Advances in common in vitro cellular models of pulmonary fibrosis.

The development of in vitro models is essential for a comprehensive understanding and investigation of pulmonary fibrosis (PF) at both cellular and molecular levels. This study presents a literature review and an analysis of various cellular models used in scientific studies, specifically focusing on their applications in elucidating the pathogenesis of PF. Our study highlights the importance of taking a comprehensive approach to studing PF, emphasizing the necessity of considering multiple cell types and organs and integrating diverse analytical perspectives. Notably, primary cells demonstrate remarkable cell growth characteristics and gene expression profiles; however, their limited availability, maintenance challenges, inability for continuous propagation and susceptibility to phenotypic changes over time significantly limit their utility in scientific investigation. By contrast, immortalized cell lines are easily accessible, cultured and continuously propagated, although they may have some phenotypic differences from primary cells. Furthermore, in vitro coculture models offer a more practical and precise method to explore complex interactions among cells, tissues and organs. Consequently, when developing models of PF, researchers should thoroughly assess the advantages, limitations and relevant mechanisms of different cell models to ensure their selection is consistent with the research objectives.

Study of Iron Complex Photosensitizer with Hollow Double-Shell Nano Structure Used to Enhance Ferroptosis and Photodynamic Therapy.

Ferroptosis therapy, which uses ferroptosis inducers to produce lethal lipid peroxides and induce tumor cell death, is considered a promising cancer treatment strategy. However, challenges remain regarding how to increase the accumulation of reactive oxygen species (ROS) in the tumor microenvironment (TME) to enhance antitumor efficacy. In this study, a hyaluronic acid (HA) encapsulated hollow mesoporous manganese dioxide (H-MnO2) with double-shell nanostructure is designed to contain iron coordinated cyanine near-infrared dye IR783 (IR783-Fe) for synergistic ferroptosis photodynamic therapy against tumors. The nano photosensitizer IR783-Fe@MnO2-HA, in which HA actively targets the CD44 receptor, subsequently dissociates and releases Fe3+ and IR783 in acidic TME. First, Fe3+ consumes glutathione to produce Fe2+, which promotes the Fenton reaction in cells to produce hydroxyl free radicals (·OH) and induce ferroptosis of tumor cells. In addition, MnO2 catalyzes the production of O2 from H2O2 and enhances the production of singlet oxygen (1O2) by IR783 under laser irradiation, thus increasing the production and accumulation of ROS to provide photodynamic therapy. The highly biocompatible IR783-Fe@MnO2-HA nano-photosensitizers have exhibited tumor-targeting ability and efficient tumor inhibition in vivo due to the synergistic effect of photodynamic and ferroptosis antitumor therapies.

Understanding the effect of moderate concentration SDS on CO2 hydrates growth in the presence of THF.

Hypothesis Additives like Tetrahydrofuran (THF) and Sodium Dodecylsulfate (SDS) improve Carbon Dioxide (CO2) hydrates thermal stability and growth rate when used separately. It has been hypothesised that combining them could improve the kinetics of growth and the thermodynamic stability of CO2 hydrates. Simulations and Experiments We exploit atomistic molecular dynamics simulations to investigate the combined impact of THF and SDS under different temperatures and concentrations. The simulation insights are verified experimentally using pendant drop tensiometry conducted at ambient pressures and high-pressure differential scanning calorimetry. Findings Our simulations revealed that the combination of both additives is synergistic at low temperatures but antagonistic at temperatures above 274.1 K due to the aggregation of SDS molecules induced by THF molecules. These aggregates effectively remove THF and CO2 from the hydrate-liquid interface, thereby reducing the driving force for hydrates growth. Experiments revealed that the critical micelle concentration of SDS in water decreases by 20% upon the addition of THF. Further experiments in the presence of THF showed that only small amounts of SDS are sufficient to increase the CO2 storage efficiency by over 40% compared to results obtained without promoters. Overall, our results provide microscopic insights into the mechanisms of THF and SDS promoters on CO2 hydrates, useful for determining the optimal conditions for hydrate growth.

