MicroRNA-15b Modulates Molecular Mediators of Blood Induced Arthropathy in Hemophilia Mice.
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ABSTRACT:
SUBMITTER: Sen D
PROVIDER: S-EPMC4848948 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundCD8+ T cells are key members of adaptive immunity against tumorigenesis. As subset of CD8+ T cells, effector T cells (Te) and memory T cells (Tm) have different biological activities. The former can kill tumor cells but come into apoptosis in a certain period and the latter is static with the ability of self-renewal. Previous studies showed that microRNAs (miRNA) played critical roles in regulating adaptive immunity. This study aimed to identify the different expression of miRNAs between Te and Tm cells in tumor-bearing mice and to sort out the target miRNAs which can be regulated to improve anti-tumor activities of CD8+ T cells.MethodsmiRNA expression profiling was performed on CD8+ Te and Tm cells from mice with Lewis lung carcinoma. Differentially expressed miRNA (miRNA-15b) was chosen and analyzed by qRT-PCR. Then, flow cytometry, ELISA, and CFSE kit were used to evaluate the biological effects of miRNA-15b on apoptosis, cytokine secretion, phenotype, and proliferation of CD8+ T cell. The possible downstream target genes of this miRNA were also analyzed.ResultsAnalysis of miRNA microarray and qRT-PCR showed that the level of miRNA-15b was higher in CD8+ Tm cells than in Te cells. Higher expression of miRNA-15b was observed in CD8+ T cells from tumor-bearing mice than those from healthy ones. Transfection of CD8+ T cells with miRNA-15b mimics could prevent T cells from apoptosis by inhibiting the translation of DEDD (Death Effector Domain-containing DNA binding protein). Moreover, ectopic miRNA-15b could inhibit the activation of CD8+ T cells (via repressing the production of IL-2 and IFN-γ and expression of CD69) and promote expression of CD44 through unknown pathways.ConclusionUp-regulation of miRNA-15b in tumor environment might negatively regulate anti-tumor immunity through inhibiting function of CD8+ T cells. miRNA-15b might be a potential therapeutic target for immunotherapy.
| S-EPMC3608092 | biostudies-literature
Project description:Doxorubicin (DOX, an anthracycline) is a widely used chemotherapy agent against various forms of cancer; however, it is also known to induce dose-dependent cardiotoxicity leading to adverse complications. Investigating the underlying molecular mechanisms and strategies to limit DOX-induced cardiotoxicity might have potential clinical implications. Our previous study has shown that expression of microRNA-377 (miR-377) increases in cardiomyocytes (CMs) after cardiac ischemia-reperfusion injury in mice, but its specific role in DOX-induced cardiotoxicity has not been elucidated. In the present study, we investigated the effect of anti-miR-377 on DOX-induced cardiac cell death, remodeling, and dysfunction. We evaluated the role of miR-377 in CM apoptosis, its target analysis by RNA sequencing, and we tested the effect of AAV9-anti-miR-377 on DOX-induced cardiotoxicity and mortality. DOX administration in mice increases miR-377 expression in the myocardium. miR-377 inhibition in cardiomyocyte cell line protects against DOX-induced cell death and oxidative stress. Furthermore, RNA sequencing and Gene Ontology (GO) analysis revealed alterations in a number of cell death/survival genes. Intriguingly, we observed accelerated mortality and enhanced myocardial remodeling in the mice pretreated with AAV9-anti-miR-377 followed by DOX administration as compared to the AAV9-scrambled-control-pretreated mice. Taken together, our data suggest that in vitro miR-377 inhibition protects against DOX-induced cardiomyocyte cell death. On the contrary, in vivo administration of AAV9-anti-miR-377 increases mortality in DOX-treated mice.
| S-EPMC8415717 | biostudies-literature
Project description:BackgroundMicroRNAs (miRNAs) are important regulators of cellular processes and are found to be deregulated in many cancers. We here analysed the miRNA expression in anal carcinomas. In a previous study, we found that our anal carcinoma tumours were divided into two groups based on the expression of E2F-regulated genes. Therefore, we searched for miRNAs that could reproduce this grouping.MethodsA global screen of the miRNA population was performed using real-time quantitative PCR (RT-qPCR) array methods and differentially expressed miRNAs were identified. Real-time-qPCR was used to verify the expression levels of selected miRNAs and genes in a larger collection of biopsies. A siRNA-mediated knockdown of human papilloma virus (HPV)16 E7 in a cervical cell line was performed to assess the effect of E7 on miR-15b.ResultsThe grouping of tumours into two groups based on the expression of E2F-controlled genes was confirmed in a larger collection of anal carcinoma tumours. The expression of miR-15b was shown to be highly correlated with that of five selected E2F-induced genes (CCNA2, CCNB1, CCNB2, MSH6 and MCM7). A knockdown of HPV16 E7 resulted in decreased levels of miR-15b in Ca Ski cells.ConclusionMiR-15b expression correlates with E2F-regulated genes in anal carcinoma and appears to be part of the E2F-regulatory network.
| S-EPMC3242611 | biostudies-literature
Project description:MicroRNAs (miRNAs or miRs) are small, non-coding RNAs that modulate mRNA stability and post-transcriptional translation. A growing body of evidence indicates that specific miRNAs can affect the cellular function of cardiomyocytes. In the present study, miRNAs that are highly expressed in the heart were overexpressed in neonatal rat ventricular myocytes, and cellular ATP levels were assessed. As a result, miR-15b, -16, -195, and -424, which have the same seed sequence, the most critical determinant of miRNA targeting, decreased cellular ATP levels. These results suggest that these miRNAs could specifically down-regulate the same target genes and consequently decrease cellular ATP levels. Through a bioinformatics approach, ADP-ribosylation factor-like 2 (Arl2) was identified as a potential target of miR-15b. It has already been shown that Arl2 localizes to adenine nucleotide transporter 1, the exchanger of ADP/ATP in mitochondria. Overexpression of miR-15b, -16, -195, and -424 suppressed the activity of a luciferase reporter construct fused with the 3'-untranslated region of Arl2. In addition, miR-15b overexpression decreased Arl2 mRNA and protein expression levels. The effects of Arl2 siRNA on cellular ATP levels were the same as those of miR-15b, and the expression of Arl2 could restore ATP levels reduced by miR-15b. A loss-of-function study of miR-15b resulted in increased Arl2 protein and cellular ATP levels. Electron microscopic analysis revealed that mitochondria became degenerated in cardiomyocytes that had been transduced with miR-15b and Arl2 siRNA. The present results suggest that miR-15b may decrease mitochondrial integrity by targeting Arl2 in the heart.
| S-EPMC2836096 | biostudies-literature
Project description:Immune responses to factor VIII remain the greatest complication in the treatment of severe hemophilia A. Recent epidemiological evidence has highlighted that recombinant factor VIII produced in baby hamster kidney cells is more immunogenic than factor VIII produced in Chinese hamster ovary cells. Glycosylation differences have been hypothesized to influence the immunogenicity of these synthetic concentrates. In two hemophilia A mouse models, baby hamster kidney cell-derived factor VIII elicited a stronger immune response compared to Chinese hamster ovary cell-derived factor VIII. Furthermore, factor VIII produced in baby hamster kidney cells exhibited accelerated clearance from circulation independent of von Willebrand factor. Lectin and mass spectrometry analysis of total N-linked glycans revealed differences in high-mannose glycans, sialylation, and the occupancy of glycan sites. Factor VIII desialylation did not influence binding to murine splenocytes or dendritic cells, nor surface co-stimulatory molecule expression. We did, however, observe increased levels of immunoglobulin M specific to baby hamster kidney-derived factor VIII in naïve hemophilia A mice. De-N-glycosylation enhanced immunoglobulin M binding, suggesting that N-glycan occupancy masks epitopes. Elevated levels of immunoglobulin M and immunoglobulin G specific to baby hamster kidney-derived factor VIII were also observed in healthy individuals, and de-N-glycosylation increased immunoglobulin G binding. Collectively, our data suggest that factor VIII produced in baby hamster kidney cells is more immunogenic than that produced in Chinese hamster ovary cells, and that incomplete occupancy of N-linked glycosylation sites leads to the formation of immunoglobulin M- and immunoglobulin G-factor VIII immune complexes that contribute to the enhanced clearance and immunogenicity in these mouse models of hemophilia A.
| S-EPMC6278987 | biostudies-literature
Project description:Osteoblast differentiation is tightly regulated by several factors including microRNAs (miRNAs). In this paper, we report that pre-mir-15b is highly expressed in differentiated osteoblasts. The functional role of miR-15b in osteoblast differentiation was determined using miR-15b mimic/inhibitor and the expression of osteoblast differentiation marker genes such as alkaline phosphatase (ALP), type I collagen genes was decreased by miR-15b inhibitor. Runx2, a bone specific transcription factor is generally required for expression of osteoblast differentiation marker genes and in response to miR-15b inhibitor treatment, Runx2 mRNA expression was not changed; whereas its protein expression was decreased. Even though Smurf1 (SMAD specific E3 ubiquitin protein ligase 1), HDAC4 (histone deacetylase 4), Smad7, and Crim1 were found to be few of miR-15b's putative target genes, there was increased expression of only Smurf1 gene at mRNA and protein levels by miR-15b inhibitor. miR-15b mimic treatment significantly increased and decreased expressions of Runx2 and Smurf1 proteins, respectively. We further identified that the Smurf1 3'UTR is directly targeted by miR-15b using the luciferase reporter gene system. This is well documented that Smurf1 interacts with Runx2 and degrades it by proteasomal pathway. Hence, based on our results we suggest that miR-15b promotes osteoblast differentiation by indirectly protecting Runx2 protein from Smurf1 mediated degradation. Thus, this study identified that miR-15b can act as a positive regulator for osteoblast differentiation.
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Project description:BackgroundDicer endonuclease, critical for maturation of miRNAs, is depleted in certain forms of cardiomyopathy which results in differential expression of certain microRNAs. We sought to elucidate the mechanisms underlying the rapid loss of cardiac function following cardiac-specific Dicer depletion in adult mice.ResultsConditional Dicer deletion in the adult murine myocardium demonstrated compromised heart function, mitochondrial dysfunction and oxidant stress. Elevated miR-15b was observed as an early response to Dicer depletion and was found to silence Pim-1 kinase, a protein responsible for maintaining mitochondrial integrity and function. Anti-miRNA based suppression of induced miRNA-15b rescued the function of Dicer-depleted adult heart and attenuated hypertrophy.ConclusionsAnti-miRNA based suppression of inducible miRNA-15b can prevent rapid loss of cardiac function in a Dicer-depleted adult heart and can be a key approach worthy of therapeutic consideration.
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Project description:Hemophilic arthropathy (HA) is a debilitating degenerative joint disease that is a major manifestation of the bleeding disorder hemophilia A. HA typically begins with hemophilic synovitis that resembles inflammatory arthritides, such as rheumatoid arthritis, and frequently results in bone loss in patients. A major cause of rheumatoid arthritis is inappropriate release of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) by the TNF-α convertase (TACE; also referred to as ADAM17) and its regulator, iRhom2. Therefore, we hypothesized that iRhom2/ADAM17-dependent shedding of TNF-α also has a pivotal role in mediating HA. Here, we show that addition of blood or its components to macrophages activates iRhom2/ADAM17-dependent TNF-α shedding, providing the premise to study the activation of this pathway by blood in the joint in vivo. For this, we turned to hemophilic FVIII-deficient mice (F8-/- mice), which develop a hemarthrosis following needle puncture injury with synovial inflammation and significant osteopenia adjacent to the affected joint. We found that needle puncture-induced bleeding leads to increased TNF-α levels in the affected joint of F8-/- mice. Moreover, inactivation of TNF-α or iRhom2 in F8-/- mice reduced the osteopenia and synovial inflammation that develops in this mouse model for HA. Taken together, our results suggest that blood entering the joint activates the iRhom2/ADAM17/TNF-α pathway, thereby contributing to osteopenia and synovitis in mice. Therefore, this proinflammatory signaling pathway could emerge as an attractive new target to prevent osteoporosis and joint damage in HA patients.
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Project description:Impaired biogenesis of microRNAs disrupts spermatogenesis and leads to infertility in male mice. Spermatogonial differentiation is a key step in spermatogenesis, yet the mechanisms that control this event remain poorly defined. In this study, we discovered microRNA 146 (Mir146) to be highly regulated during spermatogonial differentiation, a process dependent on retinoic acid (RA) signaling. Mir146 transcript levels were diminished nearly 180-fold in differentiating spermatogonia when compared with undifferentiated spermatogonia. Luciferase assays revealed the direct binding of Mir146 to the 3' untranslated region of the mediator complex subunit 1 (Med1), a coregulator of retinoid receptors (RARs and RXRs). Overexpression of Mir146 in cultured undifferentiated spermatogonia reduced Med1 transcript levels, as well as those of differentiation marker kit oncogene (Kit). MED1 protein was also diminished. Conversely, inhibition of Mir146 increased the levels of Kit. When undifferentiated spermatogonia were exposed to RA, Mir146 was downregulated along with a marker for undifferentiated germ cells, zinc finger and BTB domain containing 16 (Zbtb16; Plzf); Kit was upregulated. Overexpression of Mir146 in RA-treated spermatogonia inhibited the upregulation of Kit, stimulated by retinoic acid gene 8 (Stra8), and spermatogenesis- and oogenesis-specific basic helix-loop-helix 2 (Sohlh2). Inhibition of Mir146 in RA-treated spermatogonia greatly enhanced the upregulation of these genes. We conclude that Mir146 modulates the effects of RA on spermatogonial differentiation.
| S-EPMC4434937 | biostudies-literature
Project description:Ionizing radiation causes depletion of hematopoietic cells and enhances the risk of developing secondary hematopoietic malignancies. Vitamin E analog gamma-tocotrienol (GT3), which has anticancer properties, promotes postirradiation hematopoietic cell recovery by enhancing spleen colony-forming capacity, and provides protection against radiation-induced lethality in mice. However, the underlying molecular mechanism involved in GT3-mediated postirradiation survival is not clearly understood. Recent studies have shown that natural dietary products including vitamin E provide a benefit to biological systems by modulating microRNA (miR) expression. In this study, we show that GT3 differentially modulates the miR footprint in the spleen of irradiated mice compared to controls at early times (day 1), as well as later times (day 4 and 15) after total-body irradiation. We observed that miR expression was altered in a dose- and time-dependent manner in GT3-pretreated spleen tissues from total-body irradiated mice. GT3 appeared to affect the expression of a number of radiation-modulated miRs known to be involved in hematopoiesis and lymphogenesis. Moreover, GT3 pretreatment also suppressed the upregulation of radiation-induced p53, suggesting the function of GT3 in the prevention of radiation-induced damage to the spleen. In addition, we have shown that GT3 significantly reduced serum levels of Flt3L, a biomarker of radiation-induced bone marrow aplasia. Further in silico analyses of the effect of GT3 implied the association of p38 MAPK, ERK and insulin signaling pathways. Our study provides initial insight into the mechanism by which GT3 mediates protection of spleen after total-body irradiation.
| S-EPMC4893953 | biostudies-literature