Human lncRNA NRAV regulates antiviral response by modulating ISGs expression during influenza A virus infection
ABSTRACT: Human lncRNA NRAV-overexpression in A549 cells was found to increase the influenza virus WSN replication. To discover the mechanism underlying this promotion by NRAV, genome-wide mRNA expression was measured by using microarray. Thousands of genes were differentially expressed and some ISGs were down-regulated. Human lncRNA NRAV-overexpressioning A549 cells (Test) and empty vector control cells were infected with influenza virus A/WSN/33 (H1N1) for 16 hours at a MOI of 3. Six total RNA samples from three independent experiments were extracted and used for mRNA microarray.
Project description:Influenza virus neuraminidase (NA), a type II transmembrane glycoprotein, is transported to the virus assembly site at the plasma membrane and is a major viral envelope component that plays a critical role in the release of progeny virions and in determination of host range restriction. Although signals/sequences in NA for translocation, sorting and raft association have been identified, little is known about the host factors that are involved in regulating the intracellular and cell surface transport of NA. In this report, we have investigated the involvement of Rho family GTPases in NA transport to the cell surface. We found that expression of constitutively active or inactive mutants of RhoA or Rac1 did not significantly affect the amount of NA that reached the cell surface. Interestingly, expression of constitutively active Cdc42 or depletion of the Cdc42-specific GAP, ARHGAP21, promoted the transport of NA to the plasma membranes. By contrast, cells expressing shRNA targrting Cdc42 or overexpressing ARHGAP21 exhibited a significant decrease in the amount of cell surface-localized NA. Furthermore, silencing of Cdc42 or ARHGAP21 had significant effects on influenza A virus replication. Together, our results reveal that ARHGAP21 and Cdc42-based signaling regulates the NA transport and thereby impacts virus replication. This microarray experiment was carried out to find out whether Cdc42 and ARHGAP21 expression levels in A549 cell were changed after WSN infection. Total RNAs were extracted from three different groups of A549 cells that had been infected with or without WSN for 10 h, using TRIzol reagent (Invitrogen, Carlsbad, CA). Samples were amplified and labeled using a NimbleGen One-Color DNA Labeling Kit.
Project description:Whole-genome data was developed from influenza virus infected A549 cells to better characterize the effect of C646 on influenza virus infection A549 cells were treated with C646 or DMSO for 10 h, and then were infected with A/WSN/33 virus (WSN; H1N1) at a multiplicity of infection (MOI) 2. A549 cells for microarray studies were collected at different times. The gene expression in A549 cells was compared between C646-treated group and DMSO-treated group.
Project description:Influenza is a major cause of morbidity and mortality worldwide, and the emerging drug resistance poses an increasing challenge to the treatment of influenza virus infection. Therefore, the development of a novel antiviral drugs has become an urgent task to combat against the influenza viruses that are resistant to the current therapeutic treatment. Here, by screening a small molecule chemical compound library, we identified 3-anhydro-6-epi-ophiobolin A (named L435) as a potent anti-influenza agent. Mechanistically, L435 markedly reduced influenza virus replication in vitro and in vivo. Importantly, L435 treatment improved the survival of influenza-virus-infected mice, suggesting that L435 may be a novel therapeutic agent for treatment of influenza virus infections. This microarray experiment was carried out to explore gene expression changes in influenza-virus-infected A549 cells after L435 treatment, and find out why L435 could inhibit the replication of influenza A virus. Total RNAs were extracted from three different groups of A549 cells that were mock treated, or infected with WSN and treated with DMSO for 12h, or infected with WSN and treated with L435 for 12 h, using TRIzol reagent (Invitrogen, Carlsbad, CA). Three independent experiments were performed. Samples were amplified and labeled using the One-Color Quick Amp Labeling Kit (Agilent p/n 5190-2305).