Astragaloside IV inhibits epithelial-mesenchymal transition and pulmonary fibrosis via lncRNA-ATB/miR-200c/ZEB1 signaling pathway.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease involving multiple factors and genes. Astragaloside IV (ASV) is one of the main bioactive ingredients extracted from the root of Astragalus membranaceus, which plays an important role in anti-inflammatory, antioxidant and improve cardiopulmonary function. Epithelial-mesenchymal transition (EMT) is a key driver of the process of pulmonary fibrosis, and Zinc finger E-box-binding homeobox 1 (ZEB1) can promote pulmonary fibrosis in an EMT-dependent manner. Here, we found that ASV effectively inhibited the ZEB1 and EMT in both bleomycin (BLM)-induced rat pulmonary fibrosis and TGF-ß1-treated A549 cells. To further elucidate the molecular mechanisms underlying effects of ASV in IPF, we explored the truth using bioinformatics, plasmid construction, immunofluorescence staining, western blotting and other experiments. Dual luciferase reporter assay and bioinformatics proved that miR-200c not only acts as an upstream regulatory miRNA of ZEB1 but also has binding sites for the lncRNA-ATB. In A549 cell-based EMT models, ASV reduced the expression of lncRNA-ATB and upregulated miR-200c. Furthermore, overexpression of lncRNA-ATB and silencing of miR-200c reversed the down-regulation of ZEB1 and the inhibition of EMT processes by ASV. In addition, the intervention of ASV prevented lncRNA-ATB as a ceRNA from regulating the expression of ZEB1 through sponging miR-200c. Taken together, the results showed that ASV inhibited the EMT process through the lncRNA-ATB/miR-200c/ZEB1 signaling pathway, which provides a novel approach to the treatment of IPF.

Application of adaptive laboratory evolution to improve the tolerance of Rhodotorula strain to methanol in crude glycerol and development of an effective method for cell lysis.

Rhodotorula toruloides can utilize crude glycerol as the low-cost carbon source for lipid production, but its growth is subjected to inhibition by methanol in crude glycerol. Here, transcriptome profiling demonstrated that 1004 genes were significantly regulated in the strain R. toruloides TO2 under methanol stress. Methanol impaired the function of membrane transport and subsequently weakened the utilization of glycerol, activities of the primary metabolism and functions of nucleus and ribosome. Afterwards the tolerance of TO2 to methanol was improved by using two-round adaptive laboratory evolution (ALE). The final strain M2-ale had tolerance up to 3.5% of methanol. 1 H NMR-based metabolome analysis indicated that ALE not only improved the tolerance of M2-ale to methanol but also tuned the carbon flux towards the biosynthesis of glycerolipid-related metabolites. The biomass and lipid titer of M2-ale reached 14.63 ± 0.45 g L-1 and 7.06 ± 0.44 g L-1 at 96 h in the crude glycerol medium, which increased up to 17.69% and 31.39%, respectively, comparing with TO2. Afterwards, an effective method for cell lysis was developed by combining sonication and enzymatic hydrolysis (So-EnH). The lytic effect of So-EnH was validated by using confocal imaging and flow cytometry. At last, lipid recovery rate reached 95.4 ± 2.7% at the optimized condition.

[Retracted] Astragaloside IV inhibits oxidized low­density lipoprotein­induced endothelial damage via upregulation of miR­140­3p.

Following the publication of this paper, it was drawn to the Editors' attention by a concerned reader that the ß­actin control western blotting data featured in Fig. 3E were strikingly similar to data appearing in different form in another article by different authors. Upon asking the authors to explain this phenomenon, they were unable to provide the raw data for this experiment. Owing to the fact that the contentious data in the above article had already been published elsewhere prior to its submission to International Journal of Molecular Medicine, the Editor has decided that this paper should be retracted from the Journal. The Editor apologizes to the readership for any inconvenience caused. [International Journal of Molecular Medicine 44: 847­856, 2019; DOI: 10.3892/ijmm.2019.4257].

[Retracted] Exosome­mediated transfer of lncRNA RP11­838N2.4 promotes erlotinib resistance in non­small cell lung cancer.

