RESUMEN
BACKGROUND: Spinal cord injury (SCI) is a devastating neurological and pathological condition. Exosomal tsRNAs have reported to be promising biomarkers for cancer diagnosis and therapy. This study aimed to investigate the roles of SCI-associated exosomes, and related tsRNA mechanisms in SCI. METHODS: The serum of healthy controls and SCI patients at the acute stage were collected for exosomes isolation, and the two different exosomes were used to treat human astrocytes (HA). The cell viability, apoptosis, and cycle were determined, and the expression of the related proteins were detected by western blot. Then, the two different exosomes were sent for tsRNA sequencing, and four significant known differentially expressed tsRNAs (DE-tsRNAs) were selected for RT-qPCR validation. Finally, tRT-41 was chosen to further explore its roles and related mechanisms in SCI. RESULTS: After sequencing, 21 DE-tsRNAs were identified, which were significantly enriched in pathways of Apelin, AMPK, Hippo, MAPK, Ras, calcium, PI3K-Akt, and Rap1. RT-qPCR showed that tRF-41 had higher levels in the SCI-associated exosomes. Compared with the control HA, healthy exosomes did not significantly affect the growth of HA cells, but SCI-associated exosomes inhibited viability of HA cells, while promoted their apoptosis and increased the HA cells in G2/M phase; but tRF-41 inhibitor reversed the actions of SCI-associated exosomes. Additionally, SCI-associated exosomes, similar with tRF-41 mimics, down-regulated IGF-1, NGF, Wnt3a, and ß-catenin, while up-regulated IL-1ß and IL-6; but tRF-41 inhibitor had the opposite actions, and reversed the effects induced by SCI-associated exosomes. CONCLUSIONS: SCI-associated exosomes delivered tRF-41 may inhibit the growth of HA through regulating Wnt/ ß-catenin pathway and inflammation response, thereby facilitating the progression of SCI.
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Exosomas , Traumatismos de la Médula Espinal , Exosomas/metabolismo , Humanos , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/genética , Apoptosis , Astrocitos/metabolismo , Masculino , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Femenino , Progresión de la Enfermedad , Células Cultivadas , Midkina/metabolismo , Midkina/genética , Adulto , Proliferación Celular , Persona de Mediana EdadRESUMEN
Numerous myofibroblasts are arisen from endothelial cells (ECs) through endothelial to mesenchymal transition (EndMT) triggered by TGF-ß. However, the mechanism of ECs transforms to a different subtype, or whether there exists an intermediate state of ECs remains unclear. In present study, we demonstrate Midkine (MDK) mainly expressed by CD31 + ACTA2+ECs going through partial EndMT contribute greatly to myofibroblasts by spatial and single-cell transcriptomics. MDK is induced in TGF-ß treated ECs, which upregulates C/EBPß and increases EndMT genes, and these effects could be reversed by siMDK. Mechanistically, MDK promotes the binding ability of C/EBPß with ACTA2 promoter by stabilizing the C/EBPß protein. In vivo, knockout of Mdk or conditional knockout of Mdk in ECs reduces EndMT markers and significantly reverses fibrogenesis. In conclusion, our study provides a mechanistic link between the induction of EndMT by TGF-ß and MDK, which suggests that blocking MDK provides potential therapeutic strategies for renal fibrosis.
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Proteína beta Potenciadora de Unión a CCAAT , Transición Endotelial-Mesenquimatosa , Fibrosis , Midkina , Animales , Humanos , Masculino , Ratones , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/genética , Células Endoteliales/metabolismo , Células Endoteliales/patología , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Enfermedades Renales/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Midkina/metabolismo , Midkina/genética , Miofibroblastos/metabolismo , Miofibroblastos/patología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Cell-cell interaction factors that facilitate the progression of adenoma to sporadic colorectal cancer (CRC) remain unclear, thereby hindering patient survival. METHODS: We performed spatial transcriptomics on five early CRC cases, which included adenoma and carcinoma, and one advanced CRC. To elucidate cell-cell interactions within the tumour microenvironment (TME), we investigated the colocalisation network at single-cell resolution using a deep generative model for colocalisation analysis, combined with a single-cell transcriptome, and assessed the clinical significance in CRC patients. FINDINGS: CRC cells colocalised with regulatory T cells (Tregs) at the adenoma-carcinoma interface. At early-stage carcinogenesis, cell-cell interaction inference between colocalised adenoma and cancer epithelial cells and Tregs based on the spatial distribution of single cells highlighted midkine (MDK) as a prominent signalling molecule sent from tumour epithelial cells to Tregs. Interaction between MDK-high CRC cells and SPP1+ macrophages and stromal cells proved to be the mechanism underlying immunosuppression in the TME. Additionally, we identified syndecan4 (SDC4) as a receptor for MDK associated with Treg colocalisation. Finally, clinical analysis using CRC datasets indicated that increased MDK/SDC4 levels correlated with poor overall survival in CRC patients. INTERPRETATION: MDK is involved in the immune tolerance shown by Tregs to tumour growth. MDK-mediated formation of the TME could be a potential target for early diagnosis and treatment of CRC. FUNDING: Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Science Research; OITA Cancer Research Foundation; AMED under Grant Number; Japan Science and Technology Agency (JST); Takeda Science Foundation; The Princess Takamatsu Cancer Research Fund.
