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miRNAs associated with chemo-sensitivity in cell lines and in advanced bladder cancer.

Nordentoft I, Birkenkamp-Demtroder K, Agerbæk M, Theodorescu D, Ostenfeld MS, Hartmann A, Borre M, Ørntoft TF, Dyrskjøt L - BMC Med Genomics (2012)

Bottom Line: Three miRNAs were associated with both response and survival (886-3p, 923, 944).By changing the cellular level of the response-identified miRNAs in eight bladder cell lines with different cisplatin sensitivity we found that down-regulation of miR-27a, miR296-5p and miR-642 generally reduced the cell viability, whereas up-regulation of miR-138 and miR-886-3p reduced the viability of more than half of the cell lines.Decreasing miR-138 increased the cisplatin sensitivity in half of the cell lines and increasing miR-27a and miR-642 generally increased cisplatin sensitivity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark. iver.nordentoft@ki.au.dk

ABSTRACT

Background: MicroRNA is a naturally occurring class of non-coding RNA molecules that mediate posttranscriptional gene regulation and are strongly implicated in cellular processes such as cell proliferation, carcinogenesis, cell survival and apoptosis. Consequently there is increasing focus on miRNA expression as prognostic factors for outcome and chemotherapy response. Only approximately 50% of patients with bladder cancer respond to chemotherapy. Therefore, predictive markers, such as miRNAs, that can identify subgroups of patients who will benefit from chemotherapy will have great value for treatment guidance.

Methods: We profiled the expression of 671 miRNAs in formalin fixed paraffin embedded tumors from patients with advanced bladder cancer treated with cisplatin based chemotherapy. We delineated differentially expressed miRNAs in tumors from patients with complete response vs. patients with progressive disease and in tumors form patients with short and long overall survival time. Furthermore, we studied the effect of up- and down regulation of key miRNAs on the cisplatin sensitivity in eight bladder cancer cell lines with different sensitivities to cisplatin.

Results: miRNA expression profiling identified 15 miRNAs that correlated with response to chemotherapy and 5 miRNAs that correlated with survival time. Three miRNAs were associated with both response and survival (886-3p, 923, 944). By changing the cellular level of the response-identified miRNAs in eight bladder cell lines with different cisplatin sensitivity we found that down-regulation of miR-27a, miR296-5p and miR-642 generally reduced the cell viability, whereas up-regulation of miR-138 and miR-886-3p reduced the viability of more than half of the cell lines. Decreasing miR-138 increased the cisplatin sensitivity in half of the cell lines and increasing miR-27a and miR-642 generally increased cisplatin sensitivity.

Conclusions: MiRNAs seem to be involved in cisplatin based chemo response and may form a new target for therapy and serve as biomarkers for treatment response.

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Related in: MedlinePlus

miRNA down-regulation in bladder cell lines. LNA based antagomirs were used to silence miR-27a, miR-138, miR-296-5p, miR-642 and miR-886-3p in the bladder cell lines 253JBV, UMUC9, UMUC14, SLT4, 575A, RT4, CLR2169 and HT1197. LNA knockdown molecules were reverse transfected at 20nM (n = 8), and after 48 h incubation culture media, with or without cisplatin (GI50), was added to the cells (n = 4). The viability of the cells was determined after 48 h incubation (96 h post transfection) by MTT-assay. LF2000 designate controls using the transfection reagent alone. A: Relative cell viability expressed as the viability of the antagomiR transfected cells normalized to the negative control molecule for each cell line. B: Relative cell viability expressed as the viability of the antagomiR transfected cells treated with cisplatin (GI50) compared to the antagomiR transfected cells without cisplatin normalized to the negative control molecule for each cell line. (#: the high reduction of cell viability imposed by the antagomirs prohibits precise measure of effect of cisplatin treatment during miR down regulation).
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Figure 4: miRNA down-regulation in bladder cell lines. LNA based antagomirs were used to silence miR-27a, miR-138, miR-296-5p, miR-642 and miR-886-3p in the bladder cell lines 253JBV, UMUC9, UMUC14, SLT4, 575A, RT4, CLR2169 and HT1197. LNA knockdown molecules were reverse transfected at 20nM (n = 8), and after 48 h incubation culture media, with or without cisplatin (GI50), was added to the cells (n = 4). The viability of the cells was determined after 48 h incubation (96 h post transfection) by MTT-assay. LF2000 designate controls using the transfection reagent alone. A: Relative cell viability expressed as the viability of the antagomiR transfected cells normalized to the negative control molecule for each cell line. B: Relative cell viability expressed as the viability of the antagomiR transfected cells treated with cisplatin (GI50) compared to the antagomiR transfected cells without cisplatin normalized to the negative control molecule for each cell line. (#: the high reduction of cell viability imposed by the antagomirs prohibits precise measure of effect of cisplatin treatment during miR down regulation).

