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Journal of Thoracic Disease Feb 2024Idiopathic pulmonary fibrosis (IPF) is an unrepairable disease that results in lung dysfunction and decreased quality of life. Prevention of pulmonary fibrosis is...
BACKGROUND
Idiopathic pulmonary fibrosis (IPF) is an unrepairable disease that results in lung dysfunction and decreased quality of life. Prevention of pulmonary fibrosis is challenging, while its pathogenesis remains largely unknown. Herein, we investigated the effect and mechanism of long non-coding RNA (lncRNA) /Antisense RNA in the pathogenesis of pulmonary fibrosis.
METHODS
EdU (5-ethynyl-2'-deoxyuridine) and wound healing assays were employed to evaluate the role of DNM3OS on cell proliferation and migration. Western blot detected the proteins expressions of alpha-smooth muscle actin (α-SMA), vimentin, and fibronectin. The interactions among genes were evaluated by RNA pull-down, luciferase reporter, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP) and chromatin Isolation by RNA purification (ChIRP) assays.
RESULTS
was upregulated by transforming growth factor beta 1 (TGF-β1) in a dose- and time-dependent manner. knockdown repressed the growth and migration of lung fibroblast, and fibrotic gene expression (, , α-SMA, vimentin, and fibronectin), while suppression of accelerated the above process. DNM3OS recruited EZH2 to the promoter region of , increased the occupancy of EZH2 and H3K27me3, and thereby suppressed the expression of . promoted the transcription of .
CONCLUSIONS
-induced promoted the proliferation, migration, and expression of fibrosis-related genes in human embryo lung fibroblast via recruiting EZH2 to epigenetically suppress the expression of .
PubMed: 38505042
DOI: 10.21037/jtd-23-1145 -
Nature Communications Mar 2024Correlative light and electron microscopy (CLEM) is an important tool for the localisation of target molecule(s) and their spatial correlation with the ultrastructural...
Correlative light and electron microscopy (CLEM) is an important tool for the localisation of target molecule(s) and their spatial correlation with the ultrastructural map of subcellular features at the nanometre scale. Adoption of these advanced imaging methods has been limited in plant biology, due to challenges with plant tissue permeability, fluorescence labelling efficiency, indexing of features of interest throughout the complex 3D volume and their re-localization on micrographs of ultrathin cross-sections. Here, we demonstrate an imaging approach based on tissue processing and embedding into methacrylate resin followed by imaging of sections by both, single-molecule localization microscopy and transmission electron microscopy using consecutive CLEM and same-section CLEM correlative workflow. Importantly, we demonstrate that the use of a particular type of embedding resin is not only compatible with single-molecule localization microscopy but shows improvements in the fluorophore blinking behavior relative to the whole-mount approaches. Here, we use a commercially available Click-iT ethynyl-deoxyuridine cell proliferation kit to visualize the DNA replication sites of wild-type Arabidopsis thaliana seedlings, as well as fasciata1 and nucleolin1 plants and apply our in-section CLEM imaging workflow for the analysis of S-phase progression and nucleolar organization in mutant plants with aberrant nucleolar phenotypes.
Topics: Microscopy, Fluorescence; Microscopy, Electron; Microscopy, Electron, Transmission; Single Molecule Imaging; Electrons; Arabidopsis
PubMed: 38503728
DOI: 10.1038/s41467-024-46324-6 -
Zhongguo Xiu Fu Chong Jian Wai Ke Za... Mar 2024To explore the molecular mechanism of miR-515-5p in inhibiting chondrocyte apoptosis and alleviating inflammatory response in osteoarthritis (OA).
[miR-515-5p targeting Toll-like receptor 4 regulates myeloid differentiation primary response gene 88/nuclear factor-kappa B pathway to inhibit apoptosis and inflammatory response of osteoarthritis chondrocytes].
OBJECTIVE
To explore the molecular mechanism of miR-515-5p in inhibiting chondrocyte apoptosis and alleviating inflammatory response in osteoarthritis (OA).
