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1.
Mol Cancer ; 22(1): 21, 2023 01 31.
Artículo en Inglés | MEDLINE | ID: mdl-36721170

RESUMEN

BACKGROUND: Excessive extracellular matrix deposition and increased stiffness are typical features of solid tumors such as hepatocellular carcinoma (HCC) and pancreatic ductal adenocarcinoma (PDAC). These conditions create confined spaces for tumor cell migration and metastasis. The regulatory mechanism of confined migration remains unclear. METHODS: LC-MS was applied to determine the differentially expressed proteins between HCC tissues and corresponding adjacent tissue. Collective migration and single cell migration microfluidic devices with 6 µm-high confined channels were designed and fabricated to mimic the in vivo confined space. 3D invasion assay was created by Matrigel and Collagen I mixture treat to adherent cells. 3D spheroid formation under various stiffness environment was developed by different substitution percentage GelMA. Immunoprecipitation was performed to pull down the LH1-binding proteins, which were identified by LC-MS. Immunofluorescent staining, FRET, RT-PCR, Western blotting, FRAP, CCK-8, transwell cell migration, wound healing, orthotopic liver injection mouse model and in vivo imaging were used to evaluate the target expression and cellular phenotype. RESULTS: Lysyl hydroxylase 1 (LH1) promoted the confined migration of cancer cells at both collective and single cell levels. In addition, LH1 enhanced cell invasion in a 3D biomimetic model and spheroid formation in stiffer environments. High LH1 expression correlated with poor prognosis of both HCC and PDAC patients, while it also promoted in vivo metastasis. Mechanistically, LH1 bound and stabilized Septin2 (SEPT2) to enhance actin polymerization, depending on the hydroxylase domain. Finally, the subpopulation with high expression of both LH1 and SEPT2 had the poorest prognosis. CONCLUSIONS: LH1 promotes the confined migration and metastasis of cancer cells by stabilizing SEPT2 and thus facilitating actin polymerization.


Asunto(s)
Carcinoma Hepatocelular , Carcinoma Ductal Pancreático , Neoplasias Hepáticas , Neoplasias Pancreáticas , Animales , Ratones , Actinas , Carcinoma Hepatocelular/genética , Carcinoma Ductal Pancreático/genética , Neoplasias Hepáticas/genética , Neoplasias Pancreáticas/genética , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Septinas
2.
Anal Chem ; 94(3): 1901-1909, 2022 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-35019258

RESUMEN

Surface biotinylation has been widely adapted in profiling the cellular proteome associated with the plasma membrane. However, the workflow is subject to interference from the cytoplasmic biotin-associated proteins that compete for streptavidin-binding during purification. Here we established a bioorthogonal conjugation-assisted purification (BCAP) workflow that utilizes the Staudinger chemoselective ligation to label and isolate surface-associated proteins while minimizing the binding of endogenous biotin-associated proteins. Label-free quantitative proteomics demonstrated that BCAP is efficient in isolating cell surface proteins with excellent reproducibility. Subsequently, we applied BCAP to compare the surface proteome of proliferating and senescent mouse embryonic fibroblasts (MEFs). Among the results, EHD2 was identified and validated as a novel protein that is enhanced at the cell surface of senescent MEFs. We expect that BCAP will have broad applications in profiling cell surface proteomes in the future.


Asunto(s)
Proteoma , Proteómica , Animales , Biotinilación , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Fibroblastos/metabolismo , Espectrometría de Masas , Ratones , Proteoma/metabolismo , Proteómica/métodos , Reproducibilidad de los Resultados
3.
Anal Chem ; 94(13): 5425-5431, 2022 04 05.
Artículo en Inglés | MEDLINE | ID: mdl-35319866

RESUMEN

Cellular senescence is involved in diverse physiological processes. Accumulation of senescent cells can lead to numerous age-related diseases. Therefore, it is of great significance to develop chemical tools to effectively detect and eliminate senescent cells. Till date, a dual functional probe that could detect and eliminate senescent cells has yet been accomplished. Herein, a ß-gal-activated probe, MB-ßgal, based on the methylene blue (MB) fluorophore, was designed to detect and eliminate senescent cells. In the absence of ß-gal, the probe showed no fluorescence and its 1O2 production efficiency was suppressed simultaneously. On the other hand, MB-ßgal could be specifically activated by the high level of ß-gal in senescent cells, thus, releasing free MB with near-infrared (NIR) fluorescence and high 1O2 production efficiency under light irradiation. MB-ßgal demonstrated a fast response, high sensitivity, and high selectivity in detecting ß-gal in an aqueous solution and was further applied to visualization and ablation of senescent cells. As a proof of concept, the dual functions of MB-ßgal were successfully demonstrated in senescent HeLa cells and mouse embryonic fibroblast cells.


