No abstract

Long noncoding RNAs (lncRNAs) are involved in many aspects of cellular processes, including the antiviral immune response. To identify influenza A virus (IAV)-related lncRNAs, we performed RNA deep sequencing to compare the profiles of lncRNAs in A549 and HEK293T cells with or without IAV infection. We identified an IAV-upregulated lncRNA named lnc-ISG20 because it shares most of its sequence with ISG20. We found that lnc-ISG20 is an interferon-stimulated gene similar to ISG20. Overexpression of lnc-ISG20 inhibited IAV replication, while lnc-ISG20 knockdown favored viral replication, suggesting that lnc-ISG20 is inhibitory to IAV replication. Further study indicated that overexpression of lnc-ISG20 enhances ISG20 protein levels, while knockdown of lnc-ISG20 reduces ISG20 protein levels in A549 cells induced with poly(I·C) and Sendai virus. We demonstrated that lnc-ISG20 inhibits IAV replication in an ISG20-dependent manner. As lnc-ISG20 did not affect the mRNA level of ISG20, we postulated that lnc-ISG20 may function as endogenous RNA competing with ISG20 to enhance its translation. Indeed, we identified that microRNA 326 (miR-326) is a mutual microRNA for both ISG20 and lnc-ISG20 that targets the 3' untranslated region of ISG20 mRNA to inhibit its translation. We confirmed that lnc-ISG20 can bind miR-326, which in turn decreased the amount of miR-326 bound to ISG20 mRNA. In conclusion, we identified that the IAV-upregulated lnc-ISG20 is a novel interferon-stimulated gene that elicits its inhibitory effect on IAV replication by enhancing ISG20 expression. We demonstrated that lnc-ISG20 functions as a competitive endogenous RNA to bind miR-326 to reduce its inhibition of ISG20 translation. Our results revealed the mechanism by which lnc-ISG20 inhibits IAV replication. The replication of influenza A virus is regulated by host factors. However, the mechanisms by which lncRNAs regulate IAV infection are not well understood. We identified that lnc-ISG20 is upregulated during IAV infection and is also an interferon-stimulated gene. We demonstrated that lnc-ISG20 can enhance ISG20 expression, which in turn inhibits IAV replication. Our studies indicate that lnc-ISG20 functions as a competing endogenous RNA that binds miR-326 and reduces its inhibitory effect on ISG20. Taken together, our findings reveal the mechanistic details of lnc-ISG20 negatively regulating IAV replication. These findings indicate that lnc-ISG20 plays an important role during the host antiviral immune response.

Citrus sinensis seedlings were irrigated with nutrient solution at a concentration of 0 (Mg-deficiency) or 2 (Mg-sufficiency) mM Mg (NO3)2 for 16 weeks. Mg-deficiency-induced interveinal chlorosis, vein enlargement and corkiness, and alterations of gas exchange, pigments, chlorophyll a fluorescence (OJIP) transients and related parameters were observed in middle and lower leaves, especially in the latter, but not in upper leaves. Mg-deficiency might impair the whole photosynthetic electron transport, including structural damage to thylakoids, ungrouping of photosystem II (PSII), inactivation of oxygen-evolving complex (OEC) and reaction centers (RCs), increased reduction of primary quinone electron acceptor (QA) and plastoquinone pool at PSII acceptor side and oxidation of PSI end-electron acceptors, thus lowering energy transfer and absorption efficiency and the transfer of electrons to the dark reactions, hence, the rate of CO2 assimilation in Mg-deficiency middle and lower leaves. Although potassium, Mg, manganese and zinc concentration in blades displayed a significant and positive relationship with the corresponding element concentration in veins, respectively, great differences existed in Mg-deficiency-induced alterations of nutrient concentrations between leaf blades and veins. For example, Mg-deficiency increased boron level in the blades of upper leaves, decreased boron level in the blades of lower leaves, but did not affect boron level in the blades of middle leaves and veins of upper, middle and lower leaves. To conclude, Mg-deficiency-induced interveinal chlorosis, vein enlargement, and corkiness, and alterations to photosynthesis and related parameters increased with increasing leaf age. Mg-deficiency-induced enlargement and corkiness of veins were not caused by Mg-deficiency-induced boron-starvation.

