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Published on: October 30, 2013
MiR-133a induces apoptosis through direct regulation of GSTP1 in bladder cancer cell lines
Yousuke Uchida1, Takeshi Chiyomaru, Hideki Enokida
1Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
Objective:
We previously demonstrated that miR-133a is a tumor-suppressive microRNA (miRNA) and is commonly down-regulated in human bladder cancer (BC). The aim of this study is to determine a novel oncogenic gene targeted by miR-133a in BC.
Methods:
To identify genes targeted by miR-133a, an oligo-microarray analysis was performed using the miR-133a-transfected BC cell lines. For gain/loss-of-function studies, miR-133a/si-glutathione S-transferase π1 (GSTP1)-transfectants were subjected to XTT assay and flow cytometry to evaluate their cell viability and apoptosis status. The luciferase reporter assay was used to confirm the actual binding sites between miR-133a and GSTP1 mRNA. The mRNA and protein expression of GSTP1 in BC cell lines and clinical samples were evaluated by real-time RT-PCR and Western blot, respectively.
Results:
MiR-133a transfection induced cell viability inhibition and apoptosis in BC cell lines. We focused on the GSTP1 gene that was the top 7 down-regulated one in the gene profile from the miR-133a-transfectants. MiR-133a transfection repressed expression levels of mRNA and protein levels of GSTP1. A luciferase reporter assay suggested that the actual binding may occur between miR-133a and GSTP1 mRNA. Cell viability inhibition and apoptosis were induced in the si-GSTP1 transfectants compared with the controls (P < 0.005). GSTP1 mRNA expression levels in 43 clinical BCs were significantly higher than those in eight normal bladder epitheliums (P = 0.0277).
Conclusion:
Our data suggest that tumor suppressive miR-133a directly regulated oncogenic GSTP1 gene in BC, and that an anti-apoptotic effect mediated by GSTP1 is maintained by miR-133a down-regulation in human BC.
Insights
MicroRNA-133a (miR-133a) suppresses tumors in bladder cancer (BC) by targeting the oncogenic glutathione S-transferase pi 1 (GSTP1) gene. Down-regulation of miR-133a in BC leads to increased GSTP1, promoting cancer cell survival.
Area of Science:
- Molecular Biology
- Oncology
- Gene Regulation
Background:
- MicroRNA-133a (miRNA) is a known tumor suppressor in human bladder cancer (BC).
- MiR-133a is frequently downregulated in BC, suggesting a role in cancer progression.
Purpose of the Study:
- To identify novel oncogenic genes targeted by miR-133a in bladder cancer.
- To elucidate the regulatory relationship between miR-133a and its target gene in BC.
Main Methods:
- Oligo-microarray analysis to identify miR-133a targets in BC cell lines.
- Gain/loss-of-function studies (transfection) to assess cell viability and apoptosis.
- Luciferase reporter assay to confirm direct binding of miR-133a to target mRNA.
- Real-time RT-PCR and Western blot to evaluate gene and protein expression.
Main Results:
- MiR-133a transfection inhibited BC cell viability and induced apoptosis.
- Glutathione S-transferase pi 1 (GSTP1) was identified as a key downregulated gene.
- MiR-133a directly represses both mRNA and protein expression of GSTP1.
- Silencing GSTP1 mimicked the anti-proliferative and pro-apoptotic effects of miR-133a.
- GSTP1 expression was significantly elevated in clinical BC samples compared to normal tissues.
Conclusions:
- Tumor-suppressive miR-133a directly targets and regulates the oncogenic GSTP1 gene in bladder cancer.
- The anti-apoptotic function of GSTP1 is sustained by miR-133a downregulation in human BC.
- This miR-133a/GSTP1 axis represents a potential therapeutic target for bladder cancer.