METHODS
Human cartilage cell line C28/I2 was cultured and treated with 10 ng/mL interleukin 1β (IL-1β) for 24 hours to construct an OA model. C28/I2 cells were transfected with miR mimics, mimics negative control (NC), over expression (oe)-NC, and oe-Toll-like receptor 4 (TLR4), respectively, and then treated with 10 ng/mL IL-1β for 24 hours to establish OA model. Cell proliferation capacity was detected by cell counting kit 8 and 5-Ethynyl-2'-deoxyuridine, cell apoptosis and cell cycle were detected by flow cytometry, and B-cell lymphoma 2 protion (Bcl-2), Bcl-2-associated X protein (Bax), cleaved-Caspase-3, TLR4, myeloid differentiation primary response gene 88 (MyD88), p65 and phosphorylated p65 (p-p65) protein expression levels were detected by Western blot. Real-time fluorescence quantitative PCR was used to detect mRNA expression levels of miR-515-5p and TLR4, and ELISA was used to detect pro-inflammatory factor prostaglandin E2 (PGE2), tumor necrosis factor α (TNF -α), and IL-6 levels in cell supernatant. The potential binding sites between miR-515-5p and TLR4 were predicted by BiBiServ2 database, and the targeting relationship between miR-515-5p and TLR4 was verified by dual luciferase reporting assay.
RESULTS
After the treatment of C28/I2 cells with IL-1β, the expressions of miR-515-5p and Bcl-2 protein and the proliferation ability of C28/I2 cells significantly reduced. The expression levels of Bax and cleaved-Caspase-3 protein, the levels of pro-inflammatory factors (PGE2, TNF-α, IL-6) in the supernatant of C28/I2 cells, and the apoptosis of C28/I2 cells significantly increased. In addition, the proportion of the cells at S phase and G phase decreased significantly, and the proportion of cells at G phase increased significantly, suggesting that the cell cycle was blocked after IL-1β treatment. After transfection with miR mimics, the expression level of miR-515-5p in the cells significantly up-regulated, partially reversing the apoptosis of OA chondrocytes induced by IL-1β, and alleviating the cycle arrest and inflammatory response of OA chondrocytes. After treating C28/I2 cells with IL-1β, the mRNA and protein levels of TLR4 significantly increased. Overexpression of miR-515-5p targeted inhibition of TLR4 expression and blocked activation of MyD88/nuclear factor κB (NF-κB) pathway. Overexpression of TLR4 could partially reverse the effect of miR mimics on IL-1β-induced apoptosis and inflammation of OA chondrocytes.
CONCLUSION
miR-515-5p negatively regulates the expression of TLR4, inhibits the activation of MyD88/NF-κB pathway and apoptosis of OA chondrocytes, and effectively alleviates the inflammatory response of the cells.
Topics: Humans; Adaptor Proteins, Signal Transducing; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Chondrocytes; Dinoprostone; Interleukin-1beta; Interleukin-6; MicroRNAs; Myeloid Differentiation Factor 88; NF-kappa B; Osteoarthritis; RNA, Messenger; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha
PubMed: 38500425
DOI: 10.7507/1002-1892.202312091 -
Molecular Medicine Reports May 2024Placenta accreta spectrum (PAS) is one of the most dangerous complications in obstetrics, which can lead to severe postpartum bleeding and shock, and even necessitate...
Pigment epithelium‑derived factor inhibits proliferation, invasion and angiogenesis, and induces ferroptosis of extravillous trophoblasts by targeting Wnt‑β‑catenin/VEGF signaling in placenta accreta spectrum.
Placenta accreta spectrum (PAS) is one of the most dangerous complications in obstetrics, which can lead to severe postpartum bleeding and shock, and even necessitate uterine removal. The abnormal migration and invasion of extravillous trophoblast cells (EVTs) and enhanced neovascularization occurring in an uncontrolled manner in time and space are closely related to the abnormal expression of pro‑angiogenic and anti‑angiogenic factors. The pigment epithelium‑derived factor (PEDF) is a multifunctional regulatory factor that participates in several important biological processes and is recognized as the most efficient inhibitor of angiogenesis. The present study aimed to explore the effects of PEDF on EVT phenotypes and the underlying mechanisms in PAS. HTR‑8/SVneo cells were transfected to overexpress or knock down PEDF. Cell proliferation and invasion were assessed using Cell Counting Kit‑8, 5‑ethynyl‑2'‑deoxyuridine and Transwell assays. angiogenesis was analyzed using tube formation assays. The degree of ferroptosis was assessed by evaluating the levels of lipid reactive oxygen species, total iron, Fe, malondialdehyde and reduced glutathione using commercial kits. The expression levels of biomarkers of ferroptosis, angiogenesis, cell proliferation and Wnt signaling were examined by western blotting. PEDF overexpression decreased the proliferation, invasion and angiogenesis, and induced ferroptosis of EVTs. Activation of Wnt signaling with BML‑284 and overexpression of vascular endothelial growth factor (VEGF) reversed the PEDF overexpression‑induced suppression of cell proliferation, invasion and tube formation. PEDF overexpression‑induced ferroptosis was also decreased by Wnt agonist treatment and VEGF overexpression. It was predicted that PEDF suppressed the proliferation, invasion and angiogenesis, and increased ferroptosis in EVTs by decreasing Wnt‑β‑catenin/VEGF signaling. The findings of the present study suggested a novel regulatory mechanism of the phenotypes of EVTs and PAS.