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Neoplasias Colorrectales , Midkina , Análisis de la Célula Individual , Linfocitos T Reguladores , Microambiente Tumoral , Femenino , Humanos , Masculino , Carcinogénesis/genética , Carcinogénesis/inmunología , Comunicación Celular/inmunología , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/mortalidad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Tolerancia Inmunológica , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Transcriptoma , Microambiente Tumoral/inmunología , Midkina/inmunología , Midkina/metabolismoRESUMEN
Cholestatic liver diseases causes inflammation and fibrosis around bile ducts. However, the pathological mechanism has not been elucidated. Extracellular vesicles (EVs) are released from both the basolateral and apical sides of polarized biliary epithelial cells. We aimed to investigate the possibility that EVs released from the basolateral sides of biliary epithelial cells by bile acid stimulation induce inflammatory cells and fibrosis around bile ducts, and they may be involved in the pathogenesis of cholestatic liver disease. Human biliary epithelial cells (H69) were grown on cell culture inserts and stimulated with chenodeoxycholic acid + IFN-γ. Human THP-1-derived M1-macrophages, LX-2 cells, and KMST-6 cells were treated with the extracted basolateral EVs, and inflammatory cytokines and fibrosis markers were detected by RT-PCR. Highly expressed proteins from stimulated EVs were identified, and M1-macrophages, LX-2, KMST-6 were treated with these recombinant proteins. Stimulated EVs increased the expression of TNF, IL-1ß, and IL-6 in M1-macrophages, TGF-ß in LX-2 and KMST-6 compared with the corresponding expression levels in unstimulated EVs. Nucleophosmin, nucleolin, and midkine levels were increased in EVs from stimulated cells compared with protein expression in EVs from unstimulated cells. Leukocyte cell-derived chemotaxin-2 (LECT2) is highly expressed only in EVs from stimulated cells. Stimulation of M1-macrophages with recombinant nucleophosmin, nucleolin, and midkine significantly increased the expression of inflammatory cytokines. Stimulation of LX-2 and KMST-6 with recombinant LECT2 significantly increased the expression of fibrotic markers. These results suggest that basolateral EVs are related to the development of pericholangitis and periductal fibrosis in cholestatic liver diseases.NEW & NOTEWORTHY Our research elucidated that the composition of basolateral EVs from the biliary epithelial cells changed under bile acid exposure and the basolateral EVs contained the novel inflammation-inducing proteins NPM, NCL, and MK and the fibrosis-inducing protein LECT2. We report that these new results are possible to lead to the potential therapeutic target of cholestatic liver diseases in the future.
