RESUMEN
BACKGROUND: Hepatocellular carcinoma (HCC) arises from hepatocytes and accounts for 90% of primary liver cancer. Reasons for HCC prognosis remaining dismal are that HCC is asymptomatic in its early stages, leading to late diagnosis, and it is markedly resistant to conventional chemo- and radiotherapy. In this study, we investigated RNA interference (RNAi)-based treatment for HCC by targeting MDK. AIM: The present study aimed to evaluate MDK serum levels as a diagnostic biomarker for HCC detection and the effect of MDK silencing by RNAi on HCC. SUBJECTS AND METHODS: A total of 140 participants, including 120 patients diagnosed with HCC and 20 healthy volunteers were enrolled in this study, all patients who underwent liver resection were sampled for tumor and adjacent non-tumor liver tissues, in addition to 5 ml of blood sample. Midkine expression levels were evaluated by ELISA and by qRT-PCR. The in vitro transfection and gene knockdown efficiency of midkine by MDK-siRNA was detected by qRT-PCR and ELISA. Gene knockdown effect at the molecule level on the proliferation of HepG2 in vitro was determined by cell counting. RESULTS: The results showed that the expression of MDK was significantly increased in the serum of HCC patients compared to control serum samples with P<0.001 and significant elevated expression levels of MDK in tumor tissues compared to non-tumor ones with P<0.001. It also showed that down-regulation of MDK using RNAi can significantly inhibit HepG2 cells. CONCLUSION: Molecular targeting of MDK using RNAi interference decreases proliferation and could be a therapeutic target.
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Biomarcadores de Tumor , Carcinoma Hepatocelular , Proliferación Celular , Neoplasias Hepáticas , Midkina , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/patología , Midkina/genética , Masculino , Femenino , Persona de Mediana Edad , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/sangre , Pronóstico , Interferencia de ARN , Terapia Genética/métodos , Estudios de Casos y Controles , ARN Interferente Pequeño/genética , Células Hep G2 , Estudios de Seguimiento , Adulto , Células Tumorales CultivadasRESUMEN
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: Early diagnosis of lung adenocarcinoma (LUAD), one of the most common types of lung cancer, is very important to improve the prognosis of patients. The current methods can't meet the requirements of early diagnosis. There is a pressing need to identify novel diagnostic biomarkers. Secretory proteins are the richest source for biomarker research. This study aimed to identify candidate secretory protein biomarkers for early diagnosis of LUAD by integrated bioinformatics analysis and clinical validation. METHODS: Differentially expressed genes (DEGs) of GSE31210, gene expression data of early stage of LUAD, were analyzed by GEO2R. Upregulated DEGs predicted to encode secreted proteins were obtained by taking the intersection of the DEGs list with the list of genes encoding secreted proteins predicted by the majority decision-based method (MDSEC). The expressions of the identified secreted proteins in the lung tissues of early-stage LUAD patients were further compared with the healthy control group in mRNA and protein levels by using the UALCAN database (TCGA and CPTAC). The selected proteins expressed in plasma were further validated by using Luminex technology. The diagnostic value of the screened proteins was evaluated by receiver operating characteristic (ROC) analysis. Cell counting kit-8 assay was carried out to investigate the proliferative effects of these screened proteins. RESULTS: A total of 2183 DEGs, including 1240 downregulated genes and 943 upregulated genes, were identified in the GSE31210. Of the upregulated genes, 199 genes were predicted to encode secreted proteins. After analysis using the UALCAN database, 16 molecules were selected for further clinical validation. Plasma concentrations of three proteins, Midkine (MDK), WAP four-disulfide core domain 2 (WFDC2), and C-X-C motif chemokine ligand 14 (CXCL14), were significantly higher in LUAD patients than in healthy donors. The area under the curve values was 0.944, 0.881, and 0.809 for MDK, WFDC2, and CXCL14, 0.962 when combined them. Overexpression of the three proteins enhanced the proliferation activity of A549 cells. CONCLUSIONS: MDK, WFDC2, and CXCL14 were identified as candidate diagnostic biomarkers for early-stage LUAD and might also play vital roles in tumorigenesis.