Topics: Pregnancy; Humans; Female; Vascular Endothelial Growth Factor A; Extravillous Trophoblasts; beta Catenin; Trophoblasts; Placenta Accreta; Wnt Signaling Pathway; Angiogenesis; Ferroptosis; Cell Proliferation; Cell Movement; Placenta; Eye Proteins; Nerve Growth Factors; Serpins
PubMed: 38488028
DOI: 10.3892/mmr.2024.13199 -
International Heart Journal Mar 2024Targeting circular RNA has been a novel approach to preventing and limiting acute myocardial infarction (AMI). Here, we planned to investigate the role and mechanism of...
Targeting circular RNA has been a novel approach to preventing and limiting acute myocardial infarction (AMI). Here, we planned to investigate the role and mechanism of circ_0020887 in AMI progression.Hypoxic injury in human cardiomyocytes (AC16) was measured using cell counting kit-8 assay, 5-ethynyl-2'-deoxyuridine assay, flow cytometry, and colorimetric assay kits. RNA and protein expressions were determined using real-time quantitative PCR and western blotting. Direct interplay between RNAs was determined using dual-luciferase reporter, RNA pull-down, and RIP assays.In the plasma and hypoxia-induced AC16 cells of patients with AMI, circ_0020887 and miR-370-3p were upregulated and downregulated, respectively, concomitant with the upregulation of cytochrome P450 1B1 (CYP1B1). Circ_0020887 interference could inhibit hypoxia-induced AC16 cell apoptosis, oxidative stress, and inflammatory response. Circ_0020887 could sponge miR-370-3p, and miR-370-3p could target CYP1B1. The inhibition effect of circ_0020887 knockdown on hypoxia-induced AC16 cell injury could be reversed by the miR-370-3p inhibitor. Besides, CYP1B1 overexpression also overturned the suppressive effect of miR-370-3p on hypoxia-induced AC16 cell apoptosis, oxidative stress, and inflammatory response.In conclusion, circ_0020887 regulated the miR-370-3p/CYP1B1 axis to regulate hypoxia-induced cardiomyocyte injury, confirming that circ_0020887 might promote cardiomyocyte injury.
Topics: Humans; Myocytes, Cardiac; Apoptosis; Blotting, Western; Hypoxia; Myocardial Infarction; MicroRNAs; Cell Proliferation; Cytochrome P-450 CYP1B1
PubMed: 38479850
DOI: 10.1536/ihj.23-325 -
Acta Cirurgica Brasileira 2024This study evaluated the protective effect of hesperidin on injury induced by gastric ischemia-reperfusion.
PURPOSE
This study evaluated the protective effect of hesperidin on injury induced by gastric ischemia-reperfusion.
METHODS
Fifty male Sprague Dawley rats (250-300 g) were divided into five groups: control (C), sham (S), ischemia (I), ischemia-reperfusion (I/R) and hesperidin + ischemia-reperfusion (Hes + I/R). Hesperidin was injected intraperitoneally at the dose of 100 mg/kg one hour before the experimental stomach ischemia-reperfusion. Celiac artery was ligated. After 45 minutes ischemia and 60 minutes reperfusion period, blood samples were obtained under anesthesia. Then, animals were sacrificed, stomach tissues were excised for biochemical, and histopathological analyses were performed. Malondialdehyde levels and superoxide dismutase, glutathione peroxidase activities and total antioxidant status (TAS), total oxidant status (TOS), protein, total thiol parameters were measured in plasma, and tissue homogenate samples. H + E, periodic acid-Schiff, hypoxia inducible factor, terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end-labeling (TUNEL), and proliferating cell nuclear antigen (PCNA) for cell proliferation as immunohistochemical parameters were determined.
RESULTS
Upon biochemical and histopathological assessment, hesperidin decreased stomach tissue changes in comparison with IR group. Ischemia-reperfusion injury led to a considerably increase in malondialdehyde, protein, and TOS levels (p < 0.001) in stomach tissue. Hesperidin treatment significantly decreased malondialdehyde, protein, and TOS levels (p < 0.001). Hesperidin increased superoxide dismutase, TAS, total thiol and glutathione peroxidase activities in comparison with IR group. Hesperidin reduced damage and also increased TUNEL and PCNA immunoreactivity in stomach tissue.
