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BACKGROUND: Depression is a common mental illness with more than 280 million sufferers worldwide. Inflammation, particularly the c-Jun amino-terminal kinase (JNK) pathway, contributes to depression development and neuronal apoptosis. Gardenia is a herb with therapeutic effects on depression that has been shown to inhibit neuronal apoptosis. However, one of the components in gardenia, Genipin 1-O-ß-D-gentiobioside(GG), has been less studied for its mechanism on depression. Thus, in the current study, we investigate how Genipin 1-O-ß-D-gentiobioside improves depression and elucidate its possible mechanism of action. METHODS: In this investigation, we utilize a chronic unpredictable mild stress (CUMS) mouse model and corticosterone-induced primary cortical neurons to examine the role of GG in ameliorating depressive symptoms and neuronal apoptosis. TUNEL staining and flow cytometry assessed the effects of GG on neuronal apoptosis. Western Blot analyses and immunofluorescence assays apoptosis-related proteins in the prefrontal cortex and primary neurons. The site of action of GG in regulating homeodomain interacting protein kinase 2 (HIPK2) SUMOylation was further explored in primary neurons. We constructed siRNA-SUMO1 vectors to transfect primary neuronal cells with intracellular SUMO1 knockdown. Proximity ligation assay (PLA) experiments were performed on primary neurons according to the instructions of the assay kit to observe the physical relationship between HIPK2 and SUMO1. We predicted the HIPK2 SUMOylation modification site by an online database and constructed vectors to target and site-directed mutagenesis, then to transfected primary neuronal cells. RESULTS: The results showed that GG effectively alleviated depressive-like behaviours, down-regulated apoptosis-related proteins (p-JNK, Bax, Cleaved-Caspase-3), and inhibited neuronal apoptosis in CUMS-induced depressed mice and corticosterone-induced primary cortical neurons. We reveal a complex mechanism underlying the link between GG, SUMOylation of HIPK2, and complex pathways of neuronal apoptosis regulation. K326 and K1189 are the key SUMOylation sites regulated by GG in intricate interactions of apoptosis-related proteins. CONCLUSION: Our study demonstrated that GG exerts antidepressant-like actions through neuroprotective effects by inhibiting the apoptosis of prefrontal cortex neurons, revealing the mechanism of GG inhibition of JNK phosphorylation by enhancing HIPK2 SUMOylation.
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Apoptosis , Depresión , Ratones Endogámicos C57BL , Neuronas , Corteza Prefrontal , Proteínas Serina-Treonina Quinasas , Sumoilación , Animales , Neuronas/efectos de los fármacos , Apoptosis/efectos de los fármacos , Sumoilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Masculino , Corteza Prefrontal/metabolismo , Corteza Prefrontal/efectos de los fármacos , Ratones , Depresión/tratamiento farmacológico , Depresión/metabolismo , Células Cultivadas , Estrés Psicológico/tratamiento farmacológico , Estrés Psicológico/metabolismo , Modelos Animales de Enfermedad , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Antidepresivos/farmacología , Antidepresivos/uso terapéutico , Iridoides/farmacología , Iridoides/uso terapéutico , Glucósidos Iridoides/farmacología , Glucósidos Iridoides/uso terapéutico , CorticosteronaRESUMEN
Post-stroke anxiety (PSA) seriously affects the prognosis of patients, which is an urgent clinical problem to be addressed. However, the pathological mechanism of PSA is largely unclear. Here, we found that neuronal HIPK2 expression was upregulated in the ischemic lesion after stroke. The upregulation of HIPK2 promotes Drp1 oligomerization through the HDAC3-dependent pathway, leading to excessive mitochondrial damage. This subsequently triggers the release of cellular cytokines such as IL-18 from neurons under ischemic stress. Microglia are capable of responding to IL-18, which promotes their activation and enhances their phagocytosis, ultimately resulting in the loss of synapses and neurons, thereby exacerbating the pathological progression of PSA. HIPK2 knockdown or inhibition suppresses excessive pruning of neuronal synapses by activated microglia in the contralateral vCA1 region to compromise inactivated anxiolytic pBLA-vCA1Calb1+ circuit, relieving anxiety-like behavior after stroke. Furthermore, we discovered that early remimazolam administration can remodel HIPK2-HDAC3 axis, ameliorating the progression of PSA. In conclusion, our study revealed that the neuronal HIPK2-HDAC3 axis in the ischemic focus regulates mitochondrial fragmentation to balance inflammation stress reservoir to participate in anxiety susceptibility after stroke.
