Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 71.516
Filtrar
1.
J Transl Med ; 20(1): 395, 2022 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-36058922

RESUMO

BACKGROUND: The BCR-ABL fusion protein is the key factor that results in the occurrence of chronic myeloid leukemia (CML). Imatinib (IM) is a targeted inhibitor of BCR-ABL to achieve complete remission. However, remission failure occurs due to acquired resistance caused by secondary BCR-ABL mutations, underlining the need for novel BCR-ABL-targeting strategies. Circular RNAs (circRNAs) derived from tumor-related genes have been revealed as possible therapeutic targets for relevant cancers in recent investigations. In CML, the roles of this kind of circRNA are yet obscure. METHODS: Firstly, RT-qPCR was used for determining circCRKL expression level in cell lines and clinical samples, RNase R and Actinomycin D were employed to verify the stability of circCRKL. Then shRNAs were designed to specifically knockdown circCRKL. The function of circCRKL in vitro was investigated using CCK-8, colony formation assay, and flow cytometry, while a CML mouse model was constructed to explore the function in vivo. Finally, a dual-luciferase reporter assay, RNA pull-down, RNA immunoprecipitation, and rescue experiments were conducted to investigate the mechanism of circCRKL functioning. RESULTS: Here, we determined circCRKL, which derives from CML-relevant gene CRKL, is over-expressed in BCR-ABL+ cells. Then we noticed knocking down circCRKL using shRNA lentivirus dampens the proliferation of BCR-ABL+ cells both in vitro and in vivo, and augments susceptibility of resistant cells to IM. Intriguingly, we observed that circCRKL has a considerable impact on the expression level of BCR-ABL. Mechanistically, circCRKL could behave like a decoy for miR-877-5p to enhance the BCR-ABL level, allowing BCR-ABL+ cells to maintain viability. CONCLUSIONS: Overall, the current study uncovers that circCRKL is specifically expressed and regulates BCR-ABL expression level via decoying miR-877-5p in BCR-ABL+ cells, highlighting that targeting circCRKL along with imatinib treatment could be utilized as a potential therapeutic strategy for CML patients.


Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , MicroRNAs , Animais , Apoptose , Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos/genética , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Camundongos , MicroRNAs/genética , RNA Circular/genética , RNA Interferente Pequeno
2.
J Nanobiotechnology ; 20(1): 404, 2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36064365

RESUMO

BACKGROUND: Ulcerative colitis (UC) is a major type of inflammatory bowel disease (IBD), which could induce bloody stool, diarrhea, colon atrophy and eventually lead to colorectal cancer. The conventional daily oral administration of drugs only relieve the inflammatory response of colon in the short term, Biological agents such as antibody drugs has proven its efficiency in inhibiting colitis, while the low drug bioavailability means that large doses of antibodies are required, ultimately causing systemic toxicity. Small interfering RNA (siRNA) has significant advantages over antibody drugs in terms of safety and efficacy, and it have been widely applied as potential candidates for a variety of inflammation-related diseases. However, oral delivery of siRNA fails to overcome the degradation of the gastrointestinal environment to produce a significant therapeutic effect in ulcerative colitis. Herein, we design the hybrid delivery system that the siRNA loaded MOF encapsulated in the sodium alginate particles to overcome the barriers in the oral process. RESULTS: The hybrid delivery system (SA@MOF-siRNATNFα) was successfully constructed, and it could not only survive the low pH environment in the stomach and small intestine, but also taken up more by inflammatory macrophages, as well as released much more MOF-siRNATNFα. Moreover, SA@MOF-siRNATNFα tended to enriched and infiltrated into local colon tissues. As a result, SA@MOF-siRNATNFα significantly reduced the progression of colitis, of which the treated mice did not experience significant weight loss, bloody stools and diarrhea. CONCLUSION: We confirmed that the formulation of hydrogel-metal-organic framework hybrids could improve the protection of incorporated payload in the gastric and early small intestine, enhancing the delivery of MOF-siRNA to colon.


Assuntos
Colite Ulcerativa , Colite , Estruturas Metalorgânicas , Animais , Colite/tratamento farmacológico , Colite Ulcerativa/tratamento farmacológico , Diarreia , Hidrogéis , Camundongos , RNA Interferente Pequeno , Fator de Necrose Tumoral alfa/metabolismo
3.
Eur Rev Med Pharmacol Sci ; 26(16): 5689-5697, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36066141

RESUMO

OBJECTIVE: This study aims to summarize the role of PIWIs/piRNAs in cell apoptosis through multiple signaling pathways. The PIWI-interacting RNAs (piRNAs) are among the small non-coding RNAs (sncRNAs) and are mainly expressed in germline cells. PIWI protein is the key to the biogenesis of piRNA. With the deepening of research in recent years, the PIWIs/piRNAs are expressed in a tissue-specific way in somatic cells outside the germline. In addition, researchers have found that the PIWIs/piRNAs play a regulatory role in cell apoptosis, proliferation, and necrosis by regulating key signaling pathways, such as PI3K/Akt signaling pathway, STAT signaling pathway, TGF-ß signaling pathway, and Fas signaling pathway at the transcriptional or post-transcriptional level. However, the PIWIs/piRNAs' role in cell apoptosis and its underlying mechanisms are still not fully understood. This study reviews the regulatory functions of PIWIs/piRNAs in apoptosis from the perspective of the signal pathway. MATERIALS AND METHODS: This study is a narrative review. PubMed and MEDLINE were used as the primary sources to search the following keywords: PIWI/piRNAs, signal pathway, pro-apoptotic, anti-apoptotic, and signaling pathway. RESULTS: PIWIs/piRNAs modulated pro-apoptotic or anti-apoptotic effects in a variety of cells: PIWIs/piRNAs through PI3K/Akt signaling pathway, STAT signaling pathway, TGF-ß signaling pathway, and Fas signaling pathway for pro-apoptotic or anti-apoptotic effects in cells. CONCLUSIONS: Apoptosis is a basic biological phenomenon of cell death, and it also has a great significance and complex molecular biological mechanisms. PIWI/piRNAs are closely related to various types of diseases and play a pro-apoptotic or anti-apoptotic role through the following pathways: PI3K/Akt signaling, STAT signaling, TGF-ß signaling, and Fas signaling pathways.


