miR663 Prevents Epo Inhibition Caused by TNF-Alpha in Normoxia and Hypoxia
Objective. In chronic inflammatory diseases, proinflammatory cytokines such as TNF-α are present in high amounts in the circulation and are associated with anemia in most cases. Experimental studies have shown that TNF-α inhibits the synthesis of erythropoietin (Epo), the main stimulant of hematopoi...
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| Format: | Article |
| Language: | English |
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Wiley
2021-01-01
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| Series: | International Journal of Endocrinology |
| Online Access: | http://dx.doi.org/10.1155/2021/3670499 |
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| author | Mete Ozkurt Thomas Hellwig-Bürgel Reinhard Depping Selda Kadabere Rumeysa Ozyurt Abdullah Karadag Nilüfer Erkasap |
| author_facet | Mete Ozkurt Thomas Hellwig-Bürgel Reinhard Depping Selda Kadabere Rumeysa Ozyurt Abdullah Karadag Nilüfer Erkasap |
| author_sort | Mete Ozkurt |
| collection | DOAJ |
| description | Objective. In chronic inflammatory diseases, proinflammatory cytokines such as TNF-α are present in high amounts in the circulation and are associated with anemia in most cases. Experimental studies have shown that TNF-α inhibits the synthesis of erythropoietin (Epo), the main stimulant of hematopoiesis. Our aim was to figure out which microRNAs are involved in the Epo repression by TNF-α. Methods. First, we determined the dose of TNF-α in HepG2 cells that has no cytotoxic effect by using MTT assays and that inhibits Epo synthesis by qRT-PCR and ELISA. Then, we performed the microRNA array study with TNF-α (20 ng/ml)-treated cells, and the array results were confirmed by qRT-PCR. We transfected the miR663 group with the mimic-miR663 (30 pmol) for 24 hrs; other groups were treated with a transfection reagent followed by treatment of TNF-α for 24 hrs; miR663 groups were treated with TNF-α for 24 hrs; and the control group was incubated with normal medium. We analyzed Epo mRNA levels by qRT-PCR. If mimic-miR663 prevents the Epo repression by TNF-α, more Epo-dependent UT-7 cells would survive. Therefore, we cocultured HepG2 cells with UT-7 cells. The percentage of apoptotic UT-7 cells was determined by TUNEL assays. Results. According to our array study, TNF-α significantly decreases miR663 expression. After transfection of miR663 mimics into HepG2 cells, TNF-alpha was unable to decrease Epo mRNA amounts. Furthermore, mimic-miR663 transfection resulted in a lower apoptosis rate of UT-7 cells in coculture experiments. Conclusions. miR663 is involved in Epo mRNA production and that is able to prevent or reverse the inhibitory effect of TNF-α. In our coculture study, transfecting HepG2 cells with miR663 mimics decreased the apoptosis of UT-7 cells. |
| format | Article |
| id | doaj-art-e9ac4391f4eb411ba1c609fa92438856 |
| institution | Kabale University |
| issn | 1687-8337 1687-8345 |
| language | English |
| publishDate | 2021-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | International Journal of Endocrinology |
| spelling | doaj-art-e9ac4391f4eb411ba1c609fa924388562025-02-03T01:27:19ZengWileyInternational Journal of Endocrinology1687-83371687-83452021-01-01202110.1155/2021/36704993670499miR663 Prevents Epo Inhibition Caused by TNF-Alpha in Normoxia and HypoxiaMete Ozkurt0Thomas Hellwig-Bürgel1Reinhard Depping2Selda Kadabere3Rumeysa Ozyurt4Abdullah Karadag5Nilüfer Erkasap6Department of Physiology, Eskisehir Osmangazi University Medical Faculty, Eskisehir, TurkeyInstitute of Physiology, University of Lubeck, Lubeck, GermanyInstitute of Physiology, University of Lubeck, Lubeck, GermanyDepartment of Physiology, Eskisehir Osmangazi University Medical Faculty, Eskisehir, TurkeyDepartment of Physiology, Eskisehir Osmangazi University Medical Faculty, Eskisehir, TurkeyDepartment of Physiology, Adiyaman University Medical Faculty, Adiyaman, TurkeyDepartment of Physiology, Eskisehir Osmangazi University Medical Faculty, Eskisehir, TurkeyObjective. In chronic inflammatory diseases, proinflammatory cytokines such as TNF-α are present in high amounts in the circulation and are associated with anemia in most cases. Experimental studies have shown that TNF-α inhibits the synthesis of erythropoietin (Epo), the main stimulant of hematopoiesis. Our aim was to figure out which microRNAs are involved in the Epo repression by TNF-α. Methods. First, we determined the dose of TNF-α in HepG2 cells that has no cytotoxic effect by using MTT assays and that inhibits Epo synthesis by qRT-PCR and ELISA. Then, we performed the microRNA array study with TNF-α (20 ng/ml)-treated cells, and the array results were confirmed by qRT-PCR. We transfected the miR663 group with the mimic-miR663 (30 pmol) for 24 hrs; other groups were treated with a transfection reagent followed by treatment of TNF-α for 24 hrs; miR663 groups were treated with TNF-α for 24 hrs; and the control group was incubated with normal medium. We analyzed Epo mRNA levels by qRT-PCR. If mimic-miR663 prevents the Epo repression by TNF-α, more Epo-dependent UT-7 cells would survive. Therefore, we cocultured HepG2 cells with UT-7 cells. The percentage of apoptotic UT-7 cells was determined by TUNEL assays. Results. According to our array study, TNF-α significantly decreases miR663 expression. After transfection of miR663 mimics into HepG2 cells, TNF-alpha was unable to decrease Epo mRNA amounts. Furthermore, mimic-miR663 transfection resulted in a lower apoptosis rate of UT-7 cells in coculture experiments. Conclusions. miR663 is involved in Epo mRNA production and that is able to prevent or reverse the inhibitory effect of TNF-α. In our coculture study, transfecting HepG2 cells with miR663 mimics decreased the apoptosis of UT-7 cells.http://dx.doi.org/10.1155/2021/3670499 |
| spellingShingle | Mete Ozkurt Thomas Hellwig-Bürgel Reinhard Depping Selda Kadabere Rumeysa Ozyurt Abdullah Karadag Nilüfer Erkasap miR663 Prevents Epo Inhibition Caused by TNF-Alpha in Normoxia and Hypoxia International Journal of Endocrinology |
| title | miR663 Prevents Epo Inhibition Caused by TNF-Alpha in Normoxia and Hypoxia |
| title_full | miR663 Prevents Epo Inhibition Caused by TNF-Alpha in Normoxia and Hypoxia |
| title_fullStr | miR663 Prevents Epo Inhibition Caused by TNF-Alpha in Normoxia and Hypoxia |
| title_full_unstemmed | miR663 Prevents Epo Inhibition Caused by TNF-Alpha in Normoxia and Hypoxia |
| title_short | miR663 Prevents Epo Inhibition Caused by TNF-Alpha in Normoxia and Hypoxia |
| title_sort | mir663 prevents epo inhibition caused by tnf alpha in normoxia and hypoxia |
| url | http://dx.doi.org/10.1155/2021/3670499 |
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