SUMOylation of SMAD4 by PIAS1 in Conjunction with Vimentin Upregulation Promotes Migration Potential in Non-Small Cell Lung Cancer.

BACKGROUND: The expression of vimentin as a marker of epithelial-to-mesenchymal transition (EMT) has been speculated to be associated with tissue heterogeneity and metastases of non-small cell lung cancer (NSCLC). METHODS: This study utilized in vitro co-immunoprecipitation with small interfering RNAs (siRNAs) against protein inhibitors of STAT system type 1 (PIAS1) or SMAD4 in transforming growth factor-beta (TGF-ß) signaling pathway in combination with SUMOylation assay. RESULTS: We successfully demonstrated that PIAS1 enhanced SUMOylation of SMAD4 by forming a complex PIAS1-SUMO1-SMAD4 protein complex. This, in accordance with subsequently increased production of vimentin microfilaments, led to enhanced migration ability of non-small cell lung cancer (NSCLC) A549 line, observed from wound healing assay. CONCLUSIONS: Our results further supported the positive correlation of SUMOylated SMAD4 mediated by PIAS1 and downstream overexpression of vimentin. In addition, the observation that overexpression of vimentin in this certain cell line was not necessarily linked with accelerated relative wound closure raised concerns that further exploration will be needed to confirm if the causal relationship exists between vimentin expression and the metastases of NSCLC, and if so, to what extent vimentin contributes to it.

The diagnostic value of interleukin-36 cytokines in pleural effusions of varying etiologies.

BACKGROUND: The clinical management of pleural effusion (PE) poses challenges due to its diverse etiologies. The objective of this research was to investigate the concentrations of interleukin-36 (IL-36) cytokines in pleural fluid (PF) from different etiologies and assess their diagnostic efficacy in distinguishing the causes of PE. METHODS: This study enrolled 89 patients with confirmed PE, comprising 11 cases classified as transudate, 24 cases as malignant pleural effusion (MPE), 24 cases as tuberculous pleural effusion (TPE), and 30 cases as parapneumonic pleural effusion (PPE). The PPE group was further subdivided into 20 cases of uncomplicated parapneumonic effusion (UPPE) and 10 cases of complicated parapneumonic effusion (CPPE)/empyema. The concentrations of IL-36 cytokines in the PF of all 89 patients were quantified by the enzyme-linked immunosorbent assay (ELISA). RESULTS: IL-36α exhibited excellent diagnostic accuracy in TPE, achieving a sensitivity of 91.7 % and specificity of 83.1 %, along with a cut-off value of 435.3 pg/ml. IL-36Ra also demonstrated relatively favorable diagnostic performance in PPE, with a sensitivity of 80.0 % and specificity of 76.3 %, along with a cut-off value of 390.8 pg/ml. Multivariable logistic regression models were successfully developed for both TPE and PPE, confirming their diagnostic utility. Furthermore, the levels of IL-36Ra were notably elevated in CPPE/empyema in comparison to UPPE. Moreover, in PF, IL-36γ exhibited positive associations with both IL-36α and IL-36Ra. CONCLUSION: IL-36α and IL-36Ra may serve as novel biomarkers for diagnosing TPE and PPE, respectively. The multivariate models established significantly enhance the diagnostic efficacy of both TPE and PPE. Furthermore, IL-36Ra can function as an indicator for assessing the extent of pleural inflammation. Additionally, the interaction among IL-36 cytokines in PF may contribute to their expression modulation.

hsa_circ_0072309 Expression Profiling in Non-small-Cell Lung Carcinoma and Its Implications for Diagnosis and Prognosis.

Circular RNAs (circRNAs), which fall into the category of endogenous ncRNAs, are linked to disease progression of neoplastic diseases. Whereas, it remains uncharacterized regarding hsa_circ_0072309's function and implications in lung carcinoma (LC). Gene Expression Omnibus (GEO) database was utilized for identifying circRNAs with aberrantly expression in LC. qRT-PCR was responsible for determining hsa_circ_0072309 levels in lung adenocarcinoma (LAC). Also, its involvement in LC cell progression was investigated. Experimentally, hsa_circ_0072309 was identified as one of the most aberrantly down-regulated circRNAs in the GEO database (GSE101684 and GSE112214). qRT-PCR revealed notably down-regulated hsa_circ_0072309 in LAC tissue, which had a close association with adverse 3-year survival, as well as LNM and advanced TNM stage. Based on ROC, the AUC of hsa_circ_0072309 was determined to be 0.887, and its specificity and susceptibility can be improved by combined detection of either CYFRA21-1 or CEA. In a word, hsa_circ_0072309 is lowly expressed in lung cancer patients and the survival rate of lowly expressed patients is significantly lower, a candidate marker with prognostic utility for the disease.

