Transcriptomic Analysis of Human Astrocytes In Vitro Reveals Hypoxia-Induced Mitochondrial Dysfunction, Modulation of Metabolism, and Dysregulation of the Immune Response
Abstract
:1. Introduction
2. Results
2.1. Hypoxia Significantly Alters the Astrocyte Transcriptome
2.2. Hypoxia Modulates the Metabolic Profile of Astrocytes
2.3. Hypoxia Modulates the Astrocyte Immune Response
2.4. Validation of Candidate Gene Expression Changes
2.5. Functional Validation of Hypoxia-Induced Changes in Astrocyte Metabolism
3. Discussion
4. Materials and Methods
4.1. Primary Human Astrocytes
4.2. Microarray Analysis of Hypoxia-Induced Gene Expression Changes
4.3. Validation of Microarray Data: Quantitative Real-Time Polymerase Chain Reaction (qPCR)
4.4. XF Bioanalyser Metabolic Flux Analysis
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
AD | Alzheimer’s disease |
ADAM | a disintegrin and metalloproteinase |
ALS | amyotrophic lateral sclerosis |
ATP | adenosine tri-phosphate |
CNS | central nervous system |
CR | coupled to respiration ATP-linked flux |
DAVID | Database for Annotation Visualisation and Integrated Discovery |
ECAR | extracellular acidification rate |
ETC | electron transport chain |
FC | fold change |
FCCP | carbonyl cyanide p-trifluoromethoxy phenylhydrazone |
HIF-1 | hypoxia inducible factor-1 |
HREs | hypoxia responsive elements |
IGF | insulin-like growth factor |
IGFR | insulin-like growth factor receptors |
IL | interleukin |
IR | insulin receptor |
IRS | receptor substrates |
KEGG | Kyoto Encyclopedia of Genes and Genomes |
MMP | matrix metalloproteinases |
MR | mitochondrial respiratory flux |
NGF | non-glycolytic flux |
OCR | oxygen consumption rate |
PD | Parkinson’s disease |
PL | proton leak linked flux |
PrG | pre-glucose |
PsG | post-glucose |
QPCR | quantitative polymerase chain reaction |
RIN | RNA Integrity Number |
SRC | spare respiratory capacity |
TCA | tri-carboxylic acid cycle |
TIMP | tissue inhibitor of metalloproteinase |
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Transcript | Gene Symbol | Gene Name | FC | p-Value |
---|---|---|---|---|
202912_at | ADM | adrenomedullin | 3.4 | 0.011 |
217254_s_at | EPO | erythropoietin | 1.9 | 0.039 |
209328_x_at | HIGD2A | HIG1 hypoxia inducible domain family, member 2A | 1.3 | 0.042 |
209329_x_at | HIGD2A | HIG1 hypoxia inducible domain family, member 2A | 1.6 | 0.040 |
218507_at | HILPDA | hypoxia inducible lipid droplet-associated | 6.1 | 0.018 |
1554452_a_at | HILPDA | hypoxia inducible lipid droplet-associated | 4.4 | 0.018 |
207092_at | LEP | leptin | 1.6 | 0.030 |
229093_at | NOS3 | nitric oxide synthase 3 (endothelial cell) | 6.4 | 0.028 |
205581_s_at | NOS3 | nitric oxide synthase 3 (endothelial cell) | 1.8 | 0.035 |
217112_at | PDGFB | platelet-derived growth factor beta | 2.3 | 0.048 |
216061_x_at | PDGFB | platelet-derived growth factor beta | 1.8 | 0.028 |
203400_s_at | TF | transferrin | 1.8 | 0.031 |
212171_x_at | VEGFA | vascular endothelial growth factor A | 4.2 | 0.020 |
211527_x_at | VEGFA | vascular endothelial growth factor A | 2.7 | 0.054 |
210513_s_at | VEGFA | vascular endothelial growth factor A | 2.7 | 0.030 |
203683_s_at | VEGFB | vascular endothelial growth factor B | 2.0 | 0.052 |
209946_at | VEGFC | vascular endothelial growth factor C | 1.3 | 0.051 |
KEGG Pathway | p-Value |
---|---|
Hypoxia inducible factor-1 (HIF-1) signalling pathway | 0.002 |
Phosphatidylinositol 3-kinase and protein kinase B (PI3K-Akt) signalling pathway | 0.005 |
Renal cell carcinoma | 0.006 |
Glycolysis/Gluconeogenesis | 0.006 |
Staphylococcus aureus infection | 0.007 |
Oxytocin signalling pathway | 0.010 |