Assuntos
Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Apoptose , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta
4.
Cardiovasc Ther ; 2022: 9729018, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36082193

RESUMO

Ischemia/reperfusion (I/R) injury is accompanied by an increase of matrix metalloproteinase 2 (MMP-2) activity, which degrades heart contractile proteins. The aim of the study was to investigate the effect of MMP-2 small interfering RNA (MMP-2 siRNA) administration on I/R heart. Isolated rat hearts perfused by the Langendorff method were subjected to I/R in the presence or absence of MMP-2 siRNA. The hemodynamic parameters of heart function were monitored. Lactate dehydrogenase (LDH) activity was measured in coronary effluents. Activity and concentration of MMPs in the hearts were measured. Concentration of troponin I (TnI) in coronary effluents was examined as a target for MMP-2 degradation. Recovery of heart mechanical function was reduced after I/R; however, administration of MMP-2 siRNA resulted in restoration of proper mechanical function (p < 0.001). LDH activity was decreased after the use of MMP-2 siRNA (p = 0.02), providing evidence for reduced cardiac damage. Both MMP-2 and MMP-9 syntheses as well as their activity were inhibited in the I/R hearts after siRNA administration (p < 0.05). MMP-2 siRNA administration inhibited TnI release into the coronary effluents (p < 0.001). The use of MMP-2 siRNA contributed to the improvement of heart mechanical function and reduction of contractile proteins degradation during I/R; therefore, MMP-2 siRNA may be considered a cardioprotective agent.


Assuntos
Metaloproteinase 2 da Matriz , Traumatismo por Reperfusão Miocárdica , Animais , Coração , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Troponina I/genética
5.
Oncol Rep ; 48(4)2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36082808

RESUMO

Iron is an essential nutrient that facilitates cell proliferation and growth, and it can contribute to tumor growth. Although iron chelators have shown great potential in preclinical cancer models, they can cause adverse side­effects. The aim of the present study was to determine whether treatment with 5­aminolevurinic acid (5­ALA) has antitumor effects in bladder cancer, by reduction of mitochondrial iron without using an iron chelator, through activation of heme synthesis. T24 and MGH­U3 cells were treated with 5­ALA. Ferrochelatase uses iron to convert protoporphyrin IX into heme, thus additional groups of T24 and MGH­U3 cells were transfected with synthesized ferrochelatase small interfering RNA (siRNA) either to silence ferrochelatase or to provide a negative siRNA control group, and then cell viability, apoptosis, mitochondrial Fe2+, the cell cycle, and ferritin expression were analyzed in all groups and compared. As an in vivo assessment, mice with orthotopic bladder cancer induced using N­butyl­N­(4­hydro­oxybutyl) were treated with 5­ALA. Bladder weight and pathological findings were evaluated, and immunohistochemical analysis was performed for ferritin and proliferating cell nuclear antigen (PCNA). In the cells treated with 5­ALA, proliferation was decreased compared with the controls, and apoptosis was not detected. In addition, the expression of Fe2+ in mitochondria was decreased by 5­ALA, expression of ferritin was also reduced by 5­ALA, and the percentage of cells in the S phase of the cell cycle was significantly increased by 5­ALA. In T24 and MGH­U3 cells with silenced ferrochelatase, the inhibition of cell proliferation, decreased expression of Fe2+ in mitochondria, reduced expression of ferritin, and increased percentage of cells in the S phase by treatment with 5­ALA were weakened. In vivo, no mouse treated with 5­ALA developed muscle­invasive bladder cancer. The expression of ferritin was weaker in mice treated with 5­ALA and that of PCNA was higher than that in mice treated without 5­ALA. It was concluded that 5­ALA inhibited proliferation of bladder cancer cells by activating heme synthesis.


Assuntos
Ferroquelatase , Neoplasias da Bexiga Urinária , Ácido Aminolevulínico/farmacologia , Ácido Aminolevulínico/uso terapêutico , Animais , Proliferação de Células , Ferritinas , Ferroquelatase/genética , Ferroquelatase/metabolismo , Heme/metabolismo , Ferro/metabolismo , Camundongos , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Interferente Pequeno , Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética
6.
BMC Genomics ; 23(1): 638, 2022 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-36076187