CDKN2B antisense RNA 1 expression alleviates idiopathic pulmonary fibrosis by functioning as a competing endogenouse RNA through the miR-199a-5p/Sestrin-2 axis.

Idiopathic pulmonary fibrosis (IPF) is an idiopathic interstitial lung disease. At present, the pathogenesis of IPF has not been fully elucidated, which has affected the development of effective treatment methods. Here, we explored the function and potential mechanism of long noncoding RNA (lncRNA) CDKN2B antisense RNA 1 (CDKN2B-AS1) in IPF.Transforming growth factor-ß (TGF-ß) and bleomycin (BLM) were used to induce IPF in cells and animal models. Real Time quantitative Polymerase Chain Reaction (RT-qPCR) showed the expression of CDKN2B-AS1, miR-199a-5p and Sestrin-2 (SESN2) in cells and tissues. The double luciferase reporter gene assay confirmed the targeting relationship among CDKN2B-AS1, miR-199a-5p, and SESN2. Related protein levels were detected by Western blot combined with Cell Counting Kit-8 (CCK-8), wound healing, and flow cytometry to analyze cell proliferation, migration, and apoptosis. The pathological characteristics of mouse lung tissue were determined by Hematoxylin-eosin (HE) and Masson staining. We found that the expression of CDKN2B-AS1 was decreased in TGF-ß-treated cells and BLM-treated mice. Overexpression of CDKN2B-AS1 inhibited cell proliferation and migration, promoted apoptosis, decreased the expression of fibrosis-related proteins and promoted autophagy. In addition, overexpression of CDKN2B-AS1 alleviated pulmonary fibrosis in BLM-treated mice. Mechanistically, CDKN2B-AS1 acts as a miR-199a-5p sponge to regulate SESN2 expression. Our results indicate the importance of the CDKN2B-AS1/miR-199a-5p/SESN2 axis.

A novel mutation causes Hermansky-Pudlak syndrome type 4 with pulmonary fibrosis in 2 siblings from China.

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive multisystem disorder characterized by oculocutaneous albinism (OCA) and bleeding diathesis, although it displays both genetic and phenotypic heterogeneity. Several genetic subtypes of HPS have been identified in human; however, the characterizations of HPS type 4 (HPS-4) genotype and phenotype remain unclear. This study was aimed to identify gene mutation responsible for HPS-4 with pulmonary fibrosis (PF).Two Chinese siblings in their 50 s afflicted with OCA and progressive dyspnea were recruited and underwent clinical and genetic examinations. In both patients, chest high-resolution computerized tomography showed severe interstitial PF in bilateral lung fields, and the pulmonary function test indicated restrictive lung disease. A novel homozygous frameshift mutation (NM_022081: c.630dupC; p.A211fs) in the HPS4 gene was identified by whole-exome sequencing analysis followed by Sanger DNA sequencing, and it segregated with the phenotypes. The c.630dupC mutation was not found in unaffected healthy controls. The patients were considered as HPS-4 with interstitial PF and eventually died of respiratory failure.This is the first report on the genotype and clinical phenotype of HPS-4 in China. Our results demonstrate the association between a novel frameshift mutation in HPS4 and severe PF with poor prognosis in HPS is presented.

High-Dose Paraquat Induces Human Bronchial 16HBE Cell Death and Aggravates Acute Lung Intoxication in Mice by Regulating Keap1/p65/Nrf2 Signal Pathway.

Paraquat (PQ) intoxication seriously endangers human beings' health, however, the underlying mechanisms are still unclear. Here we found that PQ inhibits human bronchial 16HBE cell proliferation and promotes cell apoptosis, necrosis as well as ROS generation in a dose dependent manner. Of note, low-dose PQ (50 µM) induces cell autophagy, increases Nrf2 as well as p65 levels and has little impacts on Keap1, while high-dose PQ (500 µM) inhibits autophagy, upregulates Keap1 as well as downregulates p65 and Nrf2. In addition, we verified that p65 overexpression increases Nrf2 and its downstream targets in 16HBE cells, which are reversed by synergistically knocking down Nrf2. Our further results showed that high-dose PQ's effects on cell proliferation, apoptosis, ROS levels and autophagy are reversed by p65 overexpression. Besides, the protective effects of overexpressed p65 on high-dose PQ (500 µM) treated 16HBE cells are abrogated by synergistically knocking down Nrf2. In vivo experiments also showed that high-dose PQ promotes inflammatory cytokines secretion, lung fibrosis and cell apoptosis, inhibits cell proliferation in mice models by regulating Keap1/p65/Nrf2 signal pathway. Therefore, we concluded that high-dose PQ (500 µM) inhibits 16HBE cell proliferation and autophagy, promotes cell death and mice lung fibrosis by regulating Keap1/p65/Nrf2 signal pathway.

