Organ Transplant Centre, The First Affiliated Hospital of Sun Yat‑sen University, Guangzhou, Guangdong 510080, P.R. China.
Int J Mol Med. 2019 Oct;44(4):1505-1514. doi: 10.3892/ijmm.2019.4282. Epub 2019 Jul 22.
Liver regeneration (LR) is the result of a dynamic balance between the increased proliferation and decreased apoptosis of hepatocytes. However, the role of microRNA (miR)‑26a in regulating complex signalling networks involving E3 ubiquitin‑protein ligase Mdm2 (mdm2), p53, p21 and p27 in the process of LR is currently unclear. In the present study, it was hypothesized that miR‑26a may negatively regulate the mdm2/p53 signalling loop in response to LR. In vitro experiments were performed, whereby mouse liver cells were transfected with an miR‑26a vector or an anti/miR‑26a vector. Cell proliferation was analysed using an MTS assay and cell apoptosis, and cell cycle progression were analysed by flow cytometry. In addition, the expression of mdm2, p53, p21 and p27 were assessed using western blotting and reverse transcription‑quantitative polymerase chain reaction analyses. Dual‑luciferase reporter assays were also used to examine the association between mdm2 and miR‑26a. A 70% partial hepatectomy in C57BL/6J mice was then performed, which was followed by injection with an mdm2‑cDNA vector or an mdm2‑small interfering RNA vector. The liver‑to‑body weight ratio and liver function of mice were measured at 72 h following vector administration. The results demonstrated an increase in hepatocyte proliferation accompanied by decreased hepatocyte apoptosis levels. In addition, inhibition of miR‑26a expression was associated with a marked increase in mdm2 expression, while the expression of p53, p21 and p27 was decreased when compared with negative controls. The opposite effects were observed when miR‑26a was overexpressed. Notably, miR‑26a was demonstrated to target the 3'‑untranslated region of mdm2 directly. The results of the present study are the first to demonstrate as far as the authors are aware that the mdm2/p53 negative feedback loop may be targeted by miR‑26a directly in response to LR, and that mdm2 negatively regulates p53, p21 and p27 but not miR‑26a. miR‑26a may therefore function as an important factor that regulates the interaction between mdm2 and p53.
肝再生 (LR) 是肝细胞增殖增加和凋亡减少之间动态平衡的结果。然而，microRNA (miR) -26a 在调节涉及 E3 泛素-蛋白连接酶 Mdm2 (mdm2)、p53、p21 和 p27 的复杂信号网络中的作用在 LR 过程中尚不清楚。在本研究中，假设 miR-26a 可能通过负向调节 mdm2/p53 信号环来响应 LR。进行了体外实验，其中将 miR-26a 载体或抗/miR-26a 载体转染入小鼠肝实质细胞。使用 MTS 测定法分析细胞增殖，通过流式细胞术分析细胞凋亡和细胞周期进程。此外，通过 Western blot 分析和逆转录-定量聚合酶链反应分析评估 mdm2、p53、p21 和 p27 的表达。还使用双荧光素酶报告基因分析来检验 mdm2 和 miR-26a 之间的关联。然后在 C57BL/6J 小鼠中进行 70%部分肝切除术，随后注射 mdm2-cDNA 载体或 mdm2-小干扰 RNA 载体。在载体给药后 72 小时测量小鼠的肝体比和肝功能。结果表明，肝细胞增殖增加，同时肝细胞凋亡水平降低。此外，与阴性对照相比，抑制 miR-26a 表达与 mdm2 表达的显著增加相关，而 p53、p21 和 p27 的表达降低。当 miR-26a 过表达时，观察到相反的效果。值得注意的是，miR-26a 被证明可直接靶向 mdm2 的 3'-非翻译区。本研究的结果首次表明，据作者所知，mdm2/p53 负反馈环可能直接靶向 miR-26a 以响应 LR，mdm2 负向调节 p53、p21 和 p27，但不调节 miR-26a。miR-26a 因此可能作为调节 mdm2 和 p53 之间相互作用的重要因素发挥作用。