RESUMO

BACKGROUND: The strawberry fleshy fruit is actually enlarged receptacle tissue, and the successful development of the embryo and endosperm is essential for receptacle fruit set. MicroRNAs (miRNAs) and phased small interfering RNAs (phasiRNAs) play indispensable regulatory roles in plant growth and development. However, miRNAs and phasiRNAs participating in the regulation of strawberry embryo and endosperm development have yet to be explored. RESULTS: Here, we performed genome-wide identification of miRNA and phasiRNA-producing loci (PHAS) in strawberry seeds with a focus on those involved in the development of the early embryo and endosperm. We found that embryos and endosperm have different levels of small RNAs. After bioinformatics analysis, the results showed that a total of 404 miRNAs (352 known and 52 novel) and 156 PHAS genes (81 21-nt and 75 24-nt genes) could be found in strawberry seed-related tissues, of which four and nine conserved miRNA families displayed conserved expression in the endosperm and embryo, respectively. Based on refined putative annotation of PHAS loci, some auxin signal-related genes, such as CM3, TAR2, AFB2, ASA1, NAC and TAS3, were found, which demonstrates that IAA biosynthesis is important for endosperm and embryo development during early fruit growth. Additionally, some auxin signal-related conserved (miR390-TAS3) and novel (miR156-ASA1) trigger-PHAS pairs were identified. CONCLUSIONS: Taken together, these results expand our understanding of sRNAs in strawberry embryo and endosperm development and provide a genomic resource for early-stage fruit development.


Assuntos
Fragaria , MicroRNAs , Endosperma/genética , Endosperma/metabolismo , Regulação da Expressão Gênica de Plantas , Humanos , Ácidos Indolacéticos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Interferente Pequeno/genética
7.
Front Endocrinol (Lausanne) ; 13: 995499, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36120469

RESUMO

During hypertension, vascular remodeling allows the blood vessel to withstand mechanical forces induced by high blood pressure (BP). This process is well characterized in the media and intima layers of the vessel but not in the perivascular adipose tissue (PVAT). In PVAT, there is evidence for fibrosis development during hypertension; however, PVAT remodeling is poorly understood. In non-PVAT depots, mechanical forces can affect adipogenesis and lipogenic stages in preadipocytes. In tissues exposed to high magnitudes of pressure like bone, the activation of the mechanosensor PIEZO1 induces differentiation of progenitor cells towards osteogenic lineages. PVAT's anatomical location continuously exposes it to forces generated by blood flow that could affect adipogenesis in normotensive and hypertensive states. In this study, we hypothesize that activation of PIEZO1 reduces adipogenesis in PVAT preadipocytes. The hypothesis was tested using pharmacological and mechanical activation of PIEZO1. Thoracic aorta PVAT (APVAT) was collected from 10-wk old male SD rats (n=15) to harvest preadipocytes that were differentiated to adipocytes in the presence of the PIEZO1 agonist Yoda1 (10 µM). Mechanical stretch was applied with the FlexCell System at 12% elongation, half-sine at 1 Hz simultaneously during the 4 d of adipogenesis (MS+, mechanical force applied; MS-, no mechanical force used). Yoda1 reduced adipogenesis by 33% compared with CON and, as expected, increased cytoplasmic Ca2+ flux. MS+ reduced adipogenesis efficiency compared with MS-. When Piezo1 expression was blocked with siRNA [siPiezo1; NC=non-coding siRNA], the anti-adipogenic effect of Yoda1 was reversed in siPiezo1 cells but not in NC; in contrast, siPiezo1 did not alter the inhibitory effect of MS+ on adipogenesis. These data demonstrate that PIEZO1 activation in PVAT reduces adipogenesis and lipogenesis and provides initial evidence for an adaptive response to excessive mechanical forces in PVAT during hypertension.


Assuntos
Adipogenia , Hipertensão , Tecido Adiposo/metabolismo , Animais , Cálcio/metabolismo , Masculino , Mecanorreceptores/metabolismo , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley
8.
Stem Cell Res Ther ; 13(1): 460, 2022 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-36068594

RESUMO

BACKGROUND: Orthodontic tooth movement inevitably induces cementum resorption, which is an urgent problem for orthodontists to confront. Human periodontal ligament stem cells (hPDLSCs) exert an important role in the orthodontic tooth movement and exhibit multidirectional differentiation ability in cementum regeneration. Connective tissue growth factor (CTGF) is an important extracellular matrix protein for bone homeostasis and cell differentiation. The purpose of our study was to explore the role of CTGF in cementum repair and cementogenesis and to elucidate its underlying mechanism. METHODS: A cementum defect model was established by tooth movement with heavy forces, and the cementum repair effect of CTGF was observed via micro-CT, HE staining and immunohistochemical staining. RT‒qPCR, western blotting (WB), alizarin red staining and ALP activity experiments verified the mineralization ability of hPDLSCs stimulated with CTGF. The expression of Cx43 in periodontal ligament cells was detected by WB and immunofluorescence (IF) experiments after CTGF stimulation in vivo and in vitro. Subsequently, the mineralization ability of hPDLSCs was observed after application of CTGF and the small interfering RNA Si-Cx43. Additionally, co-intervention via application of the small interfering RNA Si-CTGF and the Cx43 agonist ATRA in hPDLSCs was performed to deepen the mechanistic study. Next, WB, IF experiments and co-immunoprecipitation were conducted to confirm whether CTGF triggers the Cx43/ß-catenin axis to regulate cementoblast differentiation of hPDLSCs. RESULTS: Local oral administration of CTGF to the cementum defects in vivo facilitated cementum repair. CTGF facilitated the cementogenesis of hPDLSCs in a concentration-dependent manner. Cx43 acted as a downstream effector of CTGF to regulate cementoblast differentiation. Si-Cx43 reduced CTGF-induced cementoblast differentiation. The Cx43 agonist ATRA restored the low differentiation capacity induced by Si-CTGF. Further mechanistic studies showed that CTGF triggered the activation of ß-catenin in a dose-dependent manner. In addition, co-localization IF analysis and co-immunoprecipitation demonstrated that Cx43 interacted with ß-catenin at cell‒cell connections. Si-Cx43 attenuated the substantial expression of ß-catenin induced by CTGF. The Cx43 agonist reversed the inhibition of ß-catenin induced by Si-CTGF. IF demonstrated that the nuclear importation of ß-catenin was related to the immense expression of Cx43 at cell‒cell junctions. CONCLUSIONS: Taken together, these data demonstrate that CTGF promotes cementum repair and cementogenesis through activation of the Cx43/ß-catenin signalling axis.


