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Published on: July 28, 2012
[Effect of synthetic small double-stranded RNA on the development of bladder cancer by activating P21 expression]
Qiangqiang Ge1, Chenghe Wang, Zhong Chen
1Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Institute of Urology of Hubei Province, Wuhan 430030, China.
Objective:
To investigate the effects of a synthetic small double-stranded RNA (dsRNA) dsP21-555 on the development of bladder cancer cell lines T24 and EJ.
Methods:
According to the different treatments, bladder cancer cells were divided into three groups: negative control group (transfected with dsControl), positive control group (transfected with candidate microRNA, i.e. miR-370) and experimental group (transfected with dsP21-555). Real-time fluorescent quantitative polymerase chain reaction (qPCR) was conducted to detect the expressions of p21 mRNA and cyclin-dependent kinases 4/6 (CDK4/6) mRNA; Western blot was operated to verify the expression of P21 and CDK4/6 proteins. Cell cycle distribution was measured by flow cytometry after transfection. Cell proliferation assay was performed to evaluate the proliferative capacity of transfected cells. Colony formation assay was carried out to analyze the proliferative ability of single cancer cells.
Results:
qPCR showed that, compared with the negative control group, dsP21-555 up-regulated the expressions of p21 mRNA by 2.46 times (P<0.01) in T24 cells and 2.60 times (P<0.01) in EJ cells; compared with the positive control group, the expression of p21 mRNA was no significantly different in the experimental group (P>0.05). Compared with the dsControl group, dsP21-555 suppressed the expressions of CDK4 mRNA by 43% (P<0.01) in T24 and 54% (P<0.01) in EJ cells, the expression of CDK6 mRNA by 39% (P<0.01) in T24 and 36% (P<0.01) in EJ cells; the differences in the expression of CDK4 and CDK6 mRNAs between the miR-370 and dsP21-555 groups were not statistically significant (P> 0.05). Western blot verified the differences of p21 and CDK4/6 genes expression among groups. Flow cytometry revealed that the G0/G1 phase cells significantly increased while S and G2/M phase cells decreased in the miR-370 and the dsP21-555 groups, compared with the dsControl group. Cell proliferation assay showed that, compared with the dsControl group, the proliferative capacities of cells transfected with miR-370 or dsP21-555 decreased significantly (both P<0.05), but the difference in proliferative capacities between the miR-370 and the dsP21-555 groups was no statistically significant (P>0.05). Colony formation assay showed that the numbers of colonies formed in the miR-370 and the dsP21-555 groups were both smaller than that in the dsControl group.
Conclusion:
dsP21-555 may activate the expression of P21 protein by RNA activation, thereby significantly inhibit the growth of bladder cancer cells.
Insights
Synthetic small double-stranded RNA (dsRNA) dsP21-555 effectively inhibits bladder cancer cell growth by upregulating P21 expression and downregulating CDK4/6. This dsRNA demonstrates potential as a therapeutic agent for bladder cancer.
Area of Science:
- Molecular Biology
- Cancer Research
- RNA Therapeutics
Background:
- Bladder cancer remains a significant health concern with ongoing research into novel therapeutic strategies.
- Targeting cell cycle regulators is a promising approach for cancer treatment.
- Small double-stranded RNA (dsRNA) molecules offer potential for gene regulation in cancer therapy.
Purpose of the Study:
- To evaluate the efficacy of a synthetic dsRNA, dsP21-555, in inhibiting the proliferation and development of human bladder cancer cell lines (T24 and EJ).
- To investigate the molecular mechanisms underlying the effects of dsP21-555, focusing on the p21 and cyclin-dependent kinase 4/6 (CDK4/6) pathways.
Main Methods:
- Quantitative polymerase chain reaction (qPCR) and Western blot assays were employed to assess the expression levels of p21, CDK4, and CDK6 at both mRNA and protein levels.
- Cell cycle distribution was analyzed using flow cytometry.
- Cell proliferation and colony formation assays were conducted to evaluate the impact of dsP21-555 on cancer cell growth and viability.
Main Results:
- dsP21-555 significantly upregulated p21 mRNA and protein expression in both T24 and EJ bladder cancer cells.
- Treatment with dsP21-555 led to a significant downregulation of CDK4 and CDK6 mRNA and protein expression.
- Flow cytometry indicated an increase in G0/G1 phase cells and a decrease in S and G2/M phase cells, suggesting cell cycle arrest.
- Both cell proliferation and colony formation assays demonstrated a significant inhibition of cancer cell growth following dsP21-555 treatment.
Conclusions:
- The synthetic dsRNA, dsP21-555, effectively inhibits bladder cancer cell proliferation and development.
- dsP21-555 appears to exert its anti-cancer effects by activating P21 expression and subsequently inhibiting CDK4/6, leading to cell cycle arrest.
- These findings suggest that dsP21-555 holds promise as a potential RNA-based therapeutic agent for bladder cancer.
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