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Similar to MicroRNA-214 targets COX-2 to antagonize indoxyl sulfate(IS)-induced endothelial cell apoptosis
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MicroRNA-214 targets COX-2 to antagonize indoxyl sulfate(IS)-induced endothelial cell apoptosis
- 1. ANA ISABEL MORENO GARCÍA THIRD SEMESTER 2020-1
- 2. INTRODUCTION 2 coX-2 is Used in the production of prostaglandins, prostacyclins and thromboxanes from arachidonic acid Membrane protein Located in microsomes (ER fragments) and in the endoplasmic reticulum Common dietary metabolite. Uremic toxin. Stimulates glomerular sclerosis and intestinal fibrosis. Induces endothelial dysfunction by inhibiting endothelial proliferation and migration.https://singularityhub.com/2019/04/03/synthetic-cell-co mponent-expands-the-code-of-life-in-complex-cells/
- 3. APOPTOSIS 3 X O2 DNA damage P53 Radiation Chemotherapy RO S Cytochrome C BAX APAF-1 Procaspase 9 SMAC DIABLO BH3 Only TNF-α Membrane pores Activation of: Cs 8, Cs9------Cs 3, 6 COX-2 PG TX Inflammatory RTA BCL2 BCLX
- 4. OBJECTIVE Investigate if MicroRNA-214 targets COX-2 to antagonize indoxyl sulfate(IS)-induced endothelial cell apoptosis
- 5. 5 Materials and methods MAEC DMEM Penicillin FBS Streptomycin miR-214 INH DNA sequence of miR-214, IS and COX-2 in mice aortic cells to follow the cardiac damage or damage inhibition using the mutated enzymes and inhibitors PCR-RTq Transfection ELISA Western Blot
- 6. WESTERN BLOT ✘ Allows to detect a protein of interest from a pool of proteins ✘ Uses gel, SDS page, transfer apparatus and chemical or electrical stimulation ✘ Takes lysate cells or protein extracts for adding it to the gel ✘ Separates the proteins according to their molecular weight ✘ Transferred to a filter of antibodies 6 PCR-RTq: Real time PCR ✘ Monitors the accumulation of a DNA product from a PCR reaction in real time ✘ Quantifies the amount of DNA in the sample at the start of the reaction ✘ Uses probes for denature and anneal the target sequence 1. Denaturation 2. Annealing 3. Elongation
- 7. Used for Quantify and detect antigens and antibodies in a sample ELISA Basis ✘ Antigens obtained from recombinant RNA are added ✘ They are fixed to the microtiter plate ✘ Immunocomplex reactions form ✘ Substrates and cormogens are added to demonstrate the immune reaction of the existence of complexes 7 https://www.raybiotech.com/phosphorylation-mk k3-s189-elisa/
- 8. TRANSFECTION Used for ✘ Generate cells and animals model in vitro on isolated ✘ Study gene expression and proteins function by introducing foreign material into a cell Basis ✘ Allows penetration of the cell membrane and nuclear envelope due nucleotide and cell membrane negative charge ✘ It can use high voltage electric shocks to induce nucleic acids into cells by creating pores 8 https://www.polyplus-transfection.com/about-us/what-is-transfection/
- 9. RESULTS 9 ● [IS] ● Pro-apoptotic molecules ● GAPDH Pro-apoptotic molecule activity against the inhibitor (miR-214) and the inducer (IS)
- 10. 10 COX-2 activity increases when IS concentrations are high COX-2 activity when IS and miR-214 induced activates apoptosis Anti-miR-214 and IS modificate the activity of COX-2 RESULTS
- 11. 11 NS-398 (Mutated COX-2) increases the normal COX-2 activity Caspase 3 activity increases the most when only IS concentrations predomine over other molecules RESULTS
- 12. DISCUSSION 12 AUTHOR CONTRIBUTION OR Williams KC, et al. The upregulation of endothelial cell miR-214 suppressed cell apoptosis mediated by IS possibly via targeting COX-2 for the first time Yin J, et al. IS increased COX-2 expression accompanied with miR-214 reduction suggesting COX-2 might be a possible target of miR-214 Carmona A, et al. IS has different deleterious effect on endothelial cells
- 13. CONCLUSIONS 13 miR-214 has a protective effect over the DNA of endothelial cells when they are induced to deleterious effect of IS, showing an effective and possible treatment to avoid mortality on cardiovascular diseases in patients with chronic renal failure COX-2 can be the principal targeting of miR-214 to avoid inflammation process in cells with induced apoptosis due deleterious molecules
- 15. 15 THANKS!Any questions? You can find me at anamorenog@upb.edu.co