Tao Xifeng, Fang Yafei, Huo Chen
Department of Pediatrics, The First People's Hospital of Lianyungang, Lianyungang, Jiangsu 222000, P.R. China.
Exp Ther Med. 2021 Jul;22(1):781. doi: 10.3892/etm.2021.10213. Epub 2021 May 19.
Bronchopulmonary dysplasia (BPD) is a frequent complication characterized by accelerated lung alveolarization in newborns. Long non-coding RNAs (lncRNAs) and microRNAs (miRs) are regarded as essential regulators in various diseases, including BPD. However, the detailed mechanism of the functions of RNA imprinted and accumulated in nucleus (Rian) lncRNA in the progression of BPD have remained elusive. The aim of the present study was to illustrate the interaction between miR-421 and Rian in BPD models and MLE-12 cells. The ability of Rian to protect neonatal lungs from hyperoxia-induced lung damage was examined. A mouse model of BPD and a hyperoxia-stimulated MLE-12 cell damage model were generated and treated with specific plasmid/mimics for the overexpression of Rian/miR-421. The interaction between miR-421 and Rian was predicted and verified using StarBase and a dual-luciferase reporter assay, respectively. The expression levels of miR-421 or Rian in both tissues and the MLE-12 alveolar epithelial cell line were assessed using reverse transcription-quantitative (RT-q)PCR. As parameters of alveolarization, the mean linear intercept (MLI), radial alveolar count (RAC) and the lung weight/body weight (LW/BW) ratio were measured. Furthermore, RT-qPCR was used to measure mRNA levels of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) in the lung tissue of mice, and ELISAs were performed to determine the levels of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) in the supernatant of MLE-12 cells. Cell growth and apoptosis were evaluated using an MTT assay and flow cytometry, respectively. Furthermore, caspase-3 activity was assessed using a caspase-3 activity detection kit. Prediction with StarBase and the dual-luciferase reporter assay revealed that miR-421 directly targeted Rian. RT-qPCR analysis confirmed that Rian was downregulated and miR-421 was upregulated in lung tissues of the mouse model of BPD and in hyperoxia-induced MLE-12 cells. However, the expression of miR-421 was decreased by Rian-overexpression, an effect that was reversed by miR-421 mimics. In addition, BPD was alleviated by Rian-plasmid, as confirmed by the enhanced RAC and reduced MLI and LW/BW ratio. The present results also indicated that Rian-plasmid inhibited the secretion of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) in BPD mouse serum and hyperoxia-induced MLE-12 cells. In addition, Rian-plasmid eliminated the effect of hyperoxia to inhibit cell viability and induce apoptosis in MLE-12 cells. However, all of these effects of Rian were markedly reversed by miR-421 mimics. The present results indicated that Rian may attenuate hyperoxic damage in neonatal lungs and may serve as a novel molecular target for BPD treatment.
支气管肺发育不良（BPD）是一种常见并发症，其特征为新生儿肺泡化加速。长链非编码RNA（lncRNA）和微小RNA（miR）被视为包括BPD在内的多种疾病的重要调节因子。然而，细胞核中印记并积累的RNA（Rian）lncRNA在BPD进展中的详细功能机制仍不清楚。本研究的目的是阐明miR - 421与Rian在BPD模型和MLE - 12细胞中的相互作用。检测了Rian保护新生小鼠肺免受高氧诱导的肺损伤的能力。构建了BPD小鼠模型和高氧刺激的MLE - 12细胞损伤模型，并用特异性质粒/模拟物处理以过表达Rian/miR - 421。分别使用StarBase和双荧光素酶报告基因检测法预测并验证了miR - 421与Rian之间的相互作用。使用逆转录定量（RT - q）PCR评估miR - 421或Rian在组织和MLE - 12肺泡上皮细胞系中的表达水平。作为肺泡化参数，测量了平均线性截距（MLI）、肺泡计数（RAC）和肺重量/体重（LW/BW）比值。此外，使用RT - qPCR测量小鼠肺组织中促炎细胞因子（TNF -α、IL - 6和IL - 1β）的mRNA水平，并进行酶联免疫吸附测定（ELISA）以确定MLE - 12细胞上清液中促炎细胞因子（TNF -α、IL - 6和IL - 1β）的水平。分别使用MTT法和流式细胞术评估细胞生长和凋亡。此外，使用caspase - 3活性检测试剂盒评估caspase - 3活性。StarBase预测和双荧光素酶报告基因检测显示miR - 421直接靶向Rian。RT - qPCR分析证实，在BPD小鼠模型的肺组织和高氧诱导的MLE - 12细胞中，Rian表达下调而miR - 421表达上调。然而，过表达Rian可降低miR - 421的表达，而miR - 421模拟物可逆转这种作用。此外，Rian质粒可缓解BPD，表现为RAC增加，MLI和LW/BW比值降低。本研究结果还表明，Rian质粒可抑制BPD小鼠血清和高氧诱导的MLE - 12细胞中促炎细胞因子（TNF -α、IL - 6和IL - 1β）的分泌。此外，Rian质粒消除了高氧对MLE - 12细胞活力的抑制作用并诱导其凋亡。然而，miR - 421模拟物可显著逆转Rian的所有这些作用。本研究结果表明，Rian可能减轻新生小鼠肺的高氧损伤，并可能成为BPD治疗的新分子靶点。