Assuntos
Cementogênese , beta Catenina , Diferenciação Celular , Células Cultivadas , Cementogênese/fisiologia , Fator de Crescimento do Tecido Conjuntivo/genética , Conexina 43/genética , Cemento Dentário , Humanos , Ligamento Periodontal , RNA Interferente Pequeno , beta Catenina/genética
9.
Contrast Media Mol Imaging ; 2022: 7511345, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36072628

RESUMO

Background: Recently, inflammation has become a major threat to human health. Studies have confirmed that some Chinese traditional medicine ingredients may effectively interfere with the expression of inflammatory mediators through epigenetic modification, showing a great potential of the application. Objective: To investigate the role of the PPAR/DNMT3A pathway in the reversal of galangin-mediated inflammatory lung injury, promote the development of new anti-inflammatory drugs, reduce the side effects of chemical synthetic drugs on the body, and prove the effectiveness and safety of galangin in inhibiting inflammatory response and injury. Methods: 120 rats were randomly divided into 6 groups: (Group 1) LPS group; (Group 2) LPS + galangin group; (Group 3) LPS + galangin + GW9662 group; (Group 4) LPS + galangin + DNMT3A siRNA group; (Group 5) LPS + galangin + siRNA negative group; (Group 6) control group. The model of inflammatory lung injury was established by intrathecal instillation of LPS in the first five groups and NS in the control group. SD survival rate was recorded every 24 hours after modeling, lasting for 168 hours. The lung tissues were taken 168 hours after the establishment of the model. The pathological morphology of lung tissue was observed after the staining under the light microscope, and the lung dry/wet weight ratio was calculated after drying. After NS was perfused into lung tissue, the lavage fluid was collected and the levels of IL-6 and TNF-a were measured by ELISA. The contents of PPAR, DNMT3A, phosphorylated p65, and ERK in monocytes were detected by the WB method, and the binding contents of p65 and AP-1 in the promoter regions of IL-6 and TNF-a genes were detected by the Chip-qPCR method. Results: Intraperitoneal injection of galangin could inhibit the synthesis of alveolar inflammatory factors (TFs) in the SD model of lung injury induced by LPS, reduce the degree of pathological injury of lung tissue, and improve the survival rate of the SD model. GW9662 can completely reverse the protective effect, while DNMT3A interference can only partially block its protective effect. In addition, galangin could significantly inhibit the LPS-induced expression of p65 and AP-1 in alveolar monocytes and their binding content in the promoter region of inflammatory genes by activating PPAR/DNMT3A pathway. GW9662 could completely reverse the inhibitory effect of galangin. DNMT3A interference could restore the binding content of transcription factors at the promoter of the inflammatory gene but had no significant effect on its synthesis. Conclusion: Galangin can interfere with the binding of transcription factors to inflammatory gene promoters through the methylation modification induced by PPAR/DNMT3A pathway, so as to inhibit the synthesis of inflammatory molecules and reverse inflammatory lung injury.


Assuntos
Lesão Pulmonar Aguda , Flavonoides , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Animais , Flavonoides/efeitos adversos , Interleucina-6/metabolismo , Lipopolissacarídeos , Metilação , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Fator de Transcrição AP-1/metabolismo
10.
Vopr Virusol ; 67(4): 278-289, 2022 Sep 11.
Artigo em Russo | MEDLINE | ID: mdl-36097709

RESUMO

The human immunodeficiency virus (HIV) is currently one of the most pressing global health problems. Since its discovery in 1978, HIV has claimed the lives of more than 35 million people, and the number of people infected today reaches 37 million. In the absence of highly active antiretroviral therapy (HAART), HIV infection is characterized by a steady decrease in the number of CD4+ T-lymphocytes, but its manifestations can affect the central nervous, cardiovascular, digestive, endocrine and genitourinary systems. At the same time, complications induced by representatives of pathogenic and opportunistic microflora, which can lead to the development of bacterial, fungal and viral concomitant infections, are of particular danger. It should be borne in mind that an important problem is the emergence of viruses resistant to standard therapy, as well as the toxicity of the drugs themselves for the body. In the context of this review, of particular interest is the assessment of the prospects for the creation and clinical use of drugs based on small interfering RNAs aimed at suppressing the reproduction of HIV, taking into account the experience of similar studies conducted earlier. RNA interference is a cascade of regulatory reactions in eukaryotic cells, which results in the degradation of foreign messenger RNA. The development of drugs based on the mechanism of RNA interference will overcome the problem of viral resistance. Along with this, this technology makes it possible to quickly respond to outbreaks of new viral diseases.


