ABSTRACT
This study investigates the molecular mechanisms by which extracellular vesicles (EVs) derived from adipose-derived mesenchymal stem cells (ADSCs) promote M2 polarization of macrophages and thus reduce lung injury caused by sepsis. High-throughput sequencing was used to identify differentially expressed genes related to long non-coding RNA (lncRNA) in ADSC-derived EVs (ADSC-EVs) in sepsis lung tissue. Weighted gene co-expression network analysis (WGCNA) was employed to predict the downstream target genes of the lncRNA DLEU2. The RNAInter database predicted miRNAs that interact with DLEU2 and LXN. Functional and pathway enrichment analyses were performed using GO and KEGG analysis. A mouse model of sepsis was established, and treatment with a placebo or ADSC-EVs was administered, followed by RT-qPCR analysis. ADSC-EVs were isolated and identified. In vitro cell experiments were conducted using the mouse lung epithelial cell line MLE-12, mouse macrophage cell line RAW264.7, and mouse lung epithelial cell line (LEPC). ADSC-EVs were co-cultured with RAW264.7 and MLE-12/LEPC cells to study the regulatory mechanism of the lncRNA DLEU2. Cell viability, proliferation, and apoptosis of lung injury cells were assessed using CCK-8, EdU, and flow cytometry. ELISA was used to measure the levels of inflammatory cytokines in the sepsis mouse model, flow cytometry was performed to determine the number of M1 and M2 macrophages, lung tissue pathology was evaluated by H&E staining, and immunohistochemistry was conducted to examine the expression of proliferation- and apoptosis-related proteins. High-throughput sequencing and bioinformatics analysis revealed enrichment of the lncRNA DLEU2 in ADSC-EVs in sepsis lung tissue. Animal and in vitro cell experiments showed increased expression of the lncRNA DLEU2 in sepsis lung tissue after treatment with ADSC-EVs. Furthermore, ADSC-EVs were found to transfer the lncRNA DLEU2 to macrophages, promoting M2 polarization, reducing inflammation response in lung injury cells, and enhancing their viability, proliferation, and apoptosis inhibition. Further functional experiments indicated that lncRNA DLEU2 promotes M2 polarization of macrophages by regulating miR-106a-5p/LXN, thereby enhancing the viability and proliferation of lung injury cells and inhibiting apoptosis. Overexpression of miR-106a-5p could reverse the biological effects of ADSC-EVs-DLEU2 on MLE-12 and LEPC in vitro cell models. Lastly, in vivo animal experiments confirmed that ADSC-EVs-DLEU2 promotes high expression of LXN by inhibiting the expression of miR-106a-5p, further facilitating M2 macrophage polarization and reducing lung edema, thus alleviating sepsis-induced lung injury. lncRNA DLEU2 in ADSC-EVs may promote M2 polarization of macrophages and enhance the viability and proliferation of lung injury cells while inhibiting inflammation and apoptosis reactions, thus ameliorating sepsis-induced lung injury in a mechanism involving the regulation of the miR-106a-5p/LXN axis.
Subject(s)
Lung Injury , MicroRNAs , Nerve Tissue Proteins , RNA, Long Noncoding , Sepsis , Animals , Mice , Apoptosis/genetics , Disease Models, Animal , Lung Injury/microbiology , Lung Injury/therapy , MicroRNAs/genetics , RNA, Long Noncoding/administration & dosage , RNA, Long Noncoding/genetics , Sepsis/complications , Sepsis/genetics , Nerve Tissue Proteins/genetics , Mesenchymal Stem Cells , Exosomes , Male , Mice, Inbred C57BLABSTRACT
BACKGROUND: Our study aims to investigate the role and mechanism of circular RNA_0002715 (circ_0002715) in osteoarthritis (OA) progression. METHODS: IL-1ß-induced CHON-001 cells were used to mimic OA cell model. Circ_0002715, microRNA (miR)-127-5p and Latexin (LXN) expression was detected by quantitative real-time PCR. Cell functions were determined by MTT assay, flow cytometry and ELISA assay. Protein expression was examined by western blot. RESULTS: Circ_0002715 was highly expressed in OA cartilage tissues. Circ_0002715 silencing inhibited inflammation, apoptosis, and ECM degradation in IL-1ß-interfered CHON-001 cells. Circ_0002715 could sponge miR-127-5p, and miR-127-5p could target LXN. The effect of circ_0002715 down-regulation on chondrocyte injury was partially restored by miR-127-5p inhibitor. MiR-127-5p could suppress chondrocyte injury by inhibiting LXN expression. CONCLUSION: Circ_0002715 might be a new therapeutic target for OA, which regulated miR-127-5p/LXN axis to promote IL-1ß-induced chondrocyte injury.
