RESUMO
MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression that have been implicated in a plethora of neuronal processes. Nevertheless, their role in regulating brain activity in the context of sleep has so far received little attention. To test their involvement, we deleted mature miRNAs in post-mitotic neurons at two developmental ages, i.e., in early adulthood using conditional Dicer knockout (cKO) mice and in adult mice using an inducible conditional Dicer cKO (icKO) line. In both models, electroencephalographic (EEG) activity was affected and the response to sleep deprivation (SD) altered; while the rapid-eye-movement sleep (REMS) rebound was compromised in both, the increase in EEG delta (1 to 4 Hz) power during non-REMS (NREMS) was smaller in cKO mice and larger in icKO mice compared to controls. We subsequently investigated the effects of SD on the forebrain miRNA transcriptome and found that the expression of 48 miRNAs was affected, and in particular that of the activity-dependent miR-709. In vivo inhibition of miR-709 in the brain increased EEG power during NREMS in the slow-delta (0.75 to 1.75 Hz) range, particularly after periods of prolonged wakefulness. Transcriptome analysis of primary cortical neurons in vitro revealed that miR-709 regulates genes involved in glutamatergic neurotransmission. A subset of these genes was also affected in the cortices of sleep-deprived, miR-709-inhibited mice. Our data implicate miRNAs in the regulation of EEG activity and indicate that miR-709 links neuronal activity during wakefulness to brain synchrony during sleep through the regulation of glutamatergic signaling.
Assuntos
MicroRNAs , Sono , Camundongos , Animais , Sono/fisiologia , Privação do Sono/genética , Eletroencefalografia , Vigília/fisiologia , Prosencéfalo , MicroRNAs/genética , MicroRNAs/farmacologiaRESUMO
Genome-derived microRNAs (miRNAs or miRs) govern posttranscriptional gene regulation and play important roles in various cellular processes and disease progression. While chemo-engineered miRNA mimics or biosimilars made in vitro are widely available and used, miRNA agents produced in vivo are emerging to closely recapitulate natural miRNA species for research. Our recent work has demonstrated the success of high-yield, in vivo production of recombinant miRNAs by using human tRNA (htRNA) fused precursor miRNA (pre-miR) carriers. In this study, we aim to compare the production of bioengineered RNA (BioRNA) molecules with glycyl versus leucyl htRNA fused hsa-pre-miR-34a carriers, namely, BioRNAGly and BioRNALeu, respectively, and perform the initial functional assessment. We designed, cloned, overexpressed, and purified a total of 48 new BioRNA/miRNAs, and overall expression levels, final yields, and purities were revealed to be comparable between BioRNAGly and BioRNALeu molecules. Meanwhile, the two versions of BioRNA/miRNAs showed similar activities to inhibit non-small cell lung cancer cell viability. Interestingly, functional analyses using model BioRNA/miR-7-5p demonstrated that BioRNAGly/miR-7-5p exhibited greater efficiency to regulate a known target gene expression (EGFR) than BioRNALeu/miR-7-5p, consistent with miR-7-5p levels released in cells. Moreover, BioRNAGly/miR-7-5p showed comparable or slightly greater activities to modulate MRP1 and VDAC1 expression, compared with miRCURY LNA miR-7-5p mimic. Computational modeling illustrated overall comparable 3D structures for exemplary BioRNA/miRNAs with noticeable differences in htRNA species and payload miRNAs. These findings support the utility of hybrid htRNA/hsa-pre-miR-34a as reliable carriers for RNA molecular bioengineering, and the resultant BioRNAs serve as functional biologic RNAs for research and development.
Assuntos
Antineoplásicos , Bioengenharia , MicroRNAs , RNA de Transferência de Glicina , RNA de Transferência de Leucina , RNA de Transferência de Glicina/química , RNA de Transferência de Glicina/genética , RNA de Transferência de Glicina/isolamento & purificação , RNA de Transferência de Glicina/farmacologia , RNA de Transferência de Leucina/química , RNA de Transferência de Leucina/genética , RNA de Transferência de Leucina/isolamento & purificação , RNA de Transferência de Leucina/farmacologia , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/isolamento & purificação , MicroRNAs/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Antineoplásicos/farmacologia , Expressão Gênica/efeitos dos fármacos , Simulação por Computador , Linhagem Celular TumoralRESUMO
Oligodendrocyte precursor cells are present in the adult central nervous system, and their impaired ability to differentiate into myelinating oligodendrocytes can lead to demyelination in patients with multiple sclerosis, accompanied by neurological deficits and cognitive impairment. Exosomes, small vesicles released by cells, are known to facilitate intercellular communication by carrying bioactive molecules. In this study, we utilized exosomes derived from human umbilical cord mesenchymal stem cells (HUMSCs-Exos). We performed sequencing and bioinformatics analysis of exosome-treated cells to demonstrate that HUMSCs-Exos can stimulate myelin gene expression in oigodendrocyte precursor cells. Functional investigations revealed that HUMSCs-Exos activate the Pi3k/Akt pathway and regulate the Tbr1/Wnt signaling molecules through the transfer of miR-23a-3p, promoting oligodendrocytes differentiation and enhancing the expression of myelin-related proteins. In an experimental autoimmune encephalomyelitis model, treatment with HUMSCs-Exos significantly improved neurological function and facilitated remyelination. This study provides cellular and molecular insights into the use of cell-free exosome therapy for central nervous system demyelination associated with multiple sclerosis, demonstrating its great potential for treating demyelinating and neurodegenerative diseases.