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Vesículas Extracelulares , Hepatopatías , Humanos , Midkina/metabolismo , Nucleofosmina , Células Epiteliales/metabolismo , Citocinas/metabolismo , Inflamación/metabolismo , Hepatopatías/metabolismo , Ácidos y Sales Biliares/metabolismo , Fibrosis , Vesículas Extracelulares/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismoRESUMEN
OBJECTIVE: To characterize the growth factor midkine (MDK) in the human ovary to determine whether MDK is produced locally within the ovary, examine whether different ovarian cell types are more likely to produce MDK, and determine whether there are any stage-specific variations during follicle growth. Previous studies have revealed that MDK potentially affects human follicle growth and oocyte maturation. Proteomic analyses in follicular fluid (FF) have identified MDK to functionally cluster together and follow a similar expression profile to that of well-known proteins involved in ovarian follicle development. Midkine has not yet been characterized in the human ovary. DESIGN: Descriptive study. SETTING: University Hospital. PATIENTS: The study included samples from 121 patients: 71 patients (aged 17-37 years) who underwent ovarian tissue cryopreservation provided granulosa cells (GC), cumulus cells, ovarian cortex, medulla tissue, and FF from small antral follicles (SAF); and 50 patients (aged 20-35 years) receiving in vitro fertilization treatment provided FF from preovulatory follicles before and after induction of final follicle maturation. INTERVENTIONS: None. MAIN OUTCOME MEASURES: MDK relative gene expression was quantified using a real-time quantitative polymerase chain reaction in cumulus cells, GC, and medulla tissue. Additionally, immunostaining and western blotting assays were used to detect MDK protein in the ovarian cortex, which contains preantral follicles, SAF, and medulla tissue. Furthermore, enzyme-linked immunosorbent assay analyses were performed to measure the concentration of MDK in FF aspirated from SAF and preovulatory follicles both before and 36 hours after inducing the final maturation of follicles. RESULTS: Immunostaining and reverse transcription-quantitative polymerase chain reaction revealed a more prominent expression of MDK in GC compared with other ovarian cell types. Intrafollicular MDK concentration was significantly higher in SAF compared with preovulatory follicles. In addition, different molecular weight species of MDK were detected using western blotting in various ovarian sample types: GC and FF samples presented primarily one band of approximately 15 kDa and an additional band of approximately 13 kDa, although other bands with higher molecular weight (between 30 and 38 kDa) were detected in medulla tissue. CONCLUSIONS: This is the first time that MDK has been immunolocalized in human ovarian cells at the protein level and that potentially different MDK variants have been detected in human FF, GC, and ovarian medulla tissue. Future studies are needed to sequence and identify the different potential MDK variants found to determine their functional importance for ovary and oocyte competence.
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Ovario , Proteómica , Femenino , Humanos , Líquido Folicular/metabolismo , Midkina/metabolismo , Folículo Ovárico/metabolismoRESUMEN
Midkine plays a critical role in angiogenesis by regulating the vascular endothelial growth factor (VEGF) signalling pathway, which is known to be associated with psoriasis pathogenesis. However, research on midkine-psoriasis relationship remains limited. The objective of this study was to detect midkine expression in psoriasis and investigate its potential role in the disease. Midkine expression was measured using immunohistochemistry and ELISA. Effects of midkine on HaCaT cell proliferation, VEGF-A production and signalling pathways were assessed using CCK8, RT-PCR and WB. Scratch and in vitro tube formation tests were used to evaluate the effects of HaCaT-cell-activated midkine on the migration and tube formation of human dermal microvascular endothelial cells. Murine psoriasiform models were injected with midkine recombinant protein and midkine monoclonal antibody to investigate skin lesions, tissue sections and dermal microvessel density. Levels of midkine significantly increased in both lesions and serum of patients with psoriasis. Serum expression of midkine decreased after treatment and a positive correlation was found between midkine and disease severity. Midkine promoted HaCaT cell proliferation and VEGF-A production. The Notch2/HES1/JAK2-STAT5A pathway expression increased after midkine treatment of HaCaT cells. The supernatant of HaCaT cells treated with midkine promoted HMEC-1 migration and angiogenesis in vitro. Recombinant midkine protein exacerbated psoriasiform lesions with increased expressions of VEGF-A and microvessel density, while midkine monoclonal antibody alleviated psoriasis lesions. Midkine may have a significant impact on psoriasis angiogenesis by regulating VEGF-A expression through the Notch2/HES1/JAK2-STAT5A pathway, highlighting a potential therapeutic target for psoriasis treatment.
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Psoriasis , Factor A de Crecimiento Endotelial Vascular , Humanos , Ratones , Animales , Factor A de Crecimiento Endotelial Vascular/metabolismo , Midkina/metabolismo , Midkina/farmacología , Células Endoteliales/metabolismo , Psoriasis/metabolismo , Proliferación Celular , Anticuerpos Monoclonales/uso terapéuticoRESUMEN
A total synthesis approach of CS-E oligosaccharides was established and a series of derivatives were synthesized. These oligosaccharides were evaluated for a glycosaminoglycan (GAG)-binding protein interaction against cytokines, midkine, and pleiotrophin, by surface-plasmon resonance (SPR) assay. The binding epitopes of oligosaccharides to midkine were mapped using a saturation transfer difference (STD) NMR technique. The groups on the reducing end contributed to binding affinity, and should not be ignored in biological assays. These findings contribute to the structure and activity relationship research and a foundation of understanding that will underpin potential future optimization of this class of oligosaccharides as pharmaceutical agents.
