Objective

We attempt to investigate the role of TNFRSF1A and its underlying mechanism in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury in rat pheochromocytoma PC12 cells.

Methods

Public datasets GSE61616 and GSE106680 were downloaded from GEO database. PC12 cells were used to construct OGD/R models. QRT-PCR and western blot were implemented to test the relative mRNA and protein levels, respectively. The miRNA online prediction website TargetScan was used to predict TNFRSF1A upstream regulated miRNAs, which were then confirmed by luciferase reporter assay. The changes in cell viability and apoptosis were evaluated using cell counting kit 8 (CCK-8), lactose dehydrogenase (LDH), and flow cytometry assays.

Results

Bioinformatics analysis demonstrated that the expression of TNFRSF1A was upregulated in CI/RI and middle cerebral artery occlusion models compared with control, respectively. And a significant upregulation was also observed in OGD/R-damaged PC12 cells. Depletion of TNFRSF1A can notably enhance the cells proliferation after OGD/R treatment, while enlargement of TNFRSF1A presented the opposite outcomes. Moreover, miR-29a-3p was shown to be the upstream regulatory miRNA of TNFRSF1A. The levels of TNFRSF1A were inversely mediated by miR-29a-3p. Overexpression of miR-29a-3p can raise the cell viability, decrease the LDH activity, and reduce the apoptotic ratio in OGD/R-treated cells. Besides, TNFRSF1A can attenuate the protective effect of miR-29a-3p on OGD/R-treated cells. Furthermore, miR-29a-3p mimic inhibited, while overexpression of TNFRSF1A promoted the activation of NF-κB signaling pathway, and TNFRSF1A can attenuate the suppressive effect of miR-29a-3p on the NF-κB pathway.

Conclusion

Our research illustrated that the potential regulatory role of miR-29a-3p/TNFRSF1A axis in neurons cells suffered from OGD/R, and their effects on NF-κB signaling pathway, providing a possible bio-target for protecting cells from OGD/R damage .

中文翻译：

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MiR-29a-3p通过靶向TNFRSF1A和调节NF-κB信号通路来增强被氧葡萄糖剥夺/复氧治疗所损伤的大鼠神经元细胞的活力。

目的

我们试图调查TNFRSF1A的作用及其在氧-葡萄糖剥夺/复氧（OGD / R）诱导的大鼠嗜铬细胞瘤PC12细胞损伤中的潜在机制。

方法

公共数据集GSE61616和GSE106680从GEO数据库下载。PC12细胞用于构建OGD / R模型。分别进行QRT-PCR和Western blot检测相对mRNA和蛋白水平。使用miRNA在线预测网站TargetScan预测TNFRSF1A上游调节的miRNA，然后通过萤光素酶报告基因测定法对其进行确认。使用细胞计数试剂盒8（CCK-8），乳糖脱氢酶（LDH）和流式细胞术分析评估了细胞活力和凋亡的变化。

结果

生物信息学分析表明，与对照组相比，CI / RI和大脑中动脉闭塞模型中TNFRSF1A的表达分别上调。在OGD / R损伤的PC12细胞中也观察到明显的上调。消耗OGD / R后，TNFRSF1A的消耗可以显着增强细胞增殖，而TNFRSF1A的膨胀则呈现相反的结果。此外，miR-29a-3p被证明是TNFRSF1A的上游调节性miRNA。TNFRSF1A的水平由miR-29a-3p反向介导。在OGD / R处理的细胞中，miR-29a-3p的过表达可以提高细胞活力，降低LDH活性，并降低细胞凋亡率。此外，TNFRSF1A可以减弱miR-29a-3p对OGD / R处理的细胞的保护作用。此外，miR-29a-3p模拟物也受到抑制，

结论

我们的研究表明，miR-29a-3p / TNFRSF1A轴在OGD / R神经元细胞中的潜在调控作用及其对NF-κB信号通路的影响，为保护细胞免受OGD / R损伤提供了可能的生物靶标。 。