Xiong Qian-Wen, Liu Yuntao, He Min, Shen Xiao-Die, Zhao Manli, Liu Shuang-Ai, Zhang Gensheng, Liu Qian, Wang Jinhu, Peng Wan-Xin
Department of Surgical Oncology, National Clinical Research Center for Child Health, National Children's Regional Medical Center, Children's Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Hangzhou TCM Hospital Affiliated to Zhejiang Chinese Medical University (Hangzhou Hospital of Traditional Chinese Medicine), Hangzhou, China.
Cell Biol Toxicol. 2025 Jun 7;41(1):96. doi: 10.1007/s10565-025-10049-z.
Malignant rhabdoid tumor of the kidney (MRTK) is a rare renal tumor with poor prognosis. While germline mutations of SMARCB1 are considered to be the primary cause of MRTK, emerging evidence suggests that somatic epigenetic changes also play a vital role in the development and progression of MRTK. YTHDF1, an m6A reader protein, has been implicated in regulation of tumorigenesis by influencing RNA translation and stability in several adult cancers. However, the exploration of the role of YTHDF1 in pediatric cancer, especially MRTK, remains limited.
In this study, CRISPR/Cas9 was employed to knockout (KO) YTHDF1 in G401 cells. The impact of YTHDF1 on the cell growth and chemoresistance were assessed using CCK-8 assays. Western blot and qRT-PCR were used to determine the changes in ferroptosis marker gene expression. Additionally, 4D-label free quantitative proteomics was conducted to uncover alterations by YTHDF1 deletion.
We observed that the deletion of YTHDF1 in the MRTK cell line led to a significant reduction in malignancy-associated characteristics, including decreased cell motility, invasive growth, and chemoresistance. Quantitative proteomic analysis revealed that the glutathione-related signaling pathway was notably affected by YTHDF1 KO. Specifically, YTHDF1 KO resulted in a reduction of both mRNA and protein levels of Glutathione S-Transferase Mu 2 (GSTM2), a phase II metabolizing enzyme responsible for conjugating glutathione to electrophilic compounds. The decrease in GSTM2 levels following YTHDF1 KO increased the susceptibility of MRTK cells to ferroptosis. Notably, overexpression of GSTM2 in YTHDF1 KO cells partially restored the oncogenic phenotype of MRTK cells, underscoring its role in MRTK progression.
In summary, our findings provide new insights into the molecular mechanisms driving MRTK progression, highlighting YTHDF1 and GSTM2 as potential therapeutic targets for this aggressive pediatric renal tumor.
肾恶性横纹肌样瘤（MRTK）是一种预后较差的罕见肾肿瘤。虽然SMARCB1的种系突变被认为是MRTK的主要病因，但新出现的证据表明，体细胞表观遗传变化在MRTK的发生和发展中也起着至关重要的作用。YTHDF1是一种m6A阅读蛋白，在几种成人癌症中，它通过影响RNA翻译和稳定性参与肿瘤发生的调控。然而，YTHDF1在儿童癌症，尤其是MRTK中的作用研究仍然有限。
在本研究中，采用CRISPR/Cas9技术在G401细胞中敲除（KO）YTHDF1。使用CCK-8试验评估YTHDF1对细胞生长和化疗耐药性的影响。蛋白质免疫印迹法和qRT-PCR用于测定铁死亡标记基因表达的变化。此外，进行了4D无标记定量蛋白质组学分析，以揭示YTHDF1缺失引起的变化。
我们观察到，MRTK细胞系中YTHDF1的缺失导致恶性相关特征显著降低，包括细胞运动性、侵袭性生长和化疗耐药性降低。定量蛋白质组学分析表明，谷胱甘肽相关信号通路受到YTHDF1基因敲除的显著影响。具体而言，YTHDF1基因敲除导致谷胱甘肽S-转移酶Mu 2（GSTM2）的mRNA和蛋白水平均降低，GSTM2是一种II期代谢酶，负责将谷胱甘肽与亲电子化合物结合。YTHDF1基因敲除后GSTM2水平的降低增加了MRTK细胞对铁死亡的敏感性。值得注意的是，在YTHDF1基因敲除细胞中过表达GSTM2可部分恢复MRTK细胞的致癌表型，强调了其在MRTK进展中的作用。
总之，我们的研究结果为驱动MRTK进展的分子机制提供了新的见解，突出了YTHDF1和GSTM2作为这种侵袭性儿童肾肿瘤潜在治疗靶点的作用。