Project description:A growing body of evidence suggests gene regulatory functions for the majority of non-protein-coding RNAs (ncRNAs). Besides small RNAs (sRNAs), the diverse class of long ncRNAs (lncRNAs) recently came into focus of research. So far, the relevance of lncRNAs in infection processes remains elusive. Here, we report the differential expression of several classes of lncRNAs during influenza A virus (IAV) infection in human lung epithelial cells. 2 biological replicates of each condition were hybridzed in an independent color-swap; A549 cells were washed with PBS and then infected with viruses at MOI 1 in infection buffer for for 60 min at room temperature. Cells were incubated for the indicated time periods at 37 °C in DMEM supplemented with 0.2% bovine serum albumin, 4 mM l-glutamine and antibiotics. Supernatants of A/WSN/33 (H1N1) virus infected A549 cells (MOI 5, 4hpi) were exposed to UV light for 5 min and then used to stimulate A549 cells for 4 h Supernatants of A/WSN/33 (H1N1) virus infected A549 cells (MOI 5, 8hpi) were exposed to UV light for 5 min and then used to stimulate A549 cells for 8 h
Project description:To identify miRNAs that are affected by IAV infection, we adopted a systematic approach, and performed a miRNA microarray analysis using a human alveolar adenocarcinoma cell line, A549, which was infected with a common laboratory strain of influenza A/WSN/33(H1N1) virus (WSN). We compared miRNA expression profiles in A549 cells infected with WSN for 2, 6, and 10 hours at a multiplicity of infection (MOI) of 2. Among 955 miRNAs on the microarray, 209 miRNAs were detected in all three infected A549 samples, but when compared with analysis results from mock-infected A549 cells, most expression changes were less than 1.5-fold , which was consistent with a previous report (Buggele et al., 2013). Therefore, we established 1.5-fold expression change as a threshold to select for miRNAs potentially affected by WSN infection. Further screening revealed 116 miRNAs that underwent 1.5-fold or greater expression changes at any timepoint following WSN infection. Overall design: The A549 cells were infected with WSN at an MOI of 2 in serum-free MEM medium for 2, 6 and 10 hours.
Project description:We used the microarray data to analyze host cells response on A549 cells infected with A/WSN/33 (H1N1) The A/WSN/33 (H1N1) infected A549 cells were harvested at 10 hpi and RNA extraction was performed using standard protocol as described by Affymetrix. The aim of this experiment is to analyze host response to Influenza A/WSN/33 (H1N1) infection.
Project description:We used the microarray data to analyze host cells response on A549 cells infected with A/WSN/33 (H1N1) The A/WSN/33 (H1N1) infected A549 cells were harvested at 2, 4 and 6 hpi and RNA extraction was performed using standard protocol as described by Affymetrix. The aim of this experiment is to analyze host response to Influenza A/WSN/33 (H1N1) infection.
Project description:Sustained hepatic inflammation can contribute to cancer initiation and progression via tumor-initiating cell expansion, but underlying mechanisms remains unknown. Expression profiles of lncRNAs/mRNAs were measured in normal, chronic hepatitis, cirrhotic and cancerous livers. We found abundant disease-related lncRNAs/mRNAs deregulated across different stages of inflammation-triggered liver disease and refined a transformation gene signature to distinguish pathological liver tissues. Amongst this signature, a conserved lncRNA DANCR was silenced in normal adult liver, but overexpressed in fetal and cancerous livers. Remarkably, increased DANCR significantly correlates with poor prognosis in multiple-center cohorts and is directly induced by inflammatory pathways including NF-κB and STAT3. DANCR could suppress cell differentiation and drive expansion of tumor-initiating cells, leading to chemoresistance. Moreover, in vitro and in vivo inhibition confirms the significance of DANCR as a therapeutic target when combined with other chemotherapy. We illustrate the role of DANCR relies on the regulation of CTNNB1 in a novel miRNA-blocking manner. Our studies reveal the expression of lncRNAs/mRNAs in normal and pathological livers and suggest the importance of oncofetal lncRNA DANCR in inflammation-induced malignant transformation, offering a potential prognostic marker and a therapeutic target for HCC. In the study, 10 normal livers (NL), 10 chronic inflammatory livers (IL), 10 cirrhotic livers (CL), 13 early HCC (eHCC) and 13 advanced HCC (aHCC) samples were profiled their lncRNA/mRNA expression
Project description:Human mesenchymal stem cell (MSC)-conditioned medium (CM) was previously reported to affect the biology of tumor cells; however, the precise mechanisms remain unclear. Here, we show that MSCs secreted 40- to 100-nm particles, which have the typical characteristics of exosomes, and these MSC-derived exosomes promoted migration of the breast cancer cell line MCF7. To further investigate the effect of MSC-exosomes on MCF7, we analyzed the gene expression profiles of MCF7 treated with or without MSC-exosomes for 24 h. Investigation of whole genome gene expression level changes in breast cancer cell line MCF7 which were treated with or without mesenchymal stem cell-derived exosomes. This study uses total RNA recovered from two samples. One sample is MCF7 treated with PBS for 24 hours and another one is MCF7 treated with mesenchymal stem cell-derived exosomes for 24hours. The ultimate concentration of mesenchymal stem cell-derived exosomes used in this experiment was 400ng/ul.