MiR-1244 sensitizes the resistance of non-small cell lung cancer A549 cell to cisplatin

The parental lung cancer A549 cell was purchased from Shanghai Institute of Cell Biology (Shanghai, China). The DDP-resistant cell line (A549/DDP) was established as previously published [19]. Briefly, DDP was added into A549 cells in the log phase at a concentration of 0.2 μg/ml and remained in the medium. After growth, the cells were split and treated again with progressively higher concentrations of DDP. During the treatment, the DDP concentration was increased to 15 μg/ml. All cell lines were cultured in Dulbecco’s modified Eagles medium (DMEM) containing 10 % fetal bovine serum (Gibco, NY, USA) in the humidified air with 5 % CO₂ at 37 °C.

Cells seeded in a 6-well plate (2.5 × 10⁵ per well) were transfected at 50 % confluence using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, CA, USA) with Opti-MEMI (Gibco, NY, USA) according to the manufacturer’s instructions. After 24 or 48 h, the transfected cells were harvested for downstream analyses or measured for cell wound healing assay. Both of microRNA mimics, inhibitors and their negative control were purchased from Invitrogen (Invitrogen, CA, USA).

Total RNA was isolated using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions, and A260/280 and A260/230 ratios were measured using the Nanodrop 2000 (Thermo Scientific, PA, USA). Approximately 500 ng of total RNA was converted to cDNA by First-strand cDNA synthesis kit (OriGene Technologies, MD, USA). Quantitative real-time PCR was then conducted using SYBR^® Green mastermix (cwbio, China) in a 7500 Fast PCR instrument (Applied Biosystems, CA, USA). All samples were run in triplicate in the 96-well reaction plates.

The cells (2 × 10³) in the log growth phase were seeded in a 96–well plate. After overnight growth, the cells were transfected by oligonucleotides (Invitrogen, CA, USA) as described above. After 24 h of transfection, the cells were treated with different dose of cisplatin (0, 20, 40, 80, 160 µg/ml) and incubated for 48 h. A total of 20 μl of 5 mg/ml MTT reagent (Sigma-Aldrich, MO, USA) was added and incubated in the incubator for 4 h. The plate was then read in a microplate reader at a wavelength of a 490 nm reference.

The cells were seeded in a 6-well plate (1 × 10⁶ per well). After confluent, a linear wound was made using a 10 μl pipette tip across the cell monolayer. The cells were grown in DMEM supplemented with 3 % FBS for additional 48 h. The cell motility was measured by photographing five random areas at the time of 0, 24 and 48 h after wounding.

The annexin V-FITC Apoptosis Detection Kit (MultiSciences Biotech Co, China) was used to quantify apoptosis by flow cytometry. The cells were harvested in PBS and collected by centrifugation. The cells were then re-suspended in the binding buffer, stained with FITC and PI for 5 min and immediately analyzed by flow cytometer.

The cells were harvested and lysed in RIPA buffer in the presence of protease inhibitors (Roche, Mannheim, Germany) for 10 min at 4 °C. Protein concentration in the supernatants was quantified using the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, CA, USA). Proteins (30 μg) were separated by 15 % SDS-PAGE and then transferred to PVDF membranes (Millipore, Bedford, MA). After blocking with 5 % nonfat milk in TBST for 1 h, the blot was incubated with the primary antibodies overnight at 4 °C. After three washes (15 min each) in TBST, blots were incubated with horseradish peroxidase-conjugated secondary antibodies and developed using an ECL detection system. All images showed were from a single experiment that was a representative of at least three independent replicate experiments.

MiRNA target analysis was conducted by miRDB (http://​www.​mirdb.​org/​miRDB/​), Targetscan (http://​www.​targetscan.​org/​) and CLIP-Seq. All data were presented as the mean ± SD of three or more replications, and the Student’s t test was used to test the significance between two groups. Data analysis was performed using GraphPad Prism 4.0 (Graph Pad Software, La Jolla, CA) and two-tailed P < 0.05 were considered to be significant.

Previously, we have identified by array analysis a set of miRNAs differentially expressed in NSCLC tissues compared to healthy controls (manuscript under review). In this study, we employed qRT-PCR assay to assess the expression level of the miRNAs in both A549 and A549/DDP cells. Among 37 miRNAs, we observed that 11 miRNAs were upregulated and other 26 miRNAs were down-regulated in A549/DDP cell line (Fig. 1a). Especially, miR-589 and miR-1244 were significantly decreased in A549/DDP cell (Fig. 1b), while miR-182 and miR-224 were increased in A549/DDP cell (Fig. 1c).