CONCLUSIONS
Hesperidin was able to decrease I/R injury of the stomach tissue due to inhibition of lipid peroxidation and protein oxidation, duration of antioxidant, and free radical scavenger properties. Consequently, hesperidin can provide a beneficial therapeutic choice for preventing stomach tissue ischemia-reperfusion injury in clinical application.
Topics: Male; Rats; Animals; Proliferating Cell Nuclear Antigen; Hesperidin; Antioxidants; Rats, Sprague-Dawley; Reperfusion Injury; Stomach; Superoxide Dismutase; Ischemia; Malondialdehyde; Sulfhydryl Compounds; Glutathione Peroxidase
PubMed: 38477785
DOI: 10.1590/acb391124 -
Hua Xi Kou Qiang Yi Xue Za Zhi = Huaxi... Feb 2024This study aimed to explore the effect of pituitary tumor-transforming gene 1 (PTT-G1) on the invasion and proliferation of oral squamous cell carcinoma (OSCC) cell...
OBJECTIVES
This study aimed to explore the effect of pituitary tumor-transforming gene 1 (PTT-G1) on the invasion and proliferation of oral squamous cell carcinoma (OSCC) cell lines under the action of miR-362-3p.
METHODS
The bioinformatics online database was used to query the expression of PTTG1 in head and neck squamous cell carcinoma (HNSCC). The expression of PTTG1 in the Cal-27, HN-30, and HOK cell lines was detected by Western blot. A wound-healing assay was used to determine the effect of PTTG1 on the migration ability of the OSCC cells. The Transwell assay was used to examine the changes in cell-invasion ability. 5-ethynyl-2'-deoxyuridine (EdU) cell-proliferation assay was used to detect changes in cell-proliferation ability. Bioinformatics approach predicted the upstream miRNA of PTTG1. The targeting relationship between miR-362-3p and PTTG1 was examined by the dual luciferase assay, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the expression of miRNA in OSCC tissues.
RESULTS
The ENCORI database showed that PTTG1 expression was up-regulated in OSCC tissues. Western blot confirmed that PTTG1 expression was up-regulated in Cal-27 and HN-30 cells than HOK cells. PTTG1 knockout can inhibit the migration, invasion, and proliferation of Cal-27 and HN-30 cells (<0.05). Bioinformatics prediction websites predicted that the upstream miRNA of PTTG1 was miR-362-3p, and PTTG1 can bind to miR-362-3p. Results of qRT-PCR showed that miR-362-3p expression was downregulated in OSCC tissues compared with normal tissue (<0.05). Transwell and EdU experiments confirmed that miR-362-3p knockdown can promote the invasion and proliferation of Cal-27 and HN-30 after PTTG1 knockdown.
CONCLUSIONS
miR-362-3p can inhibit the invasion and proliferation of Cal-27 and HN-30 cells by targeting PTTG1.
Topics: Humans; Squamous Cell Carcinoma of Head and Neck; Carcinoma, Squamous Cell; Mouth Neoplasms; Pituitary Neoplasms; Neoplasm Invasiveness; Cell Movement; MicroRNAs; Cell Proliferation; Head and Neck Neoplasms; Oncogenes; Cell Line, Tumor; Gene Expression Regulation, Neoplastic
PubMed: 38475950
DOI: 10.7518/hxkq.2024.2023237 -
Journal of Ginseng Research Mar 2024Cigarette smoke is generally accepted as a major contributor to chronic obstructive pulmonary disease (COPD), which is characterized by emphysematous lesions. In this...
BACKGROUND
Cigarette smoke is generally accepted as a major contributor to chronic obstructive pulmonary disease (COPD), which is characterized by emphysematous lesions. In this study, we investigated the protective effects of Korean Red Ginseng (KRG) against cigarette smoke condensate (CSC)-induced emphysema.
METHODS
Mice were instilled with 50 mg/kg of CSC intranasally once a week for 4 weeks, KRG was administered to the mice once daily for 4 weeks at doses of 100 or 300 mg/kg, and dexamethasone (DEX, positive control) was administered to the mice once daily for 2 weeks at 3 mg/kg.
RESULTS
KRG markedly decreased the macrophage population in bronchoalveolar lavage fluid and reduced emphysematous lesions in the lung tissues. KRG suppressed CSC-induced apoptosis as revealed by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling staining and Caspase 3 immunohistochemistry. Additionally, KRG effectively inhibited CSC-mediated activation of Bcl-2-associated X protein/Caspase 3 signaling, followed by the induction of cell survival signaling, including vascular endothelial growth factor/phosphoinositide 3-kinase/protein kinase B and . The DEX group also showed similar improved results and .
