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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 up-regulation in bladder cell lines. miRNA molecules were used to up-regulate the levels of miR-27a, miR-138, miR-296-5p, miR-642, miR-886-3p and miR-944 in the bladder cell lines UMUC9, UMUC14, SLT4, 253JBV, RT4, CLR2169, HT1197 and 575A. miRNAs molecules were reverse transfected at 40 nM (n = 8) and after 48 h incubation culture media with or without cisplatin (GI50) 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 miRNA 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 precursor miRs prohibits precise measure of effect of cisplatin treatment during miR down regulation).
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Figure 5: miRNA up-regulation in bladder cell lines. miRNA molecules were used to up-regulate the levels of miR-27a, miR-138, miR-296-5p, miR-642, miR-886-3p and miR-944 in the bladder cell lines UMUC9, UMUC14, SLT4, 253JBV, RT4, CLR2169, HT1197 and 575A. miRNAs molecules were reverse transfected at 40 nM (n = 8) and after 48 h incubation culture media with or without cisplatin (GI50) 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 miRNA 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 precursor miRs prohibits precise measure of effect of cisplatin treatment during miR down regulation).

Mentions: We also analyzed the effect of miRNA overexpression using synthetic precursor miRNAs (miRNA-27a, miRNA-138, miRNA-296-5p, miRNA-642, miRNA-886-3p, miRNA-944). First, we measured the effect of miRNA overexpression using MTT assays (Figure 5A). Cell viability after miRNA transfection varied extensively, however especially knock in of miR-138 and miR-886-3p had a highly deleterious effect on cell viability in half of the analyzed cell lines. Next we measured the effect of miRNA knock in on cisplatin sensitivity 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 5B). This revealed that miR-27a and miR-642 overexpression increased the cisplatin sensitivity in most of the cell lines. The effect of miR-138 and miR-886-3p overexpression on cisplatin sensitivity cannot be determined due to the highly reduced viability by the miR alone.


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 up-regulation in bladder cell lines. miRNA molecules were used to up-regulate the levels of miR-27a, miR-138, miR-296-5p, miR-642, miR-886-3p and miR-944 in the bladder cell lines UMUC9, UMUC14, SLT4, 253JBV, RT4, CLR2169, HT1197 and 575A. miRNAs molecules were reverse transfected at 40 nM (n = 8) and after 48 h incubation culture media with or without cisplatin (GI50) 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 miRNA 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 precursor miRs prohibits precise measure of effect of cisplatin treatment during miR down regulation).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3473298&req=5

Figure 5: miRNA up-regulation in bladder cell lines. miRNA molecules were used to up-regulate the levels of miR-27a, miR-138, miR-296-5p, miR-642, miR-886-3p and miR-944 in the bladder cell lines UMUC9, UMUC14, SLT4, 253JBV, RT4, CLR2169, HT1197 and 575A. miRNAs molecules were reverse transfected at 40 nM (n = 8) and after 48 h incubation culture media with or without cisplatin (GI50) 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 miRNA 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 precursor miRs prohibits precise measure of effect of cisplatin treatment during miR down regulation).
Mentions: We also analyzed the effect of miRNA overexpression using synthetic precursor miRNAs (miRNA-27a, miRNA-138, miRNA-296-5p, miRNA-642, miRNA-886-3p, miRNA-944). First, we measured the effect of miRNA overexpression using MTT assays (Figure 5A). Cell viability after miRNA transfection varied extensively, however especially knock in of miR-138 and miR-886-3p had a highly deleterious effect on cell viability in half of the analyzed cell lines. Next we measured the effect of miRNA knock in on cisplatin sensitivity 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 5B). This revealed that miR-27a and miR-642 overexpression increased the cisplatin sensitivity in most of the cell lines. The effect of miR-138 and miR-886-3p overexpression on cisplatin sensitivity cannot be determined due to the highly reduced viability by the miR alone.

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