Rapid detection of exon 2-deleted AIMP2 mutation ... - Semantic Scholar

Biosensors and Bioelectronics 46 (2013) 142–149

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Rapid detection of exon 2-deleted AIMP2 mutation as a potential biomarker for lung cancer by molecular beacons Seong-Min Jo a,1, Youngwook Kim c,1, Young-Su Jeong a, Young Hee Oh a, Keunchil Park c,d,nn, Hak-Sung Kim a,b,n a

Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea Graduate School of Nanoscience and Technology, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea c Medical Nano-Element Development Center, Samsung Biomedical Research Institute, Seoul, Republic of Korea d Division of Hematology–Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University, Seoul, Republic of Korea b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 January 2013 Received in revised form 24 February 2013 Accepted 25 February 2013 Available online 6 March 2013

Exon 2 deletion in aminoacyl tRNA synthetase complex-interacting multifunctional protein 2 (AIMP2) has been suggested to be associated with the progression of various cancers such as lung and ovarian cancers. However, few studies have been conducted regarding detection and relevance of exon 2-deleted AIMP2 (AIMP2-DX2) mutation to a specific cancer. Here, we demonstrate the rapid and simple detection of the AIMP2-DX2 mutation by molecular beacons and its relation to lung cancer. Real-time PCR with molecular beacons allowed a sensitive detection of the AIMP2-DX2 mutation as low as 0.3 pg initial template. Dual-conjugated liposomes with folate and molecular beacon enabled fluorescence imaging of cancer cells harboring the AIMP2-DX2 mutation with high resolution. Association of the AIMP2-DX2 mutation with lung cancer was shown by analyzing tissue samples from lung cancer patients using real-time PCR. Approximately, 60% of lung cancer patients harbored the AIMP2-DX2 mutation, which implies a potential of the AIMP2-DX2 mutation as a prognostic biomarker for lung cancer. Molecular beacon-based approaches will find applications in the simple and rapid detection of mutations on nucleotides for diagnosing and monitoring the progression of relevant cancer. & 2013 Elsevier B.V. All rights reserved.

Keywords: Molecular beacon AIMP2-DX2 Diagnosis Biomarker Lung cancer

1. Introduction Cancer is one of the most common life-threatening diseases worldwide (Siegel et al., 2012). Over the past decades, significant advances have been made methods to treat cancers. Along with the development of efficacious therapies, early detection and diagnosis of relevant cancers are considered to be crucial for reducing the mortality rates. Many factors cause transformation of normal to cancer cells, and genetic mutations in normal cells are the most deleterious causes. In this regard, analysis of cancerrelated mutations has generally been accepted to be the most effective way of diagnosing cancers as early as possible. Recently, exon 2 deletion in aminoacyl tRNA synthetase complex-interacting multifunctional protein 2 (AIMP2) has

n Corresponding author at: Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea. Tel.: þ82 42 350 2616; fax: þ82 42 350 2610. nn Corresponding author at: Medical Nano-Element Development Center, Samsung Biomedical Research Institute, Seoul, Republic of Korea. Tel.: þ82 2 3410 3450; fax: þ82 2 3410 1754. E-mail addresses: [email protected] (K. Park), [email protected] (H.-S. Kim). 1 These authors equally contributed to this work.

0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.02.037

attracted considerable attention, because this mutation was suggested to be associated with various cancers including lung and ovarian cancers (Choi et al., 2011, 2012; Kim et al., 2011). Wild-type AIMP2 composed of four exons was shown to play an important role in cells as a tumor-suppressor, which have proapoptotic and anti-proliferative effects (Han et al., 2008; Choi et al., 2009). On the other hand, a splicing variant of AIMP2 with exon 2 deletion (AIMP2-DX2) occurs frequently not only in various cancer cells, but also results in a loss of tumorsuppressor function. The AIMP2-DX2 mutant binds to TRAF2, which is a mediator for cell death signaling, but loses the role in triggering the interaction between TRAF2 and cIAP1, which is an ubiquitin ligase, and consequently TNF-a-mediated cell death is inhibited. Unlike wild-type AIMP2, the AIMP2-DX2 mutant is not able to protect p53 from MDM2-mediated degradation, which results in the survival and growth of abnormal cells despite serious damages or mutations. Therefore, the existence of the AIMP2-DX2 mutation provides important insight into the development and progression of cancerous cells as well as the status of cancer patients. Nonetheless, no detailed studies have been reported concerning analysis of the AIMP2-DX2 mutation and its relation with cancers. Direct sequencing has been most commonly used for detecting mutations, even though it is

