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Antiretroviral therapy has effectively suppressed HIV infection and replication and prolonged the lifespan of HIV-infected individuals. In the meantime, various complications including type 2 diabetes associated with the long-term antiviral therapy have shown steady increases. Metformin has been the front-line anti-hyperglycemic drug of choice and the most widely prescribed medication for the treatment of type 2 diabetes. However, little is known about the effects of Metformin on HIV infection and replication. In this study, we showed that Metformin treatment enhanced HIV gene expression and transcription in HIV-transfected 293T and HIV-infected Jurkat and human PBMC. Moreover, we demonstrated that Metformin treatment resulted in increased CREB expression and phosphorylation, and TBP expression. Furthermore, we showed that Metformin treatment increased the recruitment of phosphorylated CREB and TBP to the HIV LTR promoter. Lastly, we showed that inhibition of CREB phosphorylation/activation significantly abrogated Metformin-enhanced HIV gene expression. Taken together, these results demonstrated that Metformin treatment increased HIV transcription, gene expression, and production through increased CREB phosphorylation and recruitment to the HIV LTR promoter. These findings may help design the clinical management plan and HIV cure strategy of using Metformin to treat type 2 diabetes, a comorbidity with an increasing prevalence, in people living with HIV. [ABSTRACT FROM AUTHOR]

The balance of microbial communities in the gut is extremely important for normal physiological function. Disruption of the balance is often associated with various disorders and diseases. Both HIV infection and cocaine use are known to change the gut microbiota and the epithelial barrier integrity, which contribute to inflammation and immune activation. Our recent study shows that Tat expression and cocaine exposure result in changes of genome-wide DNA methylation and gene expression and lead to worsen the learning and memory impairments. In the current study, we extended the study to determine effects of Tat and cocaine on the gut microbiota composition. We found that both Tat expression and cocaine exposure increased Alteromonadaceae in 6-month-old female/male mice. In addition, we found that Tat, cocaine, or both increased Alteromonadaceae, Bacteroidaceae, Cyanobiaceae, Erysipelotrichaceae, and Muribaculaceae but decreased Clostridiales_vadinBB60_group, Desulfovibrionaceae, Helicobacteraceae, Lachnospiraceae, and Ruminococcaceae in 12-month-old female mice. Lastly, we analyzed changes of metabolic pathways and found that Tat decreased energy metabolism and nucleotide metabolism, and increased lipid metabolism and metabolism of other amino acids while cocaine increased lipid metabolism in 12-month-old female mice. These results demonstrated that Tat expression and cocaine exposure resulted in significant changes of the gut microbiota in an age- and sex-dependent manner and provide additional evidence to support the bidirectional gut–brain axis hypothesis. [ABSTRACT FROM AUTHOR]

HIV infects astrocytes in a restricted manner but leads to abundant expression of Nef, a major viral factor for HIV replication and disease progression. However, the roles of Nef in HIV gene expression and replication in astrocytes and viral transfer from astrocytes to CD4+ T cells remain largely unclear. In this study, we attempted to address these issues by transfecting human primary astrocytes with HIV molecular clones with intact Nef and without Nef (a nonsense Nef mutant) and comparing gene expression and replication in astrocytes and viral transfer from astrocytes to CD4+ T cells MT4. First, we found that lack of Nef expression led to increased extracellular virus production from astrocytes and intracellular viral protein and RNA expression in astrocytes. Using a HIV LTR-driven luciferase reporter gene assay, we showed that ectopic Nef expression alone inhibited the HIV LTR promoter activity in astrocytes. Consistent with the previously established function of Nef, we showed that the infectivity of HIV derived from astrocytes with Nef expression was significantly higher than that with no Nef expression. Next, we performed the co-culture assay to determine HIV transfer from astrocytes transfected to MT4. We showed that lack of Nef expression led to significant increase in HIV transfer from astrocytes to MT4 using two HIV clones. We also used Nef-null HIV complemented with Nef in trans in the co-culture assay and demonstrated that Nef expression led to significantly decreased HIV transfer from astrocytes to MT4. Taken together, these findings support a negative role of Nef in HIV replication and pathogenesis in astrocytes. [ABSTRACT FROM AUTHOR]

Background: Synaptodendritic damage is a pathological hallmark of HIV-associated neurocognitive disorders, and HIV-1 Tat protein is known to cause such injury in the central nervous system. In this study, we aimed to determine the molecular mechanisms of Tat-induced neurite shortening, specifically the roles of miR-132, an important regulator of neurite morphogenesis in this process.Methods: The relationship between Tat expression and miR-132 expression was first determined using reverse transcription quantitative PCR (qRT-PCR) in Tat-transfected astrocytes and neurons, astrocytes from Tat-transgenic mice, and HIV-infected astrocytes. qRT-PCR and Western blotting were performed to determine Tat effects on expression of miR-132 target genes methyl CpG-binding protein 2, Rho GTPase activator p250GAP, and brain-derived neurotrophic factor. Exosomes were isolated from Tat-expressing astrocytes, and exosomal microRNA (miRNA) uptake into neurons was studied using miRNA labeling and flow cytometry. The lactate dehydrogenase release was used to determine the cytotoxicity, while immunostaining was used to determine neurite lengths and synapse formation. Tat basic domain deletion mutant and miR-132 mimic and inhibitor were used to determine the specificity of the relationship between Tat and miR-132 and its effects on astrocytes and neurons and the underlying mechanisms of Tat-induced miR-132 expression.Results: Tat significantly induced miR-132 expression, ensuing down-regulation of miR-132 target genes in astrocytes and neurons. miR-132 induction was associated with phosphorylation of cAMP response element-binding protein and required the basic domain of Tat. miRNA-132 induction had no effects on astrocyte activation or survival but was involved in the direct neurotoxicity of Tat. miR-132 was present in astrocyte-derived exosomes and was taken up by neurons, causing neurite shortening.Conclusions: Tat-induced miR-132 expression contributes to both direct and astrocyte-mediated Tat neurotoxicity and supports the important roles of miR-132 in controlling neurite outgrowth. [ABSTRACT FROM AUTHOR]

