miR663 Prevents Epo Inhibition Caused by TNF-Alpha in Normoxia and Hypoxia

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ÖZKURT M., Hellwig-Buergel T., Depping R., Kadabere S., ÖZYURT R., Karadag A., ...More

INTERNATIONAL JOURNAL OF ENDOCRINOLOGY, vol.2021, 2021 (SCI-Expanded) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 2021
  • Publication Date: 2021
  • Doi Number: 10.1155/2021/3670499
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Academic Search Premier, CINAHL, EMBASE, Directory of Open Access Journals
  • Eskisehir Osmangazi University Affiliated: Yes


Objective. In chronic inflammatory diseases, proinflammatory cytokines such as TNF-alpha are present in high amounts in the circulation and are associated with anemia in most cases. Experimental studies have shown that TNF-alpha 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-alpha. Methods. First, we determined the dose of TNF-alpha 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-alpha (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-alpha for 24 hrs; miR663 groups were treated with TNF-alpha 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-alpha, 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-alpha 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-alpha. In our coculture study, transfecting HepG2 cells with miR663 mimics decreased the apoptosis of UT-7 cells.