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.

中文翻译：

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miR663 可防止 TNF-Alpha 在常氧和缺氧中引起的 Epo 抑制

客观。在慢性炎症性疾病中，促炎细胞因子如 TNF -α在循环中大量存在，并且在大多数情况下与贫血有关。实验研究表明，TNF -α抑制造血的主要兴奋剂促红细胞生成素（Epo）的合成。我们的目标是找出哪些 microRNA 参与了 TNF -α对 Epo 的抑制。方法。首先，我们使用 MTT 测定法确定了 HepG2 细胞中 TNF -α的剂量，该剂量没有细胞毒性作用，并通过 qRT-PCR 和 ELISA 抑制 Epo 合成。然后，我们用 TNF -α进行了 microRNA 阵列研究(20 ng/ml) 处理的细胞，阵列结果通过 qRT-PCR 确认。我们用mimic-miR663（30 pmol）转染miR663组24小时；其他组用转染试剂处理，然后用TNF -α处理24小时；miR663组用TNF -α处理24小时；对照组用普通培养基培养。我们通过 qRT-PCR 分析了 Epo mRNA 水平。如果模拟-miR663 阻止 TNF - α对 Epo 的抑制，更多的 Epo 依赖性 UT-7 细胞将存活。因此，我们将 HepG2 细胞与 UT-7 细胞共培养。通过 TUNEL 测定法确定凋亡 UT-7 细胞的百分比。结果。根据我们的阵列研究，TNF -α显着降低 miR663 的表达。将 miR663 模拟物转染到 HepG2 细胞中后，TNF-α 无法降低 Epo mRNA 的量。此外，在共培养实验中，mimic-miR663 转染导致 UT-7 细胞的凋亡率降低。结论。miR663 参与 Epo mRNA 的产生，能够阻止或逆转 TNF -α的抑制作用。在我们的共培养研究中，用 miR663 模拟物转染 HepG2 细胞可减少 UT-7 细胞的凋亡。