To date, there has not been adequate research conducted on prostate cancer tumor stem cells, which play an important role in tumor recurrence, invasion, and drug resistance. In this study, we innovatively used a tumor stem cell-associated index (mRNAsi), calculated by a single-class logistic regression machine learning algorithm (OCRL), in combination with multiple databases to identify MBNL1-AS1 as a key lncRNA for prostate cancer stem cells and elucidated via in vivo and in vitro experiments. In addition, the experiments showed that the down-regulation of MBNL1-AS1 promotes the malignance of prostate cancer cell lines through the miR-221-3p/CDKN1B/C-myc axis by stimulating the stemness of tumor stem cells. This provides the basis for theoretical studies of prostate cancer and provides targets for precision therapy.