1. Introduction
To maintain the integrity of genome, eukaryotic cells rely on a highly regulated system pathway of response to DNA damage—the DNA-Damage Response (DDR)—which encompasses damage sensors, mediators, signal transducers and effectors. Upon recognition of DNA damage, transducer kinases ATM (Ataxia-Telangiectasia Mutated), ATR (Ataxia telangiectasia and Rad3 related), and DNA-PKcs (DNA-dependent protein kinase catalytic subunit) relay and amplify the damage signal to effector proteins that in turn activate cell cycle checkpoints, regulate transcription, translation, and metabolism, and activate the appropriate DNA repair process, as well as cell fate toward apoptosis or senescence [
1,
2]. Following DNA double-strand break (DSB) induction, ATM undergoes spatial relocalization and catalytic activation; after that, it is rapidly recruited to DNA damage sites. Here ATM phosphorylates specific serines or threonines on many downstream protein substrates, including “Ser-139” of histone variant H2AXpresent in nucleosomes surrounding DSB sites, thereby regulating DDR mechanism.
Following genotoxic stress, DDR pathway is post-transcriptionally regulated through selective mRNA stabilization or decay and regulation of translation [
3]. In this context, non-coding RNAs, such as microRNAs (miRNAs), have emerged as important regulators of gene expression of key components of DDR pathway. miRNAs are natural single-stranded, small RNA molecules (18–22 nt) that regulate gene expression by binding to target mRNAs and suppress their translation or promote their cleavage [
4]. Mature miRNAs recognize their target mRNAs by base-pairing interactions between nucleotides 2–8 of the miRNA (the seed region) and complementary nucleotides in the 3′-untranslated region (3′UTR) of mRNAs. Recent evidences, however, suggest that in addition to 3′UTRs miRNAs can bind to other regions of target mRNAs, including the 5′UTRs, promoter, and open reading frames [
5]. In humans, ~800 miRNAs are predicted to exist [
6], and each single miRNA can influence the expression of up 1000 genes. miRNAs regulate many physiological processes, including differentiation, apoptosis, fat metabolism, as well as pathological processes, such as tumorigenesis. Different miRNAs are indeed dysregulated in human cancers and can function as either tumor suppressors or oncogenes, by targeting different steps of the tumorigenesis process, including initiation and progression to a metastatic phenotype [
7–
10]. Moreover, accumulating evidences have shown that miRNAs are altered following genotoxic and cytotoxic stress, and several studies suggested that miRNA expression is regulated in DDR at the transcriptional level, in a p53-dependent manner [
11] and through modulation of miRNAs’ processing and maturation steps [
12]. Moreover, more than half of the DNA repair and DNA damage checkpoint genes contained conserved miRNA target sites [
13].
By integrating the transcriptome and microRNome, we recently identified some miRNA-related genes of DDR pathway that were altered in human peripheral blood lymphocytes irradiated with γ-rays and incubated in ground gravity (1
g) and in modeled microgravity (MMG) [
14]. Several miRNAs were specifically dysregulated by IR in a dose-, time- and gravity-related manner. Among miRNAs altered by the combined action of radiation and microgravity, we identified miR-27a, which as a result, was anti-correlated to
ATM. miR-27a is classified as an
oncomir, being over-expressed in several malignancies, including breast cancer [
15], gastric and renal carcinoma [
16,
17], hepatocellular carcinoma [
18] and pediatric B-ALL [
19]. Down-regulation of miR-27a has been reported in colorectal cancer [
20], in oral squamous carcinoma [
21], and in the serum and plasma of non-small cell lung cancer patients [
22]. miR-27a is part of a cluster of three miRNAs expressed from an intergenic region of chromosome 19, whose members—miR-23a, miR-27a, miR-24-2—are involved in cell cycle control and differentiation in various cell types [
23]. Recent evidences report that miR-23a~24-2~27a cluster may possess a causal role in mammary tumorigenesis, since the expression levels of its members were significantly higher in breast cancer with lymphnode metastasis compared with that from patients without lymphnode metastasis or normal tissue [
24]. The increased expression of miR-23a~24-2~27a cluster possesses also important function in neovascular age-related macular degeneration and tumor-related angiogenesis [
25], however, the mechanisms of regulation in cancer progression is still poorly understood.
In the current study, we investigated the role of miR-27a in the DDR induced by γ-radiation in lung cancer A549 cells. We first validated the functional interaction between miR-27a and ATM by performing site-directed mutagenesis of 3′-untranslated region (UTR) of ATM gene. We then analyzed the biological effects of miR-27a over-expression on the DNA damage response to γ-rays in A549 cells by using miRNA mimics which are chemically synthesized double stranded RNAs that, when introduced into cells, efficiently mimic specific endogenous miRNAs.