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Sulfatos de Condroitina , Oligosacáridos , Sulfatos de Condroitina/farmacología , Sulfatos de Condroitina/química , Sulfatos de Condroitina/metabolismo , Midkina/metabolismo , Unión Proteica , Oligosacáridos/químicaRESUMEN
Midkine (MK)2 is an important regulatory molecule that promotes pathological angiogenesis of hepatocellular carcinoma (HCC). Although some studies have shown that its expression is increased in chronic liver disease, its effect on sinusoidal vasculopathy are still unclear. In this study, we demonstrated that MK was mainly secreted by liver sinus endothelial cells (LSECs) during the stage of precancerous lesions. Increased expression of its receptor integrin was an important mechanism by which MK participated in sinusoidal vasculopathy through autocrine and positive feedback effects. LSECs with high expression of integrin α6 (Itgα6+) and integrin α4 (Itgα4+) were used to study the mechanism of MK, and it was found that the effect of MK on LSECs was closely related to the integrin subtypes. The activation of MK /integrin α6/Src/Shc signaling pathway promoted the expression of ET-1, TXA2 and reduced the production of NO, and then induced the capillary vascularization of liver sinusoids, while the activation of MK/integrin α4/NF-κB pathway mainly induced angiogenesis by promoting the production of VEGF and Ang2. In the three-dimensional co-culture system of hepatocytes (BRL-3A) and LSECs, MK significantly increased the production of reactive oxygen species (ROS) in the co-culture system of highly expressed integrin LSECs and decreased the expression of tumor suppressor gene P53 in hepatocytes. These results suggested that MK /integrin signaling pathway, especially MK /integrin α6, was an important mechanism leaded to persistent sinusoidal hepatic vasculopathy in chronic liver disease and induced HCCï¼while MK/integrin α 4 activation was more involved in pathological angiogenesis.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Células Endoteliales/metabolismo , Integrina alfa4/metabolismo , Midkina/metabolismo , Integrina alfa6/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Hepatocitos/metabolismo , Hígado/metabolismo , Neovascularización Patológica , Integrinas/metabolismoRESUMEN
mTORC1 is hyperactive in multiple cancer types1,2. Here, we performed integrative analysis of single cell transcriptomic profiling, paired T cell receptor (TCR) sequencing, and spatial transcriptomic profiling on Tuberous Sclerosis Complex (TSC) associated tumors with mTORC1 hyperactivity, and identified a stem-like tumor cell state (SLS) linked to T cell dysfunction via tumor-modulated immunosuppressive macrophages. Rapamycin and its derivatives (rapalogs) are the primary treatments for TSC tumors, and the stem-like tumor cells showed rapamycin resistance in vitro, reminiscent of the cytostatic effects of these drugs in patients. The pro-angiogenic factor midkine (MDK) was highly expressed by the SLS population, and associated with enrichment of endothelial cells in SLS-dominant samples. Inhibition of MDK showed synergistic benefit with rapamycin in reducing the growth of TSC cell lines in vitro and in vivo. In aggregate, this study suggests an autocrine rapamycin resistance mechanism and a paracrine tumor survival mechanism via immune suppression adopted by the stem-like state tumor cells with mTORC1 hyperactivity.
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Diana Mecanicista del Complejo 1 de la Rapamicina , Midkina , Neoplasias , Células Madre Neoplásicas , Microambiente Tumoral , Células Endoteliales , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Midkina/metabolismo , Neoplasias/inmunología , Neoplasias/metabolismo , Sirolimus , Serina-Treonina Quinasas TOR , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de TumorRESUMEN
This study aimed to investigate the role of cancer-associated fibroblast (CAF)-derived midkine (MK) in cisplatin (DDP) resistance. The primary cultures of CAFs and non-cancer fibroblasts (NFs) were isolated and purified. The DDP-resistant gastric cancer (GC) cells were cultured with CAF-conditioned medium. QRT-PCR and Elisa assays were employed to determine MK expression. The expression of ST7-AS1 was measured by qRT-PCR. The impact of CAFs, MK, and ST7-AS1 silencing on DDP resistance was determined by MTT and Annexin V/PI staining assay. Expression of EMT markers and PI3K/AKT was determined by Western blot and qRT-PCR. The role of MK in DDP resistance was confirmed in a xenograft model. Incubation with CAF-conditioned medium increased the IC50 to DDP. Also, incubation with CAF-conditioned medium increased cell viability, reduced cell apoptosis, and promoted EMT in DDP-resistant GC cells, which were all blocked with MK neutralization antibody treatment. MK increased the DDP resistance and upregulated the expression of ST7-AS1 in DDP-resistant GC cells. Additionally, ST7-AS1 knockdown increased the sensitivity to DDP by inhibiting EMT. Moreover, ST7-AS1 knockdown significantly decreased the phosphorylation of PI3K and AKT, and suppressed EMT, which were restored by MK addition. Finally, MK promoted tumor growth and DDP resistance in a mice model bearing the SGC-7901/DDP xenografts. CAF-derived MK promotes EMT-mediated DDP resistance via upregulation of ST7-AS1 and activation of PI3K/AKT pathway.