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Ansiedad , Histona Desacetilasas , Ratones Endogámicos C57BL , Mitocondrias , Neuronas , Proteínas Serina-Treonina Quinasas , Animales , Histona Desacetilasas/metabolismo , Histona Desacetilasas/genética , Mitocondrias/metabolismo , Ratones , Masculino , Ansiedad/etiología , Ansiedad/metabolismo , Neuronas/metabolismo , Neuronas/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/psicología , Accidente Cerebrovascular/patología , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genéticaRESUMEN
Colon cancer, one of the most common and fatal cancers worldwide, is characterized by stepwise accumulation of specific genetic alterations in tumor suppressor genes or oncogenes, leading to tumor growth and metastasis. HIPK2 (homeodomain-interacting protein kinase 2) is a serine/threonine protein kinase and a "bona fide" oncosuppressor protein. Its activation inhibits tumor growth mainly by promoting apoptosis, while its inactivation increases tumorigenicity and resistance to therapies of many different cancer types, including colon cancer. HIPK2 interacts with many molecular pathways by means of its kinase activity or transcriptional co-repressor function modulating cell growth and apoptosis, invasion, angiogenesis, inflammation and hypoxia. HIPK2 has been shown to participate in several molecular pathways involved in colon cancer including p53, Wnt/ß-catenin and the newly identified nuclear factor erythroid 2 (NF-E2) p45-related factor 2 (NRF2). HIPK2 also plays a role in tumor-host interaction in the tumor microenvironment (TME) by inducing angiogenesis and cancer-associated fibroblast (CAF) differentiation. The aim of this review is to assess the role of HIPK2 in colon cancer and the underlying molecular pathways for a better understanding of its involvement in colon cancer carcinogenesis and response to therapies, which will likely pave the way for novel colon cancer therapies.
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Biomarcadores de Tumor , Neoplasias del Colon , Proteínas Serina-Treonina Quinasas , Humanos , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Neoplasias del Colon/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Microambiente Tumoral , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Progresión de la Enfermedad , Animales , Regulación Neoplásica de la Expresión GénicaRESUMEN
Background: This study aimed to explore the molecular mechanism of homeodomain-interacting protein kinase 2 (HIPK2) in diabetic foot ulcers (DFU).Methods: High glucose (HG)-induced human umbilical vein endothelial cells (HUVECs) were used to construct DFU cell models. Cell functions were determined using CCK8 assay, EdU assay, flow cytometry, transwell assay, wound healing assay and tube formation assay. Quantitative real-time PCR and western blot were applied to measure the gene expression.Results: HG treatment suppressed HUVECs proliferation, invasion, migration, and angiogenesis, while enhanced apoptosis. HIPK2 was overexpressed in DFU patients, and its knockdown alleviated HG-induced HUVECs dysfunctions. USP7 stabilised HIPK2 protein by reducing its ubiquitination. USP7 overexpression promoted HG-induced HUVECs dysfunctions, and HIPK2 upregulation also reversed the regulation of USP7 knockdown on HG-induced HUVECs dysfunctions. USP7/HIPK2 axis inhibited the activity of PI3K/AKT pathway.Conclusion: Our study revealed that USP7-stabilised HIPK2 contributed to HG-induced HUVECs dysfunctions, thus accelerating DFU process.
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OBJECTIVE: The study aimed to investigate the effects of verbascoside on oral squamous cell carcinoma (OSCC) cellular behaviors and underlying molecular mechanisms. DESIGN: For this purpose, SCC9 and UM1 cell lines were treated with verbascoside, and their biological behaviors, including proliferation, migration, and invasion, were evaluated using cell counting kit-8, 5-Ethynyl-2'-deoxyuridine, and Transwell assays. The expression of methyltransferase-3 (METTL3), microRNA (miR)- 31-5p, and homeodomain interacting protein kinase-2 (HIPK2) were examined using quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between METTL3 and miR-31-5p was evaluated by RNA immunoprecipitation and methylated RNA immunoprecipitation, while the interaction between miR-31-5p and HIPK2 was evaluated by dual-luciferase reporter analysis. RESULTS: The results indicated inhibition of OSCC cell proliferation, migration, and invasion post verbascoside treatment. Similarly, METTL3 was upregulated in OSCC cells and was inhibited post-verbascoside treatment. Overexpressing METTL3 promoted the cellular processes. Moreover, miR-31-5p was upregulated in OSCC cells, where METTL3 facilitated the processing of miR-31-5p in an N6-methyladenosine (m6A)-dependent manner. The HIPK2 served as miR-31-5p target, where overexpressing miR-31-5p or HIPK2 knockdown reversed the suppression of verbascoside-induced biological behaviors. CONCLUSIONS: Verbascoside inhibited the progression of OSCC by inhibiting the METTL3-regulated miR-31-5p/HIPK2 axis. These findings suggested that verbascoside might be an effective drug for OSCC therapy.