MiRNA mimic is a short sequence of oligonucleotides synthesized by chemical method which can enhance the functions of endogenous miRNA, while miRNA inhibitor is a modified oligonucleotide that can incorporate into the target miRNA thus attenuating its function. To further assess the effects of the dysregulated miRNA on DDP resistance of A549/DDP cell, we transfected the miRNA mimic for miR-589 or miR-1244, and the miRNA inhibitor for miR-182 or miR-224 into A549/DDP cells. As shown in Fig. 1c, the relative expression levels of miR-589 and miR-1244 are significantly increased in A549/DDP transfected by mimic miRNAs compared to the negative controls (Fig. 1d). Likewise, the expression level of miR-182 and miR-224 are significantly decreased in A549/DDP cell after transfection of miRNA inhibitors (Fig. 1e), indicating both mimic and inhibitory oligonucleotides could revert, at least in partial, the expression level of its corresponding miRNA.

We hypothesized that the corrected miRNA expression might sensitize A549/DDP cells to DDP. To this end, we first transfected A549/DDP cells by either miR-589 mimic or miR-1244 mimic followed by the treatment with various doses of DDP (0, 20, 40, 80 and 160 μg/ml) for 48 h. The MTT assay showed that up-regulation of miR-589 or miR-1244 led to a significant decrease in cell viability of A549/DDP in response to DDP compared to A549/DDP-NC (Fig. 2a). The IC50 value for DDP in both A549/DDP-miR-1244 (5.66 ± 2.32 μg/ml) and A549/DDP-miR-589 cell (6.53 ± 2.21 μg/ml) were significantly lower that of A549/DDP-miR-NC cell (30.1 ± 3.44 μg/ml) (P < 0.05). However, reduction of miR-182 or miR-224 has no effects on the sensitivity of A549/DDP to DDP (Fig. 2b). The IC50 in A549/DDP-anti miR-224, A549/DDP-anti miR-182 and A549/DDP-anti miR-NC cells were 83.76 ± 5.03, 28.77 ± 4.09 and 23.7 ± 2.47 μg/ml, respectively. These data suggested that up-regulation of miR-589 or miR-1244 effectively enhances the sensitivity of A549/DDP cells to DDP.

We next analyzed the effect of miR-589 or miR-1244 by flow cytometry on apoptosis of A549/DDP cell after introduction of miR-589 or miR-1244. The apoptotic cells were increased in A549/DDP cell transfected with miR-589 or miR-1244 mimics, but not miR-NC (Fig. 3a), suggesting either miR-589 or miR-1244 could induce the apoptosis of non-small cell lung cancer cells. As showed in Fig. 3b, the apoptosis rates of A549/DDP cells transfected with miR-1244 mimic, miR-589 mimic and control were 28.83 %, 38.78 versus 10.5 % (P < 0.05).

To further verify the miR-589 or miR-1244-induced cell apoptosis, we examined activation of the caspase-3, a hallmark of apoptosis. As showed by western blot (Fig. 3c), there is a significant band for cleaved caspase-3 in miR-589-transfected cells in comparison to the negative control at 24 h, indicating early events in the apoptosis pathway.

To evaluate the impact of miR-589 or miR-1244 on the migration of the DDP-resistant cells, we performed cell wound healing assay. Interestingly, the migration rate of the transfected cells by either miR-589 or miR-1244 mimics was significantly reduced compared to miR-NC at both 24 h and 48 h (Fig. 3d, e), indicated that miR-589 or miR-1244 inhibits the cell mobility of A549/DDP cells.

To gain insight into the possible molecular mechanisms underlying the striking difference in the expression of miRNA, especially miR-1244, between A549/DDP and A549 cells, we predicted the potential target of miR-1244 using miRDB (http://​www.​mirdb.​org/​miRDB/​), Targetscan (http://​www.​targetscan.​org/​) and CLIP-Seq. Among hundreds of the target genes predicted, we selected the most likely target genes and validated their expressions by qPCR. Importantly, 13 out of 17 top genes were confirmed up-regulated in the A549/DDP cells transfected by miR-1244, while 4 target genes were confirmed down-regulated, particularly NEDD4 (Fig. 4a). As an E3 ligase, knockdown of NEDD4 was showed to increase basal TP53 levels, thus causing stronger TP53 responses to DNA damage [20], we therefore analyzed the expression level of TP53 by QPCR. Interestingly, TP53 was found over-expression in the A549/DDP upon transfection of miR-1244 (Fig. 4b), indicating TP53 is one of the potential targets of miR-1244. This observation was further confirmed at protein level by western blot. As showed in Fig. 4c, while NEDD4 was down-regulated, the translational product of TP53 was significantly induced in A549/DDP-miR-1244 compared to A549/DDP-miR-NC cell. These data imply p53 protein might be involved in the process of chemoresistance of tumor cells.