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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
microRNA-27b inhibits cell proliferation and invasion in bladder cancer by targeting engrailed-2
Yunfei Li1, Qilin Duan1, Lu Gan1
1Department of Urology, Third Affilated Hospital of Shenzhen University, Shenzhen 518000, China.
Background:
Bladder cancer is considered a malignant tumour characterised by great heterogeneity. Engrailed-2 may be a gene implicated in bladder cancer. Bioinformatics analysis found base pair complementation between microRNA-27b and engrailed-2. The present study aimed to investigate the reciprocal association between microRNA-27b and engrailed-2 in bladder cancer.
Methods:
The microRNA-27b and the protein of engrailed-2 in the tissues and cells of the bladder were detected. The processes of apoptosis, proliferation, invasion, and migration of tumour cells were evaluated. The co-action between microRNA-27b and engrailed-2 was detected by a luciferase reporter system. Finally, the interaction between microRNA-27b and engrailed-2 was further verified in vivo.
Results:
The study found that the expression level of microRNA-27b is lower in bladder cancer tissues and cells than that in neighbouring ordinary tissues, whereas the opposite outcome was observed regarding the expression level of engrailed-2. Furthermore, microRNA-27b expression level is not significantly linked to the age of patients with bladder cancer; however, it is significantly associated with the clinicopathological grade of bladder cancer. Notably, engrailed-2 is negatively regulated by microRNA-27b. Transfection with microRNA-27b was associated with a significant reduction in the activity of bladder cancer cells and promoted apoptosis, while engrailed-2 restoration effectively reversed the above effects of microRNA-27b on bladder cancer in vitro and in vivo.
Conclusions:
In conclusion, engrailed-2 is engaged in the development and process of bladder cancer through the negative mediation of microRNA-27b; additionally, microRNA-27b/engrailed-2 could form a signalling pathway with a significant effect on the process of bladder cancer.
Insights
MicroRNA-27b downregulates engrailed-2, inhibiting bladder cancer progression. This microRNA-27b/engrailed-2 pathway impacts bladder cancer development and offers potential therapeutic targets.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Bladder cancer is a heterogeneous malignancy.
- Engrailed-2 (EN2) is a potential gene involved in bladder cancer.
- Bioinformatics revealed a complementarity between microRNA-27b (miR-27b) and EN2.
Purpose of the Study:
- To investigate the reciprocal association between miR-27b and EN2 in bladder cancer.
- To elucidate the role of the miR-27b/EN2 axis in bladder cancer pathogenesis.
Main Methods:
- Quantification of miR-27b and EN2 protein in bladder tissues and cells.
- Assessment of tumor cell apoptosis, proliferation, invasion, and migration.
- Luciferase reporter assays to confirm the interaction between miR-27b and EN2.
- In vitro and in vivo validation of the miR-27b/EN2 interaction.
Main Results:
- miR-27b expression was downregulated, while EN2 expression was upregulated in bladder cancer tissues and cells.
- miR-27b negatively regulated EN2 expression.
- miR-27b transfection reduced bladder cancer cell activity and promoted apoptosis.
- EN2 restoration reversed the effects of miR-27b in vitro and in vivo.
- miR-27b expression correlated significantly with bladder cancer clinicopathological grade.
Conclusions:
- EN2 is implicated in bladder cancer development via negative mediation by miR-27b.
- The miR-27b/EN2 signaling pathway significantly influences bladder cancer progression.
- Targeting the miR-27b/EN2 axis may offer therapeutic strategies for bladder cancer.
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