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Upregulation of long non-coding RNA urothelial carcinoma associated 1 by CCAAT/enhancer binding protein α contributes to bladder cancer cell growth and reduced apoptosis.

Xue M, Li X, Wu W, Zhang S, Wu S, Li Z, Chen W - Oncol. Rep. (2014)

Bottom Line: We also demonstrated that C/EBPα siRNA treatment contributed to the downregulation of lncRNA-UCA1 expression, whereas overexpression of C/EBPα enhanced lncRNA-UCA1 expression.Furthermore, lncRNA-UCA1 transcriptional repression by C/EBPα siRNA sharply reduced cell viability and induced cell apoptosis in vitro.Collectively, our results provide a novel therapeutic strategy for bladder cancer by effectively interrupting the binding of the lncRNA-UCA1 promoter and certain transcription factors, so as to reverse the upregulation of lncRNA-UCA1 and prevent bladder cancer progression.

View Article: PubMed Central - PubMed

Affiliation: Center for Translational Medicine, The First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710061, P.R. China.

ABSTRACT
Long non-coding RNA urothelial carcinoma associated 1 (lncRNA-UCA1) is upregulated in bladder cancer and plays a pivotal role in bladder cancer progression and metastasis. Recent studies and our research found that lncRNA-UCA1 may be an important biomarker and therapeutic target for bladder cancer. However, the molecular mechanism involved in the upregulation of lncRNA-UCA1 in bladder cancer is largely unknown. In the present study, we showed that lncRNA-UCA1 expression in bladder cancer cells was upregulated by transcription factor CCAAT/enhancer binding protein α (C/EBPα), which was the only candidate transcription factor simultaneously predicted by a total of five bioinformatical software programs. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay indicated that C/EBPα bound to the lncRNA-UCA1 core promoter region in vitro and in vivo. The luciferase assays further showed that there was a point mutation (A231G) in the C/EBPα binding site of the lncRNA-UCA1 core promoter in various bladder cancer cell lines, which in turn significantly increased the transcriptional activity of lncRNA-UCA1. We also demonstrated that C/EBPα siRNA treatment contributed to the downregulation of lncRNA-UCA1 expression, whereas overexpression of C/EBPα enhanced lncRNA-UCA1 expression. Furthermore, lncRNA-UCA1 transcriptional repression by C/EBPα siRNA sharply reduced cell viability and induced cell apoptosis in vitro. Collectively, our results provide a novel therapeutic strategy for bladder cancer by effectively interrupting the binding of the lncRNA-UCA1 promoter and certain transcription factors, so as to reverse the upregulation of lncRNA-UCA1 and prevent bladder cancer progression.

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C/EBPα binds to the core promoter regions of lncRNA-UCA1 and regulates its expression. (A and B) Knockdown efficiency of C/EBPα siRNA was determined by real-time PCR and western blotting. β-actin was used as the internal control (*P<0.05; n=3). (C and D) The effects of C/EBPα silencing on the lncRNA-UCA1 promoter activity and expression were detected by luciferase assay and real-time PCR. β-actin was used as the internal control (*P<0.05; n=3).
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f4-or-31-05-1993: C/EBPα binds to the core promoter regions of lncRNA-UCA1 and regulates its expression. (A and B) Knockdown efficiency of C/EBPα siRNA was determined by real-time PCR and western blotting. β-actin was used as the internal control (*P<0.05; n=3). (C and D) The effects of C/EBPα silencing on the lncRNA-UCA1 promoter activity and expression were detected by luciferase assay and real-time PCR. β-actin was used as the internal control (*P<0.05; n=3).

Mentions: To verify whether C/EBPα regulates lncRNA-UCA1 transcription in bladder cancer cells, a C/EBPα binding site deleted lncRNA-UCA1 promoter construct (mutant) was cloned. The cells transfected with the mutant promoter construct displayed a ~30% reduction in luciferase reporter activities (Fig. 3B). Therefore, the C/EBPα binding site contributes to lncRNA-UCA1 transcriptional activation. BLZ-211, 5637 and T24 cells were transfected with C/EBPα siRNA. The C/EBPα mRNA and protein levels were reduced by ~70 and ~50%, respectively (Fig. 4A and B). Silencing of C/EBPα also reduced lncRNA-UCA1 promoter activities (30–50% reduction) and expression levels (30–50% reduction) in the BLZ-211, 5637 and T24 cells (Fig. 4C and D), while overexpression of C/EBPα in the 5637 and T24 cells led to an increase in C/EBPα protein levels (~1.65-fold; Fig. 5A), followed by increases in lncRNA-UCA1 promoter activity (~2.5-fold; Fig. 5B) and expression levels (~3-fold; Fig. 5C), respectively. Taken together, these results indicate that C/EBPα strengthens lncRNA-UCA1 transcription.


