Mechanistic insights into HuR inhibitor MS-444 arresting embryonic development revealed by low-input RNA-seq and STORM,Cell Biology and Toxicology

当前位置： X-MOL 学术 › Cell Biol. Toxicol. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Mechanistic insights into HuR inhibitor MS-444 arresting embryonic development revealed by low-input RNA-seq and STORM
Cell Biology and Toxicology ( IF 5.3 ) Pub Date : 2022-09-10 , DOI: 10.1007/s10565-022-09757-7
Yongqiang Nie ₁ , Wei Xu ₂ , Geng G Tian ₁ , Xiaowei Li ₂ , Yan Guo ₂ , Xuefeng Liu ₃ , Lin He ₁ , Zhifeng Shao ₂ , Xiaoyong Li ₁ , Ji Wu _{1,

4}

Affiliation

With improvements in the survival rate of patients with cancer, fertility maintenance has become a major concern in terms of cancer treatment for women of reproductive age. Thus, it is important to examine the impact on fertility of anticancer drugs that are used clinically or are undergoing trials. The HuR small-molecule inhibitor MS-444 has been used in many cancer treatment studies, but its reproductive toxicity in females is unknown. Here, we reported that MS-444 blocked the nucleocytoplasmic transport of Agbl2 mRNA by inhibiting HuR dimerization, resulting in the developmental arrest of 2-cell stage embryos in mouse. Combining analysis of low-input RNA-seq for MS-444-treated 2-cell embryos and mapping binding sites of RNA-binding protein, Agbl2 was predicted to be the target gene of MS-444. For further confirmation, RNAi experiment in wild-type zygotes showed that Agbl2 knockdown reduced the proportion of embryos successfully developed to the blastocyst stage: from 71% in controls to 23%. Furthermore, RNA-FISH and luciferase reporter analyses showed that MS-444 blocked the nucleocytoplasmic transport of Agbl2 mRNA and reduced its stability by inhibiting HuR dimerization. In addition, optimized stochastic optical reconstruction microscopy (STORM) imaging showed that MS-444 significantly reduced the HuR dimerization, and HuR mainly existed in cluster form in 2-cell stage embryos. In conclusion, this study provides clinical guidance for maintaining fertility during the treatment of cancer with MS-444 in women of reproductive age. And also, our research provides guidance for the application of STORM in nanometer scale studies of embryonic cells.

Graphical abstract

HuR inhibitor MS-444 arrested embryonic development at 2-cell stage.

Low-input RNA-seq revealed that Agbl2 was the target gene of MS-444.

MS-444 blocked the nucleocytoplasmic transport of Agbl2 mRNA by inhibiting HuR dimerization and reduced the stability of Agbl2 mRNA.

STORM with our optimized protocol showed that HuR tended to form elliptical and dense clusters in 2-cell stage embryos.

中文翻译：

低输入 RNA-seq 和 STORM 揭示的 HuR 抑制剂 MS-444 阻止胚胎发育的机制洞察

随着癌症患者生存率的提高，生育力维持已成为育龄妇女癌症治疗的主要关注点。因此，重要的是要检查临床使用或正在进行试验的抗癌药物对生育能力的影响。HuR 小分子抑制剂 MS-444 已用于许多癌症治疗研究，但其对女性的生殖毒性尚不清楚。在这里，我们报道了 MS-444通过抑制 HuR 二聚化来阻断Agbl2 mRNA 的核质转运，导致小鼠 2 细胞期胚胎的发育停滞。结合分析 MS-444 处理的 2 细胞胚胎的低输入 RNA-seq 和绘制 RNA 结合蛋白Agbl2的结合位点被预测为MS-444的靶基因。为了进一步证实，野生型受精卵中的 RNAi 实验表明，Agbl2敲低降低了胚胎成功发育到囊胚阶段的比例：从对照的 71% 降至 23%。此外，RNA-FISH 和荧光素酶报告基因分析表明，MS-444 阻断了Agbl2的核质转运mRNA 并通过抑制 HuR 二聚化降低其稳定性。此外，优化的随机光学重建显微镜 (STORM) 成像显示 MS-444 显着降低了 HuR 二聚化，并且 HuR 主要以簇形式存在于 2 细胞期胚胎中。总之，本研究为育龄妇女在使用 MS-444 治疗癌症期间维持生育能力提供了临床指导。此外，我们的研究为 STORM 在胚胎细胞纳米尺度研究中的应用提供了指导。