scholarly journals CircRNA TADA2A relieves idiopathic pulmonary fibrosis by inhibiting proliferation and activation of fibroblasts

2020 ◽  
Author(s):  
Juan Li ◽  
Ping Li ◽  
Guojun Zhang ◽  
Pan Qin ◽  
Da Zhang ◽  
...  
Keyword(s):  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Lung Fibroblasts ◽  
Luciferase Reporter ◽  
Fibroblastic Cell ◽  
Reporter Assays ◽  
Normal Human

Abstract The excessive activation and proliferation of lung fibroblasts are responsible for the abundant deposition of extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF), while its specific mechanism is still unknown. This study focuses on the role of circRNA (circ) TADA2A in functional abnormalities of lung fibroblasts and aims to elaborate its regulatory mechanism. In the present study, circTADA2A was down-regulated in both IPF primary human lung fibroblasts and human IPF fibroblastic cell lines. Functionally, the overexpression of circTADA2A repressed the activation and proliferation of normal human fibroblastic cell line induced by several fibrogenic growth factors. Using fluorescence in situ hybridization (FISH), luciferase reporter assays, and RNA pull-down, circTADA2A was confirmed to function as sponges of miR-526b and miR-203, thus releasing the expression of Caveolin (Cav)-1 and Cav-2. The overexpression of circTADA2A suppressed lung fibroblasts activation via Cav-1 and reduced lung fibroblasts proliferation via Cav-2. In vivo experiments also confirmed that the overexpression of circTADA2A decreased fibrogenic responses induced by bleomycin in lung fibrosis mice. Collectively, circTADA2A repressed lung fibroblasts activation via miR-526b/Cav-1 and reduced lung fibroblasts proliferation via miR-203/Cav-2, thus inhibiting the excessive deposition of ECM and relieving IPF.

ID: 112: ROLE OF PHOSPHOLIPASE D IN IDIOPATHIC PULMONARY FIBROSIS

2016 ◽  
Vol 64 (4) ◽  
pp. 964.1-964
Author(s):  
V Suryadevara ◽  
T Royston ◽  
E Berdyshev ◽  
L Huang ◽  
V Natarajan ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Phospholipase D ◽  
Human Lung ◽  
Lung Fibroblasts ◽  
Human Lung Fibroblasts

Idiopathic pulmonary fibrosis (IPF) is a deadly interstitial disease that leads to scarring and fibrosis of the lung tissue. In pulmonary fibrosis, there is injury and denudation of the alveolar epithelium, which further leads to activation of fibroblasts which differentiate into myofibroblasts. This includes several mechanisms including epithelial to mesenchymal transition (EMT). In this study, we investigated the role of phospholipase D (PLD) in IPF and also its underlying mechanism like EMT and fibroblast proliferation and differentiation. An in vivo murine model of bleomycin-induced pulmonary fibrosis (PF) and in vitro models of murine alveolar type-II epithelial cells (MLE-12) and human lung fibroblasts were used. C57BL/6 and genetically engineered PLD2−/− mice were intratracheally challenged with bleomycin (1.5 U/kg animal) for 14 days and markers of inflammation, EMT and fibrosis were determined. MLE-12 cells were treated with specific PLD1 or PLD2 inhibitors prior to bleomycin (10 mU/ml) challenge, and the role of PLD in EMT and apoptosis of alveolar epithelial cells was studied. Human lung fibroblasts were serum-starved (3h), pretreated with PLD1 or PLD2 inhibitors, and the effect of TGF-β (5 ng/ml) on differentiation of lung fibroblast to myofibroblast was determined. Intra-tracheal instillation of bleomycin in the mice for 14 days leads to the progression of fibrosis in the lung. The lung tissues of the bleomycin treated mice were found to have increased PLD2 protein expression, myofibroblast markers like α-SMA, fibronectin, mesenchymal markers like vimentin, inflammatory cytokines and collagen. Genetic deletion of PLD2 in mice attenuated bleomycin-induced lung inflammation and pulmonary fibrosis. In vitro, MLE-12 cells pretreated with either PLD1 or PLD2 inhibitor did not show a profound reduction either in apoptosis or the expression of transcription factors such as SNAIL, and other markers of EMT. However, MLE-12 cells pretreated with both PLD1 (250 nM) and PLD2 (500 nM) inhibitors were resistant to bleomycin-induced apoptosis, and exhibited reduced expression of SNAIL and mesenchymal markers. On the contrary, human lung fibroblasts pretreated with PLD1 and PLD2 inhibitors showed increased fibroblast to myofibroblast differentiation mediated by TGF-β. The present study suggests a role for PLD2 in bleomycin-induced PF. In vitro, inhibition of both PLD1 and PLD2 was necessary to attenuate bleomycin-induced EMT in epithelial cells and TGF-β mediated differentiation of fibroblasts to myofibroblasts. The in vivo and in vitro results identify the mechanism by which PLD regualtes PF and suggest PLD as a potential therapeutic target in pulmonary fibrosis. This work was supported by National Institutes of Health grant P01 HL98050 to VN.

