MicroRNA-449a Enhances Radiosensitivity in CL1-0 Lung Adenocarcinoma Cells

<div><p>Lung cancer is the leading cause of cancer-related mortality worldwide. Radiotherapy is often applied for treating lung cancer, but it often fails because of the relative non-susceptibility of lung cancer cells to radiation. MicroRNAs (miRNAs) have been reported to modulate the radiosensitivity of lung cancer cells and have the potential to improve the efficacy of radiotherapy. The purpose of this study was to identify a miRNA that can adjust radiosensitivity in lung adenocarcinoma cells. Two lung adenocarcinoma cell lines (CL1-0 and CL1-5) with different metastatic ability and radiosensitivity were used. In order to understand the regulatory mechanisms of differential radiosensitivity in these isogenic tumor cells, both CL1-0 and CL1-5 were treated with 10 Gy radiation, and were harvested respectively at 0, 1, 4, and 24 h after radiation exposure. The changes in expression of miRNA upon irradiation were examined using Illumina Human microRNA BeadChips. Twenty-six miRNAs were identified as having differential expression post-irradiation in CL1-0 or CL1-5 cells. Among these miRNAs, miR-449a, which was down-regulated in CL1-0 cells at 24 h after irradiation, was chosen for further investigation. Overexpression of miR-449a in CL1-0 cells effectively increased irradiation-induced DNA damage and apoptosis, altered the cell cycle distribution and eventually led to sensitization of CL1-0 to irradiation.</p></div

Successful over-expression of miR-449a in CL1-0.

<p>(A) Expression profiling of miR-449a over-expressed in CL1-0. Relative expression levels of miR-449a in CL1-0 cells were measured at indicated time points by real-time PCR after transiently transfected with miR-449a expression plasmid. <i>RNU44</i> served as an endogenous control. Each measurement was made in triplicate. *: <i>P</i><0.001. (B) Immunoblotting of histone deacetylase 1 (HDAC1), a validated target of miR-449a. Protein levels of HDAC1 in CL1-0 were analyzed 72 h after transfection. ACTB (β-actin) was a loading control.</p

Dynamics of differentially expressed miRNA after irradiation in CL1-0 and CL1-5.

<p>(A) Hierarchical clustering of differentially expressed miRNA from CL1-0 and CL1-5 treated with 10 Gy. Cells were harvested respectively at 0, 1, 4, 24 h after irradiation. (B) Principal component analysis (PCA) of the radiation-responsive genes. The axes are the first two principal components (PC), which can explain most of the miRNA expression profiling. Three independent experiments were done at each time point. Different colors represent different cells; different shapes represent different time points. (C) Relative miR-449a expression level measured by microarray. Data were the mean ± SD for three independent experiments. *: <i>P</i><0.05. (D) Relative miR-449a expression level measured by quantitative RT-PCR. Data were the mean ± SD for four independent experiments.</p

Differential irradiation responses in CL1-0 and CL1-5.

<p>(A) Clonogenic assays of CL1-0 and CL1-5 cells treated with 0, 2, 5, and 10 Gy radiation. The surviving fraction was measured 8 days post-irradiation. (B) MTT assays for cells at 48 h after 10 Gy radiation. Cell viability is expressed as percentage relative to un-irradiated control cells. (C) Flow cytometry analysis of apoptosis using annexin V and propidium iodide in cells treated with 10 Gy. The bar chart shows the percentage of apoptotic cells induced by irradiation. (D) Flow cytometry analysis of cell cycle using propidium iodide at 24 h post-irradiation. The proportions of cells in each phase of the cell cycle were quantitated using ModFit LT software. Left: one representative diagram, with different colors indicating different phases. Right: quantitative bar chart. Data are represented as mean ± SD for three independent experiments. #: <i>P</i><0.05, *: <i>P</i><0.001.</p

Over-expression of miR-449a in CL1-0 increased radiosensitivity.

<p>(A) Immunoblotting of γH2AX in miR-449a-overexpressing cells. Cells were harvested 1 h after irradiation. ACTB (β-actin) was a loading control. (B) Immunoblotting of caspase 3 in miR-449a-overexpressing cells. Cells were harvested at 0, 24, 48, and 72 h after irradiation. β-actin was a loading control. (C) Flow cytometry analysis for apoptosis using annexin V and propidium iodide in miR-449a-overexpressing cells treated with 10 Gy. At 48 h post-irradiation, miR-449a-overexpressing cells were stained with annexin V and propidium iodide, and the percentage of apoptotic cells was calculated. One representative diagram is shown to the left, and the percentage of annexin V- positive cells after irradiation is shown to the right. Data in the bar chart are the mean ± SD for three independent experiments. (D) Flow cytometry analysis for cell cycle using propidium iodide in miR-449a-overexpressing cells after irradiation. Flow cytometry analysis of cell cycle progression was performed in miR-449a-overexpressing cells 24 h after treated with 10 Gy. The proportions of cells in each phase of cell cycle were quantitated using ModFit LT software. Left: one representative diagram, with different colors indicating different phases. Right: quantitative bar chart. Data in the bar chart are the mean ± SD for three independent experiments. #: <i>P</i><0.05,*: <i>P</i><0.001.</p