Fibrocytes Ameliorate Acute Lung Injury by Decreasing Inflammatory Cytokine and Chemokine Levels and Reducing Neutrophil Accumulation in the Lung.

BACKGROUND/AIMS: Acute lung injury (ALI) remains a severe disease that threatens human life around the world. To decrease the mortality of ALI and improve ALI treatment efficacy, the development of more ALI treatments is urgently needed. Whether fibrocytes directly participate in ALI has not been studied. Therefore, a mouse model of ALI was induced with lipopolysaccharide (LPS). METHODS: Fibrocytes were harvested from peripheral blood mononuclear cells of bleomycin mice and identified by using flow cytometry to detect the expression of molecular makers. The fibrocytes were injected for the treatment of acute lung injury mice. The curative effects were evaluated by using ELISA to determine the cytokines (including TNF-α, IL-6 and IFN-γ) concentrations in bronchoalveolar lavage fluid (BALF) supernatant. RESULTS: The concentrations of cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interferon-γ (IFN-γ) were increased in mice with ALI induced with LPS. The concentrations of TNF-α, IL-6, and IFN-γ as well as their mRNA and protein expression levels were decreased by administration of fibrocytes. The effect of fibrocytes in ameliorating ALI was time dependent. LPS treatment induced an increase in myeloperoxidase (MPO) activity, whereas the fibrocyte treatment caused inhibition of MPO activity as well as expression of the neutrophil-chemoattractant chemokine macrophage inflammatory protein 2 (MIP-2). CONCLUSION: Taken together, these data suggest that fibrocytes ameliorated ALI by suppressing inflammatory cytokines and chemokines as well as by decreasing the accumulation of neutrophils in the lung.

IL-27 inhibits the TGF-ß1-induced epithelial-mesenchymal transition in alveolar epithelial cells.

BACKGROUND: IL-27 is a multifunctional cytokine that has both pro-inflammatory and anti-inflammatory functions. Although IL-27 has been shown to potently inhibit lung fibrosis, the detailed mechanism of IL-27 in this process is poorly understood. Epithelial-mesenchymal transition (EMT) is one of the key mechanisms involved in pulmonary fibrosis. We assessed the effects of IL-27 on TGF-ß1-induced EMT in alveolar epithelial cells. METHODS: A549 cells (a human AEC cell line) were incubated with TGF-ß1, IL-27, or both TGF-ß1 and IL-27, and changes in E-cadherin, ß-catenin, vimentin and a-SMA levels were measured using real-time PCR, western blotting and fluorescence microscopy. The related proteins in the JAK/STAT and TGF-ß/Smad signalling pathways were examined by western blot. RESULTS: IL-27 increased the expression of epithelial phenotypic markers, including E-cadherin and ß-catenin, and inhibited mesenchymal phenotypic markers, including vimentin and a-SMA in A549 cells. Moreover, TGF-ß1-induced EMT was attenuated by IL-27. Furthermore, we found that TGF-ß1 activated the phosphorylation of JAK1, STAT1, STAT3, STAT5, Smad1, Smad3 and Smad5, and IL-27 partially inhibited these changes in this process. When cells were treated with the STAT3 specific inhibitor wp1006 and the Smad3 specific inhibitor SIS3, the inhibition of EMT by IL-27 was significantly strengthened. CONCLUSION: Our results suggest that IL-27 attenuates epithelial-mesenchymal transition in alveolar epithelial cells in the absence or presence of TGF-ß1 through the JAK/STAT and TGF-ß/Smad signalling pathways.

IL-27 alleviates the bleomycin-induced pulmonary fibrosis by regulating the Th17 cell differentiation.