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Adenocarcinoma del Pulmón , Quimiocinas CXC , Neoplasias Pulmonares , Midkina , Proteína 2 de Dominio del Núcleo de Cuatro Disulfuros WAP , Humanos , Adenocarcinoma del Pulmón/diagnóstico , Adenocarcinoma del Pulmón/genética , Quimiocinas CXC/genética , Detección Precoz del Cáncer , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Midkina/genética , Biomarcadores de Tumor/genética , Proteína 2 de Dominio del Núcleo de Cuatro Disulfuros WAP/genéticaRESUMEN
Thyroid cancer is the most common endocrine malignancy, and its incidence is increasing. Differentiated thyroid cancer is the most common type and papillary thyroid carcinoma is the most common type of differentiated thyroid cancer. This work aimed to study long noncoding (Lnc) RNA homeobox transcript antisense RNA (HOTAIR) expression in plasma and serum midkine, a heparin binding growth factor, as biomarkers of thyroid cancer. This study included 27 thyroid cancer patients, 29 patients with benign thyroid disease and 26 individuals as normal controls. HOTAIR expression was assessed by real time polymerase chain reaction and midkine by ELISA. These biomarkers were elevated in thyroid cancer patients than patients with benign thyroid diseases and controls. Patients with thyroid cancer stage III had higher midkine levels in comparison to those with stage-I and stage-II (p < 0.001). Patients with grade II had higher midkine in comparison to those with grade I (p < 0.001). Statistically significant elevation of HOTAIR expression was found in stage III and stage II (p=0.001), compared to stage I. However, no difference was observed between stage II and stage III (p=0.533). There was no difference in both biomarkers in different histopathological types of thyroid cancer. ROC analysis was used for detection of thyroid cancer, midkine had AUC of 0.95 at a cutoff 897.5 pg/ml with a sensitivity of 98.0%, and specificity of 81.5% (p < 0.001). HOTAIR had AUC of 1 at a cutoff 11.8-fold change with a sensitivity and specificity of 100 %, (p < 0.001). We concluded that HOTAIR has high sensitivity and specificity in detection of thyroid cancer. It was correlated with tumor stage but not with histopathological types.
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ARN Largo no Codificante , Neoplasias de la Tiroides , Humanos , ARN Largo no Codificante/genética , Genes Homeobox , Biomarcadores de Tumor/genética , ARN sin Sentido , Midkina/genética , Neoplasias de la Tiroides/diagnóstico , Neoplasias de la Tiroides/genética , Regulación Neoplásica de la Expresión Génica , PronósticoRESUMEN
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
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 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
Deubiquitinases (DUBs) have important biological functions, but their roles in breast cancer metastasis are not completely clear. In this study, through screening a series of DUBs related to breast cancer distant metastasis-free survival (DMFS) in the Kaplan-Meier Plotter database, we identified ubiquitin-specific protease 12 (USP12) as a key deubiquitinating enzyme for breast cancer metastasis. We confirmed this via an orthotopic mouse lung metastasis model. We revealed that the DMFS of breast cancer patients with high USP12 was worse than that of others. Knockdown of USP12 decreased the lung metastasis ability of 4T1 cells, while USP12 overexpression increased the lung metastasis ability of these cells in vivo. Furthermore, our results showed that the supernatant from USP12-overexpressing breast cancer cells could promote angiogenesis according to human umbilical vein endothelial cell (HUVEC) migration and tube formation assays. Subsequently, we identified midkine (MDK) as one of its substrates. USP12 could directly interact with MDK, decrease its polyubiquitination and increase its protein stability in cells. Overexpression of MDK rescued the loss of angiogenesis ability mediated by knockdown of USP12 in breast cancer cells in vitro and in vivo. There was a strong positive relationship between USP12 and MDK protein expression in clinical breast cancer samples. Consistent with the pattern for USP12, high MDK expression predicted lower DMFS and overall survival (OS) in breast cancer. Collectively, our study identified that USP12 is responsible for deubiquitinating and stabilizing MDK and leads to metastasis by promoting angiogenesis. Therefore, the USP12-MDK axis could serve as a potential target for the therapeutic treatment of breast cancer metastasis.
