Effect of METTL3 Gene on Lipopolysaccharide Induced Damage to Primary Small Intestinal Epithelial Cells in Sheep
Abstract
:1. Introduction
2. Results
2.1. Effects of Different Concentrations of LPS on IECs Activity and METTL3 Protein Expression Levels
2.2. LPS Induces Inflammatory Response, Apoptosis, and Oxidative Stress in IECs
2.3. Efficiency Detection of Overexpression and Interference of METTL3
2.4. METTL3 Promotes LPS-Induced Inflammatory Response in IECs
2.5. METTL3 Promotes LPS-Induced Apoptosis in IECs
2.6. METTL3 Promotes LPS-Induced Oxidative Stress and Inhibits Antioxidant Activity in IECs
3. Discussion
4. Materials and Methods
4.1. Immunofluorescence Identification of Sheep IECs
4.2. CCK-8 Assay
4.3. Western Blot
4.4. Total RNA Extraction, cDNA Synthesis and qRT-PCR
4.5. Construction of Overexpression Vectors and Interfering Sequences
4.6. Cell Culture and Cell Transfection
4.7. ELISA Assay
4.8. Apoptosis Detection
4.9. Reactive Oxygen Detection
4.10. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene | Primer Sequence (5′-3′) | Product Size (bp) | Annealing Temperature (°C) |
---|---|---|---|
METTL3 | F: CCCTATGGGACCCTGACAGA R: CCTGTTCGAATGATGCGCTG | 197 | 60 |
β-actin | F: CAGTCGGTTGGATCGAGCAT R: AGAAGGAGGGTGGCTTTTGG | 151 | 60 |
IL1β | F: CGTGCCTACGAACATGTC R: CACCAGGGATTTTTGCTCTC | 168 | 60 |
IL6 | F: GACTTCTGCTTTCCCTACCC R: CACACTCGTCATTCTTCTCAC | 171 | 60 |
TNFα | F: CTCGTATGCCAATGCCCTCA R: TGGTGTGGGTGAGGAACAAG | 149 | 60 |
Caspase-3 | F: ACGTTGTGGCTGAACGTAAA R: GTTTCCCTGAGGTTTGCTGC | 262 | 60 |
Caspase-9 | F: CAACGTGAACTTCTGCCGTG R: CATTTGCTTGGCAGTCAGGT | 136 | 60 |
Bax | F: GCCCTTTTGCTTCAGGGTTT R: GTCCAATGTCCAGCCCATGA | 357 | 60 |
CAT | F: CTATCCTGACACTCACCGCC R: CTTTCAGATGGCCCGCAATG | 335 | 60 |
CuZn-SOD | F: TGATCATGGGTTCCACGTCC R: CACATTGCCCAGGTCTCCAA | 139 | 60 |
Mn-SOD | F: GGATCCCCTGCAAGGAACAA R: CTTGGTGTAAGGCTGACGGT | 189 | 60 |
Primer Name | Primer Sequence (5′-3′) | Product Size (bp) | Annealing Temperature (°C) |
---|---|---|---|
OE-METTL3 | F:ctagcgtttaaacttaagcttATGTCGGACACGTGGAGCTC R:tgctggatatctgcagaattcCTATAGATTCTTAGGTTTAGAGATGATACCAT | 1782 | 62 |
Fragment Name | Sequence (5′-3′) |
---|---|
SiR-METTL3-1616 | F:GCUGAGGUUCGUUCCACUATT R:UAGUGGAACGAACCUCAGCTT |
SiR-METTL3-199 | F: GCACUUGGAUCUUCGGAAUTT R: AUUCCGAAGAUCCAAGUGCTT |
SiR-METTL3-1176 | F:GCGUUGGAGGUGACUCCAATT R:UUGGAGUCACCUCCAACGCTT |
SiR-NC | F:UUCUCCGAACGUGUCACGUTT R:ACGUGACACGUUCGGAGAATT |
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Duan, Y.; Lv, X.; Cao, X.; Sun, W. Effect of METTL3 Gene on Lipopolysaccharide Induced Damage to Primary Small Intestinal Epithelial Cells in Sheep. Int. J. Mol. Sci. 2024, 25, 9316. https://doi.org/10.3390/ijms25179316
Duan Y, Lv X, Cao X, Sun W. Effect of METTL3 Gene on Lipopolysaccharide Induced Damage to Primary Small Intestinal Epithelial Cells in Sheep. International Journal of Molecular Sciences. 2024; 25(17):9316. https://doi.org/10.3390/ijms25179316
Chicago/Turabian StyleDuan, Yanjun, Xiaoyang Lv, Xiukai Cao, and Wei Sun. 2024. "Effect of METTL3 Gene on Lipopolysaccharide Induced Damage to Primary Small Intestinal Epithelial Cells in Sheep" International Journal of Molecular Sciences 25, no. 17: 9316. https://doi.org/10.3390/ijms25179316