Background: Bladder cancer (BCa) is characterized by a high recurrence rate, and BCa stem cells (BCSCs) are widely considered the principal driver of drug resistance and metastasis. Although the transcription factor paired-related homeobox 1 (PRRX1) has been reported to regulate stemness in various solid tumors, its role and underlying mechanisms in BCa remain unclear. This study aims to investigate the function of PRRX1 in BCSCs and the potential mechanisms involved.

Methods: BCSCs were isolated from BCa cells, after which the proliferation, migration, invasion, gemcitabine resistance, and PRRX1 expression levels were detected in both BCa cells and BCSCs. Subsequently, BCSCs were transfected with si-PRRX1, and the effects of si-PRRX1 on BCSC proliferation, migration, invasion, stemness, and gemcitabine resistance were further assessed. Lastly, the impact of si-PRRX1 on the transforming growth factor-β (TGF-β)1/Smad axis in BCSCs was assessed to explore a potential mechanistic basis.

Results: Compared with BCa cells, BCSCs exhibited stronger proliferation, migration, invasion, stemness, and gemcitabine resistance, and PRRX1 expression in BCSCs was significantly higher than that in BCa cells (p < 0.05). After PRRX1 was knocked down, the proliferative, migratory, invasive, and stemness-related abilities of BCSCs were weakened, while sensitivity to gemcitabine was enhanced (p < 0.05). In addition, PRRX1 knockdown in BCSCs led to decreased protein expression of TGF-β1 and reduced phosphorylation levels of Smad2 and Smad3, as reflected by lower p-Smad2/Smad2 and p-Smad3/Smad3 ratios (p < 0.05).

Conclusion: PRRX1 knockdown suppresses BCSC proliferation, migration, invasion, stemness, and gemcitabine resistance, an effect that may be related to inhibition of the TGF-β1/Smad axis.