barrier injury in the lungs of mice after sensitisation with HDM and LPS. Similarly, the absence of ST2 exacerbated the neutrophilic inflammatory response, decreased the expression of occludin and exacerbated the severity of neutrophil-dominant allergic airway inflammation in an HDM/LPS-induced mouse model. Mechanistically, BMDMs and alveolar epithelial cells from IL-33- or ST2-deficient mice tended to produce higher levels of the neutrophilic chemokine KC. CONCLUSIONS: These results demonstrated that the IL-33/ST2 axis may play a protective role in neutrophil-dominant allergic airway inflammation.

Epithelial-Mesenchymal Transition in Asthma Airway Remodeling Is Regulated by the IL-33/CD146 Axis.

Epithelial-mesenchymal transition (EMT) is essential in asthma airway remodeling. IL-33 from epithelial cells is involved in pulmonary fibrosis. CD146 has been extensively explored in cancer-associated EMT. Whether IL-33 regulates CD146 in the EMT process associated with asthma airway remodeling is still largely unknown. We hypothesized that EMT in airway remodeling was regulated by the IL-33/CD146 axis. House dust mite (HDM) extract increased the expression of IL-33 and CD146 in epithelial cells. Increased expression of CD146 in HDM-treated epithelial cells could be blocked with an ST2-neutralizing antibody. Moreover, HDM-induced EMT was dependent on the CD146 and TGF-ß/SMAD-3 signaling pathways. IL-33 deficiency decreased CD146 expression and alleviated asthma severity. Similarly, CD146 deficiency mitigated EMT and airway remodeling in a murine model of chronic allergic airway inflammation. Furthermore, CD146 expression was significantly elevated in asthma patients. We concluded that IL-33 from HDM extract-treated alveolar epithelial cells stimulated CD146 expression, promoting EMT in airway remodeling in chronic allergic inflammation.

MiR-1165-3p Suppresses Th2 Differentiation via Targeting IL-13 and PPM1A in a Mouse Model of Allergic Airway Inflammation.

PURPOSE: CD4⁺T cells are essential in the pathogenesis of allergic asthma. We have previously demonstrated that microRNA-1165-3p (miR-1165-3p) was significantly reduced in T-helper type (Th) 2 cells and that miR-1165-3p was a surrogate marker for atopic asthma. Little is known about the mechanisms of miR-1165-3p in the regulation of Th2-dominated allergic inflammation. We aimed to investigate the associations between Th2 differentiation and miR-1165b-3p in asthma as well as the possible mechanisms. METHODS: CD4⁺ naïve T cells were differentiated into Th1 or Th2 cells in vitro. MiR-1165-3p was up-regulated or down-regulated using lentiviral systems during Th1/Th2 differentiation. In vivo, the lentiviral particles with the miR-1165-3p enhancer were administered by tail vein injection on the first day of a house dust mite -induced allergic airway inflammation model. Allergic inflammation and Th1/Th2 differentiation were routinely monitored. To investigate the potential targets of miR-1165-3p, biotin-microRNA pull-down products were sequenced, and the candidates were further verified with a dual-luciferase reporter assay. The roles of a target protein phosphatase, Mg2+/Mn2+-dependent 1A (PPM1A), in Th2 cell differentiation and allergic asthma were further explored. Plasma PPM1A was determined by ELISA in 18 subjects with asthma and 20 controls. RESULTS: The lentivirus encoding miR-1165-3p suppressed Th2-cell differentiation in vitro. In contrast, miR-1165-3p silencing promoted Th2-cell development. In the HDM-induced model of allergic airway inflammation, miR-1165-3p up-regulation was accompanied by reduced airway hyper-responsiveness, serum immunoglobulin E, airway inflammation and Th2-cell polarization. IL-13 and PPM1A were the direct targets of miR-1165-3p. The expression of IL-13 or PPM1A was inversely correlated with that of miR-1165-3p. PPM1A regulated the signal transducer and activator of transcription and AKT signaling pathways during Th2 differentiation. Moreover, plasma PPM1A was significantly increased in asthmatic patients. CONCLUSIONS: MiR-1165-3p negatively may regulate Th2-cell differentiation by targeting IL-13 and PPM1A in allergic airway inflammation.