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Carcinoma de Células Escamosas , Proteínas Portadoras , Movimiento Celular , Proliferación Celular , Glucósidos , Metiltransferasas , MicroARNs , Neoplasias de la Boca , Fenoles , Proteínas Serina-Treonina Quinasas , Humanos , Proliferación Celular/efectos de los fármacos , Metiltransferasas/metabolismo , Movimiento Celular/efectos de los fármacos , MicroARNs/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Neoplasias de la Boca/tratamiento farmacológico , Neoplasias de la Boca/patología , Neoplasias de la Boca/metabolismo , Línea Celular Tumoral , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Glucósidos/farmacología , Proteínas Portadoras/metabolismo , Fenoles/farmacología , Invasividad Neoplásica , Reacción en Cadena en Tiempo Real de la Polimerasa , Regulación hacia Arriba/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , PolifenolesRESUMEN
BACKGROUND: Pulmonary fibromatosis (PF) is a specific variant of fibromatosis, which is rarely reported occurring in the lung. PF with HIPK2-YAP1 fusion was a novel entity. CASE PRESENTATION: In this report, a 66-year-old male with PF had been smoking over 40 years. Multiple cords and small nodules in both lungs had been detected in a health examination two years earlier at our hospital. But approximately twofold enlarged in the lingual segment of the upper lobe in the left lung were disclosed in this year. Immunohistochemical analysis demonstrated that the vimentin and ß-Catenin were positive in the largest nodule. After underwent a DNA/RNA panel next-generation sequencing (NGS), missense mutations and HIPK2-YAP1 fusion were found in this sample. Ultimately, the case diagnosis as PF with HIPK2-YAP1 fusion after multidisciplinary treatment. Currently, the patient is doing well and recurrence-free at 14 months post-surgery. CONCLUSIONS: It's difficult for patients with complex morphology to make accurate diagnosis solely based on morphology and immunohistochemistry. But molecular detection is an effective method for further determining pathological subtypes.
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Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras , Neoplasias Pulmonares , Proteínas Serina-Treonina Quinasas , Factores de Transcripción , Proteínas Señalizadoras YAP , Humanos , Masculino , Factores de Transcripción/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anciano , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/diagnóstico , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Mutación MissenseRESUMEN
BACKGROUND: Doxorubicin (DOX) is widely used for the treatment of a variety of cancers. However, its clinical application is limited by dose-dependent cardiotoxicity. Recent findings demonstrated that autophagy inhibition and apoptosis of cardiomyocytes induced by oxidative stress dominate the pathophysiology of DOX-induced cardiotoxicity (DIC), however, there are no potential molecules targeting on these. PURPOSE: This study aimed to explore whether aucubin (AU) acting on inimitable crosstalk between NRF2 and HIPK2 mediated the autophagy, oxidative stress, and apoptosis in DIC, and provide a new and alternative strategy for the treatment of DIC. METHODS AND RESULTS: We first demonstrated the protection of AU on cardiac structure and function in DIC mice manifested by increased EF and FS values, decreased serum CK-MB and LDH contents and well-aligned cardiac tissue in HE staining. Furthermore, AU alleviated DOX-induced myocardial oxidative stress, mitochondrial damage, apoptosis, and autophagy flux dysregulation in mice, as measured by decreased ROS, 8-OHdG, and TUNEL-positive cells in myocardial tissue, increased SOD and decreased MDA in serum, aligned mitochondria with reduced vacuoles, and increased autophagosomes. In vitro, AU alleviated DOX-induced oxidative stress, autophagy inhibition, and apoptosis by promoting NRF2 and HIPK2 expression. We also identified crosstalk between NRF2 and HIPK2 in DIC as documented by overexpression of NRF2 or HIPK2 reversed cellular oxidative stress, autophagy blocking, and apoptosis aggravated by HIPK2 or NRF2 siRNA, respectively. Simultaneously, AU promoted the expression and nuclear localization of NRF2 protein, which was reversed by HIPK2 siRNA, and AU raised the expression of HIPK2 protein as well, which was reversed by NRF2 siRNA. Crucially, AU did not affect the antitumor activity of DOX against MCF-7 and HepG2 cells, which made up for the shortcomings of previous anti-DIC drugs. CONCLUSION: These collective results innovatively documented that AU regulated the unique crosstalk between NRF2 and HIPK2 to coordinate oxidative stress, autophagy, and apoptosis against DIC without compromising the anti-tumor effect of DOX in vitro.
