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Browsing by Author "Gu, Hongmei"
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Item 38766 Massively Parallel Reporter Assay Reveals Functional Impact of 3™-UTR SNPs Associated with Neurological and Psychiatric Disorders(Cambridge University Press, 2021) Chen, Andy B.; Thapa, Kriti; Gao, Hongyu; Reiter, Jill L.; Zhang, Junjie; Xuei, Xiaoling; Gu, Hongmei; Wang, Yue; Edenberg, Howard J.; Liu, Yunlong; Medical and Molecular Genetics, School of MedicineABSTRACT IMPACT: Screening the effect of thousands of non-coding genetic variants will help identify variants important in the etiology of diseases OBJECTIVES/GOALS: Massively parallel reporter assays (MPRAs) can experimentally evaluate the impact of genetic variants on gene expression. In this study, our objective was to systematically evaluate the functional activity of 3’-UTR SNPs associated with neurological disorders and use those results to help understand their contributions to disease etiology. METHODS/STUDY POPULATION: To choose variants to evaluate with the MPRA, we first gathered SNPs from the GWAS Catalog that were associated with any neurological disorder trait with p-value < 10-5. For each SNP, we identified the region that was in linkage disequilibrium (r2 > 0.8) and retrieved all the common 3’-UTR SNPs (allele-frequency > 0.05) within that region. We used an MPRA to measure the impact of these 3’-UTR variants in SH-SY5Y neuroblastoma cells and a microglial cell line. These results were then used to train a deep-learning model to predict the impact of variants and identify features that contribute to the predictions. RESULTS/ANTICIPATED RESULTS: Of the 13,515 3’-UTR SNPs tested, 400 and 657 significantly impacted gene expression in SH-SY5Y and microglia, respectively. Of the 84 SNPs significantly impacted in both cells, the direction of impact was the same in 81. The direction of eQTL in GTEx tissues agreed with the assay SNP effect in SH-SY5Y cells but not microglial cells. The deep-learning model predicted sequence activity level correlated with the experimental activity level (Spearman’s corr = 0.45). The deep-learning model identified several predictive motifs similar to motifs of RNA-binding proteins. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study demonstrates that MPRAs can be used to evaluate the effect of non-coding variants, and the results can be used to train a machine learning model and interpret its predictions. Together, these can help identify causal variants and further understand the etiology of diseases.Item Allele-specific expression and high-throughput reporter assay reveal functional genetic variants associated with alcohol use disorders(Springer Nature, 2021-04) Rao, Xi; Thapa, Kriti S.; Chen, Andy B.; Lin, Hai; Gao, Hongyu; Reiter, Jill L.; Hargreaves, Katherine A.; Ipe, Joseph; Lai, Dongbing; Xuei, Xiaoling; Wang, Yue; Gu, Hongmei; Kapoor, Manav; Farris, Sean P.; Tischfield, Jay; Foroud, Tatiana; Goate, Alison M.; Skaar, Todd C.; Mayfield, R. Dayne; Edenberg, Howard J.; Liu, Yunlong; Medical and Molecular Genetics, School of MedicineGenome-wide association studies (GWAS) of complex traits, such as alcohol use disorders (AUD), usually identify variants in non-coding regions and cannot by themselves distinguish whether the associated variants are functional or in linkage disequilibrium with the functional variants. Transcriptome studies can identify genes whose expression differs between alcoholics and controls. To test which variants associated with AUD may cause expression differences, we integrated data from deep RNA-seq and GWAS of four postmortem brain regions from 30 subjects with AUD and 30 controls to analyze allele-specific expression (ASE). We identified 88 genes with differential ASE in subjects with AUD compared to controls. Next, to test one potential mechanism contributing to the differential ASE, we analyzed single nucleotide polymorphisms (SNPs) in the 3′ untranslated regions (3′UTR) of these genes. Of the 88 genes with differential ASE, 61 genes contained 437 SNPs in the 3′UTR with at least one heterozygote among the subjects studied. Using a modified PASSPORT-seq (parallel assessment of polymorphisms in miRNA target-sites by sequencing) assay, we identified 25 SNPs that affected RNA levels in a consistent manner in two neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2). Many of these SNPs are in binding sites of miRNAs and RNA-binding proteins, indicating that these SNPs are likely causal variants of AUD-associated differential ASE. In sum, we demonstrate that a combination of computational and experimental approaches provides a powerful strategy to uncover functionally relevant variants associated with the risk for AUD.Item Allele-specific expression and high-throughput reporter assay reveal functional genetic variants associated with alcohol use disorders.