Mechanistic Insights Into Small-molecule Inhibitor MS-444 Arresting Embryonic Development Revealed by Low-input RNA-seq and STORM

BackgroundWith the improvement of the survival rate of cancer patients, fertility maintenance has become a major concern of cancer treatment for women of reproductive age. Thus, it is important to examine the impact on fertility of anticancer drugs that have been clinically used 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. ResultsCombining 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 develop to the blastocyst stage: from 71% in controls to 23%. Furthermore, RNA-FISH and luciferase reporter analysis 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 the form of clusters in 2-cell stage embryos.ConclusionThose results showed 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. 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 the nanometer scale study of embryonic cells.