Transcriptome analysis in an AEG‑1‑deficient neuronal HT22 cell line

Liu,Kunmei; Liu,Kunmei; Wan,Panpan; Wan,Panpan; Huang,Yue; Huang,Yue; Wang,Bin; Wang,Bin; Wang,Xuequan; Wang,Xuequan; Zhang,Rui; Zhang,Rui; Guo,Le; Guo,Le

doi:10.3892/etm.2022.11607

Transcriptome analysis in an AEG‑1‑deficient neuronal HT22 cell line

Authors:
- Kunmei Liu
- Panpan Wan
- Yue Huang
- Bin Wang
- Xuequan Wang
- Rui Zhang
- Le Guo
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Affiliations: Ningxia Key Laboratory of Cerebrocranial Disease, Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region 750004, P.R. China, Department of Microbiology and Biochemical Pharmacy, School of Pharmacy, Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region 750004, P.R. China, Ningxia Key Laboratory of Clinical and Pathogenic Microbiology, General Hospital of Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region 750004, P.R. China, Laboratory of Cellular and Molecular Radiation Oncology, The Affiliated Taizhou Hospital, Wenzhou Medical University, Taizhou, Zhejiang 317000, P.R. China
Published online on: September 13, 2022 https://doi.org/10.3892/etm.2022.11607
Article Number: 670
Copyright: © Liu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Astrocyte elevated gene‑1 (AEG‑1) is a key regulatory factor of progression in multiple types of tumor and neurodegenerative disease development. AEG‑1 is associated with glutamate excitotoxicity due to its reported function of repressing excitatory amino acid transporter 2 expression in astrocytes. Although the function of AEG‑1 has been demonstrated in neurological disorders, such as Alzheimer's disease and amyotrophic lateral sclerosis, the underlying mechanism of neuronal AEG‑1 function remains unclear. The aim of the present study was to clarify the function and related mechanism of AEG‑1 in neurons. A stable AEG‑1‑deficient HT22 neuronal cell line was constructed using CRISPR/Cas9 gene‑editing technology. Reverse transcription‑quantitative PCR and western blotting were carried out to analyze the knockdown efficiency of AEG‑1‑deficient HT22 cell line. RNA Sanger sequencing analysis was performed in AEG‑1‑deficient HT22 cells and wild‑type HT22 cells without knockout (KO). Results from RNA sequencing revealed that AEG‑1 modulated neuronal morphology and development by regulating the expression of numerous genes, such as ubiquitin C, C‑X‑C motif chemokine ligand 1, MMP9, Notch1, neuropilin 1 and ephrin type‑A receptor 4. In addition, AEG‑1 deficiency impacted several signaling pathways by mediating cell survival differentiation, apoptosis, and migration; this included the TNF‑α pathway, the NF‑κB pathway, the MAPK signaling pathway, the Notch signaling pathway and Axon guidance. Downregulation in cellular ion homeostasis, including ion channel function and neurotransmitter release, were observed after knocking out AEG‑1 expression. Collectively, the present study provides insights into AEG‑1‑dependent gene regulation and signaling pathway transduction in neurons. The results of the present study may be applied for improving the understanding of AEG‑1‑associated central nervous system diseases.

