Potential tumor-suppressive role of microRNA-99a-3p in sunitinib-resistant renal cell carcinoma cells through the regulation of RRM2

Sunitinib is the most common primary molecular-targeted agent for metastatic clear cell renal cell carcinoma (ccRCC); however, intrinsic or acquired sunitinib resistance has become a significant problem in medical practice. The present study focused on microRNA (miR)-99a-3p, which was significantly downregulated in clinical sunitinib-resistant ccRCC tissues in previous screening analyses, and investigated the molecular network associated with it. The expression levels of miR-99a-3p and its candidate target genes were evaluated in RCC cells, including previously established sunitinib-resistant 786-o (SU-R-786-o) cells, and clinical ccRCC tissues, using reverse transcription-quantitative polymerase chain reaction. Gain-of-function studies demonstrated that miR-99a-3p significantly suppressed cell proliferation and colony formation in RCC cells, including the SU-R-786-o cells, by inducing apoptosis. Based on in silico analyses and RNA sequencing data, followed by luciferase reporter assays, ribonucleotide reductase regulatory subunit-M2 (RRM2) was identified as a direct target of miR-99a-3p in the SU-R-786-o cells. Loss-of-function studies using small interfering RNA against RRM2 revealed that cell proliferation and colony growth were significantly inhibited via induction of apoptosis, particularly in the SU-R-786-o cells. Furthermore, the RRM2 inhibitor Didox (3,4-dihydroxybenzohydroxamic acid) exhibited anticancer effects in the SU-R-786-o cells and other RCC cells. To the best of our knowledge, this is the first report demonstrating that miR-99a-3p directly regulates RRM2. Identifying novel genes targeted by tumor-suppressive miR-99a-3p in sunitinib-resistant RCC cells may improve our understanding of intrinsic or acquired resistance and facilitate the development of novel therapeutic strategies.