Project description:This study aimed to determine whether the spaceflight-induced snoRNA changes in plasma extracellular vesicles (EV) and astronauts' peripheral blood mononuclear cells (PBMCs) can be utilized as potential biomarkers. Using unbiased small RNA sequencing, we evaluated the EV snoRNA changes in peripheral blood (PB) plasma of astronauts (n=5/group) who underwent median 12-day long Shuttle missions between 1998-2001. Using stringent cutoff (> log 2-fold change, FDR < 0.05), we detected 21 down-regulated snoRNAs and 9 upregulated in PB-EVs at three days after return (R+3) compared to ten days before launch (L-10). Our findings unveiled that spaceflight induced changes in EV and PBMCs snoRNA expression, thus suggesting snoRNAs may serve as novel biomarkers for monitoring astronauts' health.
Project description:We sought to determine whether the spaceflight environment can induce alterations in small extracellular vesicles (sEV) smallRNA content and their utility as biomarkers. Using small RNA sequencing (sRNAseq), we evaluated the impact of the spaceflight environment on sEV miRNA content in peripheral blood (PB) plasma of 14 astronauts, who flew STS missions between 1998-2001. Samples were collected at three-time points:10 days before the launch (L-10), the day of return (R-0), and three days post-landing (R+3).
Project description:Compared with plasma sEV (Con-sEV) from control rats, plasma sEV (DM-sEV) from 8-week diabetic rats significantly induced cardiomyocyte apoptosis as evidenced by increased percentage of apoptotic cells and activity of pro-apoptotic protein caspase 3. The proapoptotic effect of DM-sEV was blunted by RNase but not proteinase K, suggesting that DM-sEV exerted the cardiotoxic effects mainly through their contained RNAs. Increasing evidence suggests that miRNAs are the most important molecules by which sEV regulate recipient cell function. To identify the sEV-containing specific miRNAs responsible for the effects, Con-sEV and DM-sEV were subjected to miRNA sequencing.
Project description:Using Next-generation sequencing of total RNA isolated from human cytomegalovirus virions we have analyzed host (human) snoRNA molecules.
Project description:To explore the role of small nucleolar RNA (snoRNA) on self-renewal of liver cancer stem cells (CSCs), we isolated liver CSCs (CD133+CD13+) and Non-CSCs (CD133-CD13-) from huamn liver tumor tissues.
Project description:Comparison of mRNA transcriptome after differential expression of U17 snoRNA. We hypothesiaed that if U17 snoRNA regulates cholesterol homeostasis by modulating the expression of target mRNAs, genes that are differentially expressed by the loss-of-function or gain-of-function of U17 snoRNA would be candidate U17 targets. Results revealed negative regulation of target genes by U17 snoRNA, in which increasing dosage of U17 snoRNA from U17 knockdown to WT to U17 overexpression lead to decreases in target levels. Total RNA were isolated by TRIzol reagent (Invitrogen) from WT, U17 snoRNA KD (ShSnhg3 stable clone), and U17 snoRNA OE (pMDβglobinU17b stable clone) cells grown in LPDS media for 24 hours.
Project description:The Sevenless receptor (Sev) is a key component of the complex regulatory network involved in R7 photoreceptor determination. However the genetic program that responds to Sev is still unknown. Here, we explore the transcriptional profile downstream of Sev, using eye imaginal discs from third instar larvae of the sev mutants sevS11 and sevd2 compared with wild-type controls. Keywords: loss and gain of function analysis
Project description:Background: In recent years, a variety of small RNAs derived from other RNAs with well-known functions such as tRNAs and snoRNAs, have been identified. The functional relevance of these RNAs is largely unknown. To gain insight into the complexity of snoRNA processing and the functional relevance of snoRNA-derived small RNAs, we sequenced long and short RNAs, small RNAs that co-precipitate with the Argonaute 2 protein and RNA fragments obtained in photoreactive nucleotide-enhanced crosslinking and immunoprecipitation (PAR-CLIP) of core snoRNA-associated proteins. Results: Analysis of these data sets revealed that many loci in the human genome reproducibly give rise to C/D box-like snoRNAs, whose expression and evolutionary conservation are typically less pronounced relative to the snoRNAs that are currently catalogued. We further found that virtually all C/D box snoRNAs are specifically processed inside the regions of terminal complementarity, retaining in the mature form only 4-5 nucleotides upstream of the C box and 2-5 nucleotides downstream of the D box. Sequencing of the total and Argonaute 2-associated populations of small RNAs revealed that despite their cellular abundance, C/D box-derived small RNAs are not efficiently incorporated into the Ago2 protein. Conclusions: We conclude that the human genome encodes a large number of snoRNAs that are processed along the canonical pathway and expressed at relatively low levels. Generation of snoRNA-derived processing products with alternative, particularly miRNA-like, functions appears to be uncommon. PAR-CLIP profiling for snoRNP core proteins NOP56, NOP58, Fibrillarin, and Dyskerin in HEK293 cells. Small RNA profiling using RNA-seq in HEK293 and HeLa cells, small RNA profiling using IP-seq of Ago2 associated small RNAs.
Project description:Irradiated granulocyte macrophage-colony stimulating factor (GM-CSF)-transduced autologous tumor cells induce substantial antitumor immunity through the maturation and migration of dendritic cells (DCs). However, little is known about the key molecules involved in GM-CSF-sensitized DCs (GM-DCs) in tumor draining lymph nodes (TDLNs). We initially confirmed that mice subcutaneously injected with poorly immunogenic syngeneic Lewis lung carcinoma (LLC) cells transduced with Sendai virus encoding GM-CSF (LLC/SeV/GM) significantly rejected the tumor growth. Using microarray expression profiling, we obtained a large number of gene expression data files from GM-DCs and control DCs in TDLNs, and subjected them to network-based cluster analysis and unexpectedly unraveled the expression levels of type I IFNs-related genes specifically expressed in plasmacytoid DCs (pDC) were robustly up-regulated in GM-DCs. In vivo depletion assay showed that pDC-depleted mice treated with subcutaneous LLC/SeV/GM cells abrogated the antitumor effects observed in control mice. Moreover combination use of imiquimod for TLR7 triggering on pDC with irradiated LLC/SeV/GM cells induced a significant therapeutic antitumor effect with marked induction of CD9+ pDC with antitumor phenotype, whereas other control mice groups had only minimal to-modest antitumor responses, implicating that this combined vaccine strategy using imiquimod could be promising for improvement of GM-CSF-induced antitumor immunity. Mouse GM-CSF induced gene expression in mature dendritic cells in tumor draining lymph nodes from C57/BL6N female mouse was measured at 2 days after s.c. tumor challenge with GM-CSF gene-transduced LLC cells (LLC/SeV/GM) or control cells (LLC, LLC/SeV/GFP).