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1	Su ZZ, Kang DC, Chen Y, Pekarskaya O, Chao W, Volsky DJ and Fisher PB: Identification and cloning of human astrocyte genes displaying elevated expression after infection with HIV-1 or exposure to HIV-1 envelope glycoprotein by rapid subtraction hybridization, RaSH. Oncogene. 21:3592–3602. 2002.PubMed/NCBI View Article : Google Scholar
2	Thirkettle HJ, Girling J, Warren AY, Mills IG, Sahadevan K, Leung H, Hamdy F, Whitaker HC and Neal DE: LYRIC/AEG-1 is targeted to different subcellular compartments by ubiquitinylation and intrinsic nuclear localization signals. Clin Cancer Res. 15:3003–3013. 2009.PubMed/NCBI View Article : Google Scholar
3	Yoo BK, Emdad L, Lee SG, Su ZZ, Santhekadur P, Chen D, Gredler R, Fisher PB and Sarkar D: Astrocyte elevated gene-1 (AEG-1): A multifunctional regulator of normal and abnormal physiology. Pharmacol Ther. 130:1–8. 2011.PubMed/NCBI View Article : Google Scholar
4	Emdad L, Sarksr D, Su ZZ, Randolph A, Boukerche H, Valerie K and Fisher PB: Activation of the nuclear factor kappaB pathway by astrocyte elevated gene-1: Implications for tumor progression and metastasis. Cancer Res. 66:1509–1516. 2006.PubMed/NCBI View Article : Google Scholar
5	Emdad L, Sarkar D, Su ZZ, Lee SG, Kang DC, Bruce JN, Volsky DJ and Fisher PB: Astrocyte elevated gene-1: Recent insights into a novel gene involved in tumor progression, metastasis and neurodegeneration. Pharmacol Ther. 114:155–170. 2007.PubMed/NCBI View Article : Google Scholar
6	Zhang C, Li HZ, Qian BJ, Liu CM, Guo F and Lin MC: MTDH/AEG-1-based DNA vaccine suppresses metastasis and enhances chemosensitivity to paclitaxel in pelvic lymph node metastasis. Biomed Pharmacother. 70:217–226. 2015.PubMed/NCBI View Article : Google Scholar
7	Roussel BD, Kruppa AJ, Miranda E, Crowther DC, Lomas DA and Marciniak SJ: Endoplasmic reticulum dysfunction in neurological disease. Lancet Neuro1. 12:105–118. 2013.PubMed/NCBI View Article : Google Scholar
8	Anttila V, Stefansson H, Kallela M, Todt U, Terwindt GM, Calafato MS, Nyholt DR, Dimas AS, Freilinger T, Müller-Myhsok B, et al: Genome-wide association study of migraine implicates a common susceptibility variant on 8q22.1. Nat Genet. 42:869–873. 2010.PubMed/NCBI View Article : Google Scholar
9	Kang DC, Su ZZ, Sarkar D, Emdad L, Volsky DJ and Fisher PB: Cloning and characterization of HIV-1-inducible astrocyte elevated gene-1, AEG-1. Gene. 353:8–15. 2005.PubMed/NCBI View Article : Google Scholar
10	Lee SG, Kim K, Kegelman TP, Dash R, Das SK, Choi JK, Emdad L, Howlett EL, Jeon HY, Su ZZ, et al: Oncogene AEG-1 promotes glioma-induced neurodegeneration by increasing glutamate excitotoxicity. Cancer Res. 71:6514–6523. 2011.PubMed/NCBI View Article : Google Scholar
11	Vartak-Sharma N and Ghorpade A: Astrocyte elevated gene-1 regulates astrocyte responses to neural injury: Implications for reactive astrogliosis and neurodegeneration. J Neuroinflammation. 9(195)2012.PubMed/NCBI View Article : Google Scholar
12	Yin X, Ren M, Jiang H, Cui S, Wang S, Jiang H, Qi Y, Wang J, Wang X, Dong G, et al: Downregulated AEG-1 together with inhibited PI3K/Akt pathway is associated with reduced viability of motor neurons in an ALS model. Mol Cell Neurosci. 68:303–313. 2015.PubMed/NCBI View Article : Google Scholar
13	Leem E, Kim HJ, Choi M, Kim S, Oh YS, Lee KJ, Choe YS, Um JY, Shin WH, Jeong JY, et al: Upregulation of neuronal astrocyte elevated gene-1 protects nigral dopaminergic neurons in vivo. Cell Death Dis. 9(449)2018.PubMed/NCBI View Article : Google Scholar
14	Jeon HY, Choi M, Howlett EL, Vozhilla N, Yoo BK, Lloyd JA, Sarkar D, Lee SG and Fisher PB: Expression patterns of astrocyte elevated gene-1 (AEG-1) during development of the mouse embryo. Gene Expr Patterns. 10:361–367. 2010.PubMed/NCBI View Article : Google Scholar
15	Carnemolla A, Fossale E, Agostoni E, Michelazzi S, Calligaris R, De Maso L, Del Sal G, MacDonald ME and Persichetti F: Rrs1 is involved in endoplasmic reticulum stress response in Huntington disease. J Biol Chem. 284:18167–18173. 2009.PubMed/NCBI View Article : Google Scholar
16	Garneau JE, Dupuis MÈ, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadán AH and Moineau S: The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature. 468:67–71. 2010.PubMed/NCBI View Article : Google Scholar
17	Doudna JA and Charpentier E: Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science: Nov 28, 2014 (Epub ahead of print).