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Fibroblastos Asociados al Cáncer , Transición Epitelial-Mesenquimal , Midkina , ARN Largo no Codificante , Neoplasias Gástricas , Animales , Humanos , Ratones , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Línea Celular Tumoral , Proliferación Celular , Cisplatino/farmacología , Medios de Cultivo Condicionados/farmacología , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Midkina/genética , Midkina/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Transducción de Señal , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismoRESUMEN
With the emergence of antifungal resistance, systematic infections with Aspergillus are becoming the major cause of the clinical morbidity. The development of novel antifungal agents with high efficacy, low drug tolerance, and few side effects is urgent. In response to that need, we have identified NP20. Here we demonstrate clearly that NP20 has antifungal activity, capable of killing the spores of Aspergillus niger and Aspergillus fumigatus as well as causing direct damage to the surface, membrane, cytoplasm, organelle, and nucleus of the fungal spores. Interestingly, NP20 is active under temperature stress and a wide range of pH. Subsequently, MTT assay, assay for binding of NP20 to fungal cell wall components, membrane depolarization assay, confocal microscopy, ROS assay, DNA replication, and protein synthesis assay are performed to clarify the mechanisms underlying NP20 against Aspergillus. The results show that NP20 can bind with and pass through the fungal cell wall, and then interfere with the lipid membrane. Moreover, NP20 can induce intracellular ROS production, DNA fragmentation, and protein synthesis inhibition of the fungal cells. These together indicate that NP20 is a novel antifungal peptide, which has considerable potential for future development as novel peptide antibiotics against Aspergillus.
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Aspergillus fumigatus , Anfioxos , Animales , Antifúngicos/metabolismo , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Aspergillus , Aspergillus fumigatus/metabolismo , Aspergillus niger/metabolismo , Proteínas Portadoras , Citocinas , Midkina/metabolismo , Midkina/farmacología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Uveal melanoma is a rare form of melanoma that originates in the eye, exerts widespread therapeutic resistance, and displays an inherent propensity for hepatic metastases. Because metastatic disease is characterized by poor survival, there is an unmet clinical need to identify new therapeutic targets in uveal melanoma. Here, we show that the pleiotropic cytokine midkine is expressed in uveal melanoma. Midkine expression in primary uveal melanoma significantly correlates with poor survival and is elevated in patients that develop metastatic disease. Monosomy 3 and histopathologic staging parameters are associated with midkine expression. In addition, we demonstrate that midkine promotes survival, migration across a barrier of hepatic sinusoid endothelial cells and resistance to AKT/mTOR inhibition. Furthermore, midkine is secreted and mediates mTOR activation by maintaining phosphorylation of the mTOR target RPS6 in uveal melanoma cells. Therefore, midkine is identified as a uveal melanoma cell survival factor that drives metastasis and therapeutic resistance, and could be exploited as a biomarker as well as a new therapeutic target. IMPLICATIONS: Midkine is identified as a survival factor that drives liver metastasis and therapeutic resistance in melanoma of the eye.