Previously, we identified a set of long noncoding RNAs (lncRNAs) that were differentially expressed in influenza A virus (IAV)-infected cells. In this study, we focused on lnc-MxA, which is upregulated during IAV infection. We found that the overexpression of lnc-MxA facilitates the replication of IAV, while the knockdown of lnc-MxA inhibits viral replication. Further studies demonstrated that lnc-MxA is an interferon-stimulated gene. However, lnc-MxA inhibits the Sendai virus (SeV)- and IAV-induced activation of beta interferon (IFN-β). A luciferase assay indicated that lnc-MxA inhibits the activation of the IFN-β reporter upon stimulation with RIG-I, MAVS, TBK1, or active IRF3 (IRF3-5D). These data indicated that lnc-MxA negatively regulates the RIG-I-mediated antiviral immune response. A chromatin immunoprecipitation (ChIP) assay showed that the enrichment of IRF3 and p65 at the IFN-β promoter in lnc-MxA-overexpressing cells was significantly lower than that in control cells, indicating that lnc-MxA interfered with the binding of IRF3 and p65 to the IFN-β promoter. Chromatin isolation by RNA purification (ChIRP), triplex pulldown, and biolayer interferometry assays indicated that lnc-MxA can bind to the IFN-β promoter. Furthermore, an electrophoretic mobility shift assay (EMSA) showed that lnc-MxA can form complexes with the IFN-β promoter fragment. These results demonstrated that lnc-MxA can form a triplex with the IFN-β promoter to interfere with the activation of IFN-β transcription. Using a vesicular stomatitis virus (VSV) infection assay, we confirmed that lnc-MxA can repress the RIG-I-like receptor (RLR)-mediated antiviral immune response and influence the antiviral status of cells. In conclusion, we revealed that lnc-MxA is an interferon-stimulated gene (ISG) that negatively regulates the transcription of IFN-β by forming an RNA-DNA triplex. IMPORTANCE IAV can be recognized as a nonself molecular pattern by host immune systems and can cause immune responses. However, the intense immune response induced by influenza virus, known as a “cytokine storm,” can also cause widespread tissue damage (X. Z. J. Guo and P. G. Thomas, Semin Immunopathol 39:541–550, 2017, https://doi.org/10.1007/s00281-017-0636-y; S. Yokota, Nihon Rinsho 61:1953–1958, 2003; I. A. Clark, Immunol Cell Biol 85:271–273, 2007). Meanwhile, the detailed mechanisms involved in the balancing of immune responses in host cells are not well understood. Our studies reveal that, as an IFN-inducible gene, lnc-MxA functions as a negative regulator of the antiviral immune response. We uncovered the mechanism by which lnc-MxA inhibits the activation of IFN-β transcription. Our findings demonstrate that, as an ISG, lnc-MxA plays an important role in the negative-feedback loop involved in maintaining immune homeostasis.

Recent studies have indicated that a number of long noncoding RNAs (lncRNAs) are dysregulated in hepatocellular carcinoma, while their aberrant expressions are associated with tumorigenesis and poor prognosis. To identify hepatitis B virus (HBV)-related lncRNAs, we used RNA deep sequencing to quantify the abundances of lncRNAs in HepG2 cells and HBV transgenic HepG2-4D14 cells. Here, we demonstrate that lnc-HUR1 is significantly upregulated in HepG2-4D14 cells. We found that HBV-encoded hepatitis B x protein can enhance the transcription of lnc-HUR1. Overexpression of lnc-HUR1 promotes cell proliferation, whereas knockdown of lnc-HUR1 inhibits cell growth. We identified that lnc-HUR1 can interact with p53 and inhibit its transcriptional regulation on downstream genes, such as p21 and B cell lymphoma 2-associated X protein. We generated lnc-HUR1 transgenic mice and performed the partial hepatectomy (PHx) to examine liver regeneration. The data showed that the ratio of liver weight to body weight in lnc-HUR1 transgenic mice is higher than that in wild-type (WT) littermates at day 2 and day 3 following hepatectomy. Consistently, the results of bromodeoxyuridine staining on liver sections following hepatectomy indicate that the ratio of bromodeoxyuridine-positive cells in lnc-HUR1 transgenic mice is significantly higher than that in WT mice, suggesting that lnc-HUR1 promotes cell proliferation during liver regeneration. Next, we performed the experiment of diethylnitrosamine-induced tumorigenesis. The data demonstrate that tumor number in lnc-HUR1 transgenic mice is higher compared with control mice, indicating that lnc-HUR1 enhances diethylnitrosamine-induced tumorigenesis. Conclusion: We reveal that HBV-upregulated lnc-HUR1 promotes cell proliferation and tumorigenesis by interacting with p53 to block downstream gene transcription. Our findings suggest that lnc-HUR1 plays an important role in HBV-related hepatocellular carcinoma development and may serve as a therapeutic marker for hepatocellular carcinoma. (Hepatology 2018; 00:000-000).