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Neoplasias de la Mama/genética , Midkina/genética , Neovascularización Patológica/genética , Ubiquitina Tiolesterasa/metabolismo , Animales , Neoplasias de la Mama/patología , Femenino , Humanos , Ratones , PronósticoRESUMEN
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
BACKGROUND & AIMS: Our previous genomic whole-exome sequencing (WES) data identified the key ErbB pathway mutations that play an essential role in regulating the malignancy of gallbladder cancer (GBC). Herein, we tested the hypothesis that individual cellular components of the tumor microenvironment (TME) in GBC function differentially to participate in ErbB pathway mutation-dependent tumor progression. METHODS: We engaged single-cell RNA-sequencing to reveal transcriptomic heterogeneity and intercellular crosstalk from 13 human GBCs and adjacent normal tissues. In addition, we performed WES analysis to reveal the genomic variations related to tumor malignancy. A variety of bulk RNA-sequencing, immunohistochemical staining, immunofluorescence staining and functional experiments were employed to study the difference between tissues with or without ErbB pathway mutations. RESULTS: We identified 16 cell types from a total of 114,927 cells, in which epithelial cells, M2 macrophages, and regulatory T cells were predominant in tumors with ErbB pathway mutations. Furthermore, epithelial cell subtype 1, 2 and 3 were mainly found in adenocarcinoma and subtype 4 was present in adenosquamous carcinoma. The tumors with ErbB pathway mutations harbored larger populations of epithelial cell subtype 1 and 2, and expressed higher levels of secreted midkine (MDK) than tumors without ErbB pathway mutations. Increased MDK resulted in an interaction with its receptor LRP1, which is expressed by tumor-infiltrating macrophages, and promoted immunosuppressive macrophage differentiation. Moreover, the crosstalk between macrophage-secreted CXCL10 and its receptor CXCR3 on regulatory T cells was induced in GBC with ErbB pathway mutations. Elevated MDK was correlated with poor overall survival in patients with GBC. CONCLUSIONS: This study has provided valuable insights into transcriptomic heterogeneity and the global cellular network in the TME, which coordinately functions to promote the progression of GBC with ErbB pathway mutations; thus, unveiling novel cellular and molecular targets for cancer therapy. LAY SUMMARY: We employed single-cell RNA-sequencing and functional assays to uncover the transcriptomic heterogeneity and intercellular crosstalk present in gallbladder cancer. We found that ErbB pathway mutations reduced anti-cancer immunity and led to cancer development. ErbB pathway mutations resulted in immunosuppressive macrophage differentiation and regulatory T cell activation, explaining the reduced anti-cancer immunity and worse overall survival observed in patients with these mutations.
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Receptores ErbB/inmunología , Neoplasias de la Vesícula Biliar/inmunología , Huésped Inmunocomprometido/fisiología , Midkina/efectos adversos , Proliferación Celular/genética , China/epidemiología , Receptores ErbB/antagonistas & inhibidores , Neoplasias de la Vesícula Biliar/epidemiología , Neoplasias de la Vesícula Biliar/fisiopatología , Humanos , Midkina/genética , Análisis de Secuencia de ARN/métodos , Análisis de Secuencia de ARN/estadística & datos numéricos , Transducción de Señal/genética , Análisis de la Célula Individual/métodos , Análisis de la Célula Individual/estadística & datos numéricos , Secuenciación del Exoma/métodos , Secuenciación del Exoma/estadística & datos numéricosRESUMEN
Facial nerves are frequently injured during cosmetic or other types of facial surgery. However, information on the genes involved in the damage and recovery of the facial nerves is limited. Here, we aimed to identify the genes affected by facial nerve injury and repair using next-generation sequencing. We established a rat axotomy model and a parallel epineurial neurorrhaphy model, in which gene expression was analyzed from 3 days to 8 weeks after surgery. We discovered that ARRB1, SGK1, and GSK3B genes associated with neuronal cell death were upregulated in the axotomy model. In contrast, MFRP, MDK, and ACE genes involved in neural recovery and regeneration exhibited higher expression in the neurorrhaphy model. In the present study, the analysis of the big data obtained from the next-generation sequencing (RNA-seq) technology reveals that the expression of genes involved in neuronal cell death is induced during nerve damage, and those associated with neural recovery are more abundantly expressed during repair processes. These results are considered to be useful for the establishment of the treatment of related diseases and basic research in various neuroscience fields by utilizing damage and recovery mechanism of facial nerves.