Mentions: To further investigate the role of the identified miRNAs in treatment response and survival time of cancer patients, we knocked down five of these key miRNAs (miRNA-27a, miRNA-138, miRNA-296-5p, miRNA-642, miRNA-886-3p) using LNA-anti-miRs. 48 hours after transfection the cells were incubated with cisplatin (GI50) for another 48 hours followed by viability analysis. The experiments were conducted in all eight bladder cancer cell lines to avoid any single cell line-dependent phenomenon and to enable drawing of more general conclusions. miRNA down regulation was verified by qRT-PCR following knockdown using two LNA-anti-miRs (Additional file 6: Figure S1). First, the effect of miRNA knock down was measured using MTT assays (Figure 4A). The viability of the cell lines per se was clearly reduced following down-regulation of miR-296-5p and miR-642, and reduced to some extent following down-regulation of miR-27a and miR-886-3p. Next, the effect of miRNA-knock down on cisplatin sensitivity was measured by incubating Transfected cells with the estimated GI50 concentration of cisplatin. Cisplatin was added 48 h post transfection and incubated for additional 48 h followed by MTT viability measurement (Figure 4B). This revealed no clear correlation between cisplatin sensitivity and miRNA knock down, however down-regulation of miR-138 increased the cisplatin sensitivity of four of the eight tested cell lines by approximately 20% (Figure 4B). The cellular response in cisplatin treated cells to miR-296-5p and miR-642 transfection was more difficult to interpret as these miRNAs already showed reduced viability following the miRNA transfection.


miRNAs associated with chemo-sensitivity in cell lines and in advanced bladder cancer.

Nordentoft I, Birkenkamp-Demtroder K, Agerbæk M, Theodorescu D, Ostenfeld MS, Hartmann A, Borre M, Ørntoft TF, Dyrskjøt L - BMC Med Genomics (2012)

miRNA down-regulation in bladder cell lines. LNA based antagomirs were used to silence miR-27a, miR-138, miR-296-5p, miR-642 and miR-886-3p in the bladder cell lines 253JBV, UMUC9, UMUC14, SLT4, 575A, RT4, CLR2169 and HT1197. LNA knockdown molecules were reverse transfected at 20nM (n = 8), and after 48 h incubation culture media, with or without cisplatin (GI50), was added to the cells (n = 4). The viability of the cells was determined after 48 h incubation (96 h post transfection) by MTT-assay. LF2000 designate controls using the transfection reagent alone. A: Relative cell viability expressed as the viability of the antagomiR transfected cells normalized to the negative control molecule for each cell line. B: Relative cell viability expressed as the viability of the antagomiR transfected cells treated with cisplatin (GI50) compared to the antagomiR transfected cells without cisplatin normalized to the negative control molecule for each cell line. (#: the high reduction of cell viability imposed by the antagomirs prohibits precise measure of effect of cisplatin treatment during miR down regulation).
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Figure 4: miRNA down-regulation in bladder cell lines. LNA based antagomirs were used to silence miR-27a, miR-138, miR-296-5p, miR-642 and miR-886-3p in the bladder cell lines 253JBV, UMUC9, UMUC14, SLT4, 575A, RT4, CLR2169 and HT1197. LNA knockdown molecules were reverse transfected at 20nM (n = 8), and after 48 h incubation culture media, with or without cisplatin (GI50), was added to the cells (n = 4). The viability of the cells was determined after 48 h incubation (96 h post transfection) by MTT-assay. LF2000 designate controls using the transfection reagent alone. A: Relative cell viability expressed as the viability of the antagomiR transfected cells normalized to the negative control molecule for each cell line. B: Relative cell viability expressed as the viability of the antagomiR transfected cells treated with cisplatin (GI50) compared to the antagomiR transfected cells without cisplatin normalized to the negative control molecule for each cell line. (#: the high reduction of cell viability imposed by the antagomirs prohibits precise measure of effect of cisplatin treatment during miR down regulation).
Mentions: To further investigate the role of the identified miRNAs in treatment response and survival time of cancer patients, we knocked down five of these key miRNAs (miRNA-27a, miRNA-138, miRNA-296-5p, miRNA-642, miRNA-886-3p) using LNA-anti-miRs. 48 hours after transfection the cells were incubated with cisplatin (GI50) for another 48 hours followed by viability analysis. The experiments were conducted in all eight bladder cancer cell lines to avoid any single cell line-dependent phenomenon and to enable drawing of more general conclusions. miRNA down regulation was verified by qRT-PCR following knockdown using two LNA-anti-miRs (Additional file 6: Figure S1). First, the effect of miRNA knock down was measured using MTT assays (Figure 4A). The viability of the cell lines per se was clearly reduced following down-regulation of miR-296-5p and miR-642, and reduced to some extent following down-regulation of miR-27a and miR-886-3p. Next, the effect of miRNA-knock down on cisplatin sensitivity was measured by incubating Transfected cells with the estimated GI50 concentration of cisplatin. Cisplatin was added 48 h post transfection and incubated for additional 48 h followed by MTT viability measurement (Figure 4B). This revealed no clear correlation between cisplatin sensitivity and miRNA knock down, however down-regulation of miR-138 increased the cisplatin sensitivity of four of the eight tested cell lines by approximately 20% (Figure 4B). The cellular response in cisplatin treated cells to miR-296-5p and miR-642 transfection was more difficult to interpret as these miRNAs already showed reduced viability following the miRNA transfection.

