Chen Ronglin, Wang Meixia, Fu Shaopin, Cao Feng, Duan Pengkai, Lu Jiefu
Department of Critical Care Medicine, Longgang District Central Hospital, Shenzhen, Guangdong 518116, P.R. China.
Department of Intensive Care Unit, Affiliated General Hospital of Guangzhou Military Command of Southern Medical University, Guangzhou, Guangdong 510515, P.R. China.
Exp Ther Med. 2019 Nov;18(5):3299-3306. doi: 10.3892/etm.2019.7936. Epub 2019 Aug 26.
Hypoxic-ischemic encephalopathy (HIE) is a common neonatal disease that can lead to high neonatal mortality rates. Previous studies have indicated that microRNAs (miRs) may be involved in the pathogenesis of HIE; however, the specific mechanisms underlying their involvement require further investigation. The aim of the present study was to investigate the roles of miR-204 and its target gene killin p53 regulated DNA replication inhibitor (KLLN) in HIE using rat HIE models. Brain injury was induced by surgery and incubation of hypoxic incubator brain using 10-day-old pup rats. On day 3, rats were sacrificed, and the infarct size of the brain was determined using a tetrazolium chloride assay. Terminal deoxynucleotidyl transferase UTP nick-end labeling staining was performed to detect the cell death rate in the brain tissue. Following this, the brain tissues were collected, and reverse transcription-quantitative polymerase chain reaction, western blot analysis and immunohistochemistry assays were performed to examine the expression levels of miR-204 and KLLN. Furthermore, neurons were cultured and transfected with miR-204 inhibitors or mimics, and the effect of miR-204 on the proliferation and apoptosis of neurons was examined using MTT and flow cytometric assays. Finally, a dual-luciferase reporter assay was performed to confirm whether KLLN is a direct target of miR-204. The expression of miR-204 was significantly downregulated and the expression of KLLN was significantly increased in the brain tissue of HIE rats (P<0.001). In addition, the transfection with miR-204 inhibitors significantly decreased the proliferation rates and significantly increased the apoptosis rate of neurons; however, transfection with miR-204 mimics prompted the opposite results. The dual-luciferase reporter assay also confirmed that KLLN is a direct target of miR-204. Taken together, the results of the present study demonstrated that miR-204 was downregulated in HIE and that miR-204 may serve important roles in the pathogenesis of HIE through targeting KLLN.
缺氧缺血性脑病（HIE）是一种常见的新生儿疾病，可导致新生儿高死亡率。先前的研究表明，微小RNA（miRs）可能参与HIE的发病机制；然而，其具体参与机制仍需进一步研究。本研究的目的是利用大鼠HIE模型研究miR-204及其靶基因杀伤蛋白p53调控的DNA复制抑制剂（KLLN）在HIE中的作用。采用10日龄幼鼠，通过手术和缺氧培养箱孵育诱导脑损伤。第3天，处死大鼠，采用氯化三苯基四氮唑法测定脑梗死面积。进行末端脱氧核苷酸转移酶介导的缺口末端标记染色以检测脑组织中的细胞死亡率。随后，收集脑组织，进行逆转录-定量聚合酶链反应、蛋白质印迹分析和免疫组织化学分析，以检测miR-204和KLLN的表达水平。此外，培养神经元并转染miR-204抑制剂或模拟物，采用MTT法和流式细胞术检测miR-204对神经元增殖和凋亡的影响。最后，进行双荧光素酶报告基因检测以确认KLLN是否为miR-204的直接靶标。HIE大鼠脑组织中miR-204表达显著下调，KLLN表达显著上调（P<0.001）。此外，转染miR-204抑制剂显著降低神经元增殖率并显著增加凋亡率；然而，转染miR-204模拟物则产生相反结果。双荧光素酶报告基因检测也证实KLLN是miR-204的直接靶标。综上所述，本研究结果表明，miR-204在HIE中表达下调，且miR-204可能通过靶向KLLN在HIE发病机制中发挥重要作用。