(Springer, 2021-04) Rao, Xi; Thapa, Kriti S.; Chen, Andy B.; Lin, Hai; Gao, Hongyu; Reiter, Jill L.; Hargreaves, Katherine A.; Ipe, Joseph; Lai, Dongbing; Xuei, Xiaoling; Wang, Yue; Gu, Hongmei; Kapoor, Manav; Farris, Sean P.; Tischfield, Jay; Foroud, Tatiana; Goate, Alison M.; Skaar, Todd C.; Mayfield, R. Dayne; Edenberg, Howard J.; Liu, YunlongGenome-wide association studies (GWAS) of complex traits, such as alcohol use disorders (AUD), usually identify variants in non-coding regions and cannot by themselves distinguish whether the associated variants are functional or in linkage disequilibrium with the functional variants. Transcriptome studies can identify genes whose expression differs between alcoholics and controls. To test which variants associated with AUD may cause expression differences, we integrated data from deep RNA-seq and GWAS of four postmortem brain regions from 30 subjects with AUD and 30 controls to analyze allele-specific expression (ASE). We identified 88 genes with differential ASE in subjects with AUD compared to controls. Next, to test one potential mechanism contributing to the differential ASE, we analyzed single nucleotide polymorphisms (SNPs) in the 3' untranslated regions (3'UTR) of these genes. Of the 88 genes with differential ASE, 61 genes contained 437 SNPs in the 3'UTR with at least one heterozygote among the subjects studied. Using a modified PASSPORT-seq (parallel assessment of polymorphisms in miRNA target-sites by sequencing) assay, we identified 25 SNPs that affected RNA levels in a consistent manner in two neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2). Many of these SNPs are in binding sites of miRNAs and RNA-binding proteins, indicating that these SNPs are likely causal variants of AUD-associated differential ASE. In sum, we demonstrate that a combination of computational and experimental approaches provides a powerful strategy to uncover functionally relevant variants associated with the risk for AUD.Item BACE1 Inhibition Increases Susceptibility to Oxidative Stress by Promoting Mitochondrial Damage(MDPI, 2021-09-28) Francelin, Carolina; Mitter, Sayak K.; Qian, Qingwen; Barodia, Sandeep Kumar; Ip, Colin; Qi, Xiaoping; Gu, Hongmei; Quigley, Judith; Goldberg, Matthew S.; Grant, Maria B.; Boulton, Michael E.; Ophthalmology, School of MedicineBACE1 is a key enzyme facilitating the generation of neurotoxic β-amyloid (Aβ) peptide. However, given that BACE1 has multiple substrates we explored the importance of BACE1 in the maintenance of retinal pigment epithelial (RPE) cell homeostasis under oxidative stress. Inhibition of BACE1 reduced mitochondrial membrane potential, increased mitochondrial fragmentation, and increased cleaved caspase-3 expression in cells under oxidative stress. BACE1 inhibition also resulted in significantly lower levels of mitochondrial fusion proteins OPA1 and MFN1 suggesting a higher rate of mitochondrial fission while increasing the levels of mitophagic proteins Parkin and PINK1 and autophagosome numbers. In contrast, BACE2 had minimal effect on cellular response to oxidative stress. In summary, our results emphasize the importance of BACE1 in augmenting cellular defense against oxidative stress by protecting mitochondrial dynamics.Item Contribution of the anaphylatoxin receptors, C3aR and C5aR, to the pathogenesis of pulmonary fibrosis(Federation of American Societies for Experimental Biology, 2016-06) Gu, Hongmei; Fisher, Amanda J.; Mickler, Elizabeth A.; Duerson, Frank, III; Cummings, Oscar W.; Peters-Golden, Marc; Twigg, Homer L., III; Woodruff, Trent M.; Wilkes, David S.; Vittal, Ragini; Medicine, School of MedicineComplement activation, an integral arm of innate immunity, may be the critical link to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Whereas we have previously reported elevated anaphylatoxins-complement component 3a (C3a) and complement component 5a (C5a)-in IPF, which interact with TGF-β and augment epithelial injury in vitro, their role in IPF