Asunto(s)
Fibroblastos , Colorantes Fluorescentes , Animales , Senescencia Celular , Células HeLa , Humanos , Ratones , beta-Galactosidasa
4.
J Cell Biochem ; 120(8): 12369-12375, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-30848506

RESUMEN

OBJECTIVE: To study the effect of microRNA-1224 (miR-1224) on hydrogen peroxide (H2 O 2 )-induced oxidative stress injury in hepatocytes, and explore its underlying mechanism. METHODS: L02 cells were treated with H2 O 2 (100 mmol/L) to establish the model of an oxidative stress injury in hepatocytes. Quantitative reverse transcriptase polymerase chain reaction was used to detect the expression of miR-1224 and hepatocyte growth factor (HGF) in L02 cells. L02 cells were transfected with anti-miR-con (H 2 O 2 + anti-miR-con group), anti-miR-1224 (H 2 O 2 + anti-miR-1224 group), pcDNA3.1 (H 2 O 2 + ctrl group), pcDNA3.1-HGF (H 2 O 2 + HGF group), si-HGF and anti-miR-1224 (H 2 O 2 + anti-miR-1224 + HGF group), si-NC and anti-miR-1224 (H 2 O 2 + anti-miR-1224 + ctrl group) by liposome method. Cells without any treatment were regarded as a negative control (NC) group. The protein expression of HGF in each group cells was detected by Western blot analysis. Cell viability and apoptosis of each group were detected by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay or flow cytometry, respectively. The interaction between miR-1224 and HGF was measured by dual luciferase reporter gene assay. RESULTS: The expression of miR-1224 was enhanced in H2 O 2 -treated L02 cells and its knockdown alleviated H 2 O 2 -induced suppression of viability and promotion of apoptosis. HGF is a target of miR-1224 and its overexpression abated H 2 O 2 -induced injury in hepatocytes. Moreover, silencing of HGF rescued the effect of downregulation of miR-1224 on cell viability and apoptosis in H 2 O 2 -treated L02 cell. CONCLUSION: Downregulation of miR-1224 could attenuate oxidative stress-induced inhibition of viability and increase of apoptosis in hepatocytes by targeting HGF, which may provide a target for potential therapy of acute liver injury.


Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Regulación de la Expresión Génica , Hepatocitos/metabolismo , Peróxido de Hidrógeno/toxicidad , MicroARNs/antagonistas & inhibidores , Estrés Oxidativo , Sustancias Protectoras , Proliferación Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Hepatocitos/citología , Humanos , Oxidantes/toxicidad
5.
Cell Metab ; 36(7): 1586-1597.e7, 2024 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-38703762

RESUMEN

The mitochondrial genome transcribes 13 mRNAs coding for well-known proteins essential for oxidative phosphorylation. We demonstrate here that cytochrome b (CYTB), the only mitochondrial-DNA-encoded transcript among complex III, also encodes an unrecognized 187-amino-acid-long protein, CYTB-187AA, using the standard genetic code of cytosolic ribosomes rather than the mitochondrial genetic code. After validating the existence of this mtDNA-encoded protein arising from cytosolic translation (mPACT) using mass spectrometry and antibodies, we show that CYTB-187AA is mainly localized in the mitochondrial matrix and promotes the pluripotent state in primed-to-naive transition by interacting with solute carrier family 25 member 3 (SLC25A3) to modulate ATP production. We further generated a transgenic knockin mouse model of CYTB-187AA silencing and found that reduction of CYTB-187AA impairs females' fertility by decreasing the number of ovarian follicles. For the first time, we uncovered the novel mPACT pattern of a mitochondrial mRNA and demonstrated the physiological function of this 14th protein encoded by mtDNA.