scholarly journals Tumor Suppressive Role of miR-342-5p in Human Chondrosarcoma Cells and 3D Organoids

2021 ◽  
Vol 22 (11) ◽  
pp. 5590
Author(s):  
Clément Veys ◽  
Abderrahim Benmoussa ◽  
Romain Contentin ◽  
Amandine Duchemin ◽  
Emilie Brotin ◽  
...  
Keyword(s):  
Luciferase Reporter ◽  
Functional Screening ◽  
Western Blots ◽  
Egfr Expression ◽  
Reporter Assays ◽  
Direct Target ◽  
Induced Apoptosis ◽  
First Time

Chondrosarcomas are malignant bone tumors. Their abundant cartilage-like extracellular matrix and their hypoxic microenvironment contribute to their resistance to chemotherapy and radiotherapy, and no effective therapy is currently available. MicroRNAs (miRNAs) may be an interesting alternative in the development of therapeutic options. Here, for the first time in chondrosarcoma cells, we carried out high-throughput functional screening using impedancemetry, and identified five miRNAs with potential antiproliferative or chemosensitive effects on SW1353 chondrosarcoma cells. The cytotoxic effects of miR-342-5p and miR-491-5p were confirmed on three chondrosarcoma cell lines, using functional validation under normoxia and hypoxia. Both miRNAs induced apoptosis and miR-342-5p also induced autophagy. Western blots and luciferase reporter assays identified for the first time Bcl-2 as a direct target of miR-342-5p, and also Bcl-xL as a direct target of both miR-342-5p and miR-491-5p in chondrosarcoma cells. MiR-491-5p also inhibited EGFR expression. Finally, only miR-342-5p induced cell death on a relevant 3D chondrosarcoma organoid model under hypoxia that mimics the in vivo microenvironment. Altogether, our results revealed the tumor suppressive activity of miR-342-5p, and to a lesser extent of miR-491-5p, on chondrosarcoma lines. Through this study, we also confirmed the potential of Bcl-2 family members as therapeutic targets in chondrosarcomas.

scholarly journals Let-7a regulates EV secretion and mitochondrial oxidative phosphorylation by targeting SNAP23 in colorectal cancer

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
You Dong Liu ◽  
Xiao Peng Zhuang ◽  
Dong Lan Cai ◽  
Can Cao ◽  
Qi Sheng Gu ◽  
...  
Keyword(s):  
Oxidative Phosphorylation ◽  
Metabolic Reprogramming ◽  
Luciferase Reporter ◽  
Mitochondrial Oxidative Phosphorylation ◽  
In Vivo Assays ◽  
Reporter Assays ◽  
Extracellular Mirna

Abstract Background MicroRNAs (miRNAs) are abundant in tumor-derived extracellular vesicles (EVs) and the functions of extracellular miRNA to recipient cells have been extensively studied with tumorigenesis. However, the role of miRNA in EV secretion from cancer cells remains unknown. Methods qPCR and bioinformatics analysis were applied for determining extracellular let-7a expression from CRC patient serum and cells. Nanosight particle tracking analysis was performed for investigating the effect of let-7a on EV secretion. Luciferase reporter assays was used for identifying targeted genes synaptosome-associated protein 23 (SNAP23). In vitro and in vivo assays were used for exploring the function of let-7a/SNAP23 axis in CRC progression. Bioenergetic assays were performed for investigating the role of let-7a/SNAP23 in cellular metabolic reprogramming. Results let-7a miRNA was elevated in serum EVs from CRC patients and was enriched in CRC cell-derived EVs. We determined that let-7a could suppress EV secretion directly targeting SNAP23. In turn, SNAP23 promotes EV secretion of let-7a to downregulate the intracellular let-7a expression. In addition, we found a novel mechanism of let-7a/SNAP23 axis by regulating mitochondrial oxidative phosphorylation (OXPHOS) through Lin28a/SDHA signaling pathway. Conclusions Let-7a plays an essential role in not only inhibiting EV secretion, but also suppressing OXPHOS through SNAP23, resulting in the suppression of CRC progression, suggesting that let-7a/SNAP23 axis could provide not only effective tumor biomarkers but also novel targets for tumor therapeutic strategies.