Following the publication of this paper, it was drawn to the Editors' attention by a concerned reader that various panels showing the cellular data in Fig. 1B, the western blotting data shown in Figs. 2B and 3C, the TEM scanning data shown in Fig. 4B, the ChIP assay data in Fig. 2F and the flow cytometric assay data in Fig. 3D were strikingly similar to data appearing in different form in other articles by different authors from different research institutions. Owing to the fact that the contentious data in the above article had already been published, or were already under consideration for publication elsewhere, prior to its submission to International Journal of Oncology, the Editor has decided that this paper should be retracted from the Journal. The authors independently contacted the Editorial Office to request the retraction of this paper. The Editor apologizes to the readership for any inconvenience caused. [International Journal of Oncology 53: 527­538, 2018; DOI: 10.3892/ijo.2018.4412].

EpiReSIM: A Resampling Method of Epistatic Model without Marginal Effects Using Under-Determined System of Equations.

Simulation experiments are essential to evaluate epistasis detection methods, which is the main way to prove their effectiveness and move toward practical applications. However, due to the lack of effective simulators, especially for simulating models without marginal effects (eNME models), epistasis detection methods can hardly verify their effectiveness through simulation experiments. In this study, we propose a resampling simulation method (EpiReSIM) for generating the eNME model. First, EpiReSIM provides two strategies for solving eNME models. One is to calculate eNME models using prevalence constraints, and another is by joint constraints of prevalence and heritability. We transform the computation of the model into the problem of solving the under-determined system of equations. Introducing the complete orthogonal decomposition method and Newton's method, EpiReSIM calculates the solution of the underdetermined system of equations to obtain the eNME model, especially the solution of the high-order model, which is the highlight of EpiReSIM. Second, based on the computed eNME model, EpiReSIM generates simulation data by a resampling method. Experimental results show that EpiReSIM has advantages in preserving the biological properties of minor allele frequencies and calculating high-order models, and it is a convenient and effective alternative method for current simulation software.

Identification and Validation of Potential Biomarkers and Pathways for Idiopathic Pulmonary Fibrosis by Comprehensive Bioinformatics Analysis.

OBJECTIVE: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, irreversible, high-mortality lung disease, but its pathogenesis is still unclear. Our purpose was to explore potential genes and molecular mechanisms underlying IPF. METHODS: IPF-related data were obtained from the GSE99621 dataset. Differentially expressed genes (DEGs) were identified between IPF and controls. Their biological functions were analyzed. The relationships between DEGs and microRNAs (miRNAs) were predicted. DEGs and pathways were validated in a microarray dataset. A protein-protein interaction (PPI) network was constructed based on these common DEGs. Western blot was used to validate hub genes in IPF cell models by western blot. RESULTS: DEGs were identified for IPF than controls in the RNA-seq dataset. Functional enrichment analysis showed that these DEGs were mainly enriched in immune and inflammatory response, chemokine-mediated signaling pathway, cell adhesion, and other biological processes. In the miRNA-target network based on RNA-seq dataset, we found several miRNA targets among all DEGs, like RAB11FIP1, TGFBR3, and SPP1. We identified 304 upregulated genes and 282 downregulated genes in IPF compared to controls both in the microarray and RNA-seq datasets. These common DEGs were mainly involved in cell adhesion, extracellular matrix organization, oxidation-reduction process, and lung vasculature development. In the PPI network, 3 upregulated and 4 downregulated genes could be considered hub genes, which were confirmed in the IPF cell models. CONCLUSION: Our study identified several IPF-related DEGs that could become potential biomarkers for IPF. Large-scale multicentric studies are eagerly needed to confirm the utility of these biomarkers.

Metastasis-associated protein 1 promotes epithelial-mesenchymal transition in idiopathic pulmonary fibrosis by up-regulating Snail expression.

Idiopathic pulmonary fibrosis (IPF) is a progressive and usually fatal lung disease that lacking effective interventions. It is well known that aberrant activation of transforming growth factor-beta1 (TGF-ß1) frequently promotes epithelial-mesenchymal transition (EMT) in IPF. Metastasis-associated gene 1 (MTA1) has identified as an oncogene in several human tumours, and aberrant MTA1 expression has been related to the EMT regulation. However, its expression and function in IPF remain largely unexplored. Using a combination of in vitro and in vivo studies, we found that MTA1 was significantly up-regulated in bleomycin-induced fibrosis rats and TGF-ß1-treated alveolar type Ⅱ epithelial (RLE-6TN) cells. Overexpression of MTA1 induced EMT of RLE-6TN cells, as well as facilitates cell proliferation and migration. In contrast, knockdown of MTA1 reversed TGF-ß1-induced EMT of RLE-6TN cells. The pro-fibrotic action of MTA1 was mediated by increasing Snail expression through up-regulating Snail promoter activity. Moreover, inhibition of MTA1 effectively attenuated bleomycin-induced fibrosis in rats. Additionally, we preliminarily found astragaloside IV (ASV), which was previously validated having inhibitory effects on TGF-ß1-induced EMT, could inhibit MTA1 expression in TGF-ß1-treated RLE-6TN cells. These findings highlight the role of MTA1 in TGF-ß1-mediated EMT that offer novel strategies for the prevention and treatment of IPF.