Assuntos
Exossomos , Células-Tronco Mesenquimais , MicroRNAs , Esclerose Múltipla , Remielinização , Adulto , Humanos , Diferenciação Celular/genética , Exossomos/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , MicroRNAs/farmacologia , MicroRNAs/uso terapêutico , Esclerose Múltipla/genética , Esclerose Múltipla/terapia , Esclerose Múltipla/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Remielinização/efeitos dos fármacos , Remielinização/genética , Cordão Umbilical/citologia , Cordão Umbilical/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas com Domínio T/metabolismo , Modelos Animais de Doenças , Células CultivadasRESUMO
Metastatic melanoma poses significant challenges as a highly lethal disease. Despite the success of molecular targeting using BRAFV600E inhibitors (BRAFis) and immunotherapy, the emergence of early recurrence remains an issue and there is the need for novel therapeutic approaches. This study aimed at creating a targeted delivery system for the oncosuppressor microRNA 126 (miR126) and testing its effectiveness in combination with a phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT) inhibitor for treating metastatic melanoma resistant to BRAFis. To achieve this, we synthesized chitosan nanoparticles containing a chemically modified miR126 sequence. These nanoparticles were further functionalized with an antibody specific to the chondroitin sulfate proteoglycan 4 (CSPG4) melanoma marker. After evaluation in vitro, the efficacy of this treatment was evaluated through an in vivo experiment using mice bearing resistant human melanoma. The co-administration of miR126 and the PI3K/AKT inhibitor in these experiments significantly reduced tumor growth and inhibited the formation of liver and lung metastases. These results provide evidence for a strategy to target an oncosuppressive nucleic acid sequence to tumor cells while simultaneously protecting it from plasma degradation. The system described in this study exhibits encouraging potential for the effective treatment of therapy-resistant metastatic melanoma while also presenting a prospective approach for other forms of cancer.
Assuntos
Melanoma , MicroRNAs , Humanos , Animais , Camundongos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , MicroRNAs/farmacologiaRESUMO
Sodium-glucose cotransporter, type 2 inhibitors (SGLT2i) are emerging as the gold standard for treatment of type 2 diabetes (T2D) with renal protective benefits independent of glucose lowering. We took a high-level approach to evaluate the effects of the SGLT2i, empagliflozin (EMPA) on renal metabolism and function in a prediabetic model of metabolic syndrome. Male and female 12-wk-old TallyHo (TH) mice, and their closest genetic lean strain (Swiss-Webster, SW) were treated with a high-milk-fat diet (HMFD) plus/minus EMPA (@0.01%) for 12-wk. Kidney weights and glomerular filtration rate were slightly increased by EMPA in the TH mice. Glomerular feature analysis by unsupervised clustering revealed sexually dimorphic clustering, and one unique cluster relating to EMPA. Periodic acid Schiff (PAS) positive areas, reflecting basement membranes and mesangium were slightly reduced by EMPA. Phasor-fluorescent life-time imaging (FLIM) of free-to-protein bound NADH in cortex showed a marginally greater reliance on oxidative phosphorylation with EMPA. Overall, net urine sodium, glucose, and albumin were slightly increased by EMPA. In TH, EMPA reduced the sodium phosphate cotransporter, type 2 (NaPi-2), but increased sodium hydrogen exchanger, type 3 (NHE3). These changes were absent or blunted in SW. EMPA led to changes in urine exosomal microRNA profile including, in females, enhanced levels of miRs 27a-3p, 190a-5p, and 196b-5p. Network analysis revealed "cancer pathways" and "FOXO signaling" as the major regulated pathways. Overall, EMPA treatment to prediabetic mice with limited renal disease resulted in modifications in renal metabolism, structure, and transport, which may preclude and underlie protection against kidney disease with developing T2D.NEW & NOTEWORTHY Renal protection afforded by sodium glucose transporter, type 2 inhibitors (SGLT2i), e.g., empagliflozin (EMPA) involves complex intertwined mechanisms. Using a novel mouse model of obesity with insulin resistance, the TallyHo/Jng (TH) mouse on a high-milk-fat diet (HMFD), we found subtle changes in metabolism including altered regulation of sodium transporters that line the renal tubule. New potential epigenetic determinants of metabolic changes relating to FOXO and cancer signaling pathways were elucidated from an altered urine exosomal microRNA signature.