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Traumatismos del Nervio Facial/genética , Regeneración Nerviosa/genética , Neuronas/metabolismo , Transcriptoma , Animales , Muerte Celular , Traumatismos del Nervio Facial/metabolismo , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Masculino , Midkina/genética , Midkina/metabolismo , Neuronas/fisiología , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , beta-Arrestina 1/genética , beta-Arrestina 1/metabolismoRESUMEN
The substantial biological heterogeneity of metastatic prostate cancer has hindered the development of personalized therapeutic approaches. Therefore, it is difficult to predict the course of metastatic hormone-sensitive prostate cancer (mHSPC), with some men remaining on first-line androgen deprivation therapy (ADT) for several years while others progress more rapidly. Improving our ability to risk-stratify patients would allow for the optimization of systemic therapies and support the development of stratified prospective clinical trials focused on patients likely to have the greatest potential benefit. Here, we applied a liquid biopsy approach to identify clinically relevant, blood-based prognostic biomarkers in patients with mHSPC. Gene expression indicating the presence of CTCs was greater in CHAARTED high-volume (HV) patients (52% CTChigh) than in low-volume (LV) patients (23% CTChigh; * p = 0.03). HV disease (p = 0.005, q = 0.033) and CTC presence at baseline prior to treatment initiation (p = 0.008, q = 0.033) were found to be independently associated with the risk of nonresponse at 7 months. The pooled gene expression from CTCs of pre-ADT samples found AR, DSG2, KLK3, MDK, and PCA3 as genes predictive of nonresponse. These observations support the utility of liquid biomarker approaches to identify patients with poor initial response. This approach could facilitate more precise treatment intensification in the highest risk patients.
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Biomarcadores de Tumor/genética , Resistencia a Antineoplásicos , Perfilación de la Expresión Génica/métodos , Células Neoplásicas Circulantes/química , Neoplasias de la Próstata/genética , Antagonistas de Andrógenos/farmacología , Antagonistas de Andrógenos/uso terapéutico , Antígenos de Neoplasias/genética , Desmogleína 2/genética , Humanos , Calicreínas/genética , Masculino , Midkina/genética , Reacción en Cadena de la Polimerasa Multiplex , Medicina de Precisión , Pronóstico , Estudios Prospectivos , Antígeno Prostático Específico/genética , Neoplasias de la Próstata/tratamiento farmacológico , Receptores Androgénicos/genéticaRESUMEN
An open question in aggressive cancers such as melanoma is how malignant cells can shift the immune system to pro-tumorigenic functions. Here we identify midkine (MDK) as a melanoma-secreted driver of an inflamed, but immune evasive, microenvironment that defines poor patient prognosis and resistance to immune checkpoint blockade. Mechanistically, MDK was found to control the transcriptome of melanoma cells, allowing for coordinated activation of nuclear factor-κB and downregulation of interferon-associated pathways. The resulting MDK-modulated secretome educated macrophages towards tolerant phenotypes that promoted CD8+ T cell dysfunction. In contrast, genetic targeting of MDK sensitized melanoma cells to anti-PD-1/anti-PD-L1 treatment. Emphasizing the translational relevance of these findings, the expression profile of MDK-depleted tumors was enriched in key indicators of a good response to immune checkpoint blockers in independent patient cohorts. Together, these data reveal that MDK acts as an internal modulator of autocrine and paracrine signals that maintain immune suppression in aggressive melanomas.