Subject(s)
MicroRNAs , Osteoarthritis , Humans , Apoptosis , Bandages , Chondrocytes , Inflammation , Interleukin-1beta , MicroRNAs/genetics , Osteoarthritis/genetics , RNA, Circular/geneticsABSTRACT
Endothelial cells (ECs) respond to blood shear stress by changing their morphology is important for maintaining vascular homeostasis. Studies have documented a relationship between endothelial cell shape and the stress flow, and however, the mechanism underlying this cytoskeletal rearrangement due to shear stress remains uncertain. In this paper, we demonstrate that laminar shear stress (LSS) significantly reduces latexin (LXN) expression in ECs. By using siRNA and cell imaging, we demonstrated that LXN knockdown results in the morphologic change and F-actin remodelling just like what LSS does in ECs. We further demonstrate that LXN interacts with Filamin A (FLNA) and regulates FLNA proteolytic cleavage and nuclei translocation. By constructing LXN-/- mice and ApoE-/- LXN-/- double knockout mice, we evaluated the effect of LXN knockout on aortic endothelium damage in mice. We found that LXN deficiency significantly improves vascular permeability, vasodilation and atherosclerosis in mice. Our findings provide confident evidence, for the first time, that LXN is a novel regulator for morphological maintenance of ECs, and LXN deficiency has a protective effect on vascular homeostasis. This provides new strategies and drug targets for the treatment of vascular diseases.
Subject(s)
Cytoskeleton/metabolism , Endothelium, Vascular/metabolism , Filamins/metabolism , Nerve Tissue Proteins/metabolism , Animals , Aorta/cytology , Aorta/metabolism , Aorta/physiology , Apolipoproteins E/genetics , Blood Circulation , Endothelium, Vascular/cytology , Endothelium, Vascular/physiology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/genetics , Proteolysis , Stress, Mechanical , VasodilationABSTRACT
Objective To study the effects of Lxn on CD133 + PANC-1 pancreatic cancer cells.Methods CD133 + PANC-1 cell were isolated by magnetic activated cell sorting (MACS).The properties of the CD133 + PANC-1 cells and Lxn effects on CD133 + PANC-1 cell proliferation in transplanted tumor in nude mice were determined by floating spheres test and tumor xenograft assays.Cell proliferation was assayed by Cell Counting Kit-8 (CCK-8).The Bcl-2,Bax protein and mRNA expression of CD133 + PANC-1 cells treated by Lxn were analyzed by Western blot and Quantitative real-time PCR (qRT-PCR).Results We successfully isolated the CD133 + PANC-1 cells and cultured in serum free medium,CD133 + PANC-1 cells formed sphere,while CD133-PANC-1 cells grew with adherence slowly and then underwent apoptotic process.CD133 + PANC-1 cells showed high tumorigenic in athymic BALB/c mice.Lxn suppressed the growth of transplanted tumor obviously.Compared with control group [(225.52 ± 34.09) mm3],tumor volume decreased significantly (P ＜ 0.05).Significant reduction in cell proliferation was observed in response to Lxn in PANC-1 CD133 + cells by CCK-8 assay with concentration and time dependent manners (P ＜ 0.05).Treated by Lxn,Bcl-2 expression decreased,Bax expression increased.Conclusions Lxn inhibits the proliferation of CD133 + PANC-1 cells probably through a mechanism down-regualting Bcl-2 and up-regulating Bax.