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Cardiotoxicidad , Glucósidos Iridoides , Factor 2 Relacionado con NF-E2 , Ratones , Animales , Cardiotoxicidad/tratamiento farmacológico , Cardiotoxicidad/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Doxorrubicina/farmacología , Miocitos Cardíacos , Apoptosis , Estrés Oxidativo , ARN Interferente Pequeño/farmacología , AutofagiaRESUMEN
Diabetic nephropathy (DN) is one of the complications of diabetes. Long-term hyperglycemia in the kidney results in renal insufficiency, and eventually leads to end-stage renal disease. Epigenetic factor ASH2L has long been identified as a transcriptional activator, and we previously indicated that ASH2L aggravated fibrosis and inflammation in high glucose-induced glomerular mesangial cells, but the pathophysiological relevance and the mechanism of ASH2L-mediated H3K4me3 in DN is not well understood. Here we demonstrated that ASH2L is upregulated in glomeruli isolated from db/db mice. Loss of ASH2L protected glomerular injury caused by hyperglycemia, as evidenced by reduced albuminuria, preserved structure, decreased glomerular extracellular matrix deposition, and lowered renal glomerular expression of proinflammatory and profibrotic markers in db/db mice. Furthermore, we demonstrated that enrichment of ASH2L-mediated H3K4me3 on the promoter regions of ADAM17 and HIPK2 triggered their transcription, leading to aberrant activation of Notch1 signaling pathway, thereby contributing to fibrosis and inflammation in DN. The findings of this study provide compelling evidence for targeting ASH2L as a potential therapeutic strategy to prevent or slow down the progression of DN.
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Diabetes Mellitus , Nefropatías Diabéticas , Histonas , Hiperglucemia , Animales , Ratones , Diabetes Mellitus/patología , Nefropatías Diabéticas/tratamiento farmacológico , Fibrosis , Hiperglucemia/metabolismo , Inflamación/patología , Riñón/patologíaRESUMEN
Sepsis is a life-threatening syndrome that can result in multi-organ dysfunction. MicroRNA (miR)-483-3p was previously demonstrated to be upregulated in sepsis patients; however, its specific functions in sepsis-triggered intestinal injury remain unclarified. Human intestinal epithelial NCM460 cell line was stimulated with lipopolysaccharide (LPS) to mimic sepsis-induced intestinal injury in vitro. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining was utilized for examining cell apoptosis. Western blotting and real time quantitative polymerase chain reaction (RT-qPCR) were used for detecting molecular protein and RNA levels. LPS-induced cytotoxicity was determined by measuring concentrations of lactate dehydrogenase (LDH), diamine oxidase (DAO) and fatty acid binding protein 2 (FABP2). Luciferase reporter assay was utilized for verifying the interaction between miR-483-3p and homeodomain interacting protein kinase 2 (HIPK2). Inhibiting miR-483-3p alleviates LPS-triggered NCM460 cell apoptosis and cytotoxicity. miR-483-3p targeted HIPK2 in LPS-stimulated NCM460 cells. Knockdown of HIPK2 reversed the above effects mediated by miR-483-3p inhibitor. Inhibiting miR-483-3p ameliorates LPS-triggered apoptosis and cytotoxicity by targeting HIPK2.
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MicroARNs , Sepsis , Humanos , Lipopolisacáridos/farmacología , Apoptosis , Sepsis/complicaciones , Sepsis/genética , Bioensayo , MicroARNs/genética , Proteínas Portadoras , Proteínas Serina-Treonina QuinasasRESUMEN
This study aimed to investigate the role of protein kinase HIPK2 in depression and its associated mechanism. The chronic unpredictable mild stress (CUSM) model was constructed to simulate mice with depression to detect the mouse behaviors. Moreover, by using mouse microglial cells BV2 as the model. After conditional knockdown of HIPK2, the depressive behavior disorder of mice was improved, meanwhile, neuroinflammation was alleviated, and the M1 cell proportion was reduced. Similar results were obtained after applying the HIPK2 inhibitor tBID or ASO-HIPK2 treatment. HIPK2 was overexpressed in BV2 cells, which promoted M1 polarization of cells, while tBID suppressed the effect of HIPK2 and reduced the M1 polarized level in BV2 cells. Pull-down assay results indicated that HIPK2 bound to STAT3 and promoted STAT3 phosphorylation. We found that HIPK2 can bind to STAT3 to promote its phosphorylation, which accelerates M1 polarization of microglial cells, aggravates the depressive neuroinflammation, and leads to abnormal behaviors. HIPK2 is promising as the new therapeutic target of depression.