pathogenesis remains unclear. The objective of the current study is to determine the mechanistic role of the binding of C3a/C5a to their respective receptors (C3aR and C5aR) in the progression of lung fibrosis. In normal primary human fetal lung fibroblasts, C3a and C5a induces mesenchymal activation, matrix synthesis, and the expression of their respective receptors. We investigated the role of C3aR and C5aR in lung fibrosis by using bleomycin-injured mice with fibrotic lungs, elevated local C3a and C5a, and overexpression of their receptors via pharmacologic and RNA interference interventions. Histopathologic examination revealed an arrest in disease progression and attenuated lung collagen deposition (Masson's trichrome, hydroxyproline, collagen type I α 1 chain, and collagen type I α 2 chain). Pharmacologic or RNA interference-specific interventions suppressed complement activation (C3a and C5a) and soluble terminal complement complex formation (C5b-9) locally and active TGF-β1 systemically. C3aR/C5aR antagonists suppressed local mRNA expressions of tgfb2, tgfbr1/2, ltbp1/2, serpine1, tsp1, bmp1/4, pdgfbb, igf1, but restored the proteoglycan, dcn Clinically, compared with pathologically normal human subjects, patients with IPF presented local induction of C5aR, local and systemic induction of soluble C5b-9, and amplified expression of C3aR/C5aR in lesions. The blockade of C3aR and C5aR arrested the progression of fibrosis by attenuating local complement activation and TGF-β/bone morphologic protein signaling as well as restoring decorin, which suggests a promising therapeutic strategy for patients with IPF.-Gu, H., Fisher, A. J., Mickler, E. A., Duerson, F., III, Cummings, O. W., Peters-Golden, M., Twigg, H. L., III, Woodruff, T. M., Wilkes, D. S., Vittal, R. Contribution of the anaphylatoxin receptors, C3aR and C5aR, to the pathogenesis of pulmonary fibrosis.Item Crosstalk between TGF-β1 and complement activation augments epithelial injury in pulmonary fibrosis(Federation of American Societies for Experimental Biology, 2014-10) Gu, Hongmei; Mickler, Elizabeth A.; Cummings, Oscar W.; Sandusky, George E.; Weber, Daniel J.; Gracon, Adam; Woodruff, Trent; Wilkes, David S.; Vittal, Ragini; Department of Microbiology and Immunology, IU School of MedicineThe epithelial complement inhibitory proteins (CIPs) cluster of differentiation 46 and 55 (CD46 and CD55) regulate circulating immune complex-mediated complement activation in idiopathic pulmonary fibrosis (IPF). Our previous studies demonstrated that IL-17A mediates epithelial injury via transforming growth factor β1 (TGF-β1) and down-regulates CIPs. In the current study, we examined the mechanistic role of TGF-β1 in complement activation-mediated airway epithelial injury in IPF pathogenesis. We observed lower epithelial CIP expression in IPF lungs compared to normal lungs, associated with elevated levels of complement component 3a and 5a (C3a and C5a), locally and systemically. In normal primary human small airway epithelial cells (SAECs) treated with TGF-β1 (10 ng/ml), C3a, or C5a (100 nM), we observed loss of CIPs and increased poly(ADP-ribose) polymerase (PARP) activation [also observed with RNA interference (RNAi) of CD46/CD55]. TGF-β1-mediated loss of CIPs and Snail induction [SNAI1; a transcriptional repressor of E-cadherin (E-CAD)] was blocked by inhibiting mitogen-activated protein kinase (p38MAPK; SB203580) and RNAi silencing of SNAI1. C3a- and C5a-mediated loss of CIPs was also blocked by p38MAPK inhibition. While C3a upregulated TGFb transcripts, both C3a and C5a down-regulated SMAD7 (negative regulator of TGF-β), and whereas TGF-β1 induced C3a/C5a receptor (C3aR/C5aR) expression, pharmacologic C3aR/C5aR inhibition protected against C3a-/C5a-mediated loss of CIPs. Taken together, our results suggest that epithelial injury in IPF can be collectively amplified as a result of TGF-β1-induced loss of CIPs leading to complement activation that down-regulates CIPs and induces TGF-β1 expressionItem IL-17A deficiency mitigates bleomycin-induced complement activation during lung fibrosis(Federation of American Societies for Experimental Biology, 2017-12) Cipolla, Ellyse; Fisher, Amanda J.; Gu, Hongmei; Mickler, Elizabeth A.; Agarwal, Manisha; Wilke, Carol A.; Kim, Kevin K.; Moore, Bethany B.; Vittal, Ragini; Medicine, School of MedicineInterleukin 17A (IL-17A) and complement (C') activation have each been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). We