3. Experimental Section
3.1. Cell Culture
The human A549 cells (lung adenocarcinoma) were purchased from American Type Culture Collection (ATCC n. CCL-185™) and cultured in Ham’s F12-K Nutrient Mixture (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% heat inactivated fetal bovine serum (FBS, BIOCHROM, Berlin, Germany), 38 units/mL streptomycin, and 100 units/mL penicillin G, in T75 cm2 flasks (FALCON). Cells were kept at 37 °C in a humidified atmosphere of 95% air and 5% CO2, and maintained in exponential and asynchronous phase of growth by repeated trypsinization and reseeding prior to reaching subconfluency.
3.2. Construction of Recombinant Vectors and Site-Directed Mutagenesis
Luciferase reporter vectors were generated from human cDNA and cloned into the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA), immediately downstream from the stop codon of the luciferase gene. To predict base-pairing, we used PITA algorithm [
46], which predicts the targets of a miRNA by searching for the presence of 6-mer or 7-mer sites that near-perfect match the seed region of the miRNA, allowing for G:U wobbles and considering the role of site accessibility in microRNA target recognition. When indicated, the
ATM-3′UTR was mutagenized at the miR-27a recognition sites using the Quick Change Site-Directed Mutagenesis kit (Stratagene, Agilent Technologies, Santa Clara, CA, USA) according to manufacturer’s instructions. miR-27a sensor was obtained by annealing, purifying and cloning short oligonucleotides containing three perfect miR-27a binding sites into the
SacI and
XbaI sites of the pmirGLO vector (
Figure 1b). Primers used for the cloning of
ATM wild type and mutated were:
ATM Fwd-5′-ATCTAGGAGCTCAGGAGTGGAAGAAGGCACTG-3′
ATM Rev-5′-ATCTAGTCTAGAACGCTGTCCAAAGTTTTTCC-3′
ATMdel1 Fwd-5′-AGTGGAAGAAGGCACTCTCAGTGTTGGTGGAC-3′
ATMdel1 Rev-5′-GTCCACCAACACTGAGAGTGCCTTCTTCCACT-3′
ATMdel2 Fwd-5′-CAAGGACAAATGAGGAGTAGTTAGATGAAAATATTAATCATAGAATAGTTGTT-3′
ATMdel2 Rev-5′-AACAACTATTCTATGATTAATATTTTCATCTAACTACTCCTCATTTGTCCTTG-3′
3.3. Transient Transfection and Cell Irradiation
Twenty-four hours prior to transfection, cells were plated in 3.5-cm culture dishes at 40%–60% confluence. A549 cells were transfected with pre-miR™ miRNA Precursor hsa-miR-27a (PM10939, Ambion Austin, TX, USA) by using Lipofectamine™ 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) for luciferase assays, or Hiperfect Transfection Reagent (QIAGEN, Hilden, Germany) for miRNA over-expression, according to manufacturer’s protocol. Mock-transfected cells underwent the transfection process without addition of miRNA (i.e., cells were treated with transfection reagent only). The medium was replaced 4–6 h after transfection with new culture medium. Transfections were performed in triplicate for each experiment and repeated 3–4 times. Cells were tested for miR or gene over-expression 24 h later.
Cell irradiation with γ-rays was performed at 24 h after transfection at the Department of Oncological and Surgical Sciences of Padova University with a 137Cs source. miR-27a-transfected and untransfected cells were irradiated with 2 Gy (dose rate of 2.8 Gy/min). After irradiation, the medium was replaced with a fresh culture medium.
3.4. Total RNA Isolation and qRT-PCR
Total RNA was isolated by using Trizol® Reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s protocol. Total RNA quantification was performed using the ND-1000 spectrophotometer (Nanodrop, Wilmington, DE, USA); RNA integrity and the content of miRNAs were assessed by capillary electrophoresis using the Agilent Bioanalyzer 2100, with the RNA 6000 Nano and the small RNA Nano chips, respectively (Agilent Technologies, Palo Alto, CA, USA).
The endogenous levels of mature miR-27a in untreated and in transfected A549 cells were determined by qRT-PCR using TaqMan MicroRNA Assay kit (Applied Biosystems, Foster City, CA, USA), that incorporate a target-specific stem-loop reverse transcription primer to provide specificity for the mature miRNA target. In brief, each RT reaction (15 mL) contained 10 ng of total purified RNA, stem-loop RT primer, RT buffer, 0.25 mM each of dNTPs, 50 U MultiScribe™ reverse transcriptase and 3.8 U RNase inhibitor. The reactions were incubated in a Mastercycler EP gradient S (Eppendorf, Hamburg, Germany) in 0.2 mL PCR tubes for 30 min at 16 °C, 30 min at 42 °C, followed by 5 min at 85 °C, and then held at 4 °C. The resulting cDNA was quantitatively amplified in 40 cycles on an ABI 7500 Real-Time PCR System, using TaqMan Universal PCR Master Mix and TaqMan MicroRNA Assays for miR-27a, and for U48 small nuclear (RNU48) as endogenous control.
