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Chemokine Transcripts as Targets of the RNA-Binding Protein HuR in Human Airway Epithelium

ABSTRACT: HuR is a regulator of mRNA turnover or translation of inflammatory genes through binding to adenylate-uridylate-rich elements (ARE) and related motifs present in the 3’untranslated region (UTR) of mRNAs. We aimed to identify HuR targets in the human airway epithelial cell line BEAS-2B challenged with TNFa plus IFNg, a strong stimulus for inflammatory epithelial responses. Ribonucleoprotein (RNP) complexes from resting and cytokine-treated cells were immunoprecipitated (IP) using anti-HuR and isotype-control antibody, and eluted mRNAs were reverse-transcribed and hybridized to an inflammatory-focused gene array. The chemokines CCL2, CCL8, CXCL1 and CXCL2 ranked highest among 27 signaling and inflammatory genes significantly enriched in the HuR RNP-IP from stimulated cells over the control IP. Among these, 20 displayed published HuR binding motifs. Association of HuR with the four endogenous chemokine mRNAs was validated by single-gene RNP-IP, and shown to be 3’UTR-dependent by biotin pull-down assay. Cytokine treatment increased mRNA stability only for CCL2 and CCL8, and transient silencing and overexpression of HuR affected only CCL2 and CCL8 expression in primary and transformed epithelial cells. Cytokine-induced CCL2 mRNA was predominantly cytoplasmic; conversely, CXCL1 mRNA remained mostly nuclear and unaffected, as CXCL2, by changes in HuR levels. Increase in cytoplasmic HuR and HuR target expression partially relied on the inhibition of AMP-dependent kinase, a negative regulator of HuR nucleocytoplasmic shuttling.

ORGANISM(S): Homo sapiens

PROVIDER: GSE25264 | GEO | 2010/11/10

SECONDARY ACCESSION(S): PRJNA134805

REPOSITORIES: GEO

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Project description:HuR is a regulator of mRNA turnover or translation of inflammatory genes through binding to adenylate-uridylate-rich elements (ARE) and related motifs present in the 3’untranslated region (UTR) of mRNAs. We aimed to identify HuR targets in the human airway epithelial cell line BEAS-2B challenged with TNFa plus IFNg, a strong stimulus for inflammatory epithelial responses. Ribonucleoprotein (RNP) complexes from resting and cytokine-treated cells were immunoprecipitated (IP) using anti-HuR and isotype-control antibody, and eluted mRNAs were reverse-transcribed and hybridized to an inflammatory-focused gene array. The chemokines CCL2, CCL8, CXCL1 and CXCL2 ranked highest among 27 signaling and inflammatory genes significantly enriched in the HuR RNP-IP from stimulated cells over the control IP. Among these, 20 displayed published HuR binding motifs. Association of HuR with the four endogenous chemokine mRNAs was validated by single-gene RNP-IP, and shown to be 3’UTR-dependent by biotin pull-down assay. Cytokine treatment increased mRNA stability only for CCL2 and CCL8, and transient silencing and overexpression of HuR affected only CCL2 and CCL8 expression in primary and transformed epithelial cells. Cytokine-induced CCL2 mRNA was predominantly cytoplasmic; conversely, CXCL1 mRNA remained mostly nuclear and unaffected, as CXCL2, by changes in HuR levels. Increase in cytoplasmic HuR and HuR target expression partially relied on the inhibition of AMP-dependent kinase, a negative regulator of HuR nucleocytoplasmic shuttling. We postulate that HuR critically regulates the epithelial response, by associating with multiple adenylate-uridylate-rich elements (ARE)-bearing, functionally related inflammatory transcripts. On the basis of genome-wide studies probing the relationship between RNA-binding proteins and the functional profile of their associated transcripts, we combined the specific HuR immunoprecipitation of RNPs and the genome-scale microarray to profile the target mRNAs of HuR in the human airway epithelial cell line BEAS-2B challenged with very strong inflammatory stimulation, TNFa plus IFNG. The array platform we used is the Human Autoimmune and Inflammatory Response Gene Array (SuperArray Bioscience, Frederick, MD). To validate the association of HuR and its target mRNAs, we planned to apply biotin pull-down assay. HuR overexpression and knockdown assays were also planned to further verify the association of HuR and its target mRNAs. Overall, we did three biological replicates for the IP array experiments, which include both IgG1 control IP and HuR IP arrays.

Project description:Purpose: The goals of this study are to compare the difference between WT and hCD147 mice, hACE2 and hCD147 mice lung tissues Transcriptomes after infection with SARS-CoV-2 Methods: After receiving anesthesia with pentobarbital sodium, each mouse of the WT group, hCD147 group, and hACE2 group was infected with SARS-CoV-2 by nasal drip at a dose of 3 x 10^5 TCID50. The lung tissues were take for RNA-seq at 2 dpi. each mouse of the hCD147 group, and hACE2 group was infected with SARS-CoV-2 by nasal drip at a dose of 3 x 10^5 TCID50. The lung tissues were take for RNA-seq at 2 dpi. Results: RNA-seq of lung homogenates from C57BL/6 and hCD147 mice at 2 d.p.i. showed 354 upregulated and 175 downregulated genes). hCD147 mice mediated strong inflammatory responses, including Th17 cell responses, the NF-kappaB pathway and MAPK pathway, etc. The levels of cytokines and chemokines were increased in the lung of hCD147 mice, such as IL6, IL10, ILbeta, CXCL1, CXCL2, CCL2, etc. Analysis of RNA-Seq data of hACE2 mice versus hCD147 mice showed that hACE2 mice had stronger antiviral responses, including type I IFN and IFNalpha/IFNbeta responses, while hCD147 mice showed activation of multiple proinflammatory pathways, which is consistent with clinical features of COVID-19 patients. The significantly upregulated genes during SARS-CoV-2 infection of hACE2 mice enriched in cellular response to IFN-?. The upregulated genes in SARS-CoV-2-infected hCD147 mice were enriched secretory proteins, including ILbeta, IL6 and CXCL1. Conclusions: CD147 mediates potent inflammatory responses in SARS-CoV-2 infection. RNA-seq of lung homogenates from hACE2 and hCD147 mice at 2 d.p.i. showed 354 upregulated and 175 downregulated genes). hCD147 mice mediated strong inflammatory responses, including Th17 cell responses, the NF-kappaB pathway and MAPK pathway, etc. The levels of cytokines and chemokines were increased in the lung of hCD147 mice, such as IL6, IL10, ILbeta, CXCL1, CXCL2, CCL2, etc.

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Chemokine Transcripts as Targets of the RNA-Binding Protein HuR in Human Airway Epithelium

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