Department of Radiation Oncology, School of Medicine and the Winship Cancer Institute, Emory University, Atlanta, GA 30322, USA.
Int J Radiat Oncol Biol Phys. 2011 Dec 1;81(5):1524-9. doi: 10.1016/j.ijrobp.2011.05.031. Epub 2011 Oct 17.
Previously, we showed that ectopic miR-101 could sensitize human tumor cells to radiation by targeting ATM and DNA-PK catalytic subunit (DNA-PKcs) to inhibit DNA repair, as the endogenous miR-101 levels are low in tumors in general. However, the heterogeneity of human cancers may result in an exception. The purpose of this study was to test the hypothesis that a few tumor cell lines with a high level of endogenous miR-101 would prove less response to ectopic miR-101.
Fourteeen non-small-cell lung cancer (NSCLC) cell lines and one immortalized non-malignant lung epithelial cell line (NL20) were used for comparing endogenous miR-101 levels by real-time reverse transcription-polymerase chain reaction. Based on the different miR-101 levels, four cell lines with different miR-101 levels were chosen for transfection with a green fluorescent protein-lentiviral plasmid encoding miR-101. The target protein levels were measured by using Western blotting. The radiosensitizing effects of ectopic miR-101 on these NSCLC cell lines were determined by a clonogenic assay and xenograft mouse model.
The endogenous miR-101 level was similar or lower in 13 NSCLC cell lines but was 11-fold higher in one cell line (H157) than in NL20 cells. Although ectopic miR-101 efficiently decreased the ATM and DNA-PKcs levels and increased the radiosensitization level in H1299, H1975, and A549 cells, it did not change the levels of the miR-101 targets or radiosensitivity in H157 cells. Similar results were observed in xenograft mice.
A small number of NSCLC cell lines could have a high level of endogenous miR-101. The ectopic miR-101 was able to radiosensitize most NSCLC cells, except for the NSCLC cell lines that had a much higher endogenous miR-101 level. These results suggest that when we choose one miRNA as a therapeutic tool, the endogenous level of the miRNA in each tumor should be considered.
此前，我们已经证实，通过靶向 ATM 和 DNA-PK 催化亚基（DNA-PKcs），异位 miR-101 可降低人肿瘤细胞中的 miR-101 内源性水平，从而抑制 DNA 修复，使肿瘤中的 miR-101 内源性水平降低，以此来增强肿瘤细胞对辐射的敏感性。然而，人类癌症的异质性可能会导致例外情况的发生。本研究的目的是验证以下假说，即少数内源性 miR-101 水平较高的肿瘤细胞系对异位 miR-101 的反应性较低。
我们使用 14 种非小细胞肺癌（NSCLC）细胞系和 1 种永生化的非恶性肺上皮细胞系（NL20），通过实时逆转录聚合酶链反应比较内源性 miR-101 水平。根据不同的 miR-101 水平，我们选择了 4 种 miR-101 水平不同的细胞系，用转染 GFP-慢病毒质粒的方法转染 miR-101。通过 Western blot 测定靶蛋白水平。通过集落形成实验和异种移植小鼠模型来确定外源性 miR-101 对这些 NSCLC 细胞系的放射增敏作用。
13 种 NSCLC 细胞系中的内源性 miR-101 水平相似或较低，但在 1 种细胞系（H157）中比 NL20 细胞高 11 倍。尽管外源性 miR-101 能有效降低 ATM 和 DNA-PKcs 水平，并提高 H1299、H1975 和 A549 细胞的放射增敏水平，但它并没有改变 H157 细胞中 miR-101 靶标或放射敏感性的水平。在异种移植小鼠中也观察到了类似的结果。
少数 NSCLC 细胞系可能具有高水平的内源性 miR-101。外源性 miR-101 能够使大多数 NSCLC 细胞对辐射敏感，但对于内源性 miR-101 水平较高的 NSCLC 细胞系则无效。这些结果表明，当我们选择一种 miRNA 作为治疗工具时，应该考虑每个肿瘤中 miRNA 的内源性水平。
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