Radiation-induced miR-208a increases the proliferation and radioresistance by targeting p21 in human lung cancer cells

Abstract

Background: Lung cancer has long been the most dangerous malignant tumor among males in both well developed and poorly developed countries. Radiotherapy plays a critical role in the curative management of inoperable non-small cell lung cancer (NSCLC) and is also used as a post-surgical treatment in lung cancer patients. Radioresistance is an important factor that limits the efficacy of radiotherapy for NSCLC patients. Increasing evidence suggests that microRNAs (miRNAs) possess diverse cellular regulatory roles in radiation responses.

Methods: In this study, we used miRNA microarray technology to identify serum miRNAs that were differentially expressed before and after radiotherapy in lung cancer patients. We further examined the biological function of miR-208a on cell viability, apoptotic death and cell cycle distribution in human lung cancer cells and explored the probable mechanism.

Results: Nine miRNAs, including miR-29b-3p, miR-200a-3p, and miR-126-3p were significantly down-regulated, whereas miR-208a was the only miRNA that was up-regulated in the serum of the patients after radiation treatment (P < 0.05). The expression of miR-208a could be induced by X-ray irradiation in lung cancer cells. Forced expression of miR-208a promoted cell proliferation and induced radioresistance via targeting p21 with a corresponding activation of the AKT/mTOR pathway in lung cancer cells, whereas down-regulation of miR-208a resulted in the opposite effects. In addition, down-regulation of miR-208a increased the percentage of cells undergoing apoptosis and inhibited the G1 phase arrest in NSCLC cells. Moreover, miR-208a from the serum exosome fraction of lung cancer patients could shuttle to A549 cells in a time-dependent manner, which was likely to contribute to the subsequent biological effects.

Conclusions: The present study provides evidence that miR-208a can affect the proliferation and radiosensitivity of human lung cancer cells by targeting p21 and can be transported by exosomes. Thus, miR-208a may serve as a potential therapeutic target for lung cancer patients.

Fig. 1

miR-208a in pairs of serum samples from 30 lung cancer patients before and after radiotherapy and in lung cancer cells was examined by qRT-PCR. a The average serum miR-208a expression in 30 paired serum samples from lung cancer patients before and after radiotherapy. b qRT-PCR analysis of the expression of miR-208a in human lung cancer cell lines (A549, H1299, H1975 and H460) and non-cancerous bronchial epithelial cell lines (BEAS-2B and HBE). The relative expression levels of miR-208a in (c) A549, (d) H1299 and (e) HBE cells after exposure to 0, 2, 4 or 8 Gy X-ray irradiation. The relative serum miR-208a expression level normalized to miR-16 and cellular miR-208a expression normalized to U6 were calculated using the equation described in the Materials and Methods. *P < 0.05 and **P < 0.01 compared with the negative control

Fig. 2

miR-208a affected the levels of the p21/AKT/mTOR pathway in A549 cells. A549 cells were transfected with the miR-208a mimics, negative control miRNA (miRNA-NC), miR-208a inhibitor or inhibitor-NC for 24 h before lysis. a Western blot analysis the expression levels of p21. b Putative miR-208a binding sequences in the 3′-UTR of p21 mRNA. c Full-length 3′-UTR of p21 and the mutant sequences were cloned into the downstream of the luciferase reporter gene named as pGL3-UTR-WT and pGL3-UTR-MUT, respectively. Each of the plasmids and pRL-TK, were cotransfected into A549 cells, either with miR-208a mimics or inhibitor as indicated. Luciferase activity was assayed 48 h after transfection. All experiments were repeated three times independently. The results are presented as the means ± SEM (n = 3). *P < 0.05. d The membranes were incubated with antibodies to AKT, p-AKT, mTOR, or p-mTOR. The samples were derived from the same experiment and analyzed under the same experimental conditions

Fig. 3

Over-expression of miR-208a promoted cellular proliferation. a The effect of miR-208a on viability of the A549 cells was analyzed using the MTT assay, and the miRNA-NC-transfected cells were normalized as 100 %. b The proliferating A549 cells were labeled with EdU. The click-it reaction revealed the EdU staining (red). The cell nuclei were stained with Hoechst 33342 (blue). The images are representative of the results obtained. c Colony formation assays of A549 cells transfected with miR-208a mimics or miR-208a inhibitor. One thousand cells were seeded onto each plate. After 10 days, the cells were stained with crystal violet. The colonies consisting of more than 50 cells were counted. The data are presented as the means ± SEM (n = 4). *P < 0.05 and **P < 0.01 compared with the negative control

Fig. 4

miR-208a increased the cell apoptosis and disturbed the cell cycle of lung cancer cells. a A549 and (b) H1975 cells were transfected with the indicated vectors. Two days after transfection, the percentage of apoptotic cells was evaluated using flow cytometry. Western blot analyzed the apoptotic molecular component including PARP1, Bcl2 and Bax in (c) A549 and (d) H1975 cells. The cell cycle distributions of (e) A549 and (f) H1975 cells were measured using flow cytometry. *P < 0.05 and **P < 0.01 compared with the negative control

Fig. 5

miR-208a enhanced the radioresistance of A549 cells. a Colony formation assays and (b) clonogenic cell survival curves were generated. c D₀, D_q and calculated SER values of miRNA-NC- and miR-208a mimic-transfected groups are shown; (d) Colony formation assays and (e) clonogenic cell survival curves were generated. f D₀, D_q and calculated SER values of inhibitor-NC- and miR-208a inhibitor-transfected groups are shown. The SER was calculated according to the multi-target single hit model

Fig. 6

Identification and characterization of serum exosome miR-208a translocation into A549 cells. Exosomes were isolated using centrifugation, filtration and ultracentrifugation as described in the Materials and Methods. a Confirmation of the exosome marker CD63 by Western blot. b qRT-PCR analysis of miR-208a and miR-16 contained in the exosomes. c qRT-PCR analysis of miR-208a in culture medium of A549 cells. The conditional medium incubated with A549 cells collected 24 h after exposure to 0 or 4 Gy X-ray irradiation. d Microscopic analysis of the translocation of the pre-labeled exosomes. The DiI-labeled exosomes displayed red color. The cell nuclei were stained with DAPI (blue). The scale bar indicates 20 μm. *P < 0.05 compared with BR or the group treated with 0 Gy. BR: before radiotherapy; AR: after receiving 60 Gy radiotherapy