18	Caldwell JD, Shapiro RA, Jirikowski GF and Suleman F: Internalization of sex hormone-binding globulin into neurons and brain cells in vitro and in vivo. Neuroendocrinology. 86:84–93. 2007.PubMed/NCBI View Article : Google Scholar
19	Murphy TH, Miyamoto M, Sastre A, Schnaar RL and Coyle JT: Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress. Neuron. 2:1547–1558. 1989.PubMed/NCBI View Article : Google Scholar
20	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001.PubMed/NCBI View Article : Google Scholar
21	Florea L, Song L and Salzberg SL: Thousands of exon skipping events differentiate among splicing patterns in sixteen human tissues. F1000Res. 2(188)2013.PubMed/NCBI View Article : Google Scholar
22	Robinson MD, McCarthy DJ and Smyth GK: edgeR: A bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 26:139–140. 2010.PubMed/NCBI View Article : Google Scholar
23	Anders S and Huber W: Differential expression analysis for sequence count data. Genome Biol. 11(R106)2010.PubMed/NCBI View Article : Google Scholar
24	Benjamini Y and Hochberg Y: Controlling the false discovery rate: A practical and powerful approach to multiple testing. J R Stat Soc B (Methodol). 57:289–300. 1995.
25	Harris MA, Clark J, Ireland A, Lomax J, Ashburner M, Foulger R, Eilbeck K, Lewis S, Marshall B, Mungall C, et al: The gene ontology (GO) database and informatics resource. Nucleic Acids Res. 32:D258–D261. 2004.PubMed/NCBI View Article : Google Scholar
26	Yu G, Wang LG, Han Y and He QY: clusterProfiler: An R package for comparing biological themes among gene clusters. OMICS. 16:284–287. 2012.PubMed/NCBI View Article : Google Scholar
27	Kanehisa M, Sato Y, Kawashima M, Furumichi M and Tanabe M: KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res. 44:D457–D462. 2016.PubMed/NCBI View Article : Google Scholar
28	von Mering C, Jensen LJ, Snel B, Hooper SD, Krupp M, Foglierini M, Jouffre N, Huynen MA and Bork P: STRING: Known and predicted protein-protein associations, integrated and transferred across organisms. Nucleic Acids Res. 33:D433–D437. 2005.PubMed/NCBI View Article : Google Scholar
29	Xu WH, Xu Y, Wang J, Wan FN, Wang HK, Cao DL, Shi GH, Qu YY, Zhang HL and Ye DW: Prognostic value and immune infiltration of novel signatures in clear cell renal cell carcinoma microenvironment. Aging (Albany NY). 11:6999–7020. 2019.PubMed/NCBI View Article : Google Scholar
30	Chen Q, Yu D, Zhao Y, Qiu J, Xie Y and Tao M: Screening and identification of hub genes in pancreatic cancer by integrated bioinformatics analysis. J Cell Biochem. 120:19496–19508. 2019.PubMed/NCBI View Article : Google Scholar
31	Stephenson AA, Raper AT and Suo Z: Bidirectional degradation of DNA cleavage products catalyzed by CRISPR/Cas9. J Am Chem Soc. 140:3743–3750. 2018.PubMed/NCBI View Article : Google Scholar
32	Bernardino L, Agasse F, Silva B, Ferreira R, Grade S and Malva JO: Tumor necrosis factor-alpha modulates survival, proliferation, and neuronal differentiation in neonatal subventricular zone cell cultures. Stem Cells. 26:2361–2371. 2008.PubMed/NCBI View Article : Google Scholar
33	Li X, Bechara R, Zhao J, McGeachy MJ and Gaffen SL: IL-17 receptor-based signaling and implications for disease. Nat Immunol. 20:1594–1602. 2019.PubMed/NCBI View Article : Google Scholar