Asunto(s)
Citocromos b , Animales , Citocromos b/genética , Citocromos b/metabolismo , Ratones , Femenino , Ratones Transgénicos , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Humanos , Ratones Endogámicos C57BL , Genes Mitocondriales , ARN Mensajero/metabolismo , ARN Mensajero/genética , Masculino
6.
Stem Cell Res Ther ; 14(1): 333, 2023 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-38115136

RESUMEN

BACKGROUND: Recent studies demonstrated that elevated osmolarity could induce adipocyte dedifferentiation, representing an appealing procedure to generate multipotent stem cells. Here we aim to elucidate the molecular mechanisms that underlie osmotic induction of adipocyte reprogramming. METHODS: To induce dedifferentiation, the 3T3-L1 or SVF adipocytes were cultured under the hypertonic pressure in 2% PEG 300 medium. Adipocyte dedifferentiation was monitored by aspect ratio measurement, Oil Red staining and qPCR to examine the morphology, lipid droplets, and specific genes of adipocytes, respectively. The osteogenic and chondrogenic re-differentiation capacities of dedifferentiated adipocytes were also examined. To investigate the mechanisms of the osmotic stress-induced dedifferentiation, extracellular vesicles (EVs) were collected from the reprograming cells, followed by proteomic and functional analyses. In addition, qPCR, ELISA, and TNF-α neutralizing antibody (20 ng/ml) was applied to examine the activation and effects of the TNF-α signaling. Furthermore, we also analyzed the Wnt signaling by assessing the activation of ß-catenin and applying BML-284, an agonist of ß-catenin. RESULTS: Hypertonic treatment induced dedifferentiation of both 3T3-L1 and the primary stromal vascular fraction (SVF) adipocytes, characterized by morphological and functional changes. Proteomic profiling revealed that hypertonicity induced extracellular vesicles (EVs) containing mitochondrial molecules including NDUFA9 and VDAC. Functionally, the mitochondrial EVs (MEVs) stimulated TNF-α signaling that activates Wnt-ß-catenin signaling and adipocyte dedifferentiation. Neutralizing TNF-α inhibited hypertonic dedifferentiation of adipocytes. In addition, direct activation of Wnt-ß-catenin signaling using BML-284 could efficiently induce adipocyte dedifferentiation while circumventing the apoptotic effect of the hypertonic treatment. CONCLUSIONS: Hypertonicity prompts the adipocytes to release MEVs, which in turn enhances the secretion of TNF-α as a pro-inflammatory cytokine during the stress response. Importantly, TNF-α is essential for the activation of the Wnt/ß-catenin signaling that drives adipocyte dedifferentiation. A caveat of the hypertonic treatment is apoptosis, which could be circumvented by direct activation of the Wnt/ß-catenin signaling using BML-284.


Asunto(s)
Vesículas Extracelulares , Factor de Necrosis Tumoral alfa , Ratones , Animales , Factor de Necrosis Tumoral alfa/farmacología , beta Catenina/metabolismo , Proteómica , Adipocitos , Diferenciación Celular , Vía de Señalización Wnt , Vesículas Extracelulares/metabolismo , Células 3T3-L1 , Adipogénesis
7.
Artif Cells Nanomed Biotechnol ; 47(1): 1200-1206, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-30957556

RESUMEN

OBJECTIVE: To study the effect of miR-16-5p on lung cancer cell injury and apoptosis, and its mechanism. METHODS: LPS induced lung cancer cell A549 injury; qRT-PCR method was applied to detect the expression of miR-16-5p and CXCR3 in A549 cells. Con (without LPS treatment), LPS + miR-NC group (transfected negative control samples), LPS + miR-16-5p group (transfected miR-16-5p mimics); LPS + si-NC group (transfected negative control samples), LPS + si-CXCR3 group (transfected si-CXCR3); LPS + miR-16-5p + pcDNA3.1 group (co-transfected miR-16-5p mimics and pcDNA3.1), LPS + miR-16-5p + pcDNA3.1-CXCR3 group (co-transfected miR-16-5p mimics and pcDNA3.1-CXCR3) were transfected into A549 cells by liposome method. Western blot was used to detect protein expression of CXCR3, IL-6 and TNF-α in A549 cells; apoptosis of A549 cells was detected by flow cytometry. RESULTS: Compared with the control group, the expression of miR-16-5p mRNA was significantly decreased in A549 cells in LPS group, and the mRNA and protein expression of CXCR3 were significantly increased (p < .05). Overexpression of miR-16-5p and knockdown of CXCR3 both can down-regulated protein expression of IL-6 and TNF-α, and up-regulated apoptosis in LPS-induced A549 cell; CXCR3 is a target of miR-16-5p. Overexpression of CXCR3 rescued the protective effect of miR-16-5p on LPS-induced A549 cell injury. CONCLUSION: miR-16-5p can protect LPS-induced A549 cell injury, and its mechanism may be related to the targeted regulation of CXCR3, which could provide a new target for targeted therapy of lung cancer.