Assuntos
Infecções por HIV , Viroses , Terapia Antirretroviral de Alta Atividade , Linfócitos T CD4-Positivos , Infecções por HIV/tratamento farmacológico , Humanos , Interferência de RNA , RNA Interferente Pequeno
11.
Int J Mol Sci ; 23(17)2022 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-36077117

RESUMO

Interleukin-23 (IL-23) plays a pivotal role in rheumatoid arthritis (RA). IL-23 and microRNA-223 (miR-223) are both up-regulated and mediate osteoclastogenesis in mice with collagen-induced arthritis (CIA). The aim of this study was to examine the association between IL-23 and miR-223 in contributing to osteoclastogenesis and arthritis. Levels of IL-23p19 in joints of mice with CIA were determined. Lentiviral vectors expressing short hairpin RNA (shRNA) targeting IL-23p19 and lisofylline (LSF) were injected intraperitoneally into arthritic mice. Bone marrow-derived macrophages (BMMs) were treated with signal transducers and activators of transcription 4 (STAT4) specific shRNA and miR-223 sponge carried by lentiviral vectors in response to IL-23 stimulation. Treatment responses were determined by evaluating arthritis scores and histopathology in vivo, and detecting osteoclast differentiation and miR-223 levels in vitro. The binding of STAT4 to the promoter region of primary miR-223 (pri-miR-223) was determined in the Raw264.7 cell line. IL-23p19 expression was increased in the synovium of mice with CIA. Silencing IL-23p19 and inhibiting STAT4 activity ameliorates arthritis by reducing miR-223 expression. BMMs from mice in which STAT4 and miR-223 were silenced showed decreased osteoclast differentiation in response to IL-23 stimulation. IL-23 treatment increased the expression of miR-223 and enhanced the binding of STAT4 to the promoter of pri-miR-223. This study is the first to demonstrate that IL-23 promotes osteoclastogenesis by transcriptional regulation of miR-223 in murine macrophages and mice with CIA. Furthermore, our data indicate that LSF, a selective inhibitor of STAT4, should be an ideal therapeutic agent for treating RA through down-regulating miR-223-associated osteoclastogenesis.


Assuntos
Artrite Experimental , Artrite Reumatoide , MicroRNAs , Animais , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/metabolismo , Interleucina-23/genética , Interleucina-23/metabolismo , Subunidade p19 da Interleucina-23/metabolismo , Camundongos , MicroRNAs/metabolismo , Osteoclastos/metabolismo , Osteogênese/genética , RNA Interferente Pequeno/metabolismo
12.
Int J Mol Sci ; 23(17)2022 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-36077202

RESUMO

Non-viral delivery of therapeutic nucleic acids (NA), including siRNA, has potential in the treatment of diseases with high unmet clinical needs such as acute myeloid leukaemia (AML). While cationic biomaterials are frequently used to complex the nucleic acids into nanoparticles, attenuation of charge density is desirable to decrease in vivo toxicity. Here, an anionic amphiphilic CD was synthesised and the structure was confirmed by Fourier-transform infrared spectroscopy (FT-IR), Nuclear Magnetic Resonance (NMR), and high-resolution mass spectrometry (HRMS). A cationic amphiphilic cyclodextrin (CD) was initially used to complex the siRNA and then co-formulated with the anionic amphiphilic CD. Characterisation of the co-formulated NPs indicated a significant reduction in charge from 34 ± 7 mV to 24 ± 6 mV (p < 0.05) and polydispersity index 0.46 ± 0.1 to 0.16 ± 0.04 (p < 0.05), compared to the cationic CD NPs. Size was similar, 161-164 nm, for both formulations. FACS and confocal microscopy, using AML cells (HL-60), indicated a similar level of cellular uptake (60% after 6 h) followed by endosomal escape. The nano co-formulation significantly reduced the charge while maintaining gene silencing (21%). Results indicate that blending of anionic and cationic amphiphilic CDs can produce bespoke NPs with optimised physicochemical properties and potential for enhanced in vivo performance in cancer treatment.


Assuntos
Ciclodextrinas , Leucemia Mieloide Aguda , Nanopartículas , Ânions , Cátions , Ciclodextrinas/química , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Nanopartículas/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Espectroscopia de Infravermelho com Transformada de Fourier
13.
Mediators Inflamm ; 2022: 5255935, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36091665

RESUMO

Objective: Activation of toll-like receptor 9 (TLR9) has been proposed to play an inhibitory role in RANKL-induced osteoclastogenesis. A20 deubiquitinase has been found to be related to bone loss. This study investigated the role of CpG oligodeoxynucleotides (CpG-ODNs) through regulation of A20 deubiquitinase in RANKL-induced osteoclast formation. Methods: RAW 264.7 cells, a murine monocyte-macrophage cell line, were incubated with or without CpG-ODN in the presence of RANKL. Osteoclast-specific genes and their related signaling molecules were measured by real-time quantitative polymerase chain reaction and Western blot assay. Morphological assessment for osteoclast formation was performed using tartrate-resistant acid phosphatase (TRAP) staining and F-actin ring formation staining. Results: RANKL-induced osteoclast-related genes and proteins, c-Fos, NFATc1, TRAP, cathepsin K, and carbonic anhydrase II were significantly inhibited in RAW 264.7 cells stimulated with CpG-ODN. CpG-ODN attenuated TNF receptor-associated factor 6 (TRAF6), p-IκBα, and p-NF-κB expression in RAW 264 cells mediated by RANKL. CpG-ODN increased A20 gene and proteins in time-dependent manner. A20 expression under costimulation with CpG-ODN and RANKL was more decreased than under stimulation with RANKL alone. Cells transfected with A20 siRNA augmented expression of osteoclast-related genes and proteins. Number of TRAP-positive cells transfected with A20 siRNA was higher than those transfected with NC siRNA. A20 expression in cells transfected with IL-1ß siRNA in the presence of both RANKL and CpG-ODN was more decreased than those with NC siRNA. Conclusion: This study showed that CpG-ODN suppressed RANKL-induced osteoclast formation through regulation of the A20-TRAF6 axis in RAW 264.7 cells.