Upregulation of long non-coding RNA urothelial carcinoma associated 1 by CCAAT/enhancer binding protein α contributes to bladder cancer cell growth and reduced apoptosis.

Xue M, Li X, Wu W, Zhang S, Wu S, Li Z, Chen W - Oncol. Rep. (2014)

C/EBPα binds to the core promoter regions of lncRNA-UCA1 and regulates its expression. (A and B) Knockdown efficiency of C/EBPα siRNA was determined by real-time PCR and western blotting. β-actin was used as the internal control (*P<0.05; n=3). (C and D) The effects of C/EBPα silencing on the lncRNA-UCA1 promoter activity and expression were detected by luciferase assay and real-time PCR. β-actin was used as the internal control (*P<0.05; n=3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4020618&req=5

f4-or-31-05-1993: C/EBPα binds to the core promoter regions of lncRNA-UCA1 and regulates its expression. (A and B) Knockdown efficiency of C/EBPα siRNA was determined by real-time PCR and western blotting. β-actin was used as the internal control (*P<0.05; n=3). (C and D) The effects of C/EBPα silencing on the lncRNA-UCA1 promoter activity and expression were detected by luciferase assay and real-time PCR. β-actin was used as the internal control (*P<0.05; n=3).
Mentions: To verify whether C/EBPα regulates lncRNA-UCA1 transcription in bladder cancer cells, a C/EBPα binding site deleted lncRNA-UCA1 promoter construct (mutant) was cloned. The cells transfected with the mutant promoter construct displayed a ~30% reduction in luciferase reporter activities (Fig. 3B). Therefore, the C/EBPα binding site contributes to lncRNA-UCA1 transcriptional activation. BLZ-211, 5637 and T24 cells were transfected with C/EBPα siRNA. The C/EBPα mRNA and protein levels were reduced by ~70 and ~50%, respectively (Fig. 4A and B). Silencing of C/EBPα also reduced lncRNA-UCA1 promoter activities (30–50% reduction) and expression levels (30–50% reduction) in the BLZ-211, 5637 and T24 cells (Fig. 4C and D), while overexpression of C/EBPα in the 5637 and T24 cells led to an increase in C/EBPα protein levels (~1.65-fold; Fig. 5A), followed by increases in lncRNA-UCA1 promoter activity (~2.5-fold; Fig. 5B) and expression levels (~3-fold; Fig. 5C), respectively. Taken together, these results indicate that C/EBPα strengthens lncRNA-UCA1 transcription.

Bottom Line: We also demonstrated that C/EBPα siRNA treatment contributed to the downregulation of lncRNA-UCA1 expression, whereas overexpression of C/EBPα enhanced lncRNA-UCA1 expression.Furthermore, lncRNA-UCA1 transcriptional repression by C/EBPα siRNA sharply reduced cell viability and induced cell apoptosis in vitro.Collectively, our results provide a novel therapeutic strategy for bladder cancer by effectively interrupting the binding of the lncRNA-UCA1 promoter and certain transcription factors, so as to reverse the upregulation of lncRNA-UCA1 and prevent bladder cancer progression.

View Article: PubMed Central - PubMed

Affiliation: Center for Translational Medicine, The First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710061, P.R. China.

ABSTRACT
Long non-coding RNA urothelial carcinoma associated 1 (lncRNA-UCA1) is upregulated in bladder cancer and plays a pivotal role in bladder cancer progression and metastasis. Recent studies and our research found that lncRNA-UCA1 may be an important biomarker and therapeutic target for bladder cancer. However, the molecular mechanism involved in the upregulation of lncRNA-UCA1 in bladder cancer is largely unknown. In the present study, we showed that lncRNA-UCA1 expression in bladder cancer cells was upregulated by transcription factor CCAAT/enhancer binding protein α (C/EBPα), which was the only candidate transcription factor simultaneously predicted by a total of five bioinformatical software programs. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay indicated that C/EBPα bound to the lncRNA-UCA1 core promoter region in vitro and in vivo. The luciferase assays further showed that there was a point mutation (A231G) in the C/EBPα binding site of the lncRNA-UCA1 core promoter in various bladder cancer cell lines, which in turn significantly increased the transcriptional activity of lncRNA-UCA1. We also demonstrated that C/EBPα siRNA treatment contributed to the downregulation of lncRNA-UCA1 expression, whereas overexpression of C/EBPα enhanced lncRNA-UCA1 expression. Furthermore, lncRNA-UCA1 transcriptional repression by C/EBPα siRNA sharply reduced cell viability and induced cell apoptosis in vitro. Collectively, our results provide a novel therapeutic strategy for bladder cancer by effectively interrupting the binding of the lncRNA-UCA1 promoter and certain transcription factors, so as to reverse the upregulation of lncRNA-UCA1 and prevent bladder cancer progression.

Show MeSH
Related in: MedlinePlus