CONCLUSION
Taken together, KRG effectively inhibits macrophage-mediated emphysema induced by CSC exposure, possibly via the suppression of pro-apoptotic signaling, which results in cell survival pathway activation. These findings suggest that KRG has therapeutic potential for the prevention of emphysema in COPD patients.
PubMed: 38465217
DOI: 10.1016/j.jgr.2023.11.001 -
Scientific Reports Mar 2024Fluorescent molecule-based direct labeling of amplified DNA is a sensitive method employed across diverse DNA detection and diagnostics systems. However, using...
Fluorescent molecule-based direct labeling of amplified DNA is a sensitive method employed across diverse DNA detection and diagnostics systems. However, using pre-labeled primers only allows for the attachment of a single fluorophore to each DNA strand and any modifications of the system are less flexible, requiring new sets of primers. As an alternative, direct labeling of amplified products with modified nucleotides is available, but still poorly characterized. To address these limitations, we sought a direct and adaptable approach to label amplicons produced through Loop-mediated isothermal amplification (LAMP), using labeled nucleotides (dUTPs) rather than primers. The focus of this study was the development and examination of a direct labeling technique of specific genes, including those associated with drug resistance in Mycobacterium tuberculosis. We used 5-(3-Aminoallyl)-2'-deoxyuridine-5'triphosphate, tagged with 5/6-TAMRA (TAMRA-dUTP) for labeling LAMP amplicons during the amplification process and characterized amplification and incorporation efficiency. The optimal TAMRA-dUTP concentration was first determined based on amplification efficiency (0.5% to total dNTPs). Higher concentrations of modified nucleotides reduced or completely inhibited the amplification yield. Target size also showed to be determinant to the success of amplification, as longer sequences showed lower amplification rates, thus less TAMRA incorporated amplicons. Finally, we were able to successfully amplify all four M. tuberculosis target genes using LAMP and TAMRA-modified dUTPs.
Topics: Humans; Mycobacterium tuberculosis; Nucleic Acid Amplification Techniques; DNA; DNA Primers; Tuberculosis; Sensitivity and Specificity; Molecular Diagnostic Techniques
PubMed: 38454089
DOI: 10.1038/s41598-024-55289-x -
Frontiers in Cellular Neuroscience 2024During the healing process of full-thickness macular holes (FTMHs), the closure and recovery of the hole depend on the migration, proliferation, and activation of...
PURPOSE
During the healing process of full-thickness macular holes (FTMHs), the closure and recovery of the hole depend on the migration, proliferation, and activation of Müller cells to promote the closure of holes and restoration of the photosensitive layer. In this study, we investigated the ability of the epidermal growth factor (EGF), fibroblast growth factor-basic (FGF-b), and nerve growth factor (NGF) to influence this process by regulating proliferation, migration, and reprogramming of primary rat Müller cells.
METHODS
Cell proliferation was measured using CCK8 [2- (2-Methoxy-4-nitrophenyl)-3- (4-nitrophenyl)-5- (2,4-disulfophenyl)-2H-tetrazolium Sodium Salt] colorimetric assays and EdU [5-Ethynyl-2'-deoxyuridine] assays over 48 h. Cell migration was measured using scratch-wound assays and transwell migration assays over 48 h. In addition, we conducted Western blot assays and immunofluorescence assays on cells that were specially treated for 1, 3, and 5 days for cell reprogramming. The percentage of EdU-positive cells in Nestin-positive have also been tested by co-immunofluorescence (Co-IF) staining.
RESULTS
EGF and FGF-b significantly promoted the proliferation of Müller cells ( < 0.05) at a concentration of 0-50 ng/mL, but NGF did not ( > 0.05), compared to untreated controls. Exogenous FGF-b and EGF promote the reprogramming of primary rat Müller cells, significantly enhancing the neural stem cell marker Nestin after stimulation on the 1st, 3rd, and 5th days, respectively. The expression of Müller cell marker Vimentin was significantly ( < 0.05) reduced during this period compared to the control group. However, there was no significant difference between the NGF and control groups. Furthermore, the EGF group expressed stronger Nestin expression than the SCM group. The Co-IF staining showed that early 50% of activated cells came from newly proliferating cells on the 5th day.
CONCLUSION
These observations suggest that FGF-b can promote the activation of Müller cells in a short time and enhance the possessive features of neural stem cells, while EGF may act for a longer period of time. This may further the understanding of growth factor therapy in treating FTMHs, and Müller glia may be promising candidates for cell replacement therapy.
PubMed: 38450284
DOI: 10.3389/fncel.2024.1338129