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Neoplasias Hepáticas , Melanoma , Midkina , Proteína S6 Ribosómica , Serina-Treonina Quinasas TOR , Neoplasias de la Úvea , Resistencia a Antineoplásicos , Células Endoteliales/metabolismo , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Melanoma/tratamiento farmacológico , Melanoma/genética , Midkina/genética , Midkina/metabolismo , Metástasis de la Neoplasia/patología , Proteína S6 Ribosómica/genética , Proteína S6 Ribosómica/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Neoplasias de la Úvea/tratamiento farmacológico , Neoplasias de la Úvea/genéticaRESUMEN
The cytokine midkine (MK) is a growth factor that is involved in different physiological processes including tissue repair, inflammation, the development of different types of cancer and the proliferation of endothelial cells. The production of MK by primary human macrophages and monocyte-derived dendritic cells (MDDCs) was never described. We investigated whether MK is produced by primary human monocytes, macrophages and MDDCs and the capacity of macrophages and MDDCs to modulate the proliferation of endothelial cells through MK production. The TLR stimulation of human monocytes, macrophages and MDDCs induced an average of ≈200-fold increase in MK mRNA and the production of an average of 78.2, 62, 179 pg/ml MK by monocytes, macrophages and MDDCs respectively (p < 0.05). MK production was supported by its detection in CD11c+ cells, CLEC4C+ cells and CD68+ cells in biopsies of human tonsils showing reactive lymphoid follicular hyperplasia. JSH-23, which selectively inhibits NF-κB activity, decreased the TLR-induced production of MK in PMBCs, macrophages and MDDCs compared to the control (p < 0.05). The inhibition of MK production by macrophages and MDDCs using anti-MK siRNA decreased the capacity of their supernatants to stimulate the proliferation of endothelial cells (p = 0.01 and 0.04 respectively). This is the first study demonstrating that the cytokine MK is produced by primary human macrophages and MDDCs upon TLR triggering, and that these cells can stimulate endothelial cell proliferation through MK production. Our results also suggest that NF-κB plays a potential role in the production of MK in macrophages and MDDCs upon TLR stimulation. The production of MK by macrophages and MDDCs and the fact that these cells can enhance the proliferation of endothelial cells by producing MK are novel immunological phenomena that have potentially important therapeutic implications.
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Células Endoteliales , Monocitos , Proliferación Celular , Citocinas/metabolismo , Células Dendríticas , Humanos , Lectinas Tipo C/metabolismo , Macrófagos , Glicoproteínas de Membrana/metabolismo , Midkina/metabolismo , FN-kappa B/metabolismo , Receptores Inmunológicos/metabolismoRESUMEN
Midkine levels are related to various diseases, including cardiovascular disease, renal disease and autoimmune disease. The research aimed to investigate the mitigation influence of downregulation of intermediate factors on myocardial hypertrophy induced by angiotensin â ¡ (Ang), and whether downregulation of midkine could attenuate oxidative stress and autophagy. Induced myocardial hypertrophy of the mice model and treated HL-1 cells with Ang â ¡ in vitro. The expressions of midkine were increased in the model and HL-1 cells with Ang II treatment. Midkine silence alleviated cardiac hypertrophy induced by Ang II, and inhibited the increases of atrial natriuretic peptide (ANP), Brain natriuretic peptide (BNP) and beta-myosin heavy chain (ß-MHC) in the heart of mice. The raises of ANP, BNP and ß-MHC in Ang II-induced HL-1 cells were also suppressed after midkine downregulation. Downregulating of midkine inhibited the increases of oxidative stress markers 8-OHdG, superoxide anions and MDA in the heart of mice or in the Ang II-treated HL-1 cells. The raises of LC3B, Atg3, Atg5 and Beclin1 in mice heart and in the Ang II.-induced HL-1 cells were attenuated after midkine silence. These outcomes showed that midkine was upregulated in myocardial hypertrophy mice. Targeting of midkine could alleviate cardiac hypertrophy via attenuation of oxidative stress and autophagy.
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Factor Natriurético Atrial , Cardiomegalia , Midkina , Miocitos Cardíacos , Angiotensina II/metabolismo , Animales , Factor Natriurético Atrial/metabolismo , Autofagia , Cardiomegalia/inducido químicamente , Cardiomegalia/metabolismo , Ratones , Midkina/metabolismo , Miocitos Cardíacos/metabolismo , Péptido Natriurético Encefálico/metabolismo , Estrés OxidativoRESUMEN
BACKGROUND: Breast cancer (BC) is the most common malignancy in females and is the second leading cause of cancer-related death among women worldwide. Midkine (MDK) is a heparin-binding growth factor that is abnormally expressed at high levels in various human malignancies. We aimed to uncover the biological function and molecular mechanism of MDK in BC cells. METHODS AND RESULTS: MDA-MB-231-shMDK and T47D-shMDK BC cells were established. The in vitro biological functions of MDK were demonstrated by CCK-8 assays, Transwell assays and Western blotting, whereas qPCR pathway arrays were implemented to explore the mechanism of MDK in BC cells. Functionally, we verified that silencing MDK significantly suppressed BC cell proliferation and migration by inhibiting the activation of the nuclear factor kappa B (NF-κB) pathway and the nuclear distribution of NF-κB. Meanwhile, Ingenuity Pathway Analysis (IPA) and a qPCR pathway array revealed that silencing MDK decreased the expression of NR3C1, a potential downstream target of the NF-κB pathway. We also confirmed that treatment with an NF-κB inhibitor suppressed NR3C1 expression in BC cells. Finally, we demonstrated that silencing NR3C1 repressed BC cell proliferation and migration. CONCLUSIONS: Our findings highlight a novel mechanism by which MDK influences BC progression via regulation of the NF-κB-NR3C1 pathway.