have reported that IL-17A induces epithelial injury via TGF-β in murine bronchiolitis obliterans; that TGF-β and the C' cascade present signaling interactions in mediating epithelial injury; and that the blockade of C' receptors mitigates lung fibrosis. In the present study, we investigated the role of IL-17A in regulating C' in lung fibrosis. Microarray analyses of mRNA isolated from primary normal human small airway epithelial cells indicated that IL-17A (100 ng/ml; 24 h; n = 5 donor lungs) induces C' components (C' factor B, C3, and GPCR kinase isoform 5), cytokines (IL8, -6, and -1B), and cytokine ligands (CXCL1, -2, -3, -5, -6, and -16). IL-17A induces protein and mRNA regulation of C' components and the synthesis of active C' 3a (C3a) in normal primary human alveolar type II epithelial cells (AECs). Wild-type mice subjected to IL-17A neutralization and IL-17A knockout (il17a-/- ) mice were protected against bleomycin (BLEO)-induced fibrosis and collagen deposition. Further, BLEO-injured il17a-/- mice had diminished levels of circulating Krebs Von Den Lungen 6 (alveolar epithelial injury marker), local caspase-3/7, and local endoplasmic reticular stress-related genes. BLEO-induced local C' activation [C3a, C5a, and terminal C' complex (C5b-9)] was attenuated in il17a-/- mice, and IL-17A neutralization prevented the loss of epithelial C' inhibitors (C' receptor-1 related isoform Y and decay accelerating factor), and an increase in local TUNEL levels. RNAi-mediated gene silencing of il17a in fibrotic mice arrested the progression of lung fibrosis, attenuated cellular apoptosis (caspase-3/7) and lung deposition of collagen and C' (C5b-9). Compared to normals, plasma from IPF patients showed significantly higher hemolytic activity. Our findings demonstrate that limiting complement activation by neutralizing IL-17A is a potential mechanism in ameliorating lung fibrosis.-Cipolla, E., Fisher, A. J., Gu, H., Mickler, E. A., Agarwal, M., Wilke, C. A., Kim, K. K., Moore, B. B., Vittal, R. IL-17A deficiency mitigates bleomycin-induced complement activation during lung fibrosis.Item Overexpression of Decay Accelerating Factor Mitigates Fibrotic Responses to Lung Injury(American Thoracic Society, 2022) Vittal, Ragini; Fisher, Amanda J.; Thompson, Eric L.; Cipolla, Ellyse M.; Gu, Hongmei; Mickler, Elizabeth A.; Varre, Ananya; Agarwal, Manisha; Kim, Kevin K.; Vasko, Michael R.; Moore, Bethany B.; Lama, Vibha N.; Medicine, School of MedicineCD55 or decay accelerating factor (DAF), a ubiquitously expressed glycosylphosphatidylinositol (GPI)-anchored protein, confers a protective threshold against complement dysregulation which is linked to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Since lung fibrosis is associated with downregulation of DAF, we hypothesize that overexpression of DAF in fibrosed lungs will limit fibrotic injury by restraining complement dysregulation. Normal primary human alveolar type II epithelial cells (AECs) exposed to exogenous complement 3a or 5a, and primary AECs purified from IPF lungs demonstrated decreased membrane-bound DAF expression with concurrent increase in the endoplasmic reticulum (ER) stress protein, ATF6. Increased loss of extracellular cleaved DAF fragments was detected in normal human AECs exposed to complement 3a or 5a, and in lungs of IPF patients. C3a-induced ATF6 expression and DAF loss was inhibited using pertussis toxin (an enzymatic inactivator of G-protein coupled receptors), in murine AECs. Treatment with soluble DAF abrogated tunicamycin-induced C3a secretion and ER stress (ATF6 and BiP expression) and restored epithelial cadherin. Bleomycin-injured fibrotic mice subjected to lentiviral overexpression of DAF demonstrated diminished levels of local collagen deposition and complement activation. Further analyses showed diminished release of DAF fragments, as well as reduction in apoptosis (TUNEL and caspase 3/7 activity), and ER stress-related transcripts. Loss-of-function studies using Daf1 siRNA demonstrated worsened lung fibrosis detected by higher mRNA levels of Col1a1 and epithelial injury-related Muc1 and Snai1, with exacerbated local deposition of C5b-9. Our studies provide a rationale for rescuing fibrotic lungs via DAF induction that will restrain complement dysregulation and lung injury.Item Oxidative stress-mediated NFκB phosphorylation upregulates p62/SQSTM1 and promotes retinal pigmented epithelial cell survival