Ozkurt Mete, Hellwig-Bürgel Thomas, Depping Reinhard, Kadabere Selda, Ozyurt Rumeysa, Karadag Abdullah, Erkasap Nilüfer
Department of Physiology, Eskisehir Osmangazi University Medical Faculty, Eskisehir, Turkey.
Institute of Physiology, University of Lubeck, Lubeck, Germany.
Int J Endocrinol. 2021 Jul 27;2021:3670499. doi: 10.1155/2021/3670499. eCollection 2021.
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-.
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.
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.
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.
在慢性炎症性疾病中，促炎细胞因子如肿瘤坏死因子-α（TNF-α）在循环中大量存在，且在大多数情况下与贫血相关。实验研究表明，TNF-α抑制促红细胞生成素（Epo）的合成，而Epo是造血的主要刺激因子。我们的目的是弄清楚哪些微小RNA参与了TNF-α对Epo的抑制作用。
首先，我们通过MTT试验确定了在HepG2细胞中无细胞毒性且通过qRT-PCR和ELISA抑制Epo合成的TNF-α剂量。然后，我们对用TNF-α（20 ng/ml）处理的细胞进行了微小RNA芯片研究，芯片结果通过qRT-PCR进行了验证。我们用模拟物-miR663（30 pmol）转染miR663组24小时；其他组先用转染试剂处理，然后用TNF-α处理24小时；miR663组用TNF-α处理24小时；对照组用正常培养基培养。我们通过qRT-PCR分析Epo mRNA水平。如果模拟物-miR663能阻止TNF-α对Epo的抑制作用，那么更多依赖Epo的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试验确定凋亡的UT-7细胞的百分比。
根据我们的芯片研究，TNF-α显著降低miR663的表达。将miR663模拟物转染到HepG2细胞后，TNF-α无法降低Epo mRNA的量。此外，在共培养实验中，模拟物-miR663转染导致UT-7细胞的凋亡率降低。
miR663参与Epo mRNA的产生，并且能够预防或逆转TNF-α的抑制作用。在我们的共培养研究中，用miR-663模拟物转染HepG2细胞可降低UT-7细胞的凋亡。