Sirt1 antisense long non-coding RNA attenuates pulmonary fibrosis through sirt1-mediated epithelial-mesenchymal transition.

Long noncoding RNAs sirt1 antisense (sirt1 AS) was reported to play crucial roles in the progression of organ fibrosis. However, the roles of sirt1 AS in idiopathic pulmonary fibrosis (IPF) are still unknown. In addition, we have previously demonstrated that astragaloside IV (ASV), a bioactive saponin extract of the Astragalus root, significantly alleviates IPF by inhibiting transforming growth factor ß1 (TGF-ß1) induced epithelial-mesenchymal transition (EMT). Further investigations into the influence of ASV on lncRNAs expression will be helpful to delineate the complex regulatory networks underlying the biological function of ASV. Here, we found sirt1 AS expression was significantly decreased in BLM-induced pulmonary fibrosis. We further found that sirt1 AS effectively inhibited TGF-ß1-meidated EMT in vitro and alleviated the progression of IPF in vivo. Mechanistically, sirt1 AS was validate to enhance the stability of sirt1 and increased sirt1 expression, thereby to inhibit EMT in IPF. Furthermore, we demonstrated that ASV treatment increased sirt1 AS expression and silencing of sirt1 AS impaired anti-fibrosis effects of ASV on IPF. Collectively, sirt1 AS was critical for ASV-mediated inhibition of IPF progression and targeting of sirt1 AS by ASV could be a potential therapeutic approach for IPF.

Angelica Sinensis Polysaccharide Suppresses Epithelial-Mesenchymal Transition and Pulmonary Fibrosis via a DANCR/AUF-1/FOXO3 Regulatory Axis.

Idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of lung fibroblasts and extracellular matrix deposition. Angelica sinensis polysaccharide (ASP), the major bioactive component that can extracted from roots of angelica, plays functional roles in immunomodulation, anti-tumor activity, and hematopoiesis. Emerging evidence has suggested that long noncoding RNAs (lncRNAs) play important roles in pathophysiological processes in various diseases. However, the roles of lncRNAs and ASP in IPF remain poorly understood. In the present study, we investigated the effects of ASP in IPF, as well as their functional interactions with lncRNA DANCR (differentiation antagonizing non-protein coding RNA). IPF models were established by treating Sprague-Dawley rats with BLM and treating alveolar type Ⅱ epithelial (RLE-6TN) cells with TGF-ß1. Our results showed that ASP treatment suppressed pulmonary fibrosis in rats and fibrogenesis in RLE-6TN cells. The lncRNA DANCR is downregulated after ASP treatment in both rat lung tissues and RLE-6TN cells, and DANCR overexpression dramatically reversed the suppressive effects of ASP in IPF. Mechanistically, DANCR directly binds with AUF1 (AU-binding factor 1), thereby upregulating FOXO3 mRNA and protein levels. Moreover, overexpression of AUF1 or FOXO3 reversed the functional effects induced by ASP treatment. In conclusion, our findings showed that DANCR mediates ASP-induced suppression of IPF via upregulation of FOXO3 protein levels in an AUF1-dependent manner. Therefore, DANCR could serve as a promising therapeutic target in IPF treatment with ASP.

Correction to: Gingerol inhibits cisplatin-induced acute and delayed emesis in rats and minks by regulating the central and peripheral 5-HT, SP, and DA systems.

In the original publication of the article, Figures 2, 3, 5, 11 and 13 were published incorrectly. The corrections version of figures are given below.

Gingerol inhibits cisplatin-induced acute and delayed emesis in rats and minks by regulating the central and peripheral 5-HT, SP, and DA systems.