34	Liu W, Ye J and Yan H: Investigation of key genes and pathways in inhibition of oxycodone on vincristine-induced microglia activation by using bioinformatics analysis. Dis Markers. 2019(3521746)2019.PubMed/NCBI View Article : Google Scholar
35	Ng SR, Rideout WM III, Akama-Garren EH, Bhutkar A, Mercer KL, Schenkel JM, Bronson RT and Jacks T: CRISPR-mediated modeling and functional validation of candidate tumor suppressor genes in small cell lung cancer. Proc Natl Acad Sci USA. 117:513–521. 2020.PubMed/NCBI View Article : Google Scholar
36	He XY, Ren XH, Peng Y, Zhang JP, Ai SL, Liu BY, Xu C and Cheng SX: Aptamer/peptide-functionalized genome-editing system for effective immune restoration through reversal of PD-L1-mediated cancer immunosuppression. Adv Mater. 32(2000208)2020.PubMed/NCBI View Article : Google Scholar
37	Ling X, Xie B, Gao X, Chang L, Zheng W, Chen H, Huang Y, Tan L, Li M and Liu T: Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates. Sci Adv. 6(eaaz0051)2020.PubMed/NCBI View Article : Google Scholar
38	LaFountaine JS, Fathe K and Smyth HD: Delivery and therapeutic applications of gene editing technologies ZFNs, TALENs, and CRISPR/Cas9. Int J Pharm. 494:180–194. 2015.PubMed/NCBI View Article : Google Scholar
39	Zhou Z, Tan H, Li Q, Chen J, Gao S, Wang Y, Chen W and Zhang L: CRISPR/Cas9-mediated efficient targeted mutagenesis of RAS in Salvia miltiorrhiza. Phytochemistry. 148:63–70. 2018.PubMed/NCBI View Article : Google Scholar
40	Noch EK and Khalili K: The role of AEG-1/MTDH/LYRIC in the pathogenesis of central nervous system disease. Adv Cancer Res. 120:159–192. 2013.PubMed/NCBI View Article : Google Scholar
41	Wang Y, Zhang W, Zhu X and Wang Y, Mao X, Xu X and Wang Y: Upregulation of AEG-1 involves in schwann cell proliferation and migration after sciatic nerve crush. J Mol Neurosci. 60:248–257. 2016.PubMed/NCBI View Article : Google Scholar
42	Vartak-Sharma N, Nooka S and Ghorpade A: Astrocyte elevated gene-1 (AEG-1) and the A(E)Ging HIV/AIDS-HAND. Prog Neurobiol. 157:133–157. 2017.PubMed/NCBI View Article : Google Scholar
43	Serviddio G, Romano AD, Cassano T, Bellanti F, Altomare E and Vendemiale G: Principles and therapeutic relevance for targeting mitochondria in aging and neurodegenerative diseases. Curr Pharm Des. 17:2036–2055. 2011.PubMed/NCBI View Article : Google Scholar
44	Mohan H, Verhoog MB, Doreswamy KK, Eyal G, Aardse R, Lodder BN, Goriounova NA, Asamoah B, B Brakspear AB, Groot C, et al: Dendritic and axonal architecture of individual pyramidal neurons across layers of adult human neocortex. Cereb Cortex. 25:4839–4853. 2015.PubMed/NCBI View Article : Google Scholar
45	Jan YN and Jan LY: Branching out: Mechanisms of dendritic arborization. Nat Rev Neurosci. 11:316–328. 2010.PubMed/NCBI View Article : Google Scholar
46	Yoo BK, Emdad L, Su ZZ, Villanueva A, Chiang DY, Mukhopadhyay ND, Mills AS, Waxman S, Fisher RA, Llovet JM, et al: Astrocyte elevated gene-1 regulates hepatocellular carcinoma development and progression. J Clin Invest. 119:465–477. 2009.PubMed/NCBI View Article : Google Scholar
47	Yu C, Chen K, Zheng H, Guo X, Jia W, Li M, Zeng M, Li J and Song L: Overexpression of astrocyte elevated gene-1 (AEG-1) is associated with esophageal squamous cell carcinoma (ESCC) progression and pathogenesis. Carcinogenesis. 30:894–901. 2009.PubMed/NCBI View Article : Google Scholar
