miR-9 and let-7g enhance the sensitivity to ionizing radiation by suppression of NFκB1

Abstract

The activation of nuclear factor-kappa B1 (NFkB1) in cancer cells may confer resistance to ionizing radiation (IR). To enhance the therapeutic efficiency of IR in lung cancer, we screened for microRNAs (miRNAs) that suppress NFkB1 and observed their effects on radiosensitivity in a human lung cancer cell line. From time series data of miRNA expression in γ-irradiated H1299 human lung cancer cells, we found that the expression of miR-9 was inversely correlated with that of NFκB1. Overexpression of miR-9 down-regulated the level of NFκB1 in H1299 cells, and the surviving fraction of γ-irradiated cells was decreased. Interestingly, let-7g also suppressed the expression of NFκB1, although there was no canonical target site for let-7g in the NFκB1 3' untranslated region. From these results, we conclude that the expression of miR-9 and let-7g could enhance the efficiency of radiotherapy for lung cancer treatment through the inhibition of NFκB1.

Figure 1

Expression patterns of NFκB1 and microRNAs in γ-irradiated H1299 cells. (A) Heatmap analysis shows the expression of NFκB1 and microRNAs targeting NFκB1 in H1299 cells upon ionizing radiation at 0, 4, 8, and 12 h. (B) The expression of NF B1 mRNA was quantitated with real-time RT-PCR at the indicated time. The values were normalized with GAPDH mRNA. (C) The expressions of miR-9, let-7g, and miR-26b were measured by real-time RT-PCR in γ-irradiated H1299 cells at the indicated time. U6B mRNA was used as a normalization control for microRNAs. All values are presented as mean ± SD from triplicate experiments.

Figure 2

Suppression of NFκB1 by miR-9 and let-7g. (A) Target sequence of miR-9 in NF B1 3'UTR was predicted by TargetScan. (B) In miR-9, let-7g, and miR-26b transfected H1299 cells, the expression of each microRNA was confirmed by real-time RT-PCR. (C) The expression of NFκB1 mRNA in microRNA-transfected cells was measured by real-time RT-PCR. The relative NFκB1 expression levels were normalized against GAPDH and presented as mean ± SD from triplicate experiments. (D) The protein levels of NFκB1 were also examined by western blot in microRNA-transfected cells. (E) Cells were transfected with the empty renilla luciferase reporter gene (psiCHECK2) or the reporter gene fused to the NFκB1 3' UTR. In addition, the cells were co-transfected with miR-9 or without miR-9. Results are expressed as relative light units (RLU) and were normalized with the luciferase activity expressed constitutively by the psiCHECK2 vector.

Figure 3

miR-9 and let-7g enhance the sensitivity to ionizing radiation in H1299 cells. (A) miR-9, let7g, and miR-26b were overexpressed and the cell survival fraction upon γ-irradiation was measured using MTT assay in H1299 cells. (B) The expression of NFκB1 was measured in microRNA-transfected cells after γ-irradiation. (C) miR-9 was overexpressed and the cell survival fraction upon γ-irradiation was measured using MTT assay in A549 cells. (D) miR-9 can directly control the expression of NFκB1, and the indirect regulation of NFκB1 by let-7g may be proposed to control radiosensitivity.