Assuntos
Compostos Benzidrílicos , Diabetes Mellitus Tipo 2 , Glucosídeos , Nefropatias , MicroRNAs , Neoplasias , Estado Pré-Diabético , Inibidores do Transportador 2 de Sódio-Glicose , Masculino , Feminino , Camundongos , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Estado Pré-Diabético/tratamento farmacológico , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Rim , Glucose/farmacologia , MicroRNAs/farmacologia , SódioRESUMO
In the clinical setting, chemotherapy is the most widely used antitumor treatment, however, chemotherapy resistance significantly limits its efficacy. Reduced drug influx is a key mechanism of chemoresistance, and inhibition of the complexity of the tumor microenvironment (TME) may improve chemotherapy drug influx and therapeutic efficiency. In the current study, we identified that the major extracellular matrix protein collagen I is more highly expressed in lung cancer tissues than adjacent tissues in patients with lung cancer. Furthermore, Kaplan-Meier analysis suggested that COL1A1 expression was negatively correlated with the survival time of patients with lung cancer. Our previous study demonstrated that miR-29a inhibited collagen I expression in lung fibroblasts. Here, we investigated the effect of miR-29a on collagen I expression and the cellular behavior of lung cancer cells. Our results suggest that transfection with miR-29a could prevent Lewis lung carcinoma (LLC) migration by downregulating collagen I expression, but did not affect the proliferation, apoptosis, and cell cycle of LLC cells. In a 3D tumoroid model, we demonstrated that miR-29a transfection significantly increased cisplatin (CDDP) permeation and CDDP-induced cell death. Furthermore, neutral lipid emulsion-based miR-29a delivery improved the therapeutic effect of cisplatin in an LLC spontaneous tumor model in vivo. In summary, this study shows that targeting collagen I expression in the TME contributes to chemotherapy drug influx and improves therapeutic efficacy in lung cancer.
Assuntos
Neoplasias Pulmonares , MicroRNAs , Humanos , Linhagem Celular Tumoral , Proliferação de Células , Cisplatino/farmacologia , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , MicroRNAs/metabolismo , MicroRNAs/farmacologia , Permeabilidade , Microambiente TumoralRESUMO
Autism spectrum disorder (ASD) is a multifaceted neurodevelopmental disorder predominant in childhood. Despite existing treatments, the benefits are still limited. This study explored the effectiveness of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) loaded with miR-137 in enhancing autism-like behaviors and mitigating neuroinflammation. Utilizing BTBR mice as an autism model, the study demonstrated that intranasal administration of MSC-miR137-EVs ameliorates autism-like behaviors and inhibits pro-inflammatory factors via the TLR4/NF-κB pathway. In vitro evaluation of LPS-activated BV2 cells revealed that MSC-miR137-EVs target the TLR4/NF-κB pathway through miR-137 inhibits proinflammatory M1 microglia. Moreover, bioinformatics analysis identified that MSC-EVs are rich in miR-146a-5p, which targets the TRAF6/NF-κB signaling pathway. In summary, the findings suggest that the integration of MSC-EVs with miR-137 may be a promising therapeutic strategy for ASD, which is worthy of clinical adoption.
Assuntos
Comportamento Animal , Vesículas Extracelulares , Células-Tronco Mesenquimais , MicroRNAs , NF-kappa B , Transdução de Sinais , Animais , Masculino , Camundongos , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Transtorno Autístico/terapia , Vesículas Extracelulares/metabolismo , Inflamação/patologia , Lipopolissacarídeos , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Microglia/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , MicroRNAs/farmacologia , NF-kappa B/genética , NF-kappa B/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
INTRODUCTION: Di(2-ethylhexyl) phthalate (DEHP) is a common plasticizer. Studies have revealed that DEHP exposure can cause kidney damage. Green tea is among the most popular beverages in China. Green tea polyphenols (GTPs) have been proven to have therapeutic effects on organ damage induced by heavy metal exposure. However, few studies have reported on GTP-relieving DEHP-induced kidney damage. METHODS: C57BL/6J male mice aged 6-8 weeks were treated with distilled water (control group), 1,500 mg/kg/d DEHP + corn oil (model group), 1,500 mg/kg/d DEHP + corn oil + 70 mg/kg GTP (treatment group), corn oil (oil group), and 70 mg/kg GTP (GTP group) by gavage for 8 weeks, respectively. The renal function of mice and renal tissue histopathology of each group were evaluated. The renal tissues of mice in the model, treatment, and control groups were analyzed using high-throughput sequencing. We calculated the differentially expressed microRNAs (miRNAs) and messenger RNAs (mRNAs) using the limma R package, the CIBERSORT algorithm was used to predict immune infiltration, the starBase database was used to screen the miRNA-mRNA regulatory axis, and immunohistochemical analyses were performed to verify protein expression. RESULTS: GTP alleviated the deterioration of renal function, renal inflammation and fibrosis, and mitochondrial and endoplasmic reticulum lesions induced by DEHP in mice. Differential immune infiltrations of plasma, dendritic, T, and B cells were noted between the model and treatment groups. We found that three differentially expressed miRNAs (mmu-miR-383-5p, mmu-miR-152-3p, and mmu-miR-144-3p), three differentially expressed mRNAs (Ddit4, Dusp1, and Snx18), and three differentially expressed proteins (Ddit4, Dusp1, and Snx18) played crucial roles in the miRNA-mRNA-protein regulatory axes when GTPs mitigate DEHP-induced kidney damage in mice. CONCLUSION: GTP can alleviate DEHP-induced kidney damage and regulate immune cell infiltration. We screened four important miRNA-mRNA-protein regulatory axes of GTP, mitigating DEHP-induced kidney damage in mice.
Assuntos
Dietilexilftalato , MicroRNAs , Ácidos Ftálicos , Animais , Camundongos , Masculino , Dietilexilftalato/toxicidade , Óleo de Milho/farmacologia , Camundongos Endogâmicos C57BL , Antioxidantes , Rim , MicroRNAs/genética , MicroRNAs/farmacologia , RNA Mensageiro , Polifenóis/farmacologia , Polifenóis/uso terapêutico , Guanosina Trifosfato/farmacologiaRESUMO
Paternal pre-conceptual environmental experiences, such as stress and diet, can affect offspring brain and behavioral phenotypes via epigenetic modifications in sperm. Furthermore, maternal immune activation due to infection during gestation can reprogram offspring behavior and brain functioning in adulthood. However, the effects of paternal pre-conceptual exposure to immune activation on the behavior and physiology of offspring (F1) and grand-offspring (F2) are not currently known. We explored effects of paternal pre-conceptual exposure to viral-like immune activation on F1 and F2 behavioral and physiological phenotypes using a C57BL/6J mouse model. Males were treated with a single injection (intraperitoneal) of the viral mimetic polyinosinic:polycytidylic acid (Poly I:C: 12 mg/kg) then bred with naïve female mice four weeks after the Poly I:C (or 0.9% saline control) injection. The F1 offspring of Poly I:C treated fathers displayed increased depression-like behavior in the Porsolt swim test, an altered stress response in the novelty-suppressed feeding test, and significant transcriptomic changes in their hippocampus. Additionally, the F1 male offspring of Poly I:C treated F0 males showed significantly increased immune responsivity after a Poly I:C immune challenge (12 mg/kg). Furthermore, the F2 male grand-offspring took longer to enter and travelled significantly shorter distances in the light zone of the light/dark box. An analysis of the small noncoding RNA profiles in sperm from Poly I:C treated males and their male offspring revealed significant effects of Poly I:C on the sperm microRNA content at the time of conception and on the sperm PIWI-interacting RNA content of the male offspring. Notably, eight miRNAs with an FDR < 0.05 (miR-141-3p, miR-126b-5p, miR-669o-5p, miR-10b-3p, miR-471-5p, miR-463-5p, miR-148b-3p, and miR-181c-5p) were found to be significantly downregulated in the sperm of Poly I:C treated males. Collectively, we demonstrate that paternal pre-conceptual exposure to a viral immune challenge results in both intergenerational and transgenerational effects on brain and behavior that may be mediated by alterations in the sperm small noncoding RNA content.
Assuntos
MicroRNAs , Pequeno RNA não Traduzido , Masculino , Feminino , Camundongos , Animais , Humanos , Camundongos Endogâmicos C57BL , Sêmen , Espermatozoides , Pai , MicroRNAs/genética , MicroRNAs/farmacologia , Pequeno RNA não Traduzido/farmacologia , Poli I/farmacologiaRESUMO
Nanoencapsulation, employing safe materials, holds substantial promise for enhancing bioactive compounds' delivery, stability, and bioactivity. In this study, we present an innovative and safe methodology for augmenting the incorporation of the anticancer agent, curcumin, thereby inducing apoptosis by downregulating miR20a and miR21 expression. Our established methodology introduces three pivotal elements that, to our knowledge, have not undergone formal validation: (1) Novel formulation: We introduce a unique formula for curcumin incorporation. (2) Biocompatibility and biodegradability: our formulation exclusively consists of biocompatible and biodegradable constituents, ensuring the absence of detrimental residues or undesirable reactions under varying conditions. (3) Low-temperature incorporation: Curcumin is incorporated into the formulation at temperatures approximating 50 °C. The formulation comprises lecithin (LE), chitosan (CH), an eco-friendly emulsifying agent, and olive oil as the solvent for curcumin. Nanoscale conversion is achieved through ultrasonication and probe sonication (20 kHz). Transmission electron microscopy (TEM) reveals spherical nanoparticles with diameters ranging from 29.33 nm and negative zeta potentials within the -28 to -34 mV range. Molecular studies involve the design of primers for miR20a and miR21. Our findings showcase a remarkable encapsulation efficiency of 91.1% for curcumin, as determined through a linear equation. The curcumin-loaded nanoformulation demonstrates potent anticancer activity, effectively activating the apoptosis pathway in cancer cells at the minimum inhibitory concentration. These results underscore the potential of our nanoformulation as a compelling, cancer-selective treatment strategy, preserving the integrity of normal cells, and thus, warranting further exploration in the field of cancer therapy.
Assuntos
Quitosana , Curcumina , Neoplasias Esofágicas , MicroRNAs , Nanopartículas , Humanos , Curcumina/química , Quitosana/química , Lecitinas , Sobrevivência Celular , Nanopartículas/química , MicroRNAs/genética , MicroRNAs/farmacologia , Portadores de Fármacos/químicaRESUMO
This study uncovered the potential clinical value and molecular driving mechanisms of circular RNAs (circRNAs) in gallbladder cancer (GBC). Differentially expressed circRNAs in GBC cells were screened by high-throughput sequencing. CircRNA_CDKN1A (circBase ID: hsa_circ_0076194) was knocked out in BGC-SD cells through transfection with sh-circRNA_CDKN1A. Then, proliferation was investigated via CCK8 and EdU assays, apoptosis via flow cytometry, migration via wound healing assays, and invasion via Transwell assays. Bioinformatics analysis of circRNA_CDKN1A-related signaling pathways was performed using MetScape and g:Profiler. Results showed that the knockdown of circRNA_CDKN1A enhanced the proliferation, migration, and invasion of GBC cells and inhibited apoptosis. In addition, knocking out circRNA_CDKN1A promoted GBC cell proliferation and enhanced the dry indices of the OCT4 protein and CD34 expression levels. The knockdown of circRNA_CDKN1A activated the epithelial-mesenchymal transition pathway. Bioinformatics analysis revealed that the biological role of circRNA_CDKN1A in GBC cells involved the NF-κB pathway. LY2409881, which is an NF-κB inhibitor, reversed the effects induced by the knockdown of circRNA_CDKN1A in GBC-SD cells. In summary, the knockdown of circRNA_CDKN1A promoted the progression of GBC by activating the NF-κB signaling pathway. For the first time, this study revealed the mechanism of circRNA_CDKN1A-mediated regulatory action in GBC and identified the newly discovered circRNA_CDKN1A-NF-κB signaling axis as a potentially important candidate for clinical therapy and prognostic diagnosis of GBC.
Assuntos
Neoplasias da Vesícula Biliar , MicroRNAs , Humanos , NF-kappa B/metabolismo , Neoplasias da Vesícula Biliar/genética , Neoplasias da Vesícula Biliar/metabolismo , RNA Circular/genética , Linhagem Celular Tumoral , Transdução de Sinais , Proliferação de Células , Movimento Celular , MicroRNAs/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismoRESUMO
The formation of the BCR-ABL fusion gene drives human chronic myeloid leukemia (CML). The last 2 decades have witnessed that specific tyrosine kinase inhibitors (TKIs, e.g., imatinib mesylate, IM) against ABL1 improve disease treatment, although some patients still suffer from relapse and TKI resistance. Therefore, a better understanding of the molecular pathology of CML is still urgently needed. miR-181a-5p (miR-181a) acts as a tumor suppressor in CML; however, the molecular mechanism of miR-181a in CML stem/progenitor cells remains elusive. Herein, we showed that miR-181a inhibited the growth of CML CD34+ cells, including the quiescent subset, and sensitized them to IM treatment, while miR-181a inhibition by a sponge sequence collaborated with BCR-ABL to enhance the growth of normal CD34+ cells. Transcriptome data and biochemical analysis revealed that SERPINE1 was a bona fide and critical target of miR-181a, which deepened the understanding of the regulatory mechanism of SERPINE1. Genetic and pharmacological inhibition of SERPINE1 led to apoptosis mainly mediated by caspase-9 activation. The dual inhibition of SERPINE1 and BCR-ABL exhibited a significantly stronger inhibitory effect than a single agent. Taken together, this study demonstrates that a novel miR-181a/SERPINE1 axis modulates CML stem/progenitor cells, which likely provides an important approach to override TKI resistance.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , MicroRNAs , Inibidor 1 de Ativador de Plasminogênio , Humanos , Apoptose/genética , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/genética , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , MicroRNAs/farmacologia , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
BACKGROUND: Exosomes are involved in cell-to-cell communication in numerous diseases including cardiovascular diseases, neurological diseases. Little attention has been dedicated to exosomal circular RNAs in obstructive sleep apnea (OSA)-related cardiovascular diseases. The aim of this study was to explore the role of exosomal circular RNA ZNF292 (circZNF292) on AC16 cells exposure to intermittent hypoxia (IH). METHODS: Exosome release inhibitor GW4869 was used to examine the effect of exosomes on IH-induced AC16 cells apoptosis. The expression of exosomal circZNF292 was detected by qRT-PCR in AC16 cells exposure to IH, and a luciferase reporter assay was conducted to confirm the connection between circZNF292 and miR-146a-5p. Exosomal circZNF292 was stably transfected with short hairpin RNAs (shRNAs) against circZNF292 and co-cultured with AC16 cells. The expression of miR-146a-5p and apoptosis-related protein was then measured to evaluate the effect of exosomal circZNF292. RESULTS: We found that IH contributed to the AC16 cells apoptosis, and the administration of GW4869 increased the apoptosis of cardiomyocytes when exposed to IH. The expression of exosomal circZNF292 decreased and miR-146a-5p increased significantly in AC16 cells exposed to IH compared to normoxic conditions. Bioinformatics analysis predicted a circZNF292/miR-146a-5p axis in IH-induced cardiomyocytes apoptosis. The dual-luciferase reporter system validated the direct interaction of circZNF292 and miR-146a-5p. Knockdown of circZNF292 increased the expressions of miR-146a-5p and accelerated the AC16 cardiomyocytes apoptosis. CONCLUSIONS: The findings of this study suggested a novel mechanism by which exosomes transmit intrinsic regulatory signals to the myocardium through the exosomal circZNF292/miR-146a-5p axis. This finding highlights the potential of targeting this pathway as a therapeutic approach for treating cardiovascular diseases associated with OSA.
Assuntos
Compostos de Anilina , Compostos de Benzilideno , Doenças Cardiovasculares , MicroRNAs , Apneia Obstrutiva do Sono , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , MicroRNAs/farmacologia , RNA Circular/genética , RNA Circular/metabolismo , RNA Circular/farmacologia , Miócitos Cardíacos/metabolismo , Doenças Cardiovasculares/metabolismo , Apoptose/genética , Hipóxia/genética , Hipóxia/metabolismo , Luciferases/metabolismo , Luciferases/farmacologia , Apneia Obstrutiva do Sono/metabolismo , Proteínas de Transporte , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/farmacologiaRESUMO
Colon cancer is a great threat to human health. Curcumin, as a traditional Chinese medicine extract with anti-tumor and anti-inflammatory effects, can affect the development of diverse human diseases including cancer. The aim of this research was to probe the mechanism by which curcumin regulates colon cancer progression. Colon cancer cells were processed with graded concentrations of curcumin. The proliferation and apoptosis of the treated cells were determined by MTT, colony formation assay and flow cytometry. Expression of signaling pathway-related proteins and programmed death-ligand 1 (PD-L1) was measured by western blotting. The effect of curcumin on tumor cell growth was verified through T cell-mediated killing and ELISA assays. The relationship between target gene expression and the survival rate of colon cancer patients was analyzed by a survival curve. Curcumin treatment restrained proliferation and accelerated apoptosis of colon cancer cells. It elevated miR-206 expression, which in turn affected colon cancer cell function. miR-206 enhanced colon cancer cell apoptosis and inhibited PD-L1 expression; thus, curcumin enhanced the killing effect of T cells on tumor cells by suppressing PD-L1 through inhibiting the JAK/STAT3 pathway. Patients with high expression of miR-206 had better survival rates than those with low expression. Curcumin can regulate miR-206 expression and inhibit the malignant behavior of colon cancer cells and enhance T cell killing through the JAK/STAT3 pathway.
Assuntos
Neoplasias do Colo , Curcumina , MicroRNAs , Humanos , Curcumina/farmacologia , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Antígeno B7-H1/farmacologia , MicroRNAs/genética , MicroRNAs/metabolismo , MicroRNAs/farmacologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , ApoptoseRESUMO
High plasma lipid levels have been demonstrated to increase cardiovascular disease risk. Despite advances in treatments to decrease plasma lipids, additional therapeutics are still needed because many people are intolerant or nonresponsive to these therapies. We previously showed that increasing cellular levels of microRNA-30c (miR-30c) using viral vectors or liposomes reduces plasma lipids and atherosclerosis. In this study, we aimed to synthesize potent miR-30c analogs that can be delivered to hepatoma cells without the aid of viral vectors and lipid emulsions. We hypothesized that modification of the passenger strand of miR-30c would increase the stability of miR-30c and augment its delivery to liver cells. Here, we report the successful synthesis of a series of miR-30c analogs by using different chemically modified nucleosides. In these analogs, we left the active sense strand untouched so that its biological activity remained unaltered, and we modified the passenger strand of miR-30c to enhance the stability and uptake of miR-30c by hepatoma cells through phosphorothiorate linkages and the addition of GalNAc. We show that these analogs significantly reduced apolipoprotein B secretion in Huh-7 human hepatoma cells and human primary hepatocytes without affecting apolipoprotein A1 secretion and cellular lipid levels. Our results provide a proof of concept that the passenger strand of miR-30c can be modified to increase its stability and delivery to cells while retaining the potency of the sense strand. We anticipate these miR-30c analogs will be useful in the development of more efficacious analogs for the treatment of hyperlipidemias and cardiovascular diseases.