through increased autophagy(Plos, 2017-02-21) Song, Chunjuan; Mitter, Sayak K.; Qi, Xiaoping; Beli, Eleni; Rao, Haripriya V.; Ding, Jindong; Ip, Colin S.; Gu, Hongmei; Akin, Debra; Dunn, William A. Jr.; Bowes Rickman, Catherine; Lewin, Alfred S.; Grant, Maria B.; Boulton, Michael E.; Department of Ophthalmology, IU School of Medicinep62 is a scaffolding adaptor implicated in the clearance of protein aggregates by autophagy. Reactive oxygen species (ROS) can either stimulate or inhibit NFκB-mediated gene expression influencing cellular fate. We studied the effect of hydrogen peroxide (H2O2)-mediated oxidative stress and NFκB signaling on p62 expression in the retinal pigment epithelium (RPE) and investigated its role in regulation of autophagy and RPE survival against oxidative damage. Cultured human RPE cell line ARPE-19 and primary human adult and fetal RPE cells were exposed to H2O2-induced oxidative stress. The human apolipoprotein E4 targeted-replacement (APOE4) mouse model of AMD was used to study expression of p62 and other autophagy proteins in the retina. p62, NFκB p65 (total, phosphorylated, nuclear and cytoplasmic) and ATG10 expression was assessed by mRNA and protein analyses. Cellular ROS and mitochondrial superoxide were measured by CM-H2DCFDA and MitoSOX staining respectively. Mitochondrial viability was determined using MTT activity. qPCR-array system was used to investigate autophagic genes affected by p62. Nuclear and cytoplasmic levels of NFκB p65 were evaluated after cellular fractionation by Western blotting. We report that p62 is up-regulated in RPE cells under H2O2-induced oxidative stress and promotes autophagic activity. Depletion of endogenous p62 reduces autophagy by downregulation of ATG10 rendering RPE more susceptible to oxidative damage. NFκB p65 phosphorylation at Ser-536 was found to be critical for p62 upregulation in response to oxidative stress. Proteasome inhibition by H2O2 causes p62-NFκB signaling as antioxidant pre-treatment reversed p62 expression and p65 phosphorylation when RPE was challenged by H2O2 but not when by Lactacystin. p62 protein but not RNA levels are elevated in APOE4-HFC AMD mouse model, suggesting reduction of autophagic flux in disease conditions. Our findings suggest that p62 is necessary for RPE cytoprotection under oxidative stress and functions, in part, by modulating ATG10 expression. NFκB p65 activity may be a critical upstream initiator of p62 expression in RPE cells under oxidative stress.Item Potential Mechanisms Underlying TGF-β-mediated Complement Activation in Lung Fibrosis(Wiley, 2017) Fisher, Amanda J.; Cipolla, Ellyse; Varre, Ananya; Gu, Hongmei; Mickler, Elizabeth A.; Vittal, Ragini; Medicine, School of MedicineWhile our previous studies suggest that limiting bleomycin-induced complement activation suppresses TGF-β signaling, the specific hierarchical interactions between TGF-β and complement in lung fibrosis are unclear. Herein, we investigated the mechanisms underlying TGF-β-induced complement activation in the pathogenesis of lung fibrosis. C57-BL6 mice were given intratracheal instillations of adenoviral vectors overexpressing TGF-β (Ad-TGFβ) or the firefly gene-luciferase (Ad-Luc; control). Two weeks later, mice with fibrotic lungs were instilled RNAi specific to receptors for C3a or C5a-C3ar or C5ar, and sacrificed at day 28. Histopathological analyses revealed that genetic silencing of C3ar or C5ar arrested the progression of TGF-β-induced lung fibrosis, collagen deposition and content (hydroxyproline, col1a1/2); and significantly suppressed local complement activation. With genetic silencing of either C3ar or C5ar, in Ad-TGFβ-injured lungs: we detected the recovery of Smad7 (TGF-β inhibitor) and diminished local release of DAF (membrane-bound complement inhibitor); in vitro: TGF-β-mediated loss of DAF was prevented. Conversely, blockade of the TGF-β receptor prevented C3a-mediated loss of DAF in both normal primary human alveolar and small airway epithelial cells. Of the 52 miRNAs analyzed as part of the Affymetrix array, normal primary human SAECs exposed to C3a, C5a or TGF-β caused discrete and overlapping miRNA regulation related to epithelial proliferation or apoptosis (miR-891A, miR-4442, miR-548, miR-4633), cellular contractility (miR-1197) and lung fibrosis (miR-21, miR-200C, miR-31HG, miR-503). Our studies present potential mechanisms by which TGF-β activates complement and promotes lung fibrosis.