S.-M. Jo et al. / Biosensors and Bioelectronics 46 (2013) 142–149

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Scheme 1. Schematics for the detection of mutations on nucleotides using molecular beacons. (A) Structure of molecular beacons and detection principle. (B) Structure of dual-conjugated liposomes with folate and molecular beacons. (C) Cell imaging through folate receptor-mediated delivery of molecular beacons using folate-conjugated liposomes.

labor-intensive and time-consuming for a routine clinical analysis (Yatabe et al., 2006). Here we demonstrate a simple and rapid detection of the AIMP2DX2 mutation by molecular beacons and its potential as a lung cancer biomarker. Molecular beacons are nucleotide strand featuring a stem-loop structure with a fluorescence dye attached on one end and a quencher dye on the other (Tyagi and Kramer, 1996; Marras et al., 2006) (Scheme 1A). Only when hybridized to a target, the probe separates and expands its stem region, resulting in the restoration of the fluorescence. Accordingly, specific and selective detection of mutations on nucleotides or sensing target molecules can be achieved by measuring the change in the fluorescence intensity (Iwan et al., 2001; Santangelo et al., 2004; Peng et al., 2005; Yang et al., 2005; Antony et al., 2007; Oh et al., 2010; Zhang et al., 2010). We conducted real-time polymerase chain reaction (PCR) and fluorescence imaging using molecular beacons to detect the AIMP2-DX2 mutation. For clear fluorescence imaging, dualconjugated liposomes with folate and molecular beacons were synthesized and used (Schemes 1B and C). We validated the utility

and efficacy of the molecular beacon-based approaches for detecting the AIMP2-DX2 mutation in cancer cells. Furthermore, association of the AIMP2-DX2 mutation with lung cancer was evaluated by analyzing the tissue samples from lung cancer patients.

2. Experimental 2.1. Design of molecular beacons A molecular beacon for the AIMP2-DX2 mutant was designed following the guidelines (Vet and Marras, 2005), as described in Supplementary information. FAM dye was used as a reporter, and DABCYL as a quencher. 2.2. Real-time PCR using molecular beacons Real-time PCR was performed under the following conditions: 95 1C for 5 min, followed by 45 cycles of reaction at 95 1C for 30 s,

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66 1C for 30 s, and 72 1C for 30 s. The concentration of molecular beacons was 160 nM, and the total cycling time was around 2 h (Supplementary information). The PCR products were subjected to 2% agarose gel electrophoresis. Primers for amplification of AIMP2 were used as follows: forward primer, 50 -gcttcccacctgcatgtacc-30 ; reverse primer: 50 -ggtttgcgttgatcacgatgtc-30 . For two-step PCR, total RNA was extracted from cultured cells using a RNA extraction kit. Concentration of RNA was measured using a UV spectrometer (GeneQuant 1300, GE Healthcare, UK). Synthesis of complementary DNA (cDNA) was carried out using dT15 primer, and the resulting cDNA was directly used for a two-step PCR.

2.3. In situ cell imaging with molecular beacons H460 (human non-small-cell lung cancer) and WI-26 (human diploid fibroblast from embryonic lung) cells were cultured on slides. Cells were fixed with cold acetone for 7 min. Fixed cells were incubated with 200 nM of the molecular beacons solution at 60 1C for 30 min. The slides were washed three times using Dulbecco’s phosphate-buffered saline (DPBS), and mounting media with 40 ,6-diamidino-2-phenylindole (DAPI) was added. Fluorescence images were obtained by a LSM 710 confocal microscope (Carl Zeiss, NY, USA) (Oh et al., 2010).

2.4. Fabrication and characterization of dual-conjugated liposomes Folate-conjugated liposomes were firstly prepared by mixing dioleoylphosphoethanolamine (DOPE), dimethylaminoethane carbamoyl cholesterol (DC-Chol), distearoylphosphoethanolaminemethoxy polyethylene glycol2000 (DSPE-mPEG2000), and distearylphosphoethanolamine-polyethylene glycol5000-folate (DSPE-PEG5000folate) solutions in chloroform so that the molar ratio of DOPE: DC-Chol: DSPE-mPEG2000: DSPE-PEG5000-folate is 6: 4: 0.5: 0.1. Chloroform was evaporated in vacuum oven, and dried lipid film was hydrated with HEPES buffer (10 mM, pH 7.2) by hand shaking. The crude mixture was then sonicated for 10 min with a bath-type sonicator (Branson, CT, USA). The resulting liposomal suspension was kept at room temperature for 2 h, and adjusted to 1.2 mg/mL. For fabrication of dual-conjugated liposomes with folate and molecular beacons, 80 mL of the folate-conjugated liposome suspension was mixed with 10 mL of molecular beