Borna disease virus 1 (BoDV-1) is a highly neurotropic RNA virus which was recently demonstrated to cause deadly human encephalitis. Viruses can modulate microRNA expression, in turn modulating cellular immune responses and regulating viral replication. A previous study indicated that BoDV-1 infection down-regulated the expression of miR-505 in rats. However, the underlying mechanism of miR-505 during BoDV-1 infection remains unknown. In this study, we found that miR-505 can inhibit autophagy activation by down-regulating the expression of its target gene HMGB1, and ultimately inhibit the replication of BoDV-1. Specifically, we found that the expression of miR-505 was significantly down-regulated in rat primary neurons stably infected with BoDV-1. Overexpression of miR-505 can inhibit the replication of BoDV-1 in cells. Bioinformatics analysis and dual luciferase reporter gene detection confirmed that during BoDV-1 infection, the high-mobility group protein B1 (HMGB1) that mediates autophagy is the direct target gene of miR-505. The expression of HMGB1 was up-regulated after BoDV-1 infection, and overexpression of miR-505 could inhibit the expression of HMGB1. Autophagy-related detection found that after infection with BoDV-1, the expression of autophagy-related proteins and autophagy-related marker LC3 in neuronal cells was significantly up-regulated. Autophagy flow experiments and transmission electron microscopy also further confirmed that BoDV-1 infection activated HMGB1-mediated autophagy. Further regulating the expression of miR-505 found that overexpression of miR-505 significantly inhibited HMGB1-mediated autophagy. The discovery of this mechanism may provide new ideas and directions for the prevention and treatment of BoDV-1 infection in the future.
Borna disease virus (BDV) is a neurotropic RNA virus that infects the limbic system of mammals and results in behavioral disorders. The hippocampus is a core region in the limbic system, which contributes to memory and learning and is important in the regulation of emotion. However, no validated microRNA housekeeping genes have yet been identified in BDV‑infected rat primary hippocampal neurons. Proper normalization is key in accurate miRNA expression analysis. The present study used reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) to evaluate the expression stability of 10 commonly used reference genes [miR‑92a, 5S, U6, miR‑103, miR‑101a, miR-let-7a, miR‑16, E2 small nucleolar RNA (snoRNA), U87 and miR‑191] in BDV‑infected rat hippocampal neurons and non‑infected controls across 12 days post‑infection. The data was analyzed by four statistical algorithms: geNorm, NormFinder, BestKeeper, and the comparative Δ‑Ct method. Subsequently, the most suitable reference genes (miR‑101a and U87) and the least suitable (snoRNA) were determined by the RankAggreg package. miR‑155 was selected as a standard by which to evaluate the most and least suitable reference genes. When normalized to the most stable reference gene there were significant differences between the two groups. However, when the data were normalized to the less stably expressed gene, the results were not significant. miR‑101a was recommended as a suitable reference gene for BDV-infected rat primary hippocampal neurons.
This study was purposed to evaluate whether the safe concentration of magnetic nanoparticles of Fe₃O₄(MNPs-Fe₃O₄) for monocytes could induce the SKM-1 cell apoptosis. The average size and Zeta potential of MNPs-Fe₃O₄were determined by transmission electron microscopy and the Malvern Zetasizer 3000 HS, respectively. The cell viability after being exposed to MNPs-Fe₃O₄for 12, 24, 48, and 72 hours was detected by using cell count Kit-8. The cell apoptosis was evaluated by flow cytometry with Annexin V/PI double staining and Wright-Giemsa staining. The cell cycle was measured by flow cytometry. The levels of active caspase-3, survivin and bcl-rambo in cells treated with MNPs-Fe₃O₄and/or trolox for 48 hours were detected with Western blot. The results showed that the cell viability decreased in SKM-1 cells after exposure to 50 µmol/L and 100 µmol/L MNPs-Fe₃O₄(P < 0.05), but did not in monocytes (P > 0.05), compared with that of each non-MNPs-Fe₃O₄-treated group. This exposure also induced the SKM-1 cells to be arrested in G0/G1. Annexin V/PI staining assay showed that cell apoptotic rate induced by 100 µmol/L MNPs-Fe₃O₄was significantly high in SKM-1 cells while not so high in monocytes, and the pretreatment with trolox could attenuate the apoptosis. Moreover, the active caspase-3 increased in SKM-1 cells after the exposure to MNPs-Fe₃O₄, while that was not in monocytes, and the increased expression of BCL-rambo and the decreased expression of survivin involved in the process were also observed. It is concluded that MNPs-Fe₃O₄can induce the caspase 3-dependent SKM-1 cell apoptosis by increasing the BCL-rambo expression and decreasing the survivin expression, but this cytotoxic effect can not be observed in monocyte's.
MicroRNAs (miRNAs) are recognized as important regulators of gene expression via translational depression or mRNA degradation. Previously, dysregulated miRNAs have been found in neurodegenerative and neuropsychiatric disorders. Borna disease virus (BDV) is a neurotropic, negative single‑stranded RNA virus, which may be a cause of human neuropsychiatric disease. BDV is regarded as an ideal model to analyze the molecular mechanisms of mental disorders caused by viral infection. In the present study, 10 miRNAs were dysregulated in human oligodendrocytes (OL cells) infected with the BDV strain, Hu‑H1 (OL/BDV). The predicted target genes of those different miRNAs were closely associated with DNA binding, receptor activity, cytoplasm and membrane, biopolymer metabolic process and signal transduction, which were ranked highest using Gene Ontology (GO) analysis, and were predominantly involved in ‘Immune system and adaptive Immune system pathways’ on pathway analysis. Reverse transcription‑quantitative polymerase chain reaction analysis confirmed that seven miRNAs (miR‑1290, miR‑1908, miR‑146a‑5p, miR‑424‑5p, miR‑3676‑3p, miR‑296‑3p and miR‑7‑5p) were significantly downregulated in the OL/BDV cells, whereas two miRNAs (miR‑1244 and miR‑4521) showed no significant differences between the two groups. The present study revealed for the first time, to the best of our knowledge, the miRNA profile of BDV Hu‑H1‑infected human OL cells. Based on GO and pathway analyses, further investigation of the signaling processes in BDV‑infected oligodendrocytes may offer particular promise in improving understanding of the neuropathogenesis of BDV.
MicroRNA-9-1(miR-9-1) plays an important role in the mechanism that regulates the lineage fate of differentiating hematopoietic cells. Recent studies have shown that miR-9-1 is downregulated in t (8; 21) AML. However, the pathogenic mechanisms underlying miR-9-1 downregulation and the RUNX1-RUNX1T1 fusion protein, generated from the translocation of t (8; 21) in AML, remain unclear. RUNX1-RUNX1T1 can induce leukemogenesis through resides in and functions as a stable RUNX1-RUNX1T1-containing transcription factor complex. In this study, we demonstrate that miR-9-1 expression increases significantly after the treatment of RUNX1-RUNX1T1 (+) AML cell lines with decitabine (a DNMT inhibitor) and trichostatin A (an HDAC inhibitor). In addition, we show that RUNX1-RUNX1T1 triggers the heterochromatic silencing of miR-9-1 by binding to RUNX1-binding sites in the promoter region of miR-9-1 and recruiting chromatin-remodeling enzymes, DNMTs, and HDACs, contributing to hypermethylation of miR-9-1 in t (8; 21) AML. Furthermore, because RUNX1, RUNX1T1, and RUNX1-RUNX1T1 are all regulated by miR-9-1, the silencing of miR-9-1 enhances the oncogenic activity of these genes. Besides, overexpression of miR-9-1 induces differentiation and inhibits proliferation in t (8; 21) AML cell lines. In conclusion, our results indicate a feedback circuitry involving miR-9-1 and RUNX1-RUNX1T1, contributing to leukemogenesis in RUNX1-RUNX1T1 (+) AML cell lines.
The present study aimed to investigate whether artesunate (ART) could enhance the rate of apoptosis induced by decitabine (DAC) in the high-risk myelodysplastic syndrome (MDS) SKM-1 cell line, and examine the potential underlying mechanisms. The cytotoxicity and effect upon the apoptosis of ART and DAC in the SKM-1 cells was detected using the cell counting kit-8 assay and flow cytometry, respectively. The SKM-1 protein expression levels of activated caspase-3, -9 and -8, cleaved poly(ADP-ribose) polymerase and apoptosis-inducing factor (AIF) were measured by western blotting. The laser confocal microscope analysis revealed AIF transfer to the nucleus. The growth inhibition and apoptosis rates of the ART- and DAC-treated SKM-1 cells were significantly increased compared with those of the single agent-treated SKM-1 cells (P<0.05). In addition, ART and DAC induced caspase-dependent apoptosis, while ART, but not DAC, induced caspase-independent apoptosis via AIF transfer from the mitochondria to the nucleus. In addition, ART-DAC-induced cell death was not attenuated by the caspase-3/7 inhibitor, Ac-DEVD-CHO. The results of the present study suggested that the ART-DAC combination exhibited increased effectiveness compared with the single-agent therapy, in vitro. The ART-DAC combined therapy not only activated a caspase-dependent apoptotic pathway, but also a caspase-independent mitochondrial pathway.
Hematopoietic stem cell (HSC) transplantation could be of therapeutic value for aplastic anemia (AA) patients, and immunosuppressants may facilitate the efficiency of the procedure. As anti-inflammatory cytokine interleukin-11 (IL-11) has a thrombopoietic effect, its use in cases of chronic bone marrow failure, such as AA, has been proposed to induce HSC function. However, the putative mechanisms that may support this process remain poorly defined. We found that decreased miR-204-5p levels were coincident with increased proliferation in mouse HSCs following exposure to IL-11 in vitro. Through inhibiting NF-кB activity, miR-204-5p repression was demonstrated to be a downstream effect of IL-11 signaling. miR-204-5p was shown to directly target thrombopoietin (TPO) via sequence-dependent 3'-UTR repression, indicating that this microRNA-dependent pathway could serve an essential role in supporting IL-11 functions in HSCs. Increased TPO expression in HSCs following IL-11 exposure could be mimicked or blocked by inhibiting or overexpressing miR-204-5p, respectively. Consistent with these in vitro findings, IL-11 promoted HSC engraftment in a mouse model of AA, an effect that was attenuated in cells overexpressing miR-204-5p. The reduction in miR-204-5p levels is an integral component of IL-11 signaling that may play an essential role in treating AA.
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