The DNA Glycosylase NEIL2 Suppresses Fusobacterium-Infection-Induced Inflammation and DNA Damage in Colonic Epithelial Cells
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals
2.2. Bacterial Cultures
2.3. Isolation and Maintenance of Colonic Organoids
2.4. The Preparation of Enteroid-Derived Monolayers (EDMs)
2.5. RNA Isolation and qPCR for Gene Expression Analysis
2.6. Proteome Profiler Array
2.7. The Measurement of Interlekin (IL)-8 Cytokine by ELISA
2.8. Immunoblot for Detection of Base Excision Repair and Cellular Signaling Proteins
2.9. Assessment of γH2AX Phosphorylation by Immunofluorescence (IF)
2.10. Analysis of Accumulation of DNA Strand Break by Long Amplicon Quantitative PCR (LAqPCR)
2.11. Measurement of Oxidative DNA/RNA Damage
2.12. Lactate Dehydrogenase (LDH) Assay
2.13. Analysis of Datasets with the CRC Patients
2.14. Statistics
3. Results
3.1. NEIL2 is Downregulated in the Human Colonic EDMs Following Fn Infection
3.2. Fn Infection Suppressed the Expression of NEIL2 in the Murine Colonic EDMs
3.3. The Role of NEIL2 in Controlling Inflammation and DNA Strand-Break Accumulation Following Fn Infection in Colonic EDMs
3.4. NEIL2 Knockdown Exacerbated Fn-Induced DNA Double-Strand Breaks (DSBs)
3.5. Fn Infection Downregulated NEIL2 and Increased Oxidative Damage in the Murine CRC Model
3.6. NEIL2 Is Downregulated in the MSS-CRCs Patients
4. Discussion
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
EDTA | Ethylenediaminetetraacetic acid |
TGFb | Transforming Growth Factor b |
PVDF | Polyvinylidene difluoride |
NFkB | Nuclear factor kappa-light-chain enhancer of activated B-cells |
HEPES | 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid |
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Gene | Species | Forward Primer (5’ → 3’) | Reverse Primer (5′ → 3’) |
---|---|---|---|
18srRNA | GTAACCCGTTGAACCCCATT | CCATCCAATCGGTAGTAGCG | |
NEIL1 | mouse | TCGTAGACATCCGTCGCTTT | TGTCTGATAGGTTCCGAAGTACG |
NEIL2 | mouse | CTGCCGCCTTTCAGTCTCT | TCTGGATCAAACCGAAGGAA |
NTH1 | mouse | GCATGAACTCAGGGAAGGAAGA | CCTCACCATTAGCCGCTTCA |
OGG1 | mouse | TTATCATGGCTTCCCAAACC | GTACCCCAGGCCCAACTT |
NEIL1 | human | CCAGGCAGTGGGAAGTCA | AGGGAGGGTGGCAGAGTC |
NEIL2 | human | GGGGCAGCAGTAAGAAGCTA | GGAATAATTTCTTTCCATGGACCT |
NTH1 | human | GACAGCATCCTGCAGACAGA | TTGATGTATTTCACCTTGCTCCT |
OGG1 | human | CCAGACCAACAAGGAACTGG | CAAATGCATTGCCAAGGA |
IL-8/KC | mouse | CGCTTCTCTGTGCAGCGCTGCTGCT | AAGCCTCGCGACCATTCTTGAGTC |
IL-8 | Human | GAGCACTCCATAAGGCACAAA | ATGGTTCCTTCCGGTGGT |
MLH1 | Human | GATTACCCCTTCTGATTGACA | ACTGAGGCTTTCAAAACA |
MLH3 | Human | CGCACGAGCCTCAAGATCC | TCTGACTGGAAATAATTGCCTGGA |
MSH2 | Human | CAGTATATTGGAGAATCGCA | AGGGCATTTGTTTCACC |
MSH6 | Human | GCTTCTTCCCCAAGTCTCCG | AGAAGTCACAACTGGTGGGG |
PMS2 | Human | ACTGAGTCTAAGCACTGCGG | TGACATCGCTCAGTGCACAA |
Ku70 | Human | CCACAGGAAGAAGAGTTGGA | CTGCTCTGGAGTTGCCATGA |
Primer Name | Gene Name | Sequences | PCR |
---|---|---|---|
m-Polβ-LA-F | Polβ (mouse) | TAT CTC TCT TCC TCT TCA CTT CTC CCC TGG | LA-qPCR |
m-Polβ-LA-R | Polβ (mouse) | CGT GAT GCC GCC GTT GAG GGT CTC CTG | LA-qPCR |
m-β Globin-LA-F | β globin (mouse) | TTG AGA CTG TGA TTG GCA ATG CCT | LA-qPCR |
m-β Globin-LA-R | β globin (mouse) | CCT TTA ATG CCC ATC CCG GAC T | LA-qPCR |
m-Polβ-SA-F | Polβ (mouse) | TATGGACCCCCATGAGGAACA | SA-PCR |
m-Polβ-SA-R | Polβ (mouse) | AACCGTCGGCTAAAGACGTG | SA-PCR |
m-β Globin-SA-F | β globin (mouse) | ACACTACTCAGAGTGAGACCCA | SA-PCR |
m-β Globin-SA-R | β Globin (mouse) | ATACCCAATGCTGGCTCCTG | SA-PCR |
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Sayed, I.M.; Chakraborty, A.; Abd El-Hafeez, A.A.; Sharma, A.; Sahan, A.Z.; Huang, W.J.M.; Sahoo, D.; Ghosh, P.; Hazra, T.K.; Das, S. The DNA Glycosylase NEIL2 Suppresses Fusobacterium-Infection-Induced Inflammation and DNA Damage in Colonic Epithelial Cells. Cells 2020, 9, 1980. https://doi.org/10.3390/cells9091980
Sayed IM, Chakraborty A, Abd El-Hafeez AA, Sharma A, Sahan AZ, Huang WJM, Sahoo D, Ghosh P, Hazra TK, Das S. The DNA Glycosylase NEIL2 Suppresses Fusobacterium-Infection-Induced Inflammation and DNA Damage in Colonic Epithelial Cells. Cells. 2020; 9(9):1980. https://doi.org/10.3390/cells9091980
Chicago/Turabian StyleSayed, Ibrahim M., Anirban Chakraborty, Amer Ali Abd El-Hafeez, Aditi Sharma, Ayse Z. Sahan, Wendy Jia Men Huang, Debashis Sahoo, Pradipta Ghosh, Tapas K. Hazra, and Soumita Das. 2020. "The DNA Glycosylase NEIL2 Suppresses Fusobacterium-Infection-Induced Inflammation and DNA Damage in Colonic Epithelial Cells" Cells 9, no. 9: 1980. https://doi.org/10.3390/cells9091980