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Depresión , Microglía , Enfermedades Neuroinflamatorias , Proteínas Serina-Treonina Quinasas , Factor de Transcripción STAT3 , Animales , Ratones , Depresión/genética , Depresión/metabolismo , Microglía/metabolismo , Enfermedades Neuroinflamatorias/metabolismo , Fosforilación , Transducción de Señal , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Polaridad CelularRESUMEN
Circular RNAs (circRNAs) play functional roles in rheumatoid arthritis (RA) progression. Fibroblast-like synoviocytes (RASFs) are the main effectors in RA development. In this study, we explored the function and mechanism of circ_0008410 in RASFs. qRT-PCR was used to detect the expression of circ_0008410, microRNA-149-5p (miR-149-5p), and homeodomain-interacting protein kinase 2 (HIPK2). Cell counting kit-8, EdU assay, flow cytometry, and transwell assay were performed to evaluate cell proliferation, apoptosis, migration, and invasion. Western blot measured the protein levels of related markers and HIPK2. The levels of IL-1ß, TNF-α, and IL-6 were tested by corresponding ELISA kits and Western blot. The combination between miR-149-5p and circ_0008410 or HIPK2 was detected by dual-luciferase reporter assay or RNA immunoprecipitation (RIP) assay. Our data showed that circ_0008410 and HIPK2 were elevated, while miR-149-5p was downregulated in RA synovial tissues and RASFs. Circ_0008410 promoted RASF proliferation, migration, invasion, and inflammation while inhibiting apoptosis. MiR-149-5p was a target of circ_0008410, and its overexpression could reverse the promoting effects of circ_0008410 on RASF dysfunction. Moreover, miR-149-5p could target HIPK2 to suppress RASF proliferation, migration, invasion, and inflammation. Collectively, circ_0008410 promoted RASF dysfunction via miR-149-5p/HIPK2, which might provide a potential target for RA therapy.
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Artritis Reumatoide , MicroARNs , Sinoviocitos , Humanos , Membrana Sinovial , Apoptosis/genética , Artritis Reumatoide/genética , Proliferación Celular , Fibroblastos , Inflamación , MicroARNs/genética , Proteínas Portadoras , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is one of the drug target kinases involved in neurological disorders. DYRK1A phosphorylates substrate proteins related to disease progression in an intermolecular manner. Meanwhile, DYRK1A intramolecularly phosphorylates its own residues on key segments during folding process, which is required for its activation and stabilization. To reproduce the autophosphorylation in vitro, DYRK1A was expressed in Escherichia coli-based cell-free protein synthesis system. Although this system was useful for investigating autophosphorylation of serine residue at position 97 (Ser97) in DYRK1A, only a small fraction of the synthesized protein was successfully autophosphorylated. In this study, we found that the addition of DnaK, a bacterial HSP70 chaperone, to cell-free expression of DYRK1A promoted its Ser97 autophosphorylation. Structure prediction with AlphaFold2 indicates that Ser97 forms a hydrogen bond within an α-helix structure, indicating a possibility that DnaK unfolds the α-helix and maintains the structure around Ser97 in a conformation susceptible to phosphorylation. In addition, DnaK promoted phosphorylation of DYRK1B and HIPK2, but not DYRK2 and DYRK4, suggesting a sequence selectivity in the action of DnaK. This study provides a facile method for promoting autophosphorylation of DYRK family kinases in cell-free protein expression.
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Escherichia coli , Procesamiento Proteico-Postraduccional , Fosforilación , Escherichia coli/genética , Biosíntesis de ProteínasRESUMEN
While metal-based complexes are deeply investigated as anticancer chemotherapeutic drugs, fewer studies are devoted to their anti-invasive activity. Herein, two copper (Cu)(II) tropolone derivatives, [Cu(Trop)Cl] and [Cu(Trop)Sac], both containing the N,N-chelated 4,4'-bishydroxymethyl-2,2'-bipyridne ligand, were evaluated for their anticancer and anti-invasive properties. RKO (RKO-ctr) colon cancer cells and their derivatives undergoing stable small interference (si) RNA for HIPK2 protein (RKO-siHIPK2) with acquisition of pro-invasive capacity were used. The results demonstrate that while [Cu(Trop)Sac] did not show cytotoxic activity, [Cu(Trop)Cl] induced cell death in both RKO-ctr and RKO-siHIPK2 cells, indicating that structural changes on substituting the coordinated chloride ligand with saccharine (Sac) could be a key factor in suppressing mechanisms of cellular death. On the other hand, both [Cu(Trop)Sac] and [Cu(Trop)Cl] complexes counteracted RKO-siHIPK2 cell migration in the wound healing assay. The synergic effect exerted by the concomitant presence of both tropolone and saccharin ligands in [Cu(Trop)Sac] was also supported by its significant inhibition of RKO-siHIPK2 cell migration compared to the free Sac ligand. These data suggest that the two Cu(II) tropolone derivatives are also interesting candidates to be further tested in in vivo models as an anti-invasive tumor strategy.