For mRNA detection of
ATM 1 μg of total RNA was retrotranscribed with ImProm-II Reverse Transcription System (Promega, Madison, WI, USA). qRT-PCR was performed with the Go Taq qPCR Master Mix (Promega, Madison, WI, USA) and gene-specific primers for
ATM and
GAPDH as reference. The relative expression levels of ATM and miR-27a were calculated using the comparative delta Ct (threshold cycle number) method (2-ddCt) implemented in the 7500 Real Time PCR System software (Applied Biosystems
® 7500 Real-Time PCR System, Life Technologies, Carlsbad, CA, USA, 2007) [
47]. qRT-PCR reactions were always performed in quadruplicates.
3.5. Luciferase Reporter Assays
A549 cells were plated in 24-well plates (14 × 105 cells/well) and 24 h later co-transfected with 50 ng of the pmirGLO dual-luciferase constructs, containing the 3′UTR of ATM gene, and with pre-miR™ miRNA Precursor hsa-miR-27a (miR-27a mimic) or pre-miR™ miRNA Precursor Molecules- Negative Control (Control mimic) (Ambion, Austin, TX, USA), using Lipofectamine™ 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA). Lysates were collected 24 h after transfection and Firefly and Renilla Luciferase activities were consecutively measured by using Dual-Luciferase Reporter Assay (Promega, Madison, WI, USA), according to manufacturer’s instructions. Relative luciferase activity was calculated by normalizing the ratio of Firefly/Renilla luciferase to that of negative control-transfected cells.
3.6. Clonogenic Survival Assay
A549 (2 × 104–4 × 104 cells/cm2) were seeded in 3.5-cm culture dishes and allowed to attach overnight, then were subjected to transfection with miR-27a mimic and 24 h later irradiated with γ-rays. After irradiation, cells were harvested by trypsinization and counted by trypan blue dye exclusion. 250 viable cells were plated in 6-cm culture dishes in complete medium for the colony-forming assay and grown for 12 days before being stained with crystal violet for colony counting. Cell survival was calculated as percentage of cloning efficiency (CE) of transfected and untransfected cells irradiated with 2 Gy over CE of their respective non-irradiated control cells (i.e., miR-27a + 2 Gy vs. miR-27a; 2 Gy vs. CTR).
3.7. Cell Cycle Analysis
Cells (1 × 106) were harvested, fixed in 70% cold ethanol and stored at 4 °C overnight. Before analysis, cells were washed in distilled water, centrifuged and resuspended in 1 mL PBS containing 50 μg/mL propidium iodide (PI, Sigma-Aldrich, St. Louis, MO, USA) and 100 μg/mL RNAse, for DNA staining. Samples were incubated for 1 h at 37 °C and then analyzed using a BD FACSCanto™ II flow cytometer (BD Biosciences, San Jose, CA, USA). Data from 25 × 103 cells/sample were collected for acquisition and cell cycle distribution analysis using CellQuest (Version 3.0, BD Biosciences, San Jose, CA, USA, 2007) and ModFit LT 3.0 softwares (BD Biosciences, San Jose, CA, USA, 2007), respectively.
3.8. Immunofluorescence Staining
A549 cells were grown on glass coverslips for 24 h, transfected with pre-miR-27a and 24 h later, irradiated. Control cells were subjected to the same treatments except for irradiation. At 0.5 h, 2 h, and 6 h after irradiation, cells were fixed in 4% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA), at 37 °C for 15 min and washed twice with PBS. The cells were permeabilized with 0.2% Triton X-100 in PBS at 37 °C for 10 min and non-specific binding sites masked with goat serum (10% in PBS) at room temperature for 1 h. Cells were incubated for 1 h at room temperature with primary antibody anti-γ-H2AX (Ser139) (Abcam or Millipore Chemicon, Upstate Clone JBW301, 1:100). After three washes in PBS, cells were incubated with secondary antibody Alexa Fluor 488 goat anti-mouse (Life Technologies, 1:350), washed and counterstained with DAPI 0.2 μg/mL.
3.9. Statistical Analysis
Data are presented as means ± standard deviation (S.D.) from two to three independent experiments. All statistical comparisons were carried out by Student’s t-test and differences with a p-value < 0.05 considered significant.