48	Lee SG, Su ZZ, Emdad L, Sarkar D and Fisher PB: Astrocyte elevated gene-1 (AEG-1) is a target gene of oncogenic Ha-ras requiring phosphatidylinositol 3-kinase and c-Myc. Proc Natl Acad Sci USA. 103:17390–17395. 2006.PubMed/NCBI View Article : Google Scholar
49	Rohm B, Ottemeyer A, Lohrum M and Püschel AW: Plexin/neuropilin complexes mediate repulsion by the axonal guidance signal semaphorin 3A. Mech Dev. 93:95–104. 2000.PubMed/NCBI View Article : Google Scholar
50	Rolny C, Capparuccia L, Casazza A, Mazzone M, Vallario A, Cignetti A, Medico E, Carmeliet P, Comoglio PM and Tamagnone L: The tumor suppressor semaphorin 3B triggers a prometastatic program mediated by interleukin 8 and the tumor microenvironment. J Exp Med. 205:1155–1171. 2008.PubMed/NCBI View Article : Google Scholar
51	Mumm JS, Schroeter EH, Saxena MT, Griesemer A, Tian X, Pan DJ, Ray WJ and Kopan R: A ligand-induced extracellular cleavage regulates gamma-secretase-like proteolytic activation of Notch1. Mol Cell. 5:197–206. 2000.PubMed/NCBI View Article : Google Scholar
52	Liu X, Yang Z, Yin Y and Deng X: Increased expression of Notch1 in temporal lobe epilepsy: Animal models and clinical evidence. Neural Regen Res. 9:526–533. 2014.PubMed/NCBI View Article : Google Scholar
53	Feng S, Shi T, Qiu J, Yang H, Wu Y, Zhou W, Wang W and Wu H: Notch1 deficiency in postnatal neural progenitor cells in the dentate gyrus leads to emotional and cognitive impairment. FASEB J. 31:4347–4358. 2017.PubMed/NCBI View Article : Google Scholar
54	Shu Y, Xiao B, Wu Q, Liu T, Du Y, Tang H, Chen S, Feng L, Long L and Li Y: The ephrin-A5/EphA4 interaction modulates neurogenesis and angiogenesis by the p-Akt and p-ERK pathways in a mouse model of TLE. Mol Neurobiol. 53:561–576. 2016.PubMed/NCBI View Article : Google Scholar
55	Feng L, Shu Y, Wu Q, Liu T, Long H, Yang H, Li Y and Xiao B: EphA4 may contribute to microvessel remodeling in the hippocampal CA1 and CA3 areas in a mouse model of temporal lobe epilepsy. Mol Med Rep. 15:37–46. 2017.PubMed/NCBI View Article : Google Scholar
56	Salińska E and Łazarewicz JW: Role of calcium in physiology and pathology of neurons. Postepy Biochem. 58:403–417. 2012.PubMed/NCBI(In Polish).
57	Chadwick L, Gentle L, Strachan J and Layfield R: Review: Unchained maladie-a reassessment of the role of Ubb(+1)-capped polyubiquitin chains in Alzheimer's disease. Neuropathol Appl Neurobiol. 38:118–131. 2012.PubMed/NCBI View Article : Google Scholar
58	Manavalan A, Mishra M, Feng L, Sze SK, Akatsu H and Heese K: Brain site-specific proteome changes in aging-related dementia. Exp Mol Med. 45(e39)2013.PubMed/NCBI View Article : Google Scholar
59	Kothur K, Bandodkar S, Wienholt L, Chu S, Pope A, Gill D and Dale RC: Etiology is the key determinant of neuroinflammation in epilepsy: Elevation of cerebrospinal fluid cytokines and chemokines in febrile infection-related epilepsy syndrome and febrile status epilepticus. Epilepsia. 60:1678–1688. 2019.PubMed/NCBI View Article : Google Scholar
60	Silva RL, Lopes AH, Guimaraes RM and Cunha TM: CXCL1/CXCR2 signaling in pathological pain: Role in peripheral and central sensitization. Neurobiol Dis. 105:109–116. 2017.PubMed/NCBI View Article : Google Scholar
61	Ringland C, Schweig JE, Eisenbaum M, Paris D, Ait-Ghezala G, Mullan M, Crawford F, Abdullah L and Bachmeier C: MMP9 modulation improves specific neurobehavioral deficits in a mouse model of Alzheimer's disease. BMC Neurosci. 22(39)2021.PubMed/NCBI View Article : Google Scholar