KNSTRN knockdown impairs autophagy flux and inhibits bladder cancer progression through ROS- mediated lysosomal dysfunction

Bladder cancer (BLCA) is a prevalent cancer affecting the urinary tract, the mitochondria-localized Astrin-binding protein (KNSTRN) has been shown to be strongly associated with the development of several cancers. In addition, abnormal levels of autophagy have been shown to have a dramatic impact on tumor development. However, the potential mechanisms of BLCA autophagy regulation by KNSTRN remain poorly understood. In this research, we found that knockdown of KNSTRN inhibited the autophagic flux of BLCA. Mechanistically, knockdown of KNSTRN leads to reactive oxygen species (ROS)-dependent disruption of the lysosomal acidic environment and reduction of protease activity, hindering autophagosome-lysosome fusion. Clioquinol reactivates lysosomal activity by normalizing lysosomal pH, which in turn reinstates autophagic flux. Intracellular ROS production is also critical for this process, as ROS scavengers (NAC) reverse lysosomal dysfunction and reactivate autophagic flux. Additionally, the autophagy activator rapamycin (Rapa) effectively protected against KNSTRN knockdown-induced cell death in vitro and in vivo. Thus, we demonstrate that knockdown of KNSTRN leads to intracellular ROS accumulation and lysosomal dysfunction, which impairs autophagic flux and inhibits BLCA progression. Overall design: T24 cells were inoculated into 6 cm dishes and cultured for 24 hours until the cells were fully adherent to the wall, then treated with si-NC or si-KNSTRN for 24 h. subsequently, total RNA was extracted using RNA extraction solution and transcriptome sequencing was performed. Three biological replicates per group.