Department of Biochemistry and Molecular Biology, Shanxi Key Laboratory of Birth, Defect and Cell Regeneration, Shanxi Medical University, Taiyuan, Shanxi, China.
Department of Statistics, Shanxi Medical University, Taiyuan, Shanxi, China.
Cell Cycle. 2021 Nov;20(22):2372-2386. doi: 10.1080/15384101.2021.1982506. Epub 2021 Nov 15.
Previously our results showed miR-222-3p was significantly downregulated in retinoic acid-induced neural tube defect (NTD) mouse model through transcriptome. Down-regulation of miR-222-3p may be a causative biomarker in NTDs. In this study, RNA was extracted from mouse embryos at E8.5, E9.5 and E10.5, and the expression level of miR-222-3p was measured by quantitative real-time PCR analysis. The preliminary mechanism of miR-222-3p in NTDs involved in cell proliferation, apoptosis and migration was investigated in mouse HT-22 cell line. The expression of miR-222-3p was significantly decreased at E8.5, E9.5 and E10.5 developed in mouse embryos which were consistent with our transcriptome sequencing. Suppression of miR-222-3p in HT-22 cells resulted in the inhibition of cell proliferation and migration, cell cycle and apoptosis. Moreover, DNA damage transcript 4 (Ddit4) was identified as a direct and functional target of miR-222-3p. miR-222-3p is negatively regulated by Ddit4. The mutation of binding site of Ddit4 3'UTR abrogated the responsiveness of luciferase reporters to miR-222-3p and showed that Ddit4 expression partially attenuated the function of miR-222-3p. We preliminatively confirmed that low expression of miR-222-3p has reduced the expression of β-catenin, TCF4 and other related genes in the Wnt/β-catenin signaling pathway.Collectively, these results demonstrated that miR-222-3p regulates the Wnt/β-catenin signaling pathway through Ddit4 inhibition in HT-22 cells, resulted in cell proliferation and apoptosis imbalance, and thus led to neural tube defects.
先前的研究结果表明，miR-222-3p 在维甲酸诱导的神经管缺陷（NTD）小鼠模型中通过转录组显著下调。miR-222-3p 的下调可能是 NTDs 的一个致病生物标志物。在这项研究中，从 E8.5、E9.5 和 E10.5 的小鼠胚胎中提取 RNA，并通过定量实时 PCR 分析测量 miR-222-3p 的表达水平。在小鼠 HT-22 细胞系中研究了 miR-222-3p 在 NTDs 中涉及细胞增殖、凋亡和迁移的初步机制。miR-222-3p 在 E8.5、E9.5 和 E10.5 发育的小鼠胚胎中的表达明显降低，这与我们的转录组测序结果一致。HT-22 细胞中 miR-222-3p 的抑制导致细胞增殖和迁移、细胞周期和凋亡的抑制。此外，鉴定出 DNA 损伤转录物 4（DDIT4）是 miR-222-3p 的直接和功能靶标。miR-222-3p 受 DDIT4 的负调控。DDIT4 3'UTR 结合位点的突变消除了荧光素酶报告基因对 miR-222-3p 的反应性，表明 DDIT4 表达部分减弱了 miR-222-3p 的功能。我们初步证实，miR-222-3p 的低表达降低了 Wnt/β-catenin 信号通路中β-catenin、TCF4 和其他相关基因的表达。总之，这些结果表明，miR-222-3p 通过抑制 HT-22 细胞中的 DDIT4 调节 Wnt/β-catenin 信号通路，导致细胞增殖和凋亡失衡，从而导致神经管缺陷。
