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

OBJECTIVE

In chronic inflammatory diseases, proinflammatory cytokines such as TNF- are present in high amounts in the circulation and are associated with anemia in most cases. Experimental studies have shown that TNF- 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-.

METHODS

First, we determined the dose of TNF- 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- (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- for 24 hrs; miR663 groups were treated with TNF- 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-, 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- 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-. In our coculture study, transfecting HepG2 cells with miR663 mimics decreased the apoptosis of UT-7 cells.