scholarly journals MiR-125b-5p Inhibited The Progression of Hepatoblastoma As A Shared miRNA of The lncRNA NEAT1 and YES1

2020 ◽  
Author(s):  
Zhu Jin ◽  
Yutong Chen ◽  
Yuchen Mao ◽  
Mingjuan Gao ◽  
Zebing Zheng ◽  
...  
Keyword(s):  
Clinicopathological Characteristics ◽  
Luciferase Reporter ◽  
Tumor Growth Inhibition ◽  
Invasion And Migration ◽  
In Vivo Experiments ◽  
Reporter Assays ◽  
And Migration ◽  
Relationship Of

Abstract Background: microRNAs have been studied widely in hepatoblastoma. However, the role of miR-125b-5p and its relationship with the lncRNA sNEAT1 and YES1 in hepatoblastoma have not been reported previously. We aimed to reveal the role of NEAT1/miR-125b-5p/YES1 in the progression of hepatoblastoma.Methods: We collected tumor tissues and their adjacent tissues from 12 hepatoblastoma patients. qRT-PCR was applied to detect the expression of miR-125b-5p, and the relationship of miR-125b-5p with clinicopathological characteristics was analyzed. Dual luciferase reporter assays and RNA pull down assays were used to identify the relationships among NEAT1, miR-125b-5p and YES1. CCK8, Transwell assays and wound healing assays were used to examine cell viability, invasion and migration. In vivo experiments were also applied to detect the effect of miR-125b-5p on hepatoblastoma.Results: miR-125b-5p was significantly downregulated in hepatoblastoma tissue and cells. The higher the PRETEXT grade, the lower the miR-125b-5p level. NEAT1 could bind to miR-125b-5p and inhibit its expression. miR-125b-5p could target YES1 and inhibit its expression. Overexpression of miR-125b-5p decreased the proliferation, invasion, and migratory ability of hepatoblastoma cells. YES1 could rescue the above effects. At the same time, overexpression of miR-125b-5p resulted in decreased YES1 and tumor growth inhibition in vivo.Conclusion: miR-125b-5p acted as a shared miRNA of NEAT1 and YES1 in hepatoblastoma. Overexpression of miR-125b-5p could target YES1 and inhibit its expression, therefore inhibiting the progression of hepatoblastoma.

Extravascular sources of lung angiotensin peptide synthesis in idiopathic pulmonary fibrosis

2006 ◽  
Vol 291 (5) ◽  
pp. L887-L895 ◽  
Author(s):  
Xiaopeng Li ◽  
Maria Molina-Molina ◽  
Amal Abdul-Hafez ◽  
Jose Ramirez ◽  
Anna Serrano-Mollar ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Human Lung ◽  
De Novo ◽  
Alveolar Epithelial Cells ◽  
Human Lung Cells ◽  
Normal Human

Previous work from this laboratory demonstrated de novo synthesis of angiotensin (ANG) peptides by apoptotic pulmonary alveolar epithelial cells (AEC) and by lung myofibroblasts in vitro and in bleomycin-treated rats. To determine whether these same cell types also synthesize ANG peptides de novo within the fibrotic human lung in situ, we subjected paraffin sections of normal and fibrotic (idiopathic pulmonary fibrosis, IPF) human lung to immunohistochemistry (IHC) and in situ hybridization to detect ANG peptides and angiotensinogen (AGT) mRNA. These were analyzed both alone and in combination with cell-specific markers of AEC [monoclonal antibody (MAb) MNF-116] and myofibroblasts [α-smooth muscle actin (α-SMA) MAb] and an in situ DNA end labeling (ISEL) method to detect apoptosis. In normal human lung, IHC detected AGT protein in smooth muscle underlying normal bronchi and vessels, but not elsewhere. Real-time RT-PCR and Western blotting revealed that AGT mRNA and protein were 21-fold and 3.6-fold more abundant, respectively, in IPF lung biopsies relative to biopsies of normal human lung (both P < 0.05). In IPF lung, both AGT protein and mRNA were detected in AEC that double-labeled with MAb MNF-116 and with ISEL, suggesting AGT expression by apoptotic epithelia in situ. AGT protein and mRNA also colocalized to myofibroblast foci detected by α-SMA MAb, but AGT mRNA was not detected in smooth muscle. These data are consistent with earlier data from isolated human lung cells in vitro and bleomycin-induced rat lung fibrosis models, and they suggest that apoptotic AEC and myofibroblasts constitute key sources of locally derived ANG peptides in the IPF lung.