Assuntos
Osteoclastos , Fator 6 Associado a Receptor de TNF , Animais , Diferenciação Celular , Enzimas Desubiquitinantes/metabolismo , Camundongos , Oligodesoxirribonucleotídeos/metabolismo , Oligodesoxirribonucleotídeos/farmacologia , Osteoclastos/metabolismo , Células RAW 264.7 , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo
14.
Shanghai Kou Qiang Yi Xue ; 31(2): 113-119, 2022 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-36110065

RESUMO

PURPOSE: To investigate the correlation between the level of heat shock protein 90(Hsp90) and the amount of small extracellular vesicles(sEVs) in keratinocytes. METHODS: Human keratinocytes(HaCaT) were cultured in vivo and divided into wild-type group, short hairpin RNA interference group (shRNA group, low expression of Hsp90), and 17-Allylamino-17-demethoxygeldanamycin group (17-AAG group, Hsp90 protein inhibitor). sEVs were isolated from culture system by ultracentrifugation, and their morphological characteristics were observed under transmission electron microscopy (TEM). Western blotting was applied to identify the biological characteristics of sEVs. The number of sEVs particles was detected by nanoparticle tracking analysis (NTA). GraphPad Prism8.0 software was used to analyze the difference in the number of sEVs among the groups by t test (non-parametric Mann-Whitney U test). RESULTS: HaCaT-derived sEVs, obtained by ultracentrifugation, were consistent with the criteria of morphological and biological identification. No expression of Hsp90 protein was detected in HaCaT-derived sEVs. When interfered with Hsp90-shRNA, the number of sEVs were significantly increased. On day 5, the sEVs number of shRNA-interfering group was (177.4±4.18)×108(n=3), while that of vector group was (82.34±4.83)×108(n=3), and the difference was statistically significant (P<0.0001). After 5 days of inhibition with 17-AAG, the sEVs number of 17-AAG group was (652.5±26.73)×108(n=3) and that of control group was (262.22±5.44)×108(n=3), the difference was statistically significant (P<0.000 1). CONCLUSIONS: Low expression of Hsp90 protein can promote the secretion of sEVs in HaCaT cells. sEVs may be involved in the transfer of molecules between epithelial cells and immune cells.


Assuntos
Antineoplásicos , Vesículas Extracelulares , Antineoplásicos/farmacologia , Benzoquinonas , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Queratinócitos , Lactamas Macrocíclicas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
15.
Oxid Med Cell Longev ; 2022: 4674215, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36111165

RESUMO

Lipotoxicity can lead to beta-cell dysfunction and apoptosis because it induces oxidative stress. Recent studies have found that Irisin prevents pancreatic beta-cell dysfunction induced by palmitic acid (PA). However, an association between the protection against oxidative stress conferred by Irisin and beta-cell dysfunction has not been fully elucidated. In this study, we observed that Irisin treatment prevented INS-1 cell apoptosis induced by PA treatment and preserved the insulin-secreting function of INS-1 cells in vitro. These effects probably resulted from the Irisin-induced decrease in intracellular ROS levels triggered by PA treatment. In addition, PA treatment induced oxidative stress partially by inhibiting the activation of thioredoxin 2 (Trx2) through its increase of thioredoxin-interacting protein (Txnip) expression. However, Irisin administration blocked the increase in Txnip expression, which reversed the PA-induced inactivation of Trx2. Irisin also increased the nuclear translocation of Stat3, and the inhibition of Stat3 by siRNAs blocked Irisin-induced Trx2 expression, indicating that both Txnip and Stat3 are involved in Irisin-induced activation of Trx2. Furthermore, blockade of Stat3 by siRNAs led to the decreased gene expression of MafA and Ins and to cessation of glucose-induced insulin secretion that had been enhanced by Irisin. In vivo, HFD treatment led to reduced glucose tolerance and an increase in the level of the oxidative marker malondialdehyde (MDA) compared to that in the control group. However, these effects were ameliorated by Irisin injection due to the inhibition of beta-cell apoptosis and the activation of Trx2, probably through Txnip inhibition and Stat3 activation. In conclusion, our results reveal a possible mechanism for Irisin-induced beta-cell protection, which is mediated through Txnip inhibition and activation of the Stat3-Trx2 pathway.


Assuntos
Fibronectinas , Tiorredoxinas , Fibronectinas/metabolismo , Glucose/toxicidade , Insulina/metabolismo , Malondialdeído/metabolismo , Estresse Oxidativo , Ácido Palmítico/toxicidade , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Tiorredoxinas/metabolismo
16.
J Zhejiang Univ Sci B ; 23(9): 732-746, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36111570

