Shen Jianfen, Zhang Shuo, Song Yan, Yang Leiming, Huang Qi, Wang Pengyu, Zhang Youzhi
Department of Central Laboratory, The Affiliated Hospital of Jiaxing University, Jiaxing, Zhejiang, China.
School of Pharmacy, Hubei Engineering Research Center of Traditional Chinese Medicine of South Hubei Province, Xianning Medical College, Hubei University of Science and Technology, Xianning, China.
Front Pharmacol. 2025 Jan 29;15:1531220. doi: 10.3389/fphar.2024.1531220. eCollection 2024.
Non-SMC condensin II complex subunit D3 (NCAPD3) has recently been demonstrated as a crucial oncogenic factor, nevertheless, the biological role of NCAPD3 in the pathogenesis of breast cancer has not been elucidated. Evidence suggests that targeting ferroptosis can inhibit the progression of breast cancer. Moreover, 2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-glucoside (THSG) could modulate MCF-7 cell proliferation in our previous study. Therefore, we aimed to investigate the potential mechanism by which NCAPD3 mediates ferroptosis in THSG inhibition of T47D cell proliferation by full-length transcriptome sequencing.
Alternative splicing analysis was performed based on full-length transcriptome sequencing and the overlapping genes in differentially expressed transcripts (DETs) and differential alternative splicing (diAS) were obtained. Further, RT-PCR was used to validate the type of alternative splicing. And the hub genes (transcripts) were selected using the bioinformatics analysis, quantitative polymerase chain reaction (qPCR) and Western blotting (WB). Moreover, cell cycle and ferroptosis were assessed using flow cytometry analysis and WB respectively. Mechanically, cell viability and clone formation was detected using Biochemical kit. And siRNA of was transfected into T47D cells to detect the expression levels of ferroptosis-related proteins (WB) and cell viability (MTT).
40 overlapping transcripts of DETs and diAS were obtained consistent with the analysis of full-length transcriptome sequencing, and () is key gene (transcript), which was also highly expressed in breast cancer and THSG could inhibit the mRNA and protein expression. Moreover, THSG could induce cell cycle arrest in G2/M stage and reduce ferroptosis-related protein expression (xCT and GPx4). Mechanically, we found that THSG inhibits the cell proliferation and clone formation in T47D cells, and inhibition could inhibit (xCT and GPx4) proteins expression, which regulated THSG-suppressing effect in T47D cells.
THSG could inhibit the proliferation in T47D cells by NCAPD3 -dependent ferroptosis, which provided novel insights into targeted strategy for breast cancer.
非SMC凝聚素II复合体亚基D3（NCAPD3）最近被证明是一种关键的致癌因子，然而，NCAPD3在乳腺癌发病机制中的生物学作用尚未阐明。有证据表明，靶向铁死亡可以抑制乳腺癌的进展。此外，在我们之前的研究中，2,3,5,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷（THSG）可以调节MCF-7细胞的增殖。因此，我们旨在通过全长转录组测序研究NCAPD3在THSG抑制T47D细胞增殖中介导铁死亡的潜在机制。
基于全长转录组测序进行可变剪接分析，获得差异表达转录本（DETs）和差异可变剪接（diAS）中的重叠基因。此外，使用RT-PCR验证可变剪接的类型。并通过生物信息学分析、定量聚合酶链反应（qPCR）和蛋白质免疫印迹法（WB）筛选关键基因（转录本）。此外，分别使用流式细胞术分析和WB评估细胞周期和铁死亡。从机制上看，使用生化试剂盒检测细胞活力和克隆形成。将 的小干扰RNA转染到T47D细胞中，以检测铁死亡相关蛋白的表达水平（WB）和细胞活力（MTT）。
获得了40个与全长转录组测序分析一致的DETs和diAS重叠转录本， （ ）是关键基因（转录本），其在乳腺癌中也高表达，THSG可以抑制其mRNA和蛋白表达。此外，THSG可以诱导细胞周期阻滞在G2/M期，并降低铁死亡相关蛋白的表达（xCT和GPx4）。从机制上看，我们发现THSG抑制T47D细胞的增殖和克隆形成， 抑制可以抑制（xCT和GPx4）蛋白表达，这调节了THSG在T47D细胞中的抑制作用。
THSG可以通过NCAPD3依赖性铁死亡抑制T47D细胞的增殖，这为乳腺癌的靶向治疗策略提供了新的见解。