Airway invasive aspergillosis with organizing pneumonia: a case report and review of literature.

Organizing pneumonia (OP) is a distinct clinical and pathologic entity. This condition can be cryptogenic (COP) or secondary to other known causes (secondary OP, SOP). Concomitant occurrence of invasive pulmonary aspergillosis (IPA) with SOP is unusual. Here, we report a case where SOP was a presenting feature in a patient with diagnosed IPA. A previously healthy 62-year-old man presented to the hospital with a month of intermittent fever accompanied by cough and expectoration. According to computed tomography (CT), sputum culture, and transbronchial lung biopsy, he was diagnosed as IPA. Despite undergoing voriconazole and dexamethasone therapy, the patient's condition did not improve after three weeks of therapy. CT-guided percutaneous lung biopsy performed in the left upper lung showed invasive airway aspergillosis with organizing pneumonia. Two months after the combination therapy of voriconazole and methylprednisolone, the CT scan indicated the pulmonary consolidations were almost entirely resolved. To the best of our knowledge, this is the first case of successful non-surgical treatment of IPA with SOP. In a review of the literature, we aimed to highlight the possibility of invasive airway aspergillosis concurrent with secondary organizing pneumonia. Physicians should be aware of the possibility of SOP in the case of IPA.

Fine Particulate Matter (PM2.5) Promotes CD146 Expression in Alveolar Epithelial Cells and Cryptococcus neoformans Pulmonary Infection.

Air pollution is a leading cause of increasing infectious lung diseases. Pulmonary cryptococcosis is a fatal fungal pneumonia in acquired immunodeficiency syndrome patients. In some cases, the pathogen Cryptococcus neoformans also develops dormant nodules in immunocompetent individuals. In the present study, we demonstrated that fine particulate matter (PM2.5) increased CD146 expression in alveolar epithelial cells and promoted C. neoformans pulmonary infection. Aryl hydrocarbon receptor (AhR) signaling was required for increased expression of CD146 in epithelial cells treated with PM2.5. In a murine model of pulmonary infection, PM2.5 promoted fungal infection, and CD146 deficiency decreased the fugal burden of C. neoformans. Our study may highlight the importance of air pollution to lung mycosis and CD146 as a target for preventing infectious lung diseases.

Enterovirus A71 capsid protein VP1 increases blood-brain barrier permeability and virus receptor vimentin on the brain endothelial cells.

Enterovirus A71 (EV-A71) is the major cause of severe hand-foot-and-mouth diseases (HFMD), especially encephalitis and other nervous system diseases. EV-A71 capsid protein VP1 mediates virus attachment and is the important virulence factor in the EV-A71pathogenesis. In this study, we explored the roles of VP1 in the permeability of blood-brain barrier (BBB). Sera albumin, Evans blue, and dextran leaked into brain parenchyma of the 1-week-old C57BL/6J mice intracranially injected with VP1 recombinant protein. VP1 also increased the permeability of the brain endothelial cells monolayer, an in vitro BBB model. Tight junction protein claudin-5 was reduced in the brain tissues or brain endothelial cells treated with VP1. In contrast, VP1 increased the expression of virus receptor vimentin, which could be blocked with VP1 neutralization antibody. Vimentin expression in the VP1-treated brain endothelial cells was regulated by TGF-ß/Smad-3 and NF-κB signal pathways. Moreover, vimentin over-expression was accompanied with compromised BBB. From these studies, we conclude that EV-A71 virus capsid protein VP1 disrupted BBB and increased virus receptor vimentin, which both may contribute to the virus entrance into brain and EV-A71 CNS infection.

Downregulation of miR-4772-3p promotes enhanced regulatory T cell capacity in malignant pleural effusion by elevating Helios levels.