Pathways in cancer | 0.013 |
Insulin resistance | 0.014 |
Human T- cell leukemia virus, type 1 (HTLV-I) infection | 0.019 |
Biosynthesis of amino acids | 0.030 |
adenosine monophosphate-activated protein kinase (AMPK) signalling pathway | 0.032 |
Cell adhesion molecules (CAMs) | 0.035 |
Circadian rhythm | 0.038 |
Fructose and mannose metabolism | 0.043 |
Intestinal immune network for IgA production | 0.045 |
Graft-versus-host disease | 0.047 |
KEGG Pathway | p-Value |
---|---|
Cell cycle | 6.1 × 10−9 |
RNA transport | 1.9 × 10−7 |
Ubiquitin mediated proteolysis | 4.5 × 10−7 |
Oocyte meiosis | 1.9 × 10−5 |
Protein processing in endoplasmic reticulum | 5.6 × 10−5 |
RNA degradation | 1.6 × 10−4 |
Spliceosome | 3.6 × 10−4 |
Wnt signalling | 0.002 |
Endocytosis | 0.006 |
Pyramidine metabolism | 0.008 |
Progesterone-mediated oocyte maturation | 0.012 |
Peroxisome | 0.015 |
Tricarboxylic acid cycle (TCA cycle) | 0.016 |
p53 signalling | 0.016 |
DNA replication | 0.019 |
Pathways in cancer | 0.02 |
Purine metabolism | 0.02 |
Valine, leucine and isoleucine degradation | 0.02 |
RNA polymerase | 0.02 |
Fatty acid metabolism | 0.03 |
Protein export | 0.03 |
Chronic myeloid leukemia | 0.03 |
mRNA surveillance pathway | 0.04 |
HTLV-I infection | 0.04 |
Gene | Sequence | |
---|---|---|
DLAT | Probe | 56-FAM/CGCTGTGCA/ZEN/ATAACCCGACGAATG/3IABkFQ |
Primer 1 | CCAGTTCCTACAGGTGTCTTC | |
Primer 2 | TGAGGTATGGTTTGCTTTGATTG | |
HLADPB1 | Probe | 56-FAM/CCCACTCCA/ZEN/CAGATGATGAGCCC/3IABkFQ |
Primer 1 | GCTCCTCCTGTGCATGAAG | |
Primer 2 | CAAGTGGAGCACACCAG | |
MFN1 | Probe | 56-FAM/AGCTTCTAC/ZEN/TCCCACTGCTCCTACC/3IABkFQ |
Primer 1 | GAAATGCTCAAAGGGTGCTC | |
Primer 2 | GTGATGCATTATCTGGCGTTG | |
MT1X | Probe | 56-FAM/AGCTCGCCA/ZEN/TGGATCCCAACT/3IABkFQ |
Primer 1 | GCAACCTGTCCCGACTCTA | |
Primer 2 | AGCTTTTCTTGCAGGAGGTG | |
NDUFAF4 | Probe | 56-FAM/CTGTGTCTT/ZEN/CCTTGCAGGTAAAAGCTG/3IABkFQ |
Primer 1 | CCAGAAGTTAAAGGAGAGATTGCT | |
Primer 2 | GAATTCCTTCGGCTCTTGAC | |
SUCLG2 | Probe | 56-FAM/CACAGCTGA/ZEN/TCCTAAGGTTGAAGCCA/3IABkFQ |
Primer 1 | TTGGAGGTGGTGTAAAGGAAG | |
Primer 2 | GGCAATGATGGCACAGTTG | |
ACTB | Probe | 56-FAM/CTGCCTCCA/ZEN/CCCACTCCCA/3IABkFQ |
Primer 1 | GTCCCCCAACTTGAGATGTATG | |
Primer 2 | AAGTCAGTGTACAGGTAAGCC | |
GAPDH | Probe | 56-FAM/AAGGTCGGA/ZEN/GTCAACGGATTTGGTC/3IABkFQ |
Primer 1 | ACATCGCTCAGACACCATG | |
Primer 2 | TGTAGTTGAGGTCAATGAAGGG |
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Allen, S.P.; Seehra, R.S.; Heath, P.R.; Hall, B.P.C.; Bates, J.; Garwood, C.J.; Matuszyk, M.M.; Wharton, S.B.; Simpson, J.E. Transcriptomic Analysis of Human Astrocytes In Vitro Reveals Hypoxia-Induced Mitochondrial Dysfunction, Modulation of Metabolism, and Dysregulation of the Immune Response. Int. J. Mol. Sci. 2020, 21, 8028. https://doi.org/10.3390/ijms21218028
Allen SP, Seehra RS, Heath PR, Hall BPC, Bates J, Garwood CJ, Matuszyk MM, Wharton SB, Simpson JE. Transcriptomic Analysis of Human Astrocytes In Vitro Reveals Hypoxia-Induced Mitochondrial Dysfunction, Modulation of Metabolism, and Dysregulation of the Immune Response. International Journal of Molecular Sciences. 2020; 21(21):8028. https://doi.org/10.3390/ijms21218028
Chicago/Turabian StyleAllen, Scott P., Rajpinder Singh Seehra, Paul R. Heath, Benjamin P. C. Hall, Jessica Bates, Claire J. Garwood, Martyna M. Matuszyk, Stephen B. Wharton, and Julie E. Simpson. 2020. "Transcriptomic Analysis of Human Astrocytes In Vitro Reveals Hypoxia-Induced Mitochondrial Dysfunction, Modulation of Metabolism, and Dysregulation of the Immune Response" International Journal of Molecular Sciences 21, no. 21: 8028. https://doi.org/10.3390/ijms21218028