TRIM24/ZFX affects the stemness and resistance to 5-FU of colorectal cancer cells

Colorectal cancer (CRC) is the second leading cause of cancer death, and about 10% of all malignancies are CRC. Cancer stem cells are considered main culprits in CRC treatment resistance and disease recurrence. This study explored the effects of tripartite motif containing 24 (TRIM24) and zinc finger protein, X-linked (ZFX) on CRC cell stemness and 5-FU resistance. A 5-FU-resistant cell line (HT29-5-FU) was constructed for functional analysis of CRC 5-FU-resistant cells. qRT-PCR and western blot (WB) were employed to analyze mRNA and protein levels of ZFX in 5-FU resistant cells and sensitive cells. WB was also utilized to analyze the surface markers of stem cells in each group. CCK-8 assay determined the IC₅₀ values of different cell groups treated with 5-FU. The sphere-forming ability of cells in each group was determined using tumor sphere assay. Dual-luciferase reporter gene assay validated binding of ZFX to TRIM24. ZFX was highly expressed in HT29-5-FU cells. Silencing ZFX significantly reduced the 5-FU resistance and IC₅₀ value of HT29-5-FU cells, and the surface markers and cell sphere-forming ability of stem cells were also significantly reduced. The function of HT29 cells was opposite when ZFX was overexpressed. In CRC cells, TRIM24 was an upstream transcription factor of ZFX, and they interacted with each other. TRIM24 activated the expression of ZFX to influence the stemness and 5-FU resistance of cells. The TRIM24/ZFX regulatory axis affected the stemness of CRC cells and their sensitivity to 5-FU, providing potential drug targets for novel therapeutic avenues for CRC.

结直肠癌（Colorectal cancer, CRC）是癌症相关死亡的第二大诱因，全球约10%的恶性肿瘤为结直肠癌。肿瘤干细胞被认为是结直肠癌治疗耐药与疾病复发的主要元凶。本研究探讨了含三基序蛋白24（tripartite motif containing 24, TRIM24）与X连锁锌指蛋白（zinc finger protein, X-linked, ZFX）对结直肠癌细胞干性及5-氟尿嘧啶（5-FU）耐药性的影响。本研究构建了5-氟尿嘧啶耐药细胞系HT29-5-FU，用于开展结直肠癌5-氟尿嘧啶耐药细胞的功能分析。采用实时定量聚合酶链反应（qRT-PCR）与蛋白质印迹（western blot, WB）技术，分析5-氟尿嘧啶耐药细胞与敏感细胞中ZFX的mRNA及蛋白表达水平。同时采用蛋白质印迹技术检测各组细胞的干细胞表面标志物表达。采用CCK-8法检测不同细胞组经5-氟尿嘧啶处理后的半数抑制浓度（IC₅₀）值。采用肿瘤球形成实验检测各组细胞的成球能力。采用双荧光素酶报告基因实验验证了ZFX与TRIM24的结合作用。ZFX在HT29-5-FU细胞中呈高表达。沉默ZFX可显著降低HT29-5-FU细胞的5-氟尿嘧啶耐药性及其半数抑制浓度值，同时显著下调干细胞表面标志物表达并削弱细胞成球能力。过表达ZFX则对HT29细胞产生相反的效应。在结直肠癌细胞中，TRIM24是ZFX的上游转录因子，二者存在相互作用。TRIM24通过激活ZFX的表达，进而影响细胞干性与5-氟尿嘧啶耐药性。TRIM24/ZFX调控轴可影响结直肠癌细胞的干性及其对5-氟尿嘧啶的敏感性，为结直肠癌的新型治疗策略提供了潜在的药物靶点。