BACKGROUND: Malignant pleural effusion (MPE) is a complicated condition of patients with advanced tumors. Further dissecting the microenvironment of infiltrated immune cells and malignant cells are warranted to understand the immune-evasion mechanisms of tumor development and progression. METHODS: The possible involvement of microRNAs (miRNAs) in malignant pleural fluid was investigated using small RNA sequencing. Regulatory T cell (Treg) markers (CD4, CD25, forkhead box P3), and Helios (also known as IKAROS Family Zinc Finger 2 [IKZF2]) were detected using flow cytometry. The expression levels of IKZF2 and miR-4772-3p were measured using quantitative real-time reverse transcription polymerase chain reaction. The interaction between miR-4772-3p and Helios was determined using dual-luciferase reporter assays. The effects of miR-4772-3p on Helios expression were evaluated using an in vitro system. Correlation assays between miR-4772-3p and functional molecules of Tregs were performed. RESULTS: Compared with non-malignant controls, patients with non-small cell lung cancer had an increased Tregs frequency with Helios expression in the MPE and peripheral blood mononuclear cells. The verified downregulation of miR-4772-3p was inversely related to the Helios Tregs frequency and Helios expression in the MPE. Overexpression of miR-4772-3p could inhibit Helios expression in in vitro experiments. However, ectopic expression of Helios in induced Tregs reversed the effects induced by miR-4772-3p overexpression. Additionally, miR-4772-3p could regulate Helios expression by directly targeting IKZF2 mRNA. CONCLUSION: Downregulation of miR-4772-3p, by targeting Helios, contributes to enhanced Tregs activities in the MPE microenvironment.

Increased neutrophils and IL-17A in a rare organizing pneumonia secondary to extrapulmonary operation.

Organizing pneumonia (OP) is a clinical syndrome caused by various diseases. The most common causes are infection, connective tissue disease, radiation therapy, drug reaction and thoracic operation. Herein, we describe the case of a patient that developed OP after fracture internal fixation. The case was confirmed to be OP by computer tomographic (CT)-guided percutaneous needle lung biopsy, and other causes of OP were excluded. After the initiation of corticosteroid therapy, marked clinical and radiographic improvements occurred. In addition, we discovered increased neutrophils and IL-17A in the lung tissue of the patient. To the best of our knowledge, this is the first case report about OP secondary to extrapulmonary operation.

Neutrophil extracellular traps induced by VP1 contribute to pulmonary edema during EV71 infection.

Pulmonary edema is a fatal complication of EV71-associated hand, foot, and mouth disease (HFMD). The pathogenesis of EV71-induced pulmonary edema remains largely unclear. In this study, we aimed to explore the roles of the capsid protein VP1 in the occurrence of EV71-induced pulmonary edema. The intranasal inoculation of recombinant VP1 protein caused lung inflammation with an elevation of inflammatory cytokines and neutrophils infiltration. Moreover, neutrophil extracellular traps (NETs) were observed in the lung parenchyma of the mice treated with VP1. VP1 directly induced the formation of NETs, which depended on PAD4. VP1 also damaged the lung barrier via the reduction of the tight junction protein occludin. Moreover, the EV71 attachment receptor vimentin was increased upon VP1 administration. In contrast, NETs decreased vimentin levels, suggesting a novel role for NETs in viral immune defense. These results evidenced a direct role of VP1 in EV71-induced pulmonary edema and demonstrated that NETs may be both harmful and beneficial in EV71 infection.

Next Generation Sequencing for Long Non-coding RNAs Profile for CD4+ T Cells in the Mouse Model of Acute Asthma.

BACKGROUND AND AIMS: Although long non-coding RNAs (lncRNAs) have been linked to many diseases including asthma, little is known about lncRNA transcriptomes of CD4+ T cells in asthma. The present study aimed to explore the lncRNAs profile in the CD4+T cells from the mouse model of acute asthma. METHODS: Next generation sequencing for lncRNAs and mRNAs was performed on CD4+ T cells from asthma and control mice. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) pathway analyses were performed to predict the functions and signal pathways for the aberrant lncRNAs. The selected lncRNAs were further measured using quantitative real-time PCR (polymerase chain reaction) and observed in the fluorescence in situ hybridization (FISH). The lncRNA-mRNA co-expression network was constructed via Pearson's correlation coefficient and Cytoscape 3.6. RESULTS: Next generation sequencing revealed 36 up-regulated lncRNAs and 98 down-regulated lncRNAs in acute asthma compared with controls. KEGG pathway analysis showed that cytokine-cytokine receptor interaction had the highest enrichment scores. A co-expression network was constructed in which 23 lncRNAs and 301 mRNAs altered formed a total of 12424 lncRNA and mRNA pairs. To validate the RNA sequencing results, we measured the 4 different lncRNAs using qPCR. The lncRNA fantom3_9230106C11 was significantly reduced in CD4+ T cells of asthma. Bioinformatics analysis showed that lncRNA fantom3_9230106C11 had the potential to interact with many miRNAs and transcription factors related to Th2 differentiation. CONCLUSION: This study provided the first evidence for different expression of lncRNAs of CD4+T cells in asthma and may serve as a template for further, larger functional in-depth analyses regarding asthma molecular lncRNAs.