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Antineoplásicos , Complejos de Coordinación , Cobre/farmacología , Cobre/química , Ligandos , Tropolona , Línea Celular Tumoral , Antineoplásicos/farmacología , Antineoplásicos/química , Complejos de Coordinación/farmacología , Complejos de Coordinación/químicaRESUMEN
OBJECTIVE: This study aimed to investigate whether homeodomain interacting protein kinase 2 (HIPK2) polymorphism is associated with renal stone formation in a Turkish population. MATERIALS AND METHODS: A total of 129 patients with calcium nephrolithiasis and 67 sex- and age-matched healthy controls were enrolled in the study. Blood samples were collected into EDTA tubes. The DNA of patients was extracted using a QIAsymphony® automated DNA isolation system. The Chi-square test was applied in the comparisons between the patient and control groups in respect of the differences in the genotype and allele frequencies. RESULTS: No statistically significant difference was found between the groups in terms of single nucleotide polymorphism (SNP) incidence in single allele and double alleles in the rs2058265 and rs6464214 regions (p = 0.13 and 0.37, respectively). The SNP incidence in double alleles in nephrolithiasis patients at rs7456421 was statistically significantly lower than in the control group (p = 0.001). CONCLUSION: Distributions of the genotype and allele of the three polymorphisms (rs2058265, rs6464214, and rs745642 in HIPK2) were not associated with an increased risk of kidney stone in this Turkish population.
OBJETIVO: Investigar si el polimorfismo de la proteína cinasa 2 que interactúa con el homeodominio (HIPK2) está asociado con la formación de cálculos renales en una población turca. MÉTODO: Se inscribieron en el estudio 129 pacientes con nefrolitiasis cálcica y 67 sujetos control sanos, emparejados por sexo y edad. Las muestras de sangre se recogieron en tubos con EDTA. El ADN de los pacientes se extrajo mediante un sistema de aislamiento de ADN automatizado QIAsymphony®. Se aplicó la prueba χ2 en las comparaciones entre los grupos de pacientes y control con respecto a las diferencias de las frecuencias genotípicas y alélicas. RESULTADOS: No se encontraron diferencias estadísticamente significativas entre los grupos en términos de incidencia de polimorfismo de nucleótido simple (PNS) en alelo simple y alelo doble en las regiones rs2058265 y rs6464214 (p = 0.13 y 0.37, respectivamente). La incidencia de PNS en alelos dobles en pacientes con nefrolitiasis en rs7456421 fue menor que en el grupo control, con una diferencia estadísticamente significativa (p = 0.001). CONCLUSIONES: Las distribuciones de genotipo y alelo de los tres polimorfismos (rs2058265, rs6464214 y rs745642 en HIPK2) no se asociaron con un mayor riesgo de cálculos renales en esta población turca.
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Cálculos Renales , Humanos , Cálculos Renales/genética , Alelos , Genotipo , Polimorfismo de Nucleótido Simple , Proteínas Portadoras , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
BACKGROUND: Diabetic foot ulcers (DFU) are a serious complication of diabetes that lead to significant morbidity and mortality. Recent studies reported that exosomes secreted by human adipose tissue-derived mesenchymal stem cells (ADSCs) might alleviate DFU development. However, the molecular mechanism of ADSCs-derived exosomes in DFU is far from being addressed. METHODS: Human umbilical vein endothelial cells (HUVECs) were induced by high-glucose (HG), which were treated with exosomes derived from nuclear factor I/C (NFIC)-modified ADSCs. MicroRNA-204-3p (miR-204-3p), homeodomain-interacting protein kinase 2 (HIPK2), and NFIC were determined using real-time quantitative polymerase chain reaction. Cell proliferation, apoptosis, migration, and angiogenesis were assessed using cell counting kit-8, 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, wound healing, and tube formation assays. Binding between miR-204-3p and NFIC or HIPK2 was predicted using bioinformatics tools and validated using a dual-luciferase reporter assay. HIPK2, NFIC, CD81, and CD63 protein levels were measured using western blot. Exosomes were identified by a transmission electron microscope and nanoparticle tracking analysis. RESULTS: miR-204-3p and NFIC were reduced, and HIPK2 was enhanced in DFU patients and HG-treated HUVECs. miR-204-3p overexpression might abolish HG-mediated HUVEC proliferation, apoptosis, migration, and angiogenesis in vitro. Furthermore, HIPK2 acted as a target of miR-204-3p. Meanwhile, NFIC was an upstream transcription factor that might bind to the miR-204-3p promoter and improve its expression. NFIC-exosome from ADSCs might regulate HG-triggered HUVEC injury through miR-204-3p-dependent inhibition of HIPK2. CONCLUSION: Exosomal NFIC silencing-loaded ADSC sheet modulates miR-204-3p/HIPK2 axis to suppress HG-induced HUVEC proliferation, migration, and angiogenesis, providing a stem cell-based treatment strategy for DFU.
