MicroRNA-128-3p regulates mitomycin C-induced DNA damage response in lung cancer cells through repressing SPTAN1

Abstract

The DNA damage response is critical for maintaining genome integrity and preventing damage to DNA due to endogenous and exogenous insults. Mitomycin C (MMC), a potent DNA cross-linker, is used as a chemotherapeutic agent because it causes DNA inter-strand cross-links (DNA ICLs) in cancer cells. While many microRNAs, which may serve as oncogenes or tumor suppressors, are grossly dysregulated in human cancers, little is known about their roles in MMC-treated lung cancer. Here, we report that miR-128-3p can attenuate repair of DNA ICLs by targeting SPTAN1 (αII Sp), resulting in cell cycle arrest and promoting chromosomal aberrations in lung cancer cells treated with MMC. Using computational prediction and experimental validation, SPTAN1 was found to be a conserved target of miR-128-3p. We then found that miR-128-3p caused translational inhibition of SPTAN1, reducing its protein level. SPTAN1 repression via miR-128-3p also induced cell cycle arrest and chromosomal instability. Additionally, miR-128-3p significantly influenced interaction of the αII Sp/FANCA/XPF complex, thus limiting DNA repair. In summary, the results demonstrate that miR-128-3p accelerates cell cycle arrest and chromosomal instability in MMC-treated lung cancer cells by suppressing SPTAN1, and these findings could be applied for adjuvant chemotherapy of lung cancer.

Keywords: DNA repair; MicroRNA; lung cancer; mitomycin C; spectrin.

Figure 1. MiR-128-3p directly targets SPTAN1 via translational repression

(A) MMC treatment leads to an increase in chromosomal aberrations. (B, D and E) The effect of MMC on protein and mRNA levels of SPTAN1 and miR-128-3p expression. (C) Relative abundance of the αII Sp protein level in (B). (F) Schematic description of miR-128-3p binding site in the SPTAN1 3′-UTR. Paired bases are indicated by a black short line, and G:U pairs are indicated by dots. The predicted free energy of each hybrid is indicated. Sequence alignment of putative miR-128-3p binding sites across species are marked by a gray background. (G) A firefly luciferase reporter containing either wild-type (WT) or mutant SPTAN1 3′-UTR was co-transfected into A549 cells with scrambled ncRNA or miR-128-3p mimic or inhibitor. For comparison, luciferase activity in ncRNA-transfected cells was set at 1. The y-axis shows arbitrary units representing the relative luciferase activity. (H) Quantitative RT-PCR analysis of SPTAN1 mRNA levels in A549 cells transfected with scrambled ncRNA, miR-128-3p mimic or miR-128-3p inhibitor. (I) Western blot analysis of αII Sp protein levels in A549 cells transfected with scrambled ncRNAs, miR-128-3p mimic, miR-128-3p inhibitor or SPTAN1 siRNA. (J) Relative abundance of αII Sp in (I). The results are presented as the mean ± SE of three independent experiments. *p < 0.05, ^*p < 0.01, ^***p < 0.001.

Figure 2. MiR-128-3p regulates the cell cycle and chromosomal aberrations

(A) A549 cells were transfected with synthetic RNAs and subsequently treated with MMC; the percentages of cells in G0/G1, S and G2/M phases were determined by flow cytometry. (B, C and D) Bar graphs illustrate quantification of the cell cycle. (E) The effect of scrambled ncRNAs, miR-128-3p mimic and SPTAN1 siRNA on chromosomes in A549 cells. Circles indicate chromosomal aberrations. (F) One hundred metaphase spreads were scored for chromosomal aberrations, and the average number of chromosomal aberrations per metaphase from three independent experiments is shown. The results are presented as the mean ± SE of three independent experiments. *p < 0.05, ^*p < 0.01.

Figure 3. MiR-128-3p disrupts co-localization and interaction of FANCA, XPF and αII Sp

(A) Co-localization of FANCA and αII Sp. Cells were stained with anti-αII Sp (red) and anti-FANCA (green) antibodies, and nuclear DNA was counterstained with DAPI (blue). (B) Co-localization of XPF and αII Sp. Cells were stained with anti-αII Sp (red) and anti-XPF (green) antibodies, and nuclear DNA was counterstained with DAPI (blue). (C) Interaction of αII Sp, XPF and FANCA. Co-IP was carried out using an anti-αII Sp antibody or IgG (as a control), and western blot analyses were performed using an anti-αII Sp, anti-FANCA or XPF antibody. (D) Bar graphs show the relative abundances of αII Sp, FANCA or XPF. The results are presented as the mean ± SE of three independent experiments. ^*p < 0.01.