Post-translational modifications of viral proteins play important roles in regulating viral replication. Here we demonstrated that the PB2 of influenza A virus (IAV) can be modified by NEDD8. We revealed that E3 ligase HDM2 can promote PB2 NEDDylation. Overexpression of either NEDD8 or HDM2 can inhibit IAV replication, while knockdown of HDM2 has the opposite effect. Then we identified residue K699 in PB2 as the major NEDDylation site. We found that NEDDylation deficient PB2 mutant (PB2 K699R) has a longer half-life than wild-type PB2, indicating that NEDDylation of PB2 reduces its stability. We generated an IAV mutant in which PB2 was mutated to PB2 K699R (WSN-PB2 K699R) and examined the replication of WSN and WSN-PB2 K699R viruses in both MDCK and A549 cells and found that the replication of WSN-PB2 K699R was more efficient than wild-type WSN. In addition, we observed that overexpression of NEDD8 significantly inhibited the replication of WSN, but not WSN-PB2 K699R. The infection assay in mice showed that WSN-PB2 K699R exhibited enhanced virulence in mice compared to WSN, suggesting that NEDDylation of PB2 reduced IAV replication in vivo. In conclusion, we demonstrated that NEDDylation of PB2 by HDM2 negatively regulates IAV infection.

N6-methyladenosine (m6A) modification on RNA plays an important role in tumorigenesis and metastasis, which could change gene expression and even function at multiple levels such as RNA splicing, stability, translocation, and translation. In this study, we aim to conduct a comprehensive analysis on m6A RNA methylation-related genes, including m6A RNA methylation regulators and m6A RNA methylation-modified genes, in liver hepatocellular carcinoma, and their relationship with survival and clinical features. Data, which consist of the expression of widely reported m6A RNA methylation-related genes in liver hepatocellular carcinoma from The Cancer Genome Atlas (TCGA), were analyzed by one-way ANOVA, Univariate Cox regression, a protein–protein interaction network, gene enrichment analysis, feature screening, a risk prognostic model, correlation analysis, and consensus clustering analysis. In total, 405 of the m6A RNA methylation-related genes were found based on one-way ANOVA. Among them, DNA topoisomerase 2-alpha (TOP2A), exodeoxyribonuclease 1 (EXO1), ser-ine/threonine-protein kinase Nek2 (NEK2), baculoviral IAP repeat-containing protein 5 (BIRC5), hyaluronan mediated motility receptor (HMMR), structural maintenance of chromosomes protein 4 (SMC4), bloom syndrome protein (BLM), ca-sein kinase I isoform epsilon (CSNK1E), cytoskeleton-associated protein 5 (CKAP5), and inner centromere protein (INCENP), which were m6A RNA methylation-modified genes, were recognized as the hub genes based on the protein–protein interaction analysis. The risk prognostic model showed that gender, AJCC stage, grade, T, and N were significantly different between the subgroup with the high and low risk groups. The AUC, the evaluation parameter of the prediction model which was built by RandomForest, was 0.7. Furthermore, two subgroups were divided by consensus clustering analysis, in which stage, grade, and T differed. We identified the important genes expressed significantly among two clusters, including uridine-cytidine kinase 2 (UCK2), filensin (BFSP1), tubulin-specific chaperone D (TBCD), histone-lysine N-methyltransferase PRDM16 (PRDM16), phosphorylase b ki-nase regulatory subunit alpha (PHKA2), serine/threonine-protein kinase BRSK2 (BRSK2), Arf-GAP with coiled-coil (ACAP3), general transcription factor 3C polypep-tide 2 (GTF3C2), and guanine nucleotide exchange factor MSS4 (RABIF). In our study, the m6A RNA methylation-related genes in liver hepatocellular carcinoma were analyzed systematically, including the expression, interaction, function, and prognostic values, which provided an important theoretical basis for m6A RNA methylation in liver cancer. The nine important m6A-related genes could be prognostic markers in the survival time of patients.

The establishment of postmortem interval is one of the most important aspects of forensic expertise. Microbes may provide a novel way to estimate the postmortem intervals in order to avoid many of these limitations. The oral cavity harbors one of the most diverse microbiomes that play a key role in the decomposition of corpses. In this study, the oral bacterial community showed obvious changes in relative abundance during the process of mice decomposition. Meanwhile, at different taxonomic levels, specific bacteria were found to be significantly correlated with the postmortem interval. Linear regression models between relative abundance and the postmortem interval were constructed. Among these species, Gamma-proteobacteria and Proteus were the best ones that can be used to infer the postmortem interval, especially late postmortem interval. Therefore, we suggest that succession of oral microbial community can be developed as a forensic tool for estimating the postmortem interval.