[Study on the molecular mechanism of Mycobacterium tuberculosis H37Ra promoting USP33 expression through DANCR]

Objective To investigate the molecular mechanisms by which long-stranded non-coding RNA (lncRNA) differentiated antagonistic non-protein-coding RNA (DANCR) regulates ubiquitin-specific protease 33 (USP33) and the glutathione peroxidase 4 (GPX4) in the context of Mycobacterium tuberculosis (Mtb) H37Ra infection. Methods A DANCR overexpression model was established using the THP-1 cell line. By integrating bioinformatics prediction, real-time quantitative PCR, Western blot, RNA immunoprecipitation, co-immunoprecipitation-proteomics, and molecular docking techniques, the regulatory effects of DANCR on miRNA, USP33, and GPX4 were systematically analyzed, as well as the biological functions of DANCR under the infection of the attenuated Mtb strain H37Ra. Results DANCR acted as a competitive endogenous RNA (ceRNA), specifically sponging miR-19a-3p, and relieving the post-transcriptional inhibition of miR-19a-3p on USP33, thereby significantly up-regulating the mRNA and protein levels of USP33. Molecular docking and co-immunoprecipitation experiments suggested that USP33 and GPX4 interacted physically and overexpression of DANCR induced the protein level of GPX4 without affecting its mRNA, indicating that this regulation occurs at the post-translational level. Conclusion The study suggested a key DANCR-miR-19a-3p-USP33 regulatory axis, in which the long non-coding RNA DANCR acts as a ceRNA by sponging miR-19a-3p to relieve its inhibition on USP33, thereby upregulating USP33 expression. Meanwhile, the results of bioinformatics analysis and co-immunoprecipitation indicated that USP33 has a direct physical interaction with the ferroptosis key protein GPX4, and it is speculated that USP33 may stabilize GPX4 through deubiquitination modification, providing new molecular clues and therapeutic targets for understanding the metabolic regulation, oxidative stress response, and potential ferroptosis mechanism in the interaction between the host and Mtb.