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Neoplasias de la Mama , Midkina/metabolismo , FN-kappa B , Receptores de Glucocorticoides , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Humanos , FN-kappa B/genética , FN-kappa B/metabolismo , Invasividad Neoplásica , Transducción de SeñalRESUMEN
The growth factor Midkine is a heparin-binding cytokine originally discovered during the differentiation process induced by the retinoic acid in embryonal carcinoma cells. Several studies pointed out the key role of this protein in tumour progression and its elevated expression in different malignancies, including pancreatic cancer. New diagnostic and therapeutic tools are urgently required to treat this highly aggressive and incurable disease capable of metastasising, evading diagnosis, and resisting therapy. Serum midkine promises to be a very functional tumour marker and a target for cancer treatment as an elevated concentration of serum midkine is consistently reported in patients with various tumours. Here, we identified high levels of midkine in extracellular vesicles isolated from pancreatic cancer cell lines and showed that it stimulates the growth of pancreatic cancer cells not expressing midkine.
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Vesículas Extracelulares , Midkina , Neoplasias Pancreáticas , Biomarcadores de Tumor/genética , Citocinas/genética , Citocinas/metabolismo , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Humanos , Midkina/genética , Midkina/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismoRESUMEN
BACKGROUND: Activation of the insulin-like growth factor 1 receptor (IGF-1R)-mediated Janus kinase (JAK)1/2-Stat3 pathway contributes to hepatocarcinogenesis. Specifically, a previous study showed that IGF-1R inhibition downregulated Midkine expression in hepatocellular carcinoma (HCC). AIMS: The present study investigated the role of IGF-1R-JAK1/2-Stat3 and Midkine signaling in HCC, in addition to the molecular link between the IGF-1R-Stat3 pathway and Midkine. METHODS: The expression levels of IGF-1R, Stat3, and Midkine were measured using reverse transcription-quantitative PCR, following which the association of IGF-1R with Stat3 and Midkine expression was evaluated in HCC. The molecular link between the IGF-1R-Stat3 pathway and Midkine was then investigated in vitro before the effect of IGF-1R-Stat3 and Midkine signaling on HCC growth and invasion was studied in vitro and in vivo. RESULTS: IGF-1R, Stat3, and Midkine mRNA overexpressions were all found in HCC, where the levels of Stat3 and Midkine mRNA correlated positively with those of IGF-1R. In addition, Midkine mRNA level also correlated positively with Stat3 mRNA expression in HCC tissues. IGF-1R promoted Stat3 activation, which in turn led to the upregulation of Midkine expression in Huh7 cells. Similarly, Midkine also promoted Stat3 activation through potentiating JAK1/2 phosphorylation. Persistent activation of this Stat3-Midkine-Stat3 positive feedback signal loop promoted HCC growth and invasion, the inhibition of which resulted in significant antitumor activities both in vitro and in vivo. CONCLUSIONS: Constitutive activation of the IGF-1R-mediated Stat3-Midkine-Stat3 positive feedback loop is present in HCC, the inhibition of which can serve as a potential therapeutic intervention strategy for HCC.
