Liu Qin, Li Yuehong, Zhou Yong
Department of Pediatrics, Yancheng Maternal and Child Health Hospital, Yancheng, Jiangsu 224002, P.R. China.
Exp Ther Med. 2021 Mar;21(3):229. doi: 10.3892/etm.2021.9660. Epub 2021 Jan 20.
Accumulating reports have indicated that congenital hypothyroidism (CH) is an endocrine disorder caused by underdeveloped thyroid gland or thyroid dyshormonogenesis. It has been also reported that certain microRNAs (miRNAs) may exert protective effects against the development of CH. However, whether miR-489-3p regulates CH progression remains unclear. The aim of the present study was to investigate the effects of miR-489-3p on CH and elucidate the underlying mechanisms. Therefore, Sprague Dawley rats were injected with propylthiouracil (50 mg/day) to establish a CH model. Reverse transcription-quantitative PCR (RT-qPCR) assay demonstrated that miR-489-3p was upregulated in the hippocampal tissues of CH rats. Furthermore, the TargetScan software was employed to predict the target gene of miR-489-3p, and a dual luciferase reporter assay revealed that translationally controlled tumor protein 1 (TPT1) was directly targeted by miR-489-3p. Additionally, RT-qPCR and western blot assays suggested that TPT1 was markedly downregulated in the hippocampal tissues of CH rats compared with control rats. In addition, inhibitor control, miR-489-3p inhibitor, control-shRNA or TPT1-shRNA were injected into CH rats. The results of the open-field and forced swimming tests revealed that miR-489-3p inhibitor notably improved the behavior of CH rats. Flow cytometry was applied to explore the effects of miR-489-3p inhibitor on neuronal cell apoptosis, and the findings indicated that miR-489-3p inhibitor attenuated CH-induced neuronal cell apoptosis, whereas these effects were reversed by treatment with miR-489-3p inhibitor and TPT1-shRNA. Finally, the function of miR-489-3p in neuronal cells was investigated . Neuronal cell viability, apoptosis and the expression of apoptosis-related proteins were determined using MTT assay, flow cytometry and western blot analysis, respectively. The results demonstrated that miR-489-3p inhibitor enhanced cell viability, suppressed apoptosis and upregulated Pim-3, phosphorylated (p)-Bad (Ser112) and Bcl-xL expression. Rescue experiments indicated that these effects were reversed following silencing of TPT1. Taken together, the findings of the present study demonstrated that miR-489-3p inhibitor could relieve CH-induced neurological damage through regulating TPT1 expression.
越来越多的报告表明，先天性甲状腺功能减退症（CH）是一种由甲状腺发育不全或甲状腺激素合成障碍引起的内分泌疾病。也有报道称，某些微小RNA（miRNA）可能对CH的发展具有保护作用。然而，miR-489-3p是否调节CH的进展仍不清楚。本研究的目的是探讨miR-489-3p对CH的影响并阐明其潜在机制。因此，给Sprague Dawley大鼠注射丙硫氧嘧啶（50mg/天）以建立CH模型。逆转录定量PCR（RT-qPCR）分析表明，CH大鼠海马组织中miR-489-3p上调。此外，使用TargetScan软件预测miR-489-3p的靶基因，双荧光素酶报告基因分析显示翻译控制肿瘤蛋白1（TPT1）是miR-489-3p的直接靶标。另外，RT-qPCR和蛋白质印迹分析表明，与对照大鼠相比，CH大鼠海马组织中TPT1明显下调。此外，将抑制剂对照、miR-489-3p抑制剂、对照短发夹RNA（shRNA）或TPT1-shRNA注射到CH大鼠体内。旷场试验和强迫游泳试验结果显示，miR-489-3p抑制剂显著改善了CH大鼠的行为。应用流式细胞术探讨miR-489-3p抑制剂对神经元细胞凋亡的影响，结果表明miR-489-3p抑制剂减轻了CH诱导的神经元细胞凋亡，而用miR-489-3p抑制剂和TPT1-shRNA处理可逆转这些作用。最后，研究了miR-489-3p在神经元细胞中的功能。分别使用MTT法、流式细胞术和蛋白质印迹分析测定神经元细胞活力、凋亡及凋亡相关蛋白的表达。结果表明，miR-489-3p抑制剂增强了细胞活力，抑制了凋亡，并上调了Pim-3、磷酸化（p）-Bad（Ser112）和Bcl-xL的表达。拯救实验表明，沉默TPT1后这些作用被逆转。综上所述，本研究结果表明，miR-489-3p抑制剂可通过调节TPT1表达减轻CH诱导的神经损伤。
