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An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells
Published on: May 21, 2019
Tumor‑suppressive microRNA‑223 targets WDR62 directly in bladder cancer
Satoshi Sugita1, Hirofumi Yoshino1, Masaya Yonemori1
1Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890‑8520, Japan.
Abstract:
miRNA‑223 (miR‑223) has been reported to function not only as a tumor suppressor, but also as an oncogenic microRNA (miRNA or miR) in various cancer cells. Therefore, the functional role of miR‑223 has not been elucidated to date, at least to the best of our knowledge. We previously performed the deep sequencing analysis of clinical bladder cancer (BC) specimens. It was revealed that miR‑223 expression was significantly downregulated in BC, suggesting that miR‑223 functions as a tumor suppressor miRNA in BC. The aim of this study was to investigate the functional roles of miR‑223 and to identify its targets in BC. The expression levels of miR‑223 were significantly decreased in our clinical BC specimens. The Cancer Genome Atlas (TCGA) database indicated that miR‑223 expression was related to lymphovascular invasion and distant metastasis. The restoration of miR‑223 expression significantly inhibited tumor aggressiveness and induced apoptosis via caspase‑3/7 activation in BC cells. WD repeat domain 62 (WDR62), a candidate target of miR‑223 according to in silico analyses, has been previously proposed to play a role in neurodevelopment. Direct binding between WDR62 and miR‑223 was confirmed by luciferase assay. The TCGA database revealed positive associations between WDR62 mRNA expression and a higher tumor grade and stage in BC. The knockdown of WDR62 significantly inhibited tumor aggressiveness and induced the apoptosis of BC cells. On the whole, the findings of this study reveal a novel miR‑223 target, oncogenic WDR62, and provided insight into the oncogenesis of BC.
Insights
MicroRNA-223 (miR-223) acts as a tumor suppressor in bladder cancer (BC). Restoring miR-223 inhibits BC cell aggressiveness and promotes apoptosis by targeting oncogenic WDR62.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- The role of microRNA-223 (miR-223) in cancer is context-dependent, acting as both a tumor suppressor and oncogene.
- Previous deep sequencing identified downregulated miR-223 in clinical bladder cancer (BC) specimens, suggesting a tumor-suppressive role.
Purpose of the Study:
- To investigate the functional roles of miR-223 in bladder cancer.
- To identify miR-223 targets and elucidate its mechanism in BC oncogenesis.
Main Methods:
- Analysis of miR-223 expression in clinical BC specimens and TCGA database.
- Restoration of miR-223 expression and WDR62 knockdown in BC cells.
- Luciferase assay to confirm direct binding between miR-223 and WDR62.
- Assessment of tumor aggressiveness and apoptosis (caspase-3/7 activation).
Main Results:
- miR-223 expression was significantly decreased in BC, correlating with lymphovascular invasion and distant metastasis.
- Restoring miR-223 inhibited BC aggressiveness and induced apoptosis.
- WD repeat domain 62 (WDR62) was identified as a direct miR-223 target, with its expression linked to higher tumor grade and stage.
- WDR62 knockdown suppressed BC aggressiveness and induced apoptosis.
Conclusions:
- miR-223 functions as a tumor suppressor in bladder cancer.
- Oncogenic WDR62 is a novel target of miR-223, providing insights into BC oncogenesis.
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