Assuntos
Apolipoproteínas B , Carcinoma Hepatocelular , Hepatócitos , Neoplasias Hepáticas , Apolipoproteínas B/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Células Cultivadas , Hepatócitos/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , MicroRNAs/farmacologiaRESUMO
Therapeutic effects of MK-2206 are largely limited due to the complexity of the pathogenesis of nasopharyngeal cancer (NPC). Here, we aimed to investigate whether and how circLASP1 is involved in the therapeutic effects of MK-2206 on NPC. We showed circLASP1 was increased while miR-625 was decreased in NPC tissues and cell lines. CircLASP1 silence strengthened the therapeutic effects of MK-2206 via suppressing NPC cell proliferation and inducing autophagy and apoptosis in vitro. In mechanism analyses, we found that circLASP1 indirectly released AKT by directly binding to miR-625 in NPC cells, and miR-625 acted as a tumor suppressor in NPC and activated cell autophagy through inhibiting the AKT/mTOR pathway. Most importantly, knockdown of circLASP1 was revealed to enhance the therapeutic effects of MK-2206 on NPC in vivo. Our results suggest that the circLASP1/miR-625 axis is involved the therapeutic effects of MK-2206 on NPC by regulating autophagy, proliferation, and apoptosis through the AKT/mTOR pathway. miR-625 is involved in NPC tumorigenesis.
Assuntos
MicroRNAs , Neoplasias Nasofaríngeas , Humanos , Neoplasias Nasofaríngeas/tratamento farmacológico , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular Tumoral , Carcinoma Nasofaríngeo/tratamento farmacológico , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Apoptose , Proliferação de Células , MicroRNAs/genética , MicroRNAs/farmacologia , Regulação Neoplásica da Expressão GênicaRESUMO
BACKGROUND: Dysfunction of pancreatic ß-cells induced by glucocorticoids contributes to diabetes mellitus development. Long noncoding RNAs (lncRNAs) have been recognized to contain short open reading frames (ORFs) that can be translated into functional small peptides. Here, we investigated whether the short peptide encoded by the lncRNA MIR7-3 host gene (MIR7-3HG) can affect dexamethasone (DEX)-induced ß-cell dysfunction. METHODS: Bioinformatics analysis was used for selection of MIR7-3HG and prediction of its protein encoding potential. The small peptide was identified by a western blot method. The cell-permeable TAT was fused into MIR7-3HG ORF to produce the cell-permeable fusion peptide (TAT-MIR7-3HG-ORF). The effects of TAT-MIR7-3HG-ORF on DEX-induced ß-cell dysfunction were evaluated by examining cell viability, apoptosis, insulin secretion, and reactive oxygen species (ROS) generation. RESULTS: DEX induced ß-TC6 cell dysfunction by impairing cell viability, insulin secretion and promoting cell apoptosis and ROS generation. The MIR7-3HG ORF could encode a 125-amino-acid-long short peptide. TAT-MIR7-3HG-ORF effectively transduced into ß-TC6 cells and attenuated DEX-induced dysfunction in ß-TC6 cells. Moreover, transduced TAT-MIR7-3HG-ORF reversed DEX-mediated inhibition of the activation of the PI3K/AKT signaling pathway. The inhibitor of the PI3K/AKT pathway partially abolished the alleviative effect of transduced TAT-MIR7-3HG-ORF on DEX-induced ß-TC6 cell dysfunction. CONCLUSION: The lncRNA MIR7-3HG encodes a short peptide, which can protect pancreatic ß-cells from DEX-induced dysfunction by activating the PI3K/AKT pathway. Our study broadens the diversity and breadth of lncRNAs in human disorders.
Assuntos
Células Secretoras de Insulina , MicroRNAs , RNA Longo não Codificante , Humanos , Apoptose/genética , Dexametasona/efeitos adversos , MicroRNAs/genética , MicroRNAs/farmacologia , Peptídeos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Células Secretoras de Insulina/efeitos dos fármacosRESUMO
Regulatory T cells (T(reg) cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (T(eff) cell) function and gain of suppressive activity by T(reg) cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T(reg) cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3' untranslated region. Release of SATB1 from the control of Foxp3 in T(reg) cells caused loss of suppressive function, establishment of transcriptional T(eff) cell programs and induction of T(eff) cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining T(reg) cell functionality.
