Mechanistic insights into HuR inhibitor MS-444 arresting embryonic development revealed by low-input RNA-seq and STORM
Mechanistic insights into HuR inhibitor MS-444 arresting embryonic development revealed by low-input RNA-seq and STORM
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DOI：
10.1007/s10565-022-09757-7
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发表时间：
2022-09
影响因子：
6.1
通讯作者：
Yongqiang Nie;Wei Xu;Geng. G. Tian;Xiaowei Li;Yan Guo;Xuefeng Liu;Lin He;Zhifeng Shao
中科院分区：
文献类型：
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作者：
Yongqiang Nie;Wei Xu;Geng. G. Tian;Xiaowei Li;Yan Guo;Xuefeng Liu;Lin He;Zhifeng Shao
随着癌症患者存活率的提高，生育能力的维持已成为育龄妇女癌症治疗方面的一个主要问题。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。本文报道了MS-444通过抑制HuR二聚化阻断Agbl 2 mRNA的核质转运，导致小鼠2-细胞期胚胎发育停滞。结合MS-444处理的2-细胞胚胎的低输入RNA-seq分析和RNA结合蛋白的结合位点定位，预测Agbl 2是MS-444的靶基因。为了进一步证实，野生型受精卵中的RNAi实验显示Agbl 2敲低降低了成功发育到囊胚期的胚胎比例：从对照的71%降低到23%。此外，RNA-FISH和荧光素酶报告基因分析表明，MS-444阻断Agbl 2 mRNA的核质转运，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显著降低了HuR的二聚化，HuR在2-细胞期胚胎中主要以簇的形式存在。总之，本研究为育龄女性在MS-444治疗癌症期间维持生育力提供了临床指导。而且，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。图形摘要HuR抑制剂MS-444将胚胎发育阻滞在2-细胞期。低输入RNA-seq显示Agbl 2是MS-444的靶基因。MS-444的靶基因是Agbl 2。444通过抑制HuR二聚化阻断Agbl 2 mRNA的核质转运，降低Agbl 2 mRNA的稳定性在2细胞期胚胎中，HuR倾向于形成椭圆形和密集的簇。
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 ofAgbl2mRNA 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,Agbl2was predicted to be the target gene of MS-444. For further confirmation, RNAi experiment in wild-type zygotes showed thatAgbl2knockdown 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 ofAgbl2mRNA 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 abstractHuR inhibitor MS-444 arrested embryonic development at 2-cell stage.Low-input RNA-seq revealed thatAgbl2was the target gene of MS-444.MS-444 blocked the nucleocytoplasmic transport ofAgbl2mRNA by inhibiting HuR dimerization and reduced the stability ofAgbl2mRNA.STORM with our optimized protocol showed that HuR tended to form elliptical and dense clusters in 2-cell stage embryos.