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Diabetes Mellitus , Pie Diabético , Exosomas , MicroARNs , Humanos , Factores de Transcripción NFI , Pie Diabético/genética , Pie Diabético/terapia , Células Endoteliales , Células Madre , MicroARNs/genética , Proteínas Portadoras , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Keloids are fibroproliferative skin disorders caused by the improper healing of wounded skin. A growing body of evidence suggests the involvement of N6-Methyladenosine (m6A) modification in various bioprocesses; however, its role in keloid formation has not yet been investigated. The aim of this study was to determine the effect of the m6A regulator zinc finger CCCH domain containing protein 13 (ZC3H13) on the pathogenesis of keloid formation. ZC3H13 and homeodomain-interacting protein kinase 2 (HIPK2) expression was evaluated in healthy skin and keloid tissues, as well as in human dermal fibroblasts and human keloid fibroblasts (HKF), using qRT-PCR and western blotting. The effects of ZC3H13 overexpression and knockdown on the cell function of HKFs were assessed using CCK8, transwell, and flow cytometry. Furthermore, the influence of ZC3H13 on HIPK2 m6A modification was assessed using MeRIP-qPCR and mRNA stability assays. Both ZC3H13 expression and m6A RNA methylation were upregulated in keloid tissues and HKFs. Silencing of ZC3H13 inhibited proliferation and migration, while enhancing apoptosis in HKFs, whereas overexpression had the opposite effect. Furthermore, HIPK2 levels were high in keloid tissues and HKFs, and a positive correlation was observed between ZC3H13 and HIPK2. In HKFs, ZC3H13 overexpression elevated the m6A levels of HIPK2 mRNA and reduced the rate of HIPK2 mRNA degradation. Mechanically, ZC3H13-induced m6A modifications significantly improved HIPK2 mRNA stability. Collectively, ZC3H13 accelerated keloid formation by mediating the m6A modification of HIPK2 mRNA and maintaining its stability.
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MicroRNAs (miRs, miRNAs) are known players in the regulatory network of pancreatic tumorigenesis, but the downstream effectors remain poorly characterized. This study addressed this issue based on in silico prediction, in vitro experiments, and in vivo validation. The differentially expressed PCa-related miRNAs and bioinformatics tools predicted downstream regulators. The expression of miR-147b was examined in PCa cell lines. Putative targets of miR-147b were predicted by a publicly available database and confirmed by luciferase activity assay. Mimic/inhibitor, siRNA/overexpression plasmid, or pifithrin-α (p53 inhibitor) were delivered into PCa cells to assess the effect of miR-147b, HIPK2, and p53 on malignant phenotypes of PCa cells. AntagomiR-147b and shRNA targeting HIPK2 were introduced to xenograft-bearing nude mice for in vivo experiments. The expression of miR-147b was significantly increased in PCa cell lines. Ectopic expression of miR-147b promoted the malignant phenotypes of PCa cells and inhibited their apoptosis. HIPK2 was confirmed as a target gene of miR-147b. Inhibiting miR-147b could promote HIPK2 expression and potentially activate the p53 pathway, inhibiting PCa cell growth. In vivo experiments suggested that miR-147b inhibition suppressed the growth of xenograft tumors in nude mice, while HIPK2 knockdown counteracted its effect. Collectively, our work reveals a novel miR-147b-mediated carcinogenic regulatory network in PCa that may be a viable target for PCa treatment.
Asunto(s)
MicroARNs , Proteína p53 Supresora de Tumor , Humanos , Animales , Ratones , Ratones Desnudos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , MicroARNs/metabolismo , Línea Celular , Proliferación Celular/genética , Carcinogénesis/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Circular RNA (circRNA) has been confirmed to play a vital role in pancreatic ductal adenocarcinoma (PDAC) progression. However, the function and regulatory mechanism of hsa_circ_0012634 in PDAC progression remain unclear. Quantitative real-time PCR was used to measure the expression of hsa_circ_0012634, microRNA (miR)-147b and homeodomain interacting protein kinase 2 (HIPK2). Cell function was assessed by cell counting kit 8 assay, EdU assay, colony formation assay and flow cytometry. Glucose uptake and lactate production were evaluated to determine cell glycolysis ability. Protein expression was examined by western blot analysis. RNA interaction was confirmed by RNA pull-down assay and dual-luciferase reporter assay. Exosomes were isolated from serums and cell culture supernatant using ultracentrifugation and identified by transmission electron microscopy. Animal experiments were conducted using nude mice. Hsa_circ_0012634 was downregulated in PDAC tissues and cells, and its overexpression suppressed PDAC cell proliferation, glycolysis and enhanced apoptosis. MiR-147b was targeted by hsa_circ_0012634, and its inhibitors repressed PDAC cell growth and glycolysis. HIPK2 could be targeted by miR-147b, and hsa_circ_0012634 regulated miR-147b/HIPK2 to suppress PDAC cell progression. Hsa_circ_0012634 was lowly expressed in serum exosomes of PDAC patients. Exosomal hsa_circ_0012634 inhibited PDAC cell growth and glycolysis in vitro, as well as tumorigenesis in vivo. Exosomal hsa_circ_0012634 restrained PDAC progression via the miR-147b/HIPK2 pathway, confirming that hsa_circ_0012634 might serve as a diagnosis and treatment biomarker for PDAC.
