Nie Yongqiang, Xu Wei, Tian Geng G, Li Xiaowei, Guo Yan, Liu Xuefeng, He Lin, Shao Zhifeng, Li Xiaoyong, Wu Ji
Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, 200240, China.
School of Biomedical Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China.
Cell Biol Toxicol. 2022 Dec;38(6):1175-1197. doi: 10.1007/s10565-022-09757-7. Epub 2022 Sep 10.
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. 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.
随着癌症患者生存率的提高，生育力维持已成为育龄期女性癌症治疗中的一个主要关注点。因此，研究临床使用或正在进行试验的抗癌药物对生育力的影响非常重要。HuR小分子抑制剂MS-444已用于许多癌症治疗研究，但其对雌性动物的生殖毒性尚不清楚。在此，我们报道MS-444通过抑制HuR二聚化阻断了Agbl2 mRNA的核质转运，导致小鼠2细胞期胚胎发育停滞。通过对MS-444处理的2细胞胚胎进行低输入RNA测序分析并绘制RNA结合蛋白的结合位点，预测Agbl2是MS-444的靶基因。为进一步证实，在野生型受精卵中进行的RNAi实验表明，敲低Agbl2会降低成功发育到囊胚期的胚胎比例：从对照组的71%降至23%。此外，RNA-FISH和荧光素酶报告基因分析表明，MS-444通过抑制HuR二聚化阻断了Agbl2 mRNA的核质转运并降低了其稳定性。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二聚化，且HuR在2细胞期胚胎中主要以簇状形式存在。总之，本研究为育龄期女性使用MS-444治疗癌症期间维持生育力提供了临床指导。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。HuR抑制剂MS-444使胚胎发育停滞在2细胞期。低输入RNA测序显示Agbl2是MS-444的靶基因。MS-444通过抑制HuR二聚化阻断了Agbl2 mRNA的核质转运并降低了Agbl2 mRNA的稳定性。采用我们优化方案的STORM显示，HuR在2细胞期胚胎中倾向于形成椭圆形致密簇。
