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Knock-down of core proteins regulating microRNA biogenesis has no effect on sensitivity of lung cancer cells to ionizing radiation.

Surova O, Akbar NS, Zhivotovsky B - PLoS ONE (2012)

Bottom Line: We found that Drosha and Dicer were expressed at higher levels in radioresistant but not in sensitive cell lines.However, down-regulation of either Dicer or Drosha had no effect on the sensitivity of cells to irradiation.Elimination of components of the RNA-induced silencing complex Ago2 and Tudor staphylococcal nuclease also did not sensitize cells to the same treatment.

View Article: PubMed Central - PubMed

Affiliation: Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
Recent studies underline the important role of microRNAs (miRNA) in the development of lung cancer. The main regulators of miRNA biogenesis are the ribonucleases Drosha, Dicer and Ago2. Here the role of core proteins of miRNA biogenesis machinery in the response of human non-small and small cell lung carcinoma cell lines to treatment with ionizing radiation was assessed. We found that Drosha and Dicer were expressed at higher levels in radioresistant but not in sensitive cell lines. However, down-regulation of either Dicer or Drosha had no effect on the sensitivity of cells to irradiation. Elimination of components of the RNA-induced silencing complex Ago2 and Tudor staphylococcal nuclease also did not sensitize cells to the same treatment. Thus, modulation of miRNA biogenesis machinery is not sufficient to increase the radiosensitivity of lung tumors and other strategies are required to combat lung cancer.

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NSCLC and SCLC cells differ in sensitivity to radiation treatment and display differential expression of proteins involved in the regulation of miRNA biogenesis.(A) Western blot analysis of the level of protein expression of Drosha, Dicer, exportin 5 (XPO5), Tudor-SN (TSN), protein activator of the interferon-induced protein kinase (PACT), fragile X mental retardation syndrome-related protein 1 (FXR1) and Argonaute 2 (AGO2) in a panel of NSCLC (U1810, U1299, A549, H661, H157, H23) and SCLC (U1285, H82, H69, U1690, U1906, U2020) cell lines. (B) Densitometric analysis of relative levels of protein expression in H23, H1299, U1810 and H661 cell lines. Cell lines distributed according to radiosensitivity, measured as the fraction surviving at 2 Gy (SF2). Equal loading was verified using anti-β-actin antibodies. Results are representative of three independent experiments.
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pone-0033134-g001: NSCLC and SCLC cells differ in sensitivity to radiation treatment and display differential expression of proteins involved in the regulation of miRNA biogenesis.(A) Western blot analysis of the level of protein expression of Drosha, Dicer, exportin 5 (XPO5), Tudor-SN (TSN), protein activator of the interferon-induced protein kinase (PACT), fragile X mental retardation syndrome-related protein 1 (FXR1) and Argonaute 2 (AGO2) in a panel of NSCLC (U1810, U1299, A549, H661, H157, H23) and SCLC (U1285, H82, H69, U1690, U1906, U2020) cell lines. (B) Densitometric analysis of relative levels of protein expression in H23, H1299, U1810 and H661 cell lines. Cell lines distributed according to radiosensitivity, measured as the fraction surviving at 2 Gy (SF2). Equal loading was verified using anti-β-actin antibodies. Results are representative of three independent experiments.

Mentions: In order to identify molecular targets for the radiosensitization of LC cells among proteins involved in miRNA biogenesis we performed a protein expression analysis of seven core miRNA machinery components in a panel of NSCLC and SCLC (six cell lines in each panel). For each LC subtype the cell lines were selected based on their radiosensitivity, as measured by the fraction surviving after exposure to 2 Gy (SF2) in a clonogenic survival assay, and grouped into radiosensitive (RS) with SF2 < 0.3 Gy or radioresistant (RR) with SF2 ≥ 0.3 Gy [11]-[14]. The basal levels of all proteins (Drosha, Dicer, exportin-5 (XPO5), Tudor-SN (TSN), protein activator of the interferon-induced protein kinase (PACT), fragile X mental retardation syndrome-related protein 1 (FXR1) and Argonaute2 (Ago2) were assessed by Western blot in all selected cell lines prior to irradiation (Figure 1A). Both RNase III enzymes (Drosha and Dicer) were expressed at relatively high levels in NSCLC cells compared with SCLC. A member of the karyopherins protein family, XPO5, which is involved in the nuclear export of miRNAs, was expressed at a higher level in H661 while low levels of expression were seen in H69 and U1690 compared to the remaining cell lines. The level of expression of TSN, PACT, FXR1 and Ago2 proteins did not vary profoundly among the various cell lines, with the exception of H69, which exhibited lower expression of all of the studied proteins. As our panel consisted of both RS and RR cells, the H23 cell line was selected as the representative of radiosensitive cells, and U1810 and H661 were used as representatives of radioresistant cells in further investigations. Densitometric analysis of protein expression revealed that Dicer, Drosha and XPO5 were expressed at higher levels in RR cells compared to RS, whereas there was no clear correlation between the levels of expression of Ago2, TSN, PACT and FXR1 and SF2 values (Figure 1B).


