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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-21 modulates cell proliferation and sensitivity to doxorubicin in bladder cancer cells
1Department of Urology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, PR China.
Abstract:
Transitional cell carcinomas (TCCs) of the urinary bladder are common malignancies with a high recurrence rate. Since microRNA-21 (miR-21) may contribute to tumorigenesis and chemoresistance in many cancer types, we aimed to investigate its efficacy in TCCs. The expression of miR-21 and its target PTEN was determined by real-time qRT-PCR and western blotting, respectively in tumor tissues as well as adjacent non-tumor mucosa. The effect of miR-21 on cell proliferation and chemosensitivity to doxorubicin were measured using the MTT method. Cell apoptosis induced by doxorubicin was investigated using flow cytometry in the T24 cell line. BCL-2, AKT and pAKT were detected by western blotting for analysis of potential mechanisms. miR-21 was significantly up-regulated in tumor tissues while PTEN was expressed in lower levels compared to non-tumor tissues. A negative correlation between expression of miR-21 and PTEN was established in vivo. Cell proliferation and chemoresistance to doxorubicin were promoted by overexpression of miR-21 in T24 cells. BCL-2 up-regulation could be achieved by miR-21 overexpression, which prevented T24 cells from apoptosis induced by doxorubicin. Furthermore, the miR-21 induced BCL-2 up-regulation could be cancelled by the PI3K inhibitor LY294002. These data verified the oncogenic role of miR-21 in TCCs and may usher in new therapeutic strategies in treating this disease.
Insights
MicroRNA-21 (miR-21) promotes bladder cancer growth and doxorubicin resistance by downregulating PTEN and upregulating BCL-2. This suggests miR-21 as a therapeutic target for transitional cell carcinomas (TCCs).
Area of Science:
- Molecular Oncology
- Cancer Biology
- Urothelial Carcinomas Research
Background:
- Transitional cell carcinomas (TCCs) of the urinary bladder are prevalent malignancies with frequent recurrence.
- MicroRNA-21 (miR-21) is implicated in tumorigenesis and chemoresistance across various cancers.
- The role of miR-21 in bladder TCCs warrants investigation for potential therapeutic strategies.
Purpose of the Study:
- To investigate the expression and role of miR-21 in transitional cell carcinomas (TCCs) of the urinary bladder.
- To explore the impact of miR-21 on cancer cell proliferation, chemosensitivity, and apoptosis.
- To elucidate the underlying molecular mechanisms involving PTEN, BCL-2, and the PI3K/AKT pathway.
Main Methods:
- Quantitative real-time PCR (qRT-PCR) and western blotting to assess miR-21 and PTEN expression in tumor and non-tumor tissues.
- MTT assays to evaluate the effects of miR-21 on cell proliferation and doxorubicin chemosensitivity.
- Flow cytometry for apoptosis analysis and western blotting for BCL-2, AKT, and pAKT detection in T24 cell lines.
Main Results:
- miR-21 was significantly upregulated in TCC tissues, inversely correlated with PTEN expression.
- Overexpression of miR-21 enhanced T24 cell proliferation and conferred resistance to doxorubicin.
- miR-21 promoted BCL-2 upregulation, inhibiting doxorubicin-induced apoptosis, an effect reversed by PI3K inhibition.
Conclusions:
- miR-21 plays a significant oncogenic role in urinary bladder TCCs.
- miR-21 contributes to chemoresistance, potentially through the PI3K/AKT/BCL-2 pathway.
- Targeting miR-21 may represent a novel therapeutic strategy for bladder cancer treatment.
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