Asunto(s)
Carcinogénesis/efectos de los fármacos , Melanoma Experimental/terapia , Midkina/genética , Microambiente Tumoral/genética , Animales , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/genética , Linfocitos T CD8-positivos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Terapia Genética , Humanos , Melanoma Experimental/genética , Melanoma Experimental/patología , Ratones , Midkina/farmacología , FN-kappa B/genética , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Transcriptoma/genéticaRESUMEN
BACKGROUND: Epidermal growth factor receptor (EGFR) is overexpressed in head and neck squamous cell carcinomas (HNSCCs). Midkine expression is restricted in adult tissues but is increased in several malignant tumors, including HNSCCs. AIM: Here, we evaluated the antitumor effect of Midkine promoter-based conditionally replicative adenovirus expressing siRNA against EGFR for targeting HNSCCs expressing Midkine. METHODS AND RESULTS: A conditionally replicative adenovirus vector controlled by the Midkine promoter, Ad-MK-siEGFR, was generated by integrating gene-expressing siRNA against EGFR. Antitumor effect of Ad-MK-siEGFR was tested in vitro using established HNSCC cell line, T891 with strong Midkine expression. Expression of EGFR in T891 infected with Ad-MK-siEGFR was significantly lower than that of T891 infected with control. Cytotoxicity assays showed significant growth suppression of Ad-MK-siEGFR in T891 cells. CONCLUSIONS: This study demonstrated the possibility of oncolytic therapy using the Midkine promoter-based conditional replication-selective adenovirus containing siRNA against EGFR in HNSCC cell line T891. Further validation of the findings in more cell lines and in vivo should be performed to clarify the potential clinical application.
Asunto(s)
Adenoviridae/genética , Neoplasias de Cabeza y Cuello/terapia , Midkina/genética , Viroterapia Oncolítica/métodos , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Humanos , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/terapia , Células Tumorales CultivadasRESUMEN
To identify candidate tissue regeneration enhancer elements (TREEs) important for zebrafish fin regeneration, we performed ATAC-seq from bulk tissue or purified fibroblasts of uninjured and regenerating caudal fins. We identified tens of thousands of DNA regions from each sample type with dynamic accessibility during regeneration, and assigned these regions to proximal genes with corresponding expression changes by RNA-seq. To determine whether these profiles reveal bona fide TREEs, we tested the sufficiency and requirements of several sequences in stable transgenic lines and mutant lines with homozygous deletions. These experiments validated new non-coding regulatory sequences near induced and/or essential genes during fin regeneration, including fgf20a, mdka and cx43, identifying distinct domains of directed expression for each confirmed TREE. Whereas deletion of the previously identified LEN enhancer abolished detectable induction of the nearby leptin b gene during regeneration, deletions of enhancers linked to fgf20a, mdka and cx43 had no effect or partially reduced gene expression. Our study generates a new resource for dissecting the regulatory mechanisms of appendage generation and reveals a range of requirements for individual TREEs in control of regeneration programs.
Asunto(s)
Aletas de Animales/metabolismo , Elementos de Facilitación Genéticos/genética , Regeneración/fisiología , Pez Cebra/metabolismo , Aletas de Animales/fisiología , Animales , Animales Modificados Genéticamente/metabolismo , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Conexina 43/genética , Conexina 43/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica , Leptina/genética , Leptina/metabolismo , Midkina/genética , Midkina/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Zebrafish have the ability to regenerate damaged cells and tissues by activating quiescent stem and progenitor cells or reprogramming differentiated cells into regeneration-competent precursors. Proliferation among the cells that will functionally restore injured tissues is a fundamental biological process underlying regeneration. Midkine-a is a cytokine growth factor, whose expression is strongly induced by injury in a variety of tissues across a range of vertebrate classes. Using a zebrafish Midkine-a loss of function mutant, we evaluated regeneration of caudal fin, extraocular muscle and retinal neurons to investigate the function of Midkine-a during epimorphic regeneration. In wildtype zebrafish, injury among these tissues induces robust proliferation and rapid regeneration. In Midkine-a mutants, the initial proliferation in each of these tissues is significantly diminished or absent. Regeneration of the caudal fin and extraocular muscle is delayed; regeneration of the retina is nearly completely absent. These data demonstrate that Midkine-a is universally required in the signaling pathways that convert tissue injury into the initial burst of cell proliferation. Further, these data highlight differences in the molecular mechanisms that regulate epimorphic regeneration in zebrafish.
