CD44通过调节癌细胞转移、干性和化疗敏感性在肿瘤发生中起到关键作用，并被认为是人类肿瘤，包括结直肠癌（CRC）的有希望的治疗靶点。因此，本研究旨在检查CD44沉默和5-氟尿嘧啶（5-FU）对CRC细胞体外肿瘤发生的同时治疗效应。首先在TCGA数据库和CRC组织中评估了CD44的表达。此外，在过表达CD44的HT-29 CRC细胞上进行了功能分析。将细胞转染CD44 siRNA，然后用5-FU处理。因此，为探索联合治疗对细胞活力、迁移、凋亡和染色质断裂的影响，我们进行了MTT实验、划痕实验、Annexin V/PI染色和DAPI染色实验。细胞球和集落形成实验进一步用于研究干性特征。利用Western blotting和qPCR探索了蛋白和mRNA水平的基因表达。我们的发现表明，与正常样本相比，CRC组织中CD44显著过表达。抑制CD44显著提高了HT-29细胞对5-FU的化疗敏感性，并通过诱导凋亡实现。此外，联合治疗导致凋亡基因（包括P53、caspase-3和caspase-9）的过表达，以及AKT1表达的下调。此外，抑制CD44，无论单独还是与5-FU联合，通过下调Sox2和Nanog的表达抑制了HT-29细胞的干性特性。此外，联合治疗显著下调了MMPs并抑制了CRC细胞的迁移。考虑到CD44在对5-FU的化疗敏感性中的作用，可以建议将其作为改善CRC化疗效果的潜在靶点。©2023 The Authors.

CD44 plays a pivotal role through tumorigenesis by regulating cancer cell metastasis, stemness, and chemosensitivity and is considered a promising therapeutic target for human cancers, including colorectal cancer (CRC). Therefore, the present research aimed to examine the simultaneous therapeutic effect of CD44 silencing and 5-fluorouracil (5-FU) on in vitro tumorigenesis of CRC cells.CD44 expression was initially evaluated in TCGA datasets and CRC tissues. Furthermore, functional analysis was performed on HT-29 CRC cells overexpressing CD44. The cells were transfected with CD44 siRNA and then treated with 5-FU. Consequently, to explore the combination therapy effect on cell viability, migration, apoptosis, and chromatin fragmentation, we performed MTT assay, scratch assay, Annexin V/PI staining and DAPI staining assays, respectively. The spheroid and colony formation assays were further employed to investigate stemness features. The gene expression at protein and mRNA levels were explored using western blotting and qPCR.Our findings illustrated that CD44 was significantly overexpressed in CRC tissues compared to normal samples. The suppression of CD44 considerably promoted the chemosensitivity of HT-29 cells to 5-FU by apoptosis induction. Also, the combination therapy led to overexpression of apoptotic genes, including P53, caspase-3, and caspase-9, as well as downregulation of AKT1 expression. Furthermore, CD44 suppression, separately or combined with 5-FU, hindered stemness properties in HT-29 cells via downregulation of Sox2 and Nanog expression. Besides, the combination therapy remarkably downregulated MMPs and suppressed CRC cell migration.Considering its involvement in chemosensitivity to 5-FU, CD44 could be suggested as a potential target for improving the efficiency of CRC chemotherapy.©2023 The Authors.