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Carcinoma Hepatocelular/genética , Janus Quinasa 1/genética , Neoplasias Hepáticas/genética , Midkina/genética , Receptor IGF Tipo 1/genética , Factor de Transcripción STAT3/genética , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Femenino , Humanos , Técnicas In Vitro , Janus Quinasa 1/metabolismo , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Desnudos , Midkina/metabolismo , Trasplante de Neoplasias , Receptor IGF Tipo 1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/metabolismoRESUMEN
Lung adenocarcinomas (LUAD) arise from precancerous lesions such as atypical adenomatous hyperplasia, which progress into adenocarcinoma in situ and minimally invasive adenocarcinoma, then finally into invasive adenocarcinoma. The cellular heterogeneity and molecular events underlying this stepwise progression remain unclear. In this study, we perform single-cell RNA sequencing of 268,471 cells collected from 25 patients in four histologic stages of LUAD and compare them to normal cell types. We detect a group of cells closely resembling alveolar type 2 cells (AT2) that emerged during atypical adenomatous hyperplasia and whose transcriptional profile began to diverge from that of AT2 cells as LUAD progressed, taking on feature characteristic of stem-like cells. We identify genes related to energy metabolism and ribosome synthesis that are upregulated in early stages of LUAD and may promote progression. MDK and TIMP1 could be potential biomarkers for understanding LUAD pathogenesis. Our work shed light on the underlying transcriptional signatures of distinct histologic stages of LUAD progression and our findings may facilitate early diagnosis.
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Adenocarcinoma del Pulmón/metabolismo , Neoplasias Pulmonares/metabolismo , Ribosomas/metabolismo , Adenocarcinoma del Pulmón/genética , Linaje de la Célula , Metabolismo Energético/genética , Metabolismo Energético/fisiología , Humanos , Neoplasias Pulmonares/genética , Midkina/genética , Midkina/metabolismo , Ribosomas/genética , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismoRESUMEN
Dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy and main indication for heart transplantation in children. Therapies specific to pediatric DCM remain limited due to lack of a disease model. Our previous study showed that treatment of neonatal rat ventricular myocytes (NRVMs) with serum from nonfailing or DCM pediatric patients activates the fetal gene program (FGP). Here we show that serum treatment with proteinase K prevents activation of the FGP, whereas RNase treatment exacerbates it, suggesting that circulating proteins, but not circulating miRNAs, promote these pathological changes. Evaluation of the protein secretome showed that midkine (MDK) is upregulated in DCM serum, and NRVM treatment with MDK activates the FGP. Changes in gene expression in serum-treated NRVMs, evaluated by next-generation RNA-Seq, indicated extracellular matrix remodeling and focal adhesion pathways were upregulated in pediatric DCM serum and in DCM serum-treated NRVMs, suggesting alterations in cellular stiffness. Cellular stiffness was evaluated by Atomic Force Microscopy, which showed an increase in stiffness in DCM serum-treated NRVMs. Of the proteins increased in DCM sera, secreted frizzled-related protein 1 (sFRP1) was a potential candidate for the increase in cellular stiffness, and sFRP1 treatment of NRVMs recapitulated the increase in cellular stiffness observed in response to DCM serum treatment. Our results show that serum circulating proteins promoted pathological changes in gene expression and cellular stiffness, and circulating miRNAs were protective against pathological changes.
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Cardiomiopatía Dilatada/genética , Matriz Extracelular/efectos de los fármacos , Adhesiones Focales/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacos , Adolescente , Animales , Animales Recién Nacidos , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/patología , Niño , Preescolar , Endopeptidasa K/farmacología , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Femenino , Adhesiones Focales/metabolismo , Adhesiones Focales/patología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/farmacología , Masculino , Microscopía de Fuerza Atómica , Midkina/metabolismo , Midkina/farmacología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , RNA-Seq , Ratas , Ribonucleasas/farmacología , Secretoma , Remodelación Ventricular/genéticaRESUMEN
Pleiotrophin (PTN) and midkine (MK) are growth factors that modulate alcohol consumption and reward. Since both PTN and MK limit the rewarding effects of alcohol, pharmacological potentiation of the PTN and MK signaling pathways has been proposed for the treatment of alcohol use disorders (AUD). Although the use of this therapy in the prevention of alcohol relapse is important, the potential role of these cytokines in extinguishing alcohol-induced seeking behavior is a key question that remains unanswered. To fill this gap, we have now studied the extinction of the conditioned place preference (CPP) induced by different doses of alcohol in Ptn knockout (Ptn-/-) and Mk knockout (Mk-/-) mice. The data confirm a higher sensitivity of Ptn-/- mice to the conditioning effects of a low dose (1 g/kg) and a rewarding dose (2 g/kg) of alcohol, while Mk-/- mice are only more susceptible to the conditioning effects of the low dose of this drug. More importantly, the percentage of Mk-/- mice, not Ptn-/- mice, that efficiently extinguished alcohol-induced CPP was significantly higher than that of Wt mice. Taken together, the data presented here confirm that Ptn and Mk are genetic factors that determine the conditioning effects of alcohol in mice and that Mk is a novel factor that plays an important role in the extinction of alcohol-induced CPP.