Gingerol, a biologically active component in ginger, has shown antiemetic properties. Our study aimed to explore the underlying mechanisms of gingerol on protecting rats and minks from chemotherapy-induced nausea and vomiting. The preventive impact of gingerol was evaluated in the pica model of rats and the vomiting model of minks induced by cisplatin at every 6 h continuously for a duration of 72 h. Animals were arbitrarily separated into blank control group, simple gingerol control group, cisplatin control group, cisplatin + metoclopramide group, cisplatin + three different doses gingerol group (low-dose; middle-dose; high-dose). The area postrema as well as ileum damage were assessed using H&E stain. The levels of 5-TH, 5-HT3 receptor, TPH, SERT, SP, NK1 receptor, PPT, NEP, DA, D2R, TH, and DAT were determined using immunohistochemistry or qRT-PCR in rats and minks. All indicators were measured in the area postrema along with ileum. The kaolin intake by rats and the incidence of CINV of minks were significantly decreased after pretreatment with gingerol in a dosage-dependent way for the duration of 0-24-h and 24-72-h. Gingerol markedly decreased the levels of 5-TH, 5-HT3 receptor, TPH, SP, NK1 receptor, PPT, DA, D2R, TH, alleviated area postrema as well as ileum damage, and increased the accumulation of SERT, NEP, DAT in the area postrema along with ileum of rats and minks. Gingerol alleviates cisplatin-induced kaolin intake of rats and emesis of minks possibly by regulating central and peripheral 5-HT system, SP system and DA system.

Astragaloside IV protects endothelial progenitor cells from the damage of ox-LDL via the LOX-1/NLRP3 inflammasome pathway.

Purpose: Functional impairment of endothelial progenitor cells (EPCs) is frequently observed in patients with diabetic vascular complications. Astragaloside IV (ASV) has a significant protective effect against vascular endothelial dysfunction. Thus, this study aimed to investigate the role of ASV on oxidized low-density lipoprotein (ox-LDL)-induced EPCs dysfunction and its potential mechanisms. Methods: EPCs were isolated from the peripheral blood of mice and treated with different concentration of ASV (10, 20, 40, 60, 80, 100 and 200 µM). ox-LDL was served as a stimulus for cell model. The proliferation and migration, and improved tube formation ability of EPCs were determined. Reactive oxygen species (ROS) production and the levels of inflammatory cytokines, including interleukin 1ß (IL-1ß), IL-6, IL-10 and tumor necrosis factor (TNF-α) were measured. The expression oflectin-like oxidized LDL receptor (LOX-1) andNod-like receptor nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3) inflammasome were detected by Western blot analysis. Results: We found ASV treatment alleviated ox-LDL-induced cellular dysfunction, as evidenced by promoted proliferation and migration, and improved tube formation ability. Besides, ASV treatment significantly suppressed ox-LDL-induced ROS production and the levels of inflammatory cytokines. ASV inhibited ox-LDL-induced expression of LOX-1 in a concentration-dependent manner. Overexpression of LOX-1 in EPCs triggered NLRP3inflammasome activation, while inhibition of LOX-1 or treatment with ASV suppressed ox-LDL-induced NLRP3 inflammasome activation. Furthermore, overexpression of LOX-1 in ox-LDL-induced EPCs furtherly impaired cellular function, which could be ameliorated by ASV treatment. Conclusion: Our study showed that ASV may protect EPCs against ox-LDL-induced dysfunction via LOX-1/NLRP3 pathway.

Astragaloside IV inhibits oxidized low­density lipoprotein­induced endothelial damage via upregulation of miR­140­3p.