Knock-down of core proteins regulating microRNA biogenesis has no effect on sensitivity of lung cancer cells to ionizing radiation.

Surova O, Akbar NS, Zhivotovsky B - PLoS ONE (2012)

NSCLC and SCLC cells differ in sensitivity to radiation treatment and display differential expression of proteins involved in the regulation of miRNA biogenesis.(A) Western blot analysis of the level of protein expression of Drosha, Dicer, exportin 5 (XPO5), Tudor-SN (TSN), protein activator of the interferon-induced protein kinase (PACT), fragile X mental retardation syndrome-related protein 1 (FXR1) and Argonaute 2 (AGO2) in a panel of NSCLC (U1810, U1299, A549, H661, H157, H23) and SCLC (U1285, H82, H69, U1690, U1906, U2020) cell lines. (B) Densitometric analysis of relative levels of protein expression in H23, H1299, U1810 and H661 cell lines. Cell lines distributed according to radiosensitivity, measured as the fraction surviving at 2 Gy (SF2). Equal loading was verified using anti-β-actin antibodies. Results are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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pone-0033134-g001: NSCLC and SCLC cells differ in sensitivity to radiation treatment and display differential expression of proteins involved in the regulation of miRNA biogenesis.(A) Western blot analysis of the level of protein expression of Drosha, Dicer, exportin 5 (XPO5), Tudor-SN (TSN), protein activator of the interferon-induced protein kinase (PACT), fragile X mental retardation syndrome-related protein 1 (FXR1) and Argonaute 2 (AGO2) in a panel of NSCLC (U1810, U1299, A549, H661, H157, H23) and SCLC (U1285, H82, H69, U1690, U1906, U2020) cell lines. (B) Densitometric analysis of relative levels of protein expression in H23, H1299, U1810 and H661 cell lines. Cell lines distributed according to radiosensitivity, measured as the fraction surviving at 2 Gy (SF2). Equal loading was verified using anti-β-actin antibodies. Results are representative of three independent experiments.
Mentions: In order to identify molecular targets for the radiosensitization of LC cells among proteins involved in miRNA biogenesis we performed a protein expression analysis of seven core miRNA machinery components in a panel of NSCLC and SCLC (six cell lines in each panel). For each LC subtype the cell lines were selected based on their radiosensitivity, as measured by the fraction surviving after exposure to 2 Gy (SF2) in a clonogenic survival assay, and grouped into radiosensitive (RS) with SF2 < 0.3 Gy or radioresistant (RR) with SF2 ≥ 0.3 Gy [11]-[14]. The basal levels of all proteins (Drosha, Dicer, exportin-5 (XPO5), Tudor-SN (TSN), protein activator of the interferon-induced protein kinase (PACT), fragile X mental retardation syndrome-related protein 1 (FXR1) and Argonaute2 (Ago2) were assessed by Western blot in all selected cell lines prior to irradiation (Figure 1A). Both RNase III enzymes (Drosha and Dicer) were expressed at relatively high levels in NSCLC cells compared with SCLC. A member of the karyopherins protein family, XPO5, which is involved in the nuclear export of miRNAs, was expressed at a higher level in H661 while low levels of expression were seen in H69 and U1690 compared to the remaining cell lines. The level of expression of TSN, PACT, FXR1 and Ago2 proteins did not vary profoundly among the various cell lines, with the exception of H69, which exhibited lower expression of all of the studied proteins. As our panel consisted of both RS and RR cells, the H23 cell line was selected as the representative of radiosensitive cells, and U1810 and H661 were used as representatives of radioresistant cells in further investigations. Densitometric analysis of protein expression revealed that Dicer, Drosha and XPO5 were expressed at higher levels in RR cells compared to RS, whereas there was no clear correlation between the levels of expression of Ago2, TSN, PACT and FXR1 and SF2 values (Figure 1B).

Bottom Line: We found that Drosha and Dicer were expressed at higher levels in radioresistant but not in sensitive cell lines.However, down-regulation of either Dicer or Drosha had no effect on the sensitivity of cells to irradiation.Elimination of components of the RNA-induced silencing complex Ago2 and Tudor staphylococcal nuclease also did not sensitize cells to the same treatment.

View Article: PubMed Central - PubMed

Affiliation: Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
Recent studies underline the important role of microRNAs (miRNA) in the development of lung cancer. The main regulators of miRNA biogenesis are the ribonucleases Drosha, Dicer and Ago2. Here the role of core proteins of miRNA biogenesis machinery in the response of human non-small and small cell lung carcinoma cell lines to treatment with ionizing radiation was assessed. We found that Drosha and Dicer were expressed at higher levels in radioresistant but not in sensitive cell lines. However, down-regulation of either Dicer or Drosha had no effect on the sensitivity of cells to irradiation. Elimination of components of the RNA-induced silencing complex Ago2 and Tudor staphylococcal nuclease also did not sensitize cells to the same treatment. Thus, modulation of miRNA biogenesis machinery is not sufficient to increase the radiosensitivity of lung tumors and other strategies are required to combat lung cancer.

Show MeSH
Related in: MedlinePlus