A novel microRNA miR-1165-3p as a potential diagnostic biomarker for allergic asthma.

CONTEXT: A further examination of a novel miRNA,miR-1165-3p as a biomarker for asthma, which was previously implicated in helper T cells (Th2) in a murine asthma model. OBJECTIVE: To determine whether serum miR-1165-3p can serve as a potential diagnostic biomarker for allergic asthma. METHODS: Serum miR-1165-3p was quantified via quantitative real-time PCR (qRT-PCR) in asthmatic and control samples. Serum miR-1165-3p levels were compared between groups and the clinical diagnostic abilities of miR-1165-3p were evaluated. The analyses utilized included a student's t test, one-way ANOVA, and the generation of receiver operating characteristic (ROC) curves. RESULTS: Serum miRNA-1165-3p levels were significantly elevated in asthmatics when compared to the healthy controls. Furthermore, the sensitivity and specificity of serum miR-1165-3p were found to be 83% and 68.2%. Additionally, serum miR-1165-3p levels were also found to be significantly elevated in patients with allergic rhinitis (AR) or allergic bronchopulmonary aspergillosis (ABPA). CONCLUSIONS: This study showed that serum miR-1165-3p can potentially be utilized as a noninvasive biomarker that is able to aid in the diagnosis and characterization of allergic asthma.

Fine Particulate Matter (PM2.5) Promoted the Invasion of Lung Cancer Cells via an ARNT2/PP2A/STAT3/MMP2 Pathway.

Accumulating evidence indicates that fine particulate matter (PM2.5) exposure is associated with many cardiopulmonary diseases, particularly lung carcinoma. Nevertheless, the underlying biological mechanisms by which PM2.5 exposure initiates and aggravates lung carcinoma remain elusive. In the present study, we collected PM2.5 in Nanjing and explored the mechanisms underlying the oncogenic roles of PM2.5 in the murine lung carcinoma cell line LLC in vitro and in vivo. PM2.5 was closely attached to and internalized by lung cancer cells. Moreover, PM2.5 increased the production of ARNT2 and the inactivation of the tumor suppressor B56γ-PP2A, which was followed by the activation of ps727STAT3 and the enhancement of invasive ability by MMP-2. Furthermore, we took advantage of an orthotopic lung carcinoma metastasis mouse model to illustrate the prometastatic effect of PM2.5 in vivo; our results suggested that the ARNT2/PP2A/STAT3/MMP-2 cascade played a key role in PM2.5-related oncogenicity. Finally, we observed that PM2.5 was deposited in human lung carcinoma tissues, indicating that this potential pathway may also be involved in human lung carcinoma. These findings demonstrated that fine particulate matter, or PM2.5, promoted the invasion of lung cancer cells via an ARNT2/PP2A/STAT3/MMP2 pathway, which may be targeted to alleviate the tumorigenic effect of PM2.5 in lung cancer.

Next generation sequencing for miRNA profile of spleen CD4+ T cells in the murine model of acute asthma.

AIM: To explore the miRNAs profile of CD4+ T lymphocytes in asthma via next generation sequencing. METHODS: In the murine model of acute asthma, spleen CD4+ T lymphocytes were sorted, in which small RNAs were extracted and sequenced. Novel miRNAs were measured with real time quantitative reverse transcription polymerase chain reaction (qRT-PCR). RESULTS: A total of 127 miRNAs were found to exhibit at least twofold change. In the 262 predicted novel miRNAs, 14 novel miRNAs were measured in qRT-PCR in the sorted CD4+ T cells or in the differentiated Th1/Th2 cells and novel miR-11 (xxx-m0228-3p) was significantly decreased in the sorted CD4+ T cells from the murine model of asthma and in the Th2 cells. CONCLUSION: Aberrant miRNAs profile in the CD4+ T lymphocytes from acute asthma was documented.