Asunto(s)
Adenocarcinoma , Carcinoma Ductal Pancreático , Exosomas , MicroARNs , Neoplasias Pancreáticas , Animales , Ratones , Exosomas/genética , Ratones Desnudos , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , Proliferación Celular , MicroARNs/genética , Línea Celular Tumoral , Neoplasias PancreáticasRESUMEN
AIMS: Diabetic cardiomyopathy (DC) is one of serious complications of diabetic patients. This study investigated the biological function of activating transcription factor 4 (ATF4) in DC. METHODS AND RESULTS: Streptozotocin-treated mice and high glucose (HG)-exposed HL-1 cells were used as the in vivo and in vitro models of DC. Myocardial infarction (MI) was induced by left coronary artery ligation in mice. Cardiac functional parameters were detected by echocardiography. Target molecule expression was determined by real time quantitative PCR and western blotting. Cardiac fibrosis was observed by haematoxylin and eosin and Masson's staining. Cardiac apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling. Activities of superoxide dismutase, glutathione peroxidase, and levels of malonic dialdehyde and reactive oxygen species were used to assess oxidative stress damage. Molecular mechanisms were evaluated by chromatin immunoprecipitation, dual luciferase assay, and co-immunoprecipitation. ATF4 was up-regulated in the DC and MI mice (P < 0.01). Down-regulation of ATF4 improved cardiac function as evidenced by changes in cardiac functional parameters (P < 0.01), inhibited myocardial collagen I (P < 0.001) and collagen III (P < 0.001) expression, apoptosis (P < 0.001), and oxidative stress (P < 0.001) in diabetic mice. Collagen I (P < 0.01) and collagen III (P < 0.01) expression was increased in MI mice, which was reversed by ATF4 silencing (P < 0.05). ATF4 depletion enhanced viability (P < 0.01), repressed apoptosis (P < 0.001), oxidative damage (P < 0.001), and collagen I (P < 0.001), and collagen III (P < 0.001) expression of HG-stimulated HL-1 cells. ATF4 transcriptionally activated Smad ubiquitin regulatory factor 2 (Smurf2, P < 0.001) to promote ubiquitination and degradation of homeodomain interacting protein kinase-2 (P < 0.001) and subsequently caused inactivation of nuclear factor erythroid 2-related factor 2/heme oxygenase 1 pathway (P < 0.001). The inhibitory effects of ATF4 silencing on HG-induced apoptosis (P < 0.01), oxidative injury (P < 0.01), collagen I (P < 0.001), and collagen III (P < 0.001) expression were reversed by Smurf2 overexpression. CONCLUSIONS: ATF4 facilitates diabetic cardiac fibrosis and oxidative stress by promoting Smurf2-mediated ubiquitination and degradation of homeodomain interacting protein kinase-2 and then inactivation of nuclear factor erythroid 2-related factor 2/heme oxygenase 1 pathway, suggesting ATF4 as a treatment target for DC.
Asunto(s)
Diabetes Mellitus Experimental , Cardiomiopatías Diabéticas , Infarto del Miocardio , Animales , Ratones , Factor de Transcripción Activador 4/genética , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Cardiomiopatías Diabéticas/genética , Cardiomiopatías Diabéticas/metabolismo , Fibrosis , Hemo-Oxigenasa 1 , Proteínas QuinasasRESUMEN
Lysosomotropic agent chloroquine was shown to sensitize non-stem glioblastoma cells to radiation in vitro with p53-dependent apoptosis implicated as one of the underlying mechanisms. The in vivo outcomes of chloroquine or its effects on glioblastoma stem cells have not been previously addressed. This study undertakes a combinatorial approach encompassing in vitro, in vivo and in silico investigations to address the relationship between chloroquine-mediated radiosensitization and p53 status in glioblastoma stem cells. Our findings reveal that chloroquine elicits antagonistic impacts on signaling pathways involved in the regulation of cell fate via both transcription-dependent and transcription-independent mechanisms. Evidence is provided that transcriptional impacts of chloroquine are primarily determined by p53 with chloroquine-mediated activation of pro-survival mevalonate and p21-DREAM pathways being the dominant response in the background of wild type p53. Non-transcriptional effects of chloroquine are conserved and converge on key cell fate regulators ATM, HIPK2 and AKT in glioblastoma stem cells irrespective of their p53 status. Our findings indicate that pro-survival responses elicited by chloroquine predominate in the context of wild type p53 and are diminished in cells with transcriptionally impaired p53. We conclude that p53 is an important determinant of the balance between pro-survival and pro-death impacts of chloroquine and propose that p53 functional status should be taken into consideration when evaluating the efficacy of glioblastoma radiosensitization by chloroquine.