Asunto(s)
Midkina/metabolismo , Regeneración/fisiología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Aletas de Animales/fisiología , Animales , Animales Modificados Genéticamente/metabolismo , Diferenciación Celular , Proliferación Celular , Midkina/genética , Mutagénesis , Neuroglía/citología , Neuroglía/metabolismo , Músculos Oculomotores/fisiología , Neuronas Retinianas/fisiología , Proteínas de Pez Cebra/genéticaRESUMEN
Pulmonary arterial hypertension (PAH) is a progressive fatal disease caused by pulmonary arterial remodeling. Midkine regulates cell proliferation and migration, and it is induced by hypoxia, but its roles in pulmonary arterial remodeling remain unclear. Serum midkine levels were significantly increased in PAH patients compared with control patients. Midkine expression was increased in lungs and sera of hypoxia-induced PAH mice. Hypoxia-induced pulmonary arterial remodeling and right ventricular hypertrophy were attenuated in midkine-knockout mice. Midkine-induced proliferation and migration of pulmonary arterial smooth muscle cells (PASMC) and epidermal growth factor receptor (EGFR) signaling were significantly increased under hypoxia, which also induced cell-surface translocation of nucleolin. Nucleolin siRNA treatment suppressed midkine-induced EGFR activation in vitro, and nucleolin inhibitor AS1411 suppressed proliferation and migration of PASMC induced by midkine. Furthermore, AS1411 significantly prevented the development of PAH in Sugen hypoxia rat model. Midkine plays a crucial role in PAH development through interaction with surface nucleolin. These data define a role for midkine in PAH development and suggest midkine-nucleolin-EGFR axis as a novel therapeutic target for PAH.
Asunto(s)
Hipoxia/complicaciones , Midkina/metabolismo , Fosfoproteínas/metabolismo , Hipertensión Arterial Pulmonar/patología , Proteínas de Unión al ARN/metabolismo , Remodelación Vascular/fisiología , Anciano , Animales , Aptámeros de Nucleótidos , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Receptores ErbB/metabolismo , Femenino , Humanos , Hipoxia/fisiopatología , Pulmón/patología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Midkina/sangre , Midkina/genética , Músculo Liso Vascular/citología , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/patología , Oligodesoxirribonucleótidos/farmacología , Oligodesoxirribonucleótidos/uso terapéutico , Fosfoproteínas/antagonistas & inhibidores , Cultivo Primario de Células , Hipertensión Arterial Pulmonar/sangre , Hipertensión Arterial Pulmonar/prevención & control , Arteria Pulmonar/citología , Arteria Pulmonar/patología , Proteínas de Unión al ARN/antagonistas & inhibidores , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , NucleolinaRESUMEN
NEW FINDINGS: What is the central question of this study? The aim was to investigate the function of microRNA-188 in the biological characteristics of lung cancer stem cells and the molecular mechanisms involved. What is the main finding and its importance? This study highlights a new molecular mechanism involving microRNA-188, MDK and the Hippo signalling pathway that plays a suppressive role in biological activity of lung cancer stem cells. This finding might offer new insights into gene-based therapy for lung cancer. ABSTRACT: MicroRNAs (miRNAs) have been implicated in lung cancer and reported as new promising diagnostic and therapeutic tools for cancer control. Here, we investigated the action of microRNA-188 (miR-188) in lung cancer stem cells. We first tested miR-188 expression in clinical samples of lung cancer patients, and a low expression profile of miR-188 was found. Next, we analysed the role of miR-188 in lung cancer stem cells with cell growth assays. To verify the in vitro results, we used a xenograft model to validate the capability of miR-188 in tumorigenesis. Overexpression of miR-188 reduced viability and metastasis of cancer stem cells. Similar results were reproduced in vivo, where overexpression of miR-188 retarded tumour growth in mice. We also identified MDK as a target of miR-188, and overexpression of MDK was found in lung cancer samples. Overexpressed MDK promoted the malignant behaviours of lung cancer stem cells. In addition, the Hippo pathway was found to be inactivated in lung cancer tissues, presenting as increased levels of YAP and TAZ. Suppression of the Hippo pathway also enhanced lung cancer stem cell activity and promoted the growth of xenograft tumours. To sum up, our results reveal that miR-188 inhibits the malignant behaviours of lung cancer stem cells and the growth of xenograft tumours. This study might offer new insights into gene-based therapies for cancer.