Oxidized low­density lipoprotein (ox­LDL)­mediated endothelial cell injury has an important role in the vascular complications of type 2 diabetes. Astragaloside IV (ASV) is an active component of Radix Astragali, which has been demonstrated to exert protective effects against endothelial damage. The present study explored whether microRNAs (miRNAs) are involved in mediating the protective effects of ASV on ox­LDL­induced damage in human umbilical vein endothelial cells (HUVECs). RNA sequencing and reverse transcription­quantitative PCR analyses revealed that ox­LDL treatment significantly downregulated miR­140­3p expression in HUVECs. miR­140­3p overexpression promoted cell proliferation and inhibited apoptosis in ox­LDL­induced HUVECs. However, inhibition of miR­140­3p expression could reverse the effects of ASV on ox­LDL­induced HUVECs and reactivate ASV­inhibited PI3K/Akt signaling in ox­LDL­induced HUVECs. In addition, Krüppel­like factor 4 (KLF4) was identified as a target of miR­140­3p in ox­LDL­treated HUVECs. Subsequent experiments revealed that KLF4 overexpression partially counteracted the protective effects of miR­140­3p or ASV treatment in ox­LDL­induced HUVECs. Taken together, the current findings demonstrated that the protective effects of ASV on HUVECs were dependent on miR­140­3p upregulation and subsequent inhibition of KLF4 expression, which in turn suppressed the PI3K/Akt signaling pathway. The present results shed light to the molecular mechanism by which ASV alleviated ox­LDL­induced endothelial cell damage.

lncRNA ZEB1-AS1 promotes pulmonary fibrosis through ZEB1-mediated epithelial-mesenchymal transition by competitively binding miR-141-3p.

Long non-coding RNAs (lncRNAs) have been reported to be involved in various pathophysiological processes in many diseases. However, the role and mechanism of lncRNAs in pulmonary fibrosis have not been explicitly delineated. In the present study, we found that lncRNA ZEB1 antisense RNA 1 (ZEB1-AS1) is upregulated in the lungs of BLM-induced rats and TGF-ß1-induced RLE-6TN cells, and positively correlated with the levels of ZEB1, an epithelial-mesenchymal transition (EMT) master regulator. Knockdown of ZEB1-AS1 alleviated BLM-induced fibrogenesis, in vivo, via inhibiting EMT progress. Mechanistically, we identified that ZEB1-AS1 promoted fibrogenesis in RLE-6TN cells and ZEB1-AS1 silencing inhibited TGF-ß1-induced fibrogenesis through modulation of miR-141-3p. Further experiments revealed that ZEB1-AS1 acted as competing endogenous RNA (ceRNA) of miR-141-3p: forced expression of ZEB1-AS1 reduced the expression of miR-141-3p to activate Zinc-finger Ebox Binding Homeobox 1 (ZEB1) in RLE-6TN cells. In addition, we found that upregulation of miR-141-3p prevented fibrogenesis by targeting ZEB1. Therefore, our finding suggested lncRNA ZEB1-AS1 as a new profibrotic molecule that acts as a regulator of miR-141-3p/ZEB1 axis during lung fibrosis and demonstrated ZEB1-AS1 as a potential therapeutic target for the prevention and treatment of pulmonary fibrosis.

Astragaloside IV modulates TGF-ß1-dependent epithelial-mesenchymal transition in bleomycin-induced pulmonary fibrosis.

Epithelial-mesenchymal transition (EMT) plays an important role in idiopathic pulmonary fibrosis (IPF). Astragaloside IV (ASV), a natural saponin from astragalus membranaceus, has shown anti-fibrotic property in bleomycin (BLM)-induced pulmonary fibrosis. The current study was undertaken to determine whether EMT was involved in the beneficial of ASV against BLM-induced pulmonary fibrosis and to elucidate its potential mechanism. As expected, in BLM-induced IPF, ASV exerted protective effects on pulmonary fibrosis and ASV significantly reversed BLM-induced EMT. Intriguing, transforming growth factor-ß1 (TGF-ß1) was found to be up-regulated, whereas Forkhead box O3a (FOXO3a) was hyperphosphorylated and less expressed. However, ASV treatment inhibited increased TGF-ß1 and activated FOXO3a in lung tissues. TGF-ß1 was administered to alveolar epithelial cells A549 to induce EMT in vitro. Meanwhile, stimulation with TGF-ß1-activated phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) pathway and induced FOXO3a hyperphosphorylated and down-regulated. It was found that overexpression of FOXO3a leading to the suppression of TGF-ß1-induced EMT. Moreover, ASV treatment, similar with the TGF-ß1 or PI3K/Akt inhibitor, reverted these cellular changes and inhibited EMT in A549 cells. Collectively, the results suggested that ASV significantly inhibited TGF-ß1/PI3K/Akt-induced FOXO3a hyperphosphorylation and down-regulation to reverse EMT during the progression of fibrosis.