Asunto(s)
Lipopolisacáridos/farmacología , MicroARNs/genética , Receptores CXCR3/genética , Células A549 , Apoptosis/efectos de los fármacos , Apoptosis/genética , Secuencia de Bases , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Humanos , Interleucina-6/genética , Receptores CXCR3/deficiencia , Factor de Necrosis Tumoral alfa/genética
8.
Stem Cells Int ; 2019: 7043890, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30936924

RESUMEN

Stem/progenitor cells have recently been demonstrated to play key roles in the maturation, injury repair, and regeneration of distinct organs or tissues. Porcine has spurred an increased interest in biomedical research models and xenotransplantation, owing to most of its organs share similarities in physiology, cellular composition and size to humans. Therefore, characterization of stem/progenitor cells in porcine organs or tissues may provide a novel avenue to better understand the biology and function of stem cells in humans. In the present study, potential stem/progenitor cells in conducting airway epithelium of a porcine lung were characterized by morphometric analysis of bromodeoxyuridine (BrdU) label-retaining cells (LRCs) during the maturation of the lung. The results showed a pseudostratified mucociliary epithelium comprised of basal, ciliated, goblet, and columnar cells in the conducting airway of a porcine lung. In addition, the majority of primary epithelial cells able to proliferate in vitro expressed keratin 5, a subpopulation of these keratin 5-positive cells, also expressed CD117 (c-Kit) or CD49f (integrin alpha 6, ITGA6), implying that they might be potential epithelial stem/progenitor cells in conducting airway of a porcine lung. Lineage tracing analysis with a BrdU-labeled neonatal piglet showed that the proportion of BrdU-labeled cells in conducting airways decreased over the 90-day period of lung maturation. The BrdU-labeled epithelial cells also expressed keratin 14, mucin 5AC, or prosurfactant protein C (ProSP-C); among them, the keratin 14-positive cells were the most frequent BrdU-labeled epithelial cell type as determined by immunohistochemical and immunofluorescence staining. This study may provide valuable information on the biology and function of epithelial stem/progenitor cells in conducting airway of pigs and humans.

9.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 34(2): 105-109, 2018 Feb.
Artículo en Zh | MEDLINE | ID: mdl-29673451

RESUMEN

Objective To investigate the influence of human placenta mesenchymal stem cells (hPMSCs) on the expression levels of inflammatory factors in the lung tissues of lipopolysaccharide (LPS)-induced acute lung injury (ALI) mice. Methods Six-week-old healthy C57BL/6 male mice were randomly divided into control group, ALI model group and hPMSC-treated group, with 8 mice in each group. LPS was trickled in ALI model group by the trachea. The hPMSC-treated mice were injected with hPMSCs by tail vein at 12 hours after administration of LPS. The control group was managed with the corresponding dose of normal saline. The surface markers of hPMSCs were identificated by flow cytometry. The mice were sacrificed after 24 hours, and the histopathological changes of lung tissues were detected by HE staining before and after injection of hPMSCs. The dry to wet mass ratio (W/D) and the myeloperoxidase (MPO) activity of the lung tissues were tested, and the levels of inflammatory factors in bronchoalveolar lavage fluid (BALF) were measured by ELISA, including tumor necrosis factor (TNF-α), interleukin-1 (IL-1) and interleukin-6 (IL-6). Results Phenotypic identification of hPMSCs showed the positive expression for CD105, CD90 and CD73, but negative expression for CD14, CD34, CD45. Compared with the control group, the pathological injury of the lung tissues was severe in the ALI model group, the W/D ratio and MPO activity in the lung tissues increased, and the levels of TNF-α, IL-1, and IL-6 in the BALF were significantly elevated. Compared with the ALI model group, the treatment with hPMSCs alleviated the lung pathological damage, decreased the W/D ratio and MPO activity in the lung tissues, and down-regulated the expressions of TNF-α, IL-1 and IL-6 in the BALF. Conclusion Treatment with hPMSCs can reduce the levels of inflammatory cytokines in the lung tissues and attenuate LPS-induced ALI.


Asunto(s)
Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/terapia , Tratamiento Basado en Trasplante de Células y Tejidos , Trasplante de Células Madre Mesenquimatosas , Placenta/citología , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/patología , Animales , Femenino , Humanos , Interleucina-1/genética , Interleucina-1/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Pulmón/inmunología , Pulmón/patología , Masculino , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos C57BL , Peroxidasa/genética , Peroxidasa/inmunología , Placenta/trasplante , Embarazo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología
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