Introduction: Patients with advanced bladder cancer are generally treated with a combination of chemotherapeutics, including gemcitabine, but the effect is limited due to acquisition of drug resistance. Several microRNAs (miRNAs) have been proven to be associated with drug resistance. Thus, in this study, we focused on miRNAs and investigated the mechanism of gemcitabine resistance. Methods: First, human BC cell lines (BOY, T24) were cultured in gemcitabine supplemented culture medium for 12 months to establish gemcitabine-resistant cells (GEM-R-BOY, GEM-R-T24). Small RNA sequencing analyses were performed to search for miRNAs involved in gemcitabine resistance. The candidate miRNA was transduced into gemcitabine-resistant cells for functional analysis. Target genes of the candidate miRNA were subjected to RNA sequencing analysis, and functional analyses were performed by knockdown of target genes. Results: Established gemcitabine-resistant cells showed more than 3-fold resistance to gemcitabine in IC50 compared to the parental cells in vitro, and also showed significant gemcitabine resistance in vivo. miR-99a-5p, selected as a candidate miRNA for gemcitabine resistance, was down-regulated compared to its parental cells. In gain-of-function studies, miR-99a-5p inhibited proliferation, migration and invasion both in parent and gemcitabine-resistant cells. In addition, miR-99a-5p restored the susceptibility to gemcitabine in gemcitabine-resistant cells. As a target of miR-99a-5p, we focused on SMARCD1 based on RNA sequence analysis. Dual luciferase reporter assays showed that miR-99a-5p directly regulated SMARCD1. Loss-of-function studies with si-RNAs revealed suppression of proliferative, migratory, and invasive abilities of cancer cells both in parent and gemcitabine-resistant cells, and restored their sensitivity to gemcitabine in gemcitabine-resistant cells. Furthermore, ß-galactosidase staining showed that miR-99a-5p induction and SMARCD1 suppression contributed to cellular senescence through p21 induction. Conclusions: We found that miR-99a-5p overexpression or SMARCD1 down regulation induced cellular senescence with recovery of gemcitabine sensitivity. The identification of miR-99a-5p and SMARCD1 as key molecules to overcome gemcitabine resistance may lead to a better understanding of BC and the development of new therapeutic strategies to treat this disease. SOURCE OF Funding: none
