miR-212-5p 通过靶向 FZD5 调节 AML 细胞的增殖和凋亡。
MiR-212-5p regulates the proliferation and apoptosis of AML cells through targeting FZD5.
机构信息
Department of Hematology and Rheumatology, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.
出版信息
Eur Rev Med Pharmacol Sci. 2018 Dec;22(23):8415-8422. doi: 10.26355/eurrev_201812_16540.
OBJECTIVE
To explore the effects of microRNA-212-5p (miR-212-5p) on biological functions of acute myeloid leukemia (AML) and to find the potential molecular mechanism.
PATIENTS AND METHODS
We measured the expression level of miR-212-5p in 35 AML patients and 20 patients with idiopathic thrombocytopenic purpura (ITP) as control cases. Besides, the miR-212-5p expression at cellular level was checked as well. In order to screen the functional targets of miR-212-5p, online prediction software was used and gene frizzled class receptor 5 (FZD5) attracted our attention. The effects of miR-212-5p on biological functions of AML cell line (Kasumi-1) were analyzed by subsequent experiments. The mRNA and protein expressions of FZD5 were detected by quantitative Real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB) analysis, respectively. Cell proliferation was tested by cell counting kit-8 (CCK-8) assay. Cell cycle and apoptosis were measured by flow cytometry. Finally, protein expression of β-catenin was analyzed by WB assay.
RESULTS
In AML cases and cells, miR-212-5p was found to be lowly expressed. The potential target of miR-212-5p was predicated in three public databases. Through a series of experiments including qRT-PCR, WB and luciferase assay, we identified FZD5 as a functional target of miR-212-5p. In further cellular functional experiments on Kasumi-1, we found overexpression of miR-212-5p in Kasumi-1 cells greatly inhibited the cell viability and proliferation. The ratio of cells in G0/G1 phase and the proportion of apoptotic cells increased after miR-212-5p overexpression. Furthermore, Wnt/β-catenin signal pathway was the most apparent pathway that was regulated by miR-212-5p according to WB results. However, the effects of miR-212-5p were suppressed after restoring the expression of FZD5.
CONCLUSIONS
Expression of miR-212-5p was significantly lower in AML patients and cell lines, indicating that miR-212-5p served as a tumor-suppressor gene in AML. According to our in vitro experiments, miR-212-5p/FZD5 was likely to become a new therapeutic target for AML.
目的
探讨微小 RNA-212-5p（miR-212-5p）对急性髓系白血病（AML）生物学功能的影响，并寻找潜在的分子机制。
患者和方法
我们测量了 35 例 AML 患者和 20 例特发性血小板减少性紫癜（ITP）患者作为对照的 miR-212-5p 的表达水平。此外，还检查了细胞水平的 miR-212-5p 表达。为了筛选 miR-212-5p 的功能靶点，我们使用了在线预测软件，基因卷曲受体 5（FZD5）引起了我们的注意。通过后续实验分析 miR-212-5p 对 AML 细胞系（Kasumi-1）生物学功能的影响。通过定量实时聚合酶链反应（qRT-PCR）和 Western blot（WB）分析分别检测 FZD5 的 mRNA 和蛋白表达。通过细胞计数试剂盒-8（CCK-8）测定细胞增殖。通过流式细胞术测量细胞周期和凋亡。最后，通过 WB 分析测定 β-连环蛋白的蛋白表达。
结果
在 AML 病例和细胞中，发现 miR-212-5p 表达水平较低。在三个公共数据库中预测了 miR-212-5p 的潜在靶点。通过一系列包括 qRT-PCR、WB 和荧光素酶测定在内的实验，我们鉴定 FZD5 是 miR-212-5p 的功能靶点。在对 Kasumi-1 的进一步细胞功能实验中，我们发现 miR-212-5p 在 Kasumi-1 细胞中的过表达极大地抑制了细胞活力和增殖。过表达 miR-212-5p 后，G0/G1 期细胞比例和凋亡细胞比例增加。此外，根据 WB 结果，Wnt/β-连环蛋白信号通路是受 miR-212-5p 调节最明显的通路。然而，在恢复 FZD5 的表达后，miR-212-5p 的作用受到抑制。
结论
AML 患者和细胞系中 miR-212-5p 的表达明显降低，表明 miR-212-5p 在 AML 中作为肿瘤抑制基因发挥作用。根据我们的体外实验结果，miR-212-5p/FZD5 可能成为 AML 的新治疗靶点。
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