RESUMO

BACKGROUND: Circular RNAs (circRNAs) are covalently closed single-stranded RNAs with multiple biological functions. CircRNA.0007127 is derived from the carbon catabolite repression 4-negative on TATA-less (CCR4-NOT) complex subunit 2 (CNOT2), which was found to regulate tumor cell apoptosis through caspase pathway. METHODS: Potential circRNA.0007127 target microRNAs (miRNAs) were analyzed by miRanda, TargetScan, and RNAhybrid software, and the miRNAs with binding sites for apoptosis-related genes were screened. The roles of circRNA.0007127 and its downstream target, microRNA (miR)|-513a-5p, were validated by quantitative real-time polymerase chain reaction (qPCR), flow cytometry, mitochondrial membrane potential, immunofluorescence, western blot, and caspase-8 (CASP8) protein activity in vitro in H2O2-induced K-562 cells. The circRNA.|0007127|‒|miR-513a-5p and CASP8|‒|miR-513a-5p interactions were verified by luciferase reporter assays. RESULTS: Silencing circRNA.0007127 decreased cell apoptosis by inhibiting CASP8 pathway activation in K-562 cells. Compared with the control group, the expression of CASP8 was reduced by 50% and the 43-kD fragment of CASP8 protein was significantly reduced (P≤0.05). The luciferase reporting assay showed that circRNA.0007127 combined with miR-513a-5p or CASP8, with extremely significant differences (P≤0.001). The overexpression of miR-513a-5p inhibited the gene expression level of CASP8 in a human myeloid leukemia cell model (75% change) and the level of a 43-kD fragment of CASP8 protein (P≤0.01). The rescue experiment showed that cotransfection with circRNA.0007127 small-interfering RNA (siRNA) and the miR-513a-5p inhibitor increased CASP8 gene expression and the apoptosis rate, suggesting that the miR-513a-5p inhibitor is a circRNA.0007127 siRNA antagonist. CONCLUSIONS: CircRNA.0007127 regulates K-562 cell apoptosis through the miR-513a-5p/CASP8 axis, which can serve as a novel powerful molecular target for K-562 cells.


Assuntos
MicroRNAs , RNA Circular , Apoptose , Caspase 8/genética , Caspase 8/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Circular/genética , RNA Interferente Pequeno/genética , Proteínas Repressoras
17.
Zhongguo Dang Dai Er Ke Za Zhi ; 24(9): 1053-1060, 2022.
Artigo em Chinês | MEDLINE | ID: mdl-36111726

RESUMO

OBJECTIVES: To observe the change in ferroptosis in hippocampal neurons after hypoxia-ischemia (HI) in neonatal rats and investigate the related mechanism based on the TXNIP/Trx-1/GPX4 signaling pathway. METHODS: Healthy neonatal Sprague-Dawley rats, aged 7 days, were randomly divided into three groups: sham-operation (n=30), hypoxic-ischemic brain damage (HIBD) (n=30) and siRNA (TXNIP siRNA) (n=12). The classic Rice-Vannucci method was used to establish a neonatal rat model of HIBD. At 6 hours, 24 hours, 72 hours, and 7 days after modeling, Western blot was used to measure the protein expression of GPX4 in the hippocampal tissue at the injured side; at 24 hours after modeling, laser speckle imaging combined with hematoxylin-eosin staining was used to determine whether the model was established successfully; NeuN/GPX4 and GFAP/GPX4 immunofluorescence staining combined with Western blot and other methods was used to measure the protein expression of GPX4 and the signal molecules TXNIP and Trx-1 in the hippocampal tissue at the injured side; the kits for determining the content of serum iron and tissue iron were used to measure the change in iron content; quantitative real-time PCR was used to measure the mRNA expression of TXNIP, Trx-1, and GPX4. RESULTS: At 6 hours, 24 hours, 72 hours, and 7 days after modeling, the HIBD group had a significantly lower protein expression level of GPX4 than the sham-operation group (P<0.05). At 24 hours after modeling, the HIBD group had a significantly lower cerebral blood flow of the injured side than the sham-operation group (P<0.05), with loose and disordered arrangement and irregular morphology of hippocampal CA1 neurons at the injured side. Compared with the sham-operation group, the HIBD group had a significantly higher number of TXNIP+ cells and significantly lower numbers of Trx-1+ cells and NeuN+GPX4+/NeuN+ cells in the hippocampal CA1 region at the injured side (P<0.05), with almost no GFAP+GPX4+ cells in the hippocampal CA1 region. Compared with the sham-operation group, the HIBD group and the siRNA group had significantly higher levels of serum iron and tissue iron in the hippocampus at the injured side (P<0.05). Compared with the HIBD group, the siRNA group had significantly lower levels of serum iron and tissue iron in the hippocampus at the injured side (P<0.05). The HIBD group and the siRNA group had significantly higher mRNA and protein expression levels of TXNIP than the sham-operation group (P<0.05), and the siRNA group had significantly lower expression levels than the HIBD group (P<0.05). The HIBD group and the siRNA group had significantly lower mRNA and protein expression levels of Trx-1 and GPX4 in the hippocampus at the injured side than the sham-operation group (P<0.05), and the siRNA group had significantly higher expression levels than the HIBD group (P<0.05). CONCLUSIONS: HI induces ferroptosis of hippocampal neurons in neonatal rats by activating the TXNIP/Trx-1/GPX4 pathway, thereby resulting in HIBD.


Assuntos
Ferroptose , Hipóxia-Isquemia Encefálica , Animais , Animais Recém-Nascidos , Proteínas de Ciclo Celular/metabolismo , Amarelo de Eosina-(YS)/metabolismo , Hematoxilina/metabolismo , Hipocampo/química , Hipóxia-Isquemia Encefálica/metabolismo , Ferro/metabolismo , Isquemia/metabolismo , Neurônios/metabolismo , RNA Mensageiro/análise , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley , Salicilamidas
18.
Eur J Pharmacol ; 932: 175237, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-36063871