Bottom Line: Three miRNAs were associated with both response and survival (886-3p, 923, 944).By changing the cellular level of the response-identified miRNAs in eight bladder cell lines with different cisplatin sensitivity we found that down-regulation of miR-27a, miR296-5p and miR-642 generally reduced the cell viability, whereas up-regulation of miR-138 and miR-886-3p reduced the viability of more than half of the cell lines.Decreasing miR-138 increased the cisplatin sensitivity in half of the cell lines and increasing miR-27a and miR-642 generally increased cisplatin sensitivity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark. iver.nordentoft@ki.au.dk

ABSTRACT

Background: MicroRNA is a naturally occurring class of non-coding RNA molecules that mediate posttranscriptional gene regulation and are strongly implicated in cellular processes such as cell proliferation, carcinogenesis, cell survival and apoptosis. Consequently there is increasing focus on miRNA expression as prognostic factors for outcome and chemotherapy response. Only approximately 50% of patients with bladder cancer respond to chemotherapy. Therefore, predictive markers, such as miRNAs, that can identify subgroups of patients who will benefit from chemotherapy will have great value for treatment guidance.

Methods: We profiled the expression of 671 miRNAs in formalin fixed paraffin embedded tumors from patients with advanced bladder cancer treated with cisplatin based chemotherapy. We delineated differentially expressed miRNAs in tumors from patients with complete response vs. patients with progressive disease and in tumors form patients with short and long overall survival time. Furthermore, we studied the effect of up- and down regulation of key miRNAs on the cisplatin sensitivity in eight bladder cancer cell lines with different sensitivities to cisplatin.

Results: miRNA expression profiling identified 15 miRNAs that correlated with response to chemotherapy and 5 miRNAs that correlated with survival time. Three miRNAs were associated with both response and survival (886-3p, 923, 944). By changing the cellular level of the response-identified miRNAs in eight bladder cell lines with different cisplatin sensitivity we found that down-regulation of miR-27a, miR296-5p and miR-642 generally reduced the cell viability, whereas up-regulation of miR-138 and miR-886-3p reduced the viability of more than half of the cell lines. Decreasing miR-138 increased the cisplatin sensitivity in half of the cell lines and increasing miR-27a and miR-642 generally increased cisplatin sensitivity.

Conclusions: MiRNAs seem to be involved in cisplatin based chemo response and may form a new target for therapy and serve as biomarkers for treatment response.

Show MeSH
Related in: MedlinePlus