Assuntos
Montagem e Desmontagem da Cromatina/imunologia , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica , Proteínas de Ligação à Região de Interação com a Matriz/imunologia , Tolerância a Antígenos Próprios , Linfócitos T Reguladores/imunologia , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Citometria de Fluxo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Lentivirus , Ativação Linfocitária/efeitos dos fármacos , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/imunologia , MicroRNAs/metabolismo , MicroRNAs/farmacologia , Interferência de RNA , RNA Interferente Pequeno/imunologia , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tolerância a Antígenos Próprios/efeitos dos fármacos , Tolerância a Antígenos Próprios/genética , Tolerância a Antígenos Próprios/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Transdução GenéticaRESUMO
BACKGROUND: The interaction between transthyretin (TTR) and heterogeneous nuclear ribonucleoprotein (hnRNP)A2B1 is involved in the neovascularization of human retinal microvascular endothelial cells (hRECs) under hyperglycemic conditions. However, whether the TTR-hnRNPA2B1 interface can be altered and how this protein-protein interaction and associated downstream pathways are regulated is unclear. METHODS: We performed homologous sequential analysis and binding energy assays using Discovery Studio and designed substitution targeting three fragments of the interface (fragment 1: aa 34-39, -RKAADD-; fragment 2, aa 61-68, -EEEFVEGI-; and fragment 3, aa 96-102, -TANDSGP-) to disrupt or stabilize the TTR-hnRNPA2B1 complex and were subjected to Co-immunoprecipitation analysis. To investigate the effect of TTR-hnRNPA2B1 interface alterations on the physiological properties of hRECs, we performed CCK-8, EdU, migration, wound healing and tube formation assays. To study the downstream genes, we performed qRT-PCR and western blot. RESULTS: Nineteen TTR substitutions were recombinantly expressed in soluble form, results indicated that reducing the binding energy stabilized the TTR-hnRNPA2B1, while increasing the binding energy had the opposite effect. The native TTR significantly prohibited the proliferation, DNA synthesis, migration and tube formation capacities of hRECs, while fragment 1 always reduced these effects. However, the I68R and D99R substitutions in fragments 2 and 3, respectively, increased the inhibitory effect of TTR. Furthermore, our qRT-PCR and western blot results showed that the expression and protein levels of STAT-4, miR-223-3p and FBXW7 were also regulated by the alteration of the TTR-hnRNPA2B1 interface. CONCLUSION: This work suggests that the formation of the TTR-hnRNPA2B1 complex plays vital role in hyperglycemia, and modification of this interface regulates the TTR-mediated inhibition of hREC neovascularization via the STAT-4/miR-223-3p/FBXW7 pathway. This mechanism could have important implications for diabetic retinopathy treatment.
Assuntos
Retinopatia Diabética , Hiperglicemia , MicroRNAs , Humanos , Células Endoteliais , Proteína 7 com Repetições F-Box-WD/metabolismo , Pré-Albumina/genética , Pré-Albumina/metabolismo , Pré-Albumina/farmacologia , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Hiperglicemia/metabolismo , MicroRNAs/metabolismo , MicroRNAs/farmacologia , Glucose/farmacologia , Glucose/metabolismoRESUMO
AIMS: Experiments confirmed that circular RNAs contributed to the pathogenesis of diabetic foot ulcers (DFUs). CircHIPK3 was upregulated in type 2 diabetes mellitus (T2DM), but its role in DFU remained unknown. Our study aimed to investigate the regulatory functions of exosomal circHIPK3 and its potential mechanisms in DFU. METHODS: Exosomal size and distribution, marker proteins, and circHIPK3 levels were evaluated by transmission electron microscope, ExoView R200, western blot, and qRT-PCR. Flow cytometry, MTT, Wound healing assays, and tube formation assays were used to assess the roles of exosomal circHIPK3 in high glucose (HG)-treated human umbilical vein endothelial cells (HUVECs). The relationships between Nrf2/VEGFA/circHIPK3 and miR-20b-5p, and between Nrf2 and VEGFA were determined by luciferase reporter assay and RNA immunoprecipitation. We used cell and mice models to investigate the mechanisms of exosomal circHIPK3 under diabetic conditions. RESULTS: CircHIPK3 was significantly upregulated in exo-circHIPK3 rather than exo-vector. Exo-circHIPK3 remarkably inhibited cell apoptosis but promoted cell proliferation, migration, and tube formation in HG-treated HUVECs. Luciferase reporter and RIP assays showed that miR-20b-5p targeted and inhibited Nrf2 and VEGFA, and circHIPK3 acted as a ceRNA of miR-20b-5p to inhibit the binding to its downstream genes Nrf2 and VEGFA. Mechanistically, circHIPK3 promoted cell proliferation, migration, and angiogenesis via downregulating miR-20b-5p to upregulate Nrf2 and VEGFA. However, the overexpressed miR-20b-5p could abolish the promoting effects of circHIPK3 overexpression on cell proliferation, migration, and tube formation under HG conditions. CONCLUSION: UCMSCs-derived exosomal circHIPK3 protected HG-treated HUVECs via miR-20b-5p/Nrf2/VEGFA axis. The exosomal circHIPK3 might be a therapeutic candidate to treat DFU.