Background: RNA-protein interactions play important roles in gene expression control. These interactions are mediated by several recurring RNA-binding motifs including a well-known and characterized ribonucleoprotein motif or so-called RNA recognition motif (RRM). Results: In the current study, we set out to identify the RNA ligand(s) of a RRM-containing protein Tip110, also known as p110nrb, SART3, or p110, using a RNA-based yeast three-hybrid cloning strategy. Six putative RNA targets were isolated and found to contain a consensus sequence that was identical to nucleotides 34-46 of U6 small nuclear RNA. Tip110 binding to U6 was confirmed to be specific and RRM-dependent in an electrophoretic mobility shift assay. Both in vitro pre-mRNA splicing assay and in vivo splicing-dependent reporter gene assay showed that the pre-mRNA splicing was correlated with Tip110 expression. Moreover, Tip110 was found in the spliceosomes containing prespliced pre-mRNA and spliced mRNA products. Nonetheless, the RRM-deleted mutant (ΔRRM) that did not bind to U6 showed promotion in vitro pre-mRNA splicing, whereas the nuclear localization signal (NLS)-deleted mutant ΔNLS that bound to U6 promoted the pre-mRNA splicing both in vitro and in vivo. Lastly, RNA-Seq analysis confirmed that Tip110 regulated a number of gene pre-mRNA splicing including several splicing factors. Conclusions: Taken together, these results demonstrate that Tip110 is directly involved in constitutive eukaryotic pre-mRNA splicing, likely through its binding to U6 and regulation of other splicing factors, and provide further evidence to support the global roles of Tip110 in regulation of host gene expression. [ABSTRACT FROM AUTHOR]

Tip110, an important regulator of several oncogenic proteins, was significantly downregulated in human metastatic melanoma cells exposed to a hypoxic condition. Therefore, in this study, we set to determine whether differential expression of Tip110 could be an important indicator for melanoma tumorigenesis and metastasis. We found that in melanoma, but not in other cancer types, Tip110 knockdown enhanced significant expression and secretion of IL-8 and melanoma cells invasions. This induction was further potentiated under hypoxia and by inflammatory cytokine and found independent of TNF-α autocrine signaling. We further showed that Tip110 knockdown-mediated IL-8 induction involved IL-8 mRNA stability. Furthermore, the transcriptomic profiling data and survival from 455 melanoma patients demonstrated that the correlation between Tip110 expression and the clinical outcomes in melanoma was stage-dependent. These findings uncover important roles of Tip110 in melanoma tumorigenesis and metastasis through regulation of IL-8 and hope to provide new clues for future therapeutic strategies. [ABSTRACT FROM AUTHOR]

MicroRNA (miR) regulates hematopoiesis through targeting different genes post-transcriptionally. We have recently shown that Tip110 expression is downregulated during hematopoietic stem cell differentiation. However, the underlying mechanisms are not known. In this study, we identified a conserved miR-124-binding site on the Tip110 3′-untranslated region (3′-UTR) and showed that Tip110 was downregulated by miR-124 through its 3′-UTR. We then examined the relationship among miR-124 and Tip110 expression and differentiation of human cord blood CD34+ cells. We found that miR-124 was expressed in a low level in human cord blood CD34+ cells, but it was considerably upregulated during culturing and differentiation of these cells. Moreover, we demonstrated that miR-124 expression decreased Tip110 expression and promoted differentiation of human cord blood CD34+ cells, while miR-124 knockdown increased Tip110 expression, slowed down differentiation of human cord blood CD34+ cells, and caused an expansion of hematopoietic progenitor cells in vitro. Finally, we used mouse embryonic fibroblasts derived from Tip110 transgenic mice, performed the exon array analysis, and found that Tip110 altered a number of genes in the hematopoiesis pathways. Dnmt3a as de novo methyltransferase was also significantly upregulated. That miR-124 was markedly upregulated during human cord blood CD34+ cell differentiation could be the result of direct loss of its promoter methylation from Dnmt3a. Taken together, our study demonstrates that miR-124 regulates Tip110 expression and differentiation of human cord blood CD34+ cells and suggests important roles of miR-124/ Tip110 in hematopoiesis. [ABSTRACT FROM AUTHOR]