BACKGROUND: Interleukin-27 (IL-27) is a multifunctional cytokine with both pro-inflammatory and immunoregulatory functions. At present, the role of IL-27 in pulmonary fibrosis remains unknown. METHODS: In this study, we observed the expression of IL-27/IL-27R in a mouse model of bleomycin (BLM)-induced pulmonary fibrosis. We verified the role of IL-27 using hematoxylin and eosin as well as Masson's staining methods and measuring the content of hydroxyproline as well as collagen I and III. We assessed the differentiation of T lymphocytes in the spleen and measured the concentration of cytokines in bronchoalveolar lavage fluid (BALF) and the expression level of relevant proteins in the JAK/STAT and TGF-ß/Smad signaling pathways in lung tissue. RESULTS: Increased IL-27 expression in BLM-induced pulmonary fibrosis was noted. IL-27 treatment may alleviate pulmonary fibrosis and increase the survival of mice. IL-27 inhibited the development of CD4(+) IL-17(+), CD4(+) IL-4(+) T, and CD4(+) Foxp3(+) cells and the secretion of IL-17, IL-4, IL-6, and TGF-ß. IL-27 induced the production of CD4(+) IL-10(+) and CD4(+) INF-γ(+) T cells. IL-27 decreased the levels of phosphorylated STAT1, STAT3, STAT5, Smad1, and Smad3 but increased the level of SOCS3. CONCLUSIONS: This study demonstrates that IL-27 potentially attenuates BLM-induced pulmonary fibrosis by regulating Th17 differentiation and cytokine secretion.

Therapeutic effect of intravenous bone marrow-derived mesenchymal stem cell transplantation on early-stage LPS-induced acute lung injury in mice.

OBJECTIVE: To investigate the effect of intravenous bone marrow-derived mesenchymal stem cell (MSC) transplantation for early intervention of lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. METHODS: Thirty-six mice were randomized into control group, PBS-treated ALI group, and MSC-treated ALI group. In the latter two groups, mouse models of ALI were established by intranasal instillation of LPS, and 1 h later, the 4th passage of MSCs isolated from the bone marrow of mice or PBS were administered via the tail vein. The histological findings, lung wet/dry (W/D) weight ratio, neutrophil count and protein and cytokine contents in the bronchoalveolar lavage fluid (BALF), and myeloperoxidase (MPO) level in the lung tissue were analyzed at 24 h after MSC administration. Engraftment of MSCs in the recipient lung was determined by fluorescent PKH26 staining and flow cytometry. RESULTS: Compared with the control group, PBS-treated ALI group showed significantly higher protein levels, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and neutrophil count in the BALF and MPO content in the lung tissue, with also severe damage of lung histology. MSCs administration significantly reduced the lung W/D weight ratio, the levels of protein, TNF-α, IL-6 and neutrophil count in the BALF and MPO content in the lung tissue, and obviously lessened the lung injury 24 h after the transplantation. MSC administration also significantly increased the level of IL-10 in the BALF. CONCLUSION: Intravenous MSC transplantation can effectively improve the lung histology, attenuate the inflammatory response, reduce pulmonary edema in the early stage of LPS-induced ALI in mice, and such effects are independent of MSC engraftment in the lungs.

The role of all-trans retinoic acid in bleomycin-induced pulmonary fibrosis in mice.

Much evidence suggests that immune imbalance in the lung plays a crucial role in the development of pulmonary fibrosis. Recently, all-trans retinoic acid (ATRA) shifting the regulatory T/T-helper 17 (Treg/Th17) profile had been proven in some diseases. However, to date, the effect of ARTA of pulmonary fibrosis has not been examined from this aspect. The objective of this study was to study the effect of ATRA on bleomycin-induced pulmonary fibrosis in mice and its possible mechanism. Pulmonary fibrosis was induced in C57BL/6 male mice by intratracheal instillation of bleomycin (5 mg.kg(-1)), which were randomly divided into control, bleomycin, and ATRA groups. Five mice in each group were sacrificed on day 28 after intratracheal instillation. Hemotoxylin and eosin (H&E) and Masson staining were used for pathological examination, and hydroxyproline in lung tissue was measured. Interleukin (IL)-17A protein expression was observed in lung with immunohistochemistry. The expression of IL-17A, IL-10, IL-6, and transforming growth factor (TGF)-ß mRNAs were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Th17 and Treg expression in spleen lymphocytes were measured by flow cytometry. H&E and masson staining and expression of hydroxyproline showed that ATRA significantly alleviated lung fibrosis than in the bleomycin group. The expression of IL-17A, IL-10, IL-6, and TGF-ß mRNAs were higher in the bleomycin group than in the normal group. ATRA can decrease these cytokines except for IL-10. CD4+CD25+ Treg cell ratio in the bleomycin group was significantly lower than normal, but CD4+IL-17+ T cells was higher; ARTA reversed this kind of expression. ATRA may ease the bleomycin-induced pulmonary fibrosis by inhibiting the expression of IL-6 and TGF-ß, shifting the Treg/Th17 ratio and reducing the secretion of IL-17A.