RESUMO

Increasing evidence indicates that hyperuricaemia (HUA) is not only a result of decreased renal urate excretion but also a contributor to kidney disease. Na+-K+-ATPase (NKA), which establishes the sodium gradient for urate transport in proximal tubular epithelial cells (PTECs), its impairment leads to HUA-induced nephropathy. However, the specific mechanism underlying NKA impairment-mediated renal tubular injury and increased urate reabsorption in HUA is not well understood. In this study, we investigated whether autophagy plays a key role in the NKA impairment signalling and increased urate reabsorption in HUA-induced renal tubular injury. Protein spectrum analysis of exosomes from the urine of HUA patients revealed the activation of lysosomal processes, and exosomal expression of lysosomal-associated membrane protein-2 was associated with increased serum levels and decreased renal urate excretion in patients. We demonstrated that high uric acid (UA) induced lysosome dysfunction, autophagy and inflammation in a time- and dose-dependent manner and that high UA and/or NKA α1 siRNA significantly increased mitochondrial abnormalities, such as reductions in mitochondrial respiratory complexes and cellular ATP levels, accompanied by increased apoptosis in cultured PTECs. The autophagy inhibitor hydroxychloroquine (HCQ) ameliorated NKA impairment-mediated mitochondrial dysfunction, Nod-like receptor pyrin domain-containing protein 3 (NLRP3)-interleukin-1ß (IL-1ß) production, and abnormal urate reabsorption in PTECs stimulated with high UA and in rats with oxonic acid (OA)-induced HUA. Our findings suggest that autophagy plays a pivotal role in NKA impairment-mediated signalling and abnormal urate reabsorption in HUA-induced renal tubular injury and that inhibition of autophagy by HCQ could be a promising treatment for HUA.


Assuntos
Hiperuricemia , Adenosina Trifosfatases , Trifosfato de Adenosina , Animais , Autofagia , Hidroxicloroquina , Hiperuricemia/complicações , Hiperuricemia/tratamento farmacológico , Hiperuricemia/metabolismo , Interleucina-1beta , Glicoproteínas de Membrana Associadas ao Lisossomo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ácido Oxônico , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley , Sódio , Ácido Úrico/metabolismo
19.
Cytokine ; 159: 156013, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36067712

RESUMO

BACKGROUND: Gastric cancer (GC) is one of the most common malignant tumours and has a high fatality rate worldwide. This study investigated the role of the Notch-1 signalling pathway in the pathogenesis and progression of GC. METHODS: A total of 64 patients with GC were included in this study. Immunohistochemistry staining was used to detect Notch-1 expression in tumour tissues and adjacent non-tumour tissues, and Notch-1 knockdown in GC cells was identified using short hairpin RNA. A cell scratch assay, transwell assay and flow cytometry analysis were used to analyse the effect of Notch-1 knockdown on cell proliferation, migration and cell cycle distribution. The expression of Notch-1, PTEN, Akt, ERK1/2, E-cadherin and other proteins was detected using Western blotting. RESULTS: The expression level of Notch-1 in GC tissues was higher than that in adjacent non-tumour tissues (P < 0.05). High levels of Notch-1 were also found to be associated with sex (male) and lymph node metastasis (P < 0.05). Notch-1 knockdown in the AGS and BGC-823 GC cell lines inhibited the migration and proliferation of GC cells, and Notch-1 knockdown arrested the cell cycle in the G0/G1 phase. PTEN protein expression was elevated in the presence of Notch-1 knockdown, resulting in the inhibition of phosphorylated Akt protein expression. In addition, phosphorylated ERK protein levels decreased in the presence of Notch-1 knockdown. Further inhibition of ERK1/2 signalling by the MEK1/2 inhibitor U0126 decreased the proliferation of AGS cells. The results of in vivo experiments with xenotransplantation in nude mice are consistent with these results. CONCLUSIONS: Notch-1 plays a key role in the development of GC and was found to promote the lymph node metastasis of GC. Notch-1 knockdown can effectively attenuate the progression of GC cells, which may function in part through the Notch-1-PTEN-ERK1/2 signalling axis.


Assuntos
Neoplasias Gástricas , Animais , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/fisiologia , Regulação Neoplásica da Expressão Gênica , Metástase Linfática , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Nus , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo , Neoplasias Gástricas/patologia
20.
Int J Med Microbiol ; 312(6): 151561, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36087399

RESUMO

Klebsiella pneumoniae is a gram-negative bacterium that can cause many diseases in hospitals and communities. Intestinal K. pneumoniae infections are relatively rare. Most K. pneumoniae infections begin with the colonization of the gastrointestinal system. In this study, clinically isolated K. pneumoniae strains were used to infect intestinal epithelial Caco-2 cells to study the possible intestinal translocation mechanism of K. pneumoniae. We found that of the three K. pneumoniae strains tested, KP1821 exhibited the strongest adhesive and invasive abilities and that the adhesion to Caco-2 intestinal epithelial cells was affected by the acidic environment of the stomach. Transcriptome sequencing revealed the involvement of molecules associated with the extracellular matrix and cell adhesion, inflammatory response, calcium ion and transforming growth factor ß (TGF-ß) signaling pathways, and other abnormalities in biological processes and cell signaling pathways. Additionally, tolloid-like protein 1 (TLL1) was significantly upregulated. Knocking down TLL1 with shRNA significantly reduced KP1821's ability to invade and adhere to intestinal epithelial cells. TLL1 is involved in the activation of the TGF-ß signaling pathway. Inhibition of this pathway using the inhibitor SB431542 induced significantly reduced adhesion and invasion capabilities of KP1821. Our findings demonstrate that TLL1 participates in K. pneumoniae adhesion and invasion of intestinal epithelial cells by activating the TGF-ß signaling pathway.


Assuntos
Cálcio , Klebsiella pneumoniae , Células CACO-2 , Células Epiteliais/microbiologia , Humanos , Klebsiella pneumoniae/fisiologia , RNA Interferente Pequeno , Transdução de Sinais , Metaloproteases Semelhantes a Toloide , Fator de Crescimento Transformador beta
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...