miR-144-5p Enhances the Radiosensitivity of Non-Small-Cell Lung Cancer Cells via Targeting ATF2

Abstract

MicroRNAs (miRNAs or miRs) regulate gene expression at the posttranscriptional level and are involved in many biological processes such as cell proliferation and migration, stem cell differentiation, inflammation, and apoptosis. In particular, miR-144-3p is downregulated in various cancers, and its overexpression inhibits the proliferation and metastasis of cancer cells. However, the role of miR-144-5p in non-small-cell lung cancer (NSCLC), especially radiosensitivity, is unknown. In this study, we found that miR-144-5p was downregulated in NSCLC clinical specimens as well as NSCLC cell lines exposed to radiation. Enhanced expression of miR-144-5p promoted the radiosensitivity of NSCLC cells in vitro and A549 cell mouse xenografts in vivo. Furthermore, we identified activating transcription factor 2 (ATF2) as the direct and functional target of miR-144-5p using integrated bioinformatics analysis and a luciferase reporter assay. In addition, restoration of ATF2 expression inhibited miR-144-5p-induced NSCLC cell sensitivity to radiation in vitro and in vivo. Our findings suggest that deregulation of the miR-144-5p/ATF2 axis plays an important role in NSCLC cell radiosensitivity, thus representing a new potential therapeutic target for NSCLC.

Figure 1

miR-144-5p expression in lung cancer patient samples and cell lines treated with irradiation. (a) Relative expression levels of miR-144-5p in normal lung tissue and lung cancer specimens were measured by real-time polymerase chain reaction. NLT, normal lung tissue (n = 6); AC, adenocarcinoma (n = 12); SC, squamous carcinoma (n = 10); SCLC, small cell lung cancer (n = 8). ^∗∗P < 0.01 versus NLT, ^#P < 0.01 versus SCLC. (b) miR-144-5p expression in the indicated NSCLC cell lines. Data are representative images or expressed as the mean ± standard deviation of each group of cells from three separate experiments. ^∗P < 0.05 versus 16-HBE, ^∗∗P < 0.01 versus 16-HBE, ^&P < 0.05 versus H1417. (c) miR-144-5p expression in A549 cells and (d) H460 cells after radiation treatment at different doses (0 Gy, 2 Gy, 4 Gy, and 8 Gy). ^∗∗P < 0.01 versus 0 Gy.

Figure 2

miR-144-5p regulated A549 and H460 cell viability and apoptosis after irradiation. (a) The expression of miR-144-5p in control A549 and H460 cells (nontransfected cells), as well as cells transfected with agomir-144 or agomir-NC, was determined using qRT-PCR. (b) Control A549 and H460 cells as well as cells transfected with agomir-144 or agomir-NC were exposed to varying doses of radiation (0, 2, 4, 6, and 8 Gy). MTT assay was used to determine the cell viability 48 h after IR. Cell viability is expressed as the percentage relative to the control at 0 Gy. (c) A549 and H460 cells with or without agomir-144 or agomir-NC transfection were subjected to 8 Gy radiation. Cell apoptosis was assessed by staining with annexin V and propidium iodide 48 h after IR. The percentage of apoptotic cells was determined using flow cytometric analysis. Data are representative images or expressed as the mean ± standard deviation of each group of cells from three separate experiments. ^∗P < 0.05 versus agomir-NC. ^∗∗P < 0.01 versus agomir-NC.

Figure 3

miR-144-5p enhanced the tumor suppression capability of IR in vitro and in vivo. (a) A549 or H460 cells transfected with agomir-144 or agomiR-NC and the parental cells (control) were subjected to 8 Gy radiation, followed by a colony formation assay. Colony formation was suppressed in agomiR-144-5p-transfected A549 cells. ^∗∗P < 0.01 versus agomir-NC. (b) Seven days after A549 cell injection in mice (six in each group), agomir-144 or agomir-NC was intratumorally injected into the implanted tumor every 3 days for seven times. Mice were irradiated with 4 Gy once per day for the following 5 days. Tumor volumes were measured every 3 days after injection. (c) Tumors were dissected, and the tumor weights were measured on day 28 after inoculation. ^∗P < 0.05 versus agomir-NC. ^∗∗P < 0.01 versus agomir-NC.

Figure 4

miR-144-5p targeted ATF2 in lung cancer cells. (a) The sequence alignment of human miR-144-5p with the 3′UTR of ATF2 is shown. The luciferase reporter constructs hsa-ATF2-wt and hsa-ATF2-mut were made using the seed sequence of miR-144-5p matching the 3′UTR of ATF2 mRNA. HEK293 cells were transiently cotransfected with the indicated plasmids and agomir-144 or agomir-NC for 48 h, and then they were subjected to the luciferase assay. ^∗∗P < 0.01 versus agomir-NC. (b, c) A549 and H450 cells were transfected with agomir-144-5p or agomir-NC. The ATF2 protein (b) and mRNA (c) levels were determined by immunoblotting and RT-PCR, respectively. (d) The relative ATF2 mRNA levels in normal lung tissues (NLT, n = 6) or lung cancer (LC, n = 40) tissues were assessed using quantitative RT-PCR. (e) Correlation between the expression levels of ATF2 and that of mature miR-144-5p in 6 NLT and 40 LC tissues. ^∗∗P < 0.01 versus agomir-NC. ^&P < 0.01 versus NLT.

Figure 5

Restoration of ATF2 expression inhibited miR-144-5p-mediated radiosensitivity of lung cancer cells. A549 cells with or without ATF2 overexpression were transfected with agomir-144. (a) The levels of miR-144-5p in nontransfected A549 cells (control) and cells transfected with ATF2 lentiviral particles or mock particles (vector) were determined using western blot analysis. (b) After exposure to varying doses of radiation (0, 2, 4, 6, and 8 Gy), MTT assay was used to determine the cell viability. After 8 Gy radiation, cell apoptosis (c) and survival (d) were then determined using Annexin V/propidium iodide staining and the colony formation assay, respectively. (e) A total of 2 × 10⁶ A549 cells stably transfected with ATF2-overexpressing plasmid or vector were injected subcutaneously into mice (6 in each group) to establish a xenograft model. At 7 days after inoculation, agomir-144 was intratumorally injected into the implanted tumor every 3 days for seven times. At the same time, mice were irradiated with 4 Gy once per day for the following 5 days. Tumor volumes were measured every 3 days after injection. (f) Tumors were dissected, and the weights were measured on day 28 after inoculation. Data are representative images or expressed as the mean ± standard deviation of each group of cells from three separate experiments. ^∗P < 0.05 versus vector + miR-144. ^∗∗P < 0.01 versus vector + miR-144. & represents P < 0.01 versus vector.