Exosome-mediated transfer of lncRNA RP11­838N2.4 promotes erlotinib resistance in non-small cell lung cancer.

Currently, resistance to tyrosine kinase inhibitors, such as erlotinib, has become a major obstacle for improving the clinical outcome of patients with metastatic and advanced­stage non-small cell lung cancer (NSCLC). While cell behavior can be modulated by long non-coding RNAs (lncRNAs), the roles of lncRNAs within extracellular vesicles (exosomes) are largely unknown. To this end, in this study, the involvement and regulatory functions of potential lncRNAs wrapped by exosomes during the development of chemoresistance in human NSCLC were investigated. Erlotinib-resistant cell lines were established by grafting HCC827 and HCC4006 cells into mice and which were treated with erlotinib. After one treatment course, xenografted NSCLC cells were isolated and transplanted into nude mice again followed by erlotinib treatment. This process was repeated until 4th generation xenografts were isolated and confirmed to be erlotinib-resistant NSCLC cells. lncRNA microarray assays followed by RT­qPCR were then performed which identified that lncRNA RP11­838N2.4 was upregulated in erlotinib-resistant cells when compared to normal NSCLC cells. Furthermore, bioinformatics analysis and chromatin immunoprecipitation revealed that forkhead box protein O1 (FOXO1) could bind to the promoter region of lncRNA RP11­838N2.4, resulting in its silencing through the recruitment of histone deacetylase. Functional experiments demonstrated that the knockdown of lncRNA RP11­838N2.4 potently promoted erlotinib-induced cytotoxicity. Furthermore, extracellular lncRNA RP11­838N2.4 could be incorporated into exosomes and transmitted to sensitive cells, thus disseminating erlotinib resistance. Treatment-sensitive cells with exosomes containing lncRNA RP11­838N2.4 induced erlotinib resistance, while the knockdown of lncRNA RP11­838N2.4 abrogated this effect. In addition, the serum expression levels of exosomal lncRNA RP11­838N2.4 were upregulated in patients exhibiting resistance to erlotinib treatment. On the whole, exosomal lncRNA RP11­838N2.4 may serve as a therapeutic target for patients with NSCLC.

LncRNA FUNDC2P4 down-regulation promotes epithelial-mesenchymal transition by reducing E-cadherin expression in residual hepatocellular carcinoma after insufficient radiofrequency ablation.

PURPOSE: Hepatocellular carcinoma (HCC) after insufficient radiofrequency ablation (RFA) could induce epithelial-mesenchymal transition (EMT) in residual tumours, resulting in rapid and aggressive recurrence. However, the role of EMT-related Long noncoding RNAs (lncRNAs) in residual tumour progression remains unclear. METHODS: Insufficient RFA was simulated in vitro by heating Huh7 cells in water bath at 47 °C, named as Huh7-H. Cell invasion, migration assays and wound healing assay were conducted for functional analysis. Cell proliferation was determined by CCK8 assay. Differential expression profile of EMT-related lncRNAs between Huh7-H and Huh7 was analysed by LncPath human EMT array, and validated by qRT-PCR. Gain/loss-of-function assays of selected lncRNA were conducted by over-expressing or silencing its expression. RESULTS: Huh7-H presented characteristic EMT morphological changes. WB analysis showed significantly decreased E-cadherin in Huh7-H cells. Transwell assays indicated the abilities of Huh7-H cells in migration and invasion were evidently strengthened. A new lncRNA, FUNDC2P4, was identified by LncPath human EMT array to be significantly down-regulated in Huh7-H cells. In vitro studies showed overexpression of FUNDC2P4 inhibited proliferation, invasion and migration potential and up-regulated E-cadherin expression in SMMC-7721 cells, whereas silencing FUNDC2P4 promoted these potentials and down-regulated E-cadherin expression in Huh7 cells. CONCLUSIONS: We explored that lncRNA FUNDC2P4 down-regulation promoted EMT leading to tumour proliferation, invasion and migration by reducing E-cadherin expression in residual HCC after insufficient RFA in vitro. These results suggest that FUNDC2P4 may have potentially therapeutic value for prevention and treatment of HCC recurrence after RFA in the future.