scholarly journals LncRNA-URHC Functions as a ceRNA to Regulate Danjb9 Expression by Competitively Binding to miR-5007-3p in Hepatocellular Carcinoma

2021 ◽  
Author(s):  
kunwei niu ◽  
Shibin Qu ◽  
Xuan Zhang ◽  
Jimin Dai ◽  
Jianlin Wang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Biological Effects ◽  
Luciferase Reporter ◽  
Underlying Mechanisms ◽  
Proliferation In Vitro ◽  
Hcc Cells

Abstract Background: Hepatocellular carcinoma (HCC) is often diagnosed at a late stage, when the prognosis is poor. The regulation of long non-coding RNAs (lncRNAs) plays a crucial role in HCC. However, the precise regulatory mechanisms of lncRNA signaling in HCC remain largely unknown. We study aim to investigate the underlying mechanisms of lncRNA (upregulated in hepatocellular carcinoma) URHC in HCC. Methods: RT-qPCR, fluorescence in situ hybridization (FISH) staining, EdU, colony formation, and tumor xenografts experiments were used to identify localized and biological effects of URHC on HCC cells in vitro and in vivo. The bioinformatics analysis, Dual-luciferase reporter assay, and rescue experiments revealed the potential mechanism of URHC.Results: URHC silencing may inhibit the HCC cells proliferation in vitro and in vivo. We found that URHC was mainly localized in the cytoplasm. The expression of miR-5007-3p was negatively regulated by URHC. And miR-5007-3p could reverse the effect of URHC in HCC cells. The expression of DNAJB9 was negatively regulated by miR-5007-3p but positively regulated by URHC. These suggesting of lncRNA-URHC positively regulated the level of DNAJB9 by sponging miR-5007-3p.Conclusion: Together, our study elucidated the role of URHC as a miRNA sponge in HCC, and shed new light on lncRNA-directed diagnostics and therapeutics in HCC.

scholarly journals Itaconate controls the severity of pulmonary fibrosis

2020 ◽  
Vol 5 (52) ◽  
pp. eabc1884 ◽  
Author(s):  
Patricia P. Ogger ◽  
Gesa J. Albers ◽  
Richard J. Hewitt ◽  
Brendan J. O’Sullivan ◽  
Joseph E. Powell ◽  
...  
Keyword(s):  
Gene Expression ◽  
Wound Healing ◽  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Lung Fibrosis ◽  
Therapeutic Strategy ◽  
Lung Fibroblasts ◽  
Disease Phenotype ◽  
Wild Type

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease in which airway macrophages (AMs) play a key role. Itaconate has emerged as a mediator of macrophage function, but its role during fibrosis is unknown. Here, we reveal that itaconate is an endogenous antifibrotic factor in the lung. Itaconate levels are reduced in bronchoalveolar lavage, and itaconate-synthesizing cis-aconitate decarboxylase expression (ACOD1) is reduced in AMs from patients with IPF compared with controls. In the murine bleomycin model of pulmonary fibrosis, Acod1−/− mice develop persistent fibrosis, unlike wild-type (WT) littermates. Profibrotic gene expression is increased in Acod1−/− tissue-resident AMs compared with WT, and adoptive transfer of WT monocyte-recruited AMs rescued mice from disease phenotype. Culture of lung fibroblasts with itaconate decreased proliferation and wound healing capacity, and inhaled itaconate was protective in mice in vivo. Collectively, these data identify itaconate as critical for controlling the severity of lung fibrosis, and targeting this pathway may be a viable therapeutic strategy.

scholarly journals SIRT3 blocks myofibroblast differentiation and pulmonary fibrosis by preventing mitochondrial DNA damage

2017 ◽  
Vol 312 (1) ◽  
pp. L68-L78 ◽  
Author(s):  
Samik Bindu ◽  
Vinodkumar B. Pillai ◽  
Abhinav Kanwal ◽  
Sadhana Samant ◽  
Gökhan M. Mutlu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mitochondrial Dna ◽  
Pulmonary Fibrosis ◽  
Lung Fibrosis ◽  
Lung Fibroblasts ◽  
Whole Body ◽  
Myofibroblast Differentiation ◽  
Mitochondrial Dna Damage

Myofibroblast differentiation is a key process in the pathogenesis of fibrotic diseases. Transforming growth factor-β1 (TGF-β1) is a powerful inducer of myofibroblast differentiation and is implicated in pathogenesis of tissue fibrosis. This study was undertaken to determine the role of mitochondrial deacetylase SIRT3 in TGF-β1-induced myofibroblast differentiation in vitro and lung fibrosis in vivo. Treatment of human lung fibroblasts with TGF-β1 resulted in increased expression of fibrosis markers, smooth muscle α-actin (α-SMA), collagen-1, and fibronectin. TGF-β1 treatment also caused depletion of endogenous SIRT3, which paralleled with increased production of reactive oxygen species (ROS), DNA damage, and subsequent reduction in levels of 8-oxoguanine DNA glycosylase (OGG1), an enzyme that hydrolyzes oxidized guanine (8-oxo-dG) and thus protects DNA from oxidative damage. Overexpression of SIRT3 by adenovirus-mediated transduction reversed the effects of TGF-β1 on ROS production and mitochondrial DNA damage and inhibited TGF-β1-induced myofibroblast differentiation. To determine the antifibrotic role of SIRT3 in vivo, we used the bleomycin-induced mouse model of pulmonary fibrosis. Compared with wild-type controls, Sirt3-knockout mice showed exacerbated fibrosis after intratracheal instillation of bleomycin. Increased lung fibrosis was associated with decreased levels of OGG1 and concomitant accumulation of 8-oxo-dG and increased mitochondrial DNA damage. In contrast, the transgenic mice with whole body Sirt3 overexpression were protected from bleomycin-induced mtDNA damage and development of lung fibrosis. These data demonstrate a critical role of SIRT3 in the control of myofibroblast differentiation and lung fibrosis.

scholarly journals LncRNA PFAR contributes to fibrogenesis in lung fibroblasts through competitively binding to miR-15a

2019 ◽  
Vol 39 (7) ◽  
Author(s):  
Jian Sun ◽  
Wei Su ◽  
Xiaoguang Zhao ◽  
Tianjiao Shan ◽  
Tongzhu Jin ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Treatment Method ◽  
Biological Information ◽  
Lung Fibroblasts ◽  
Luciferase Reporter ◽  
Migration Ability ◽  
Related Factors ◽  
Reverse Transcription Pcr ◽  
Twist Expression ◽  
Reporter Assays

Abstract Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, debilitating disease with unknown etiopathogenesis. Previous reports have reported that long non-coding RNAs (lncRNAs) were involved in various pathophysiological processes. However, the role of lncRNAs in IPF has not been fully described. We aimed to explore the relationship between miR-15a and lncRNA PFAR and its function in pulmonary fibrosis. Biological information analysis and luciferase were used to identify targeted binding of lncRNA PFAR and miR-15a. Western blot, quantitative reverse transcription-PCR (qRT-PCR) and immunofluorescence staining were conducted to detect fibrosis-related factors. Fibroblasts proliferation were analyzed using 5-ethynyl-2′-deoxyuridine (EdU) staining and fibroblasts migration ability were measured using wound-healing scratch assay. We identified that lncRNA PFAR has a binding site with miR-15a and luciferase reporter assays demonstrated their combinative relationship. Our results showed that silencing PFAR attenuated TGF-β1 induced fibrogenesis in primary lung fibroblasts. And miR-15a antagonized the function of PFAR and inhibited PFAR induced extracellular collagen deposition, fibroblasts proliferation, migration and differentiation. In conclusion, our results revealed that PFAR functions as a competitive endogenous RNA (ceRNA) by acting as a sponge for miR-15a, revealing a potential regulatory network involving PFAR and miR-15a with a role in the modulation of YAP1-Twist expression. This mechanism may contribute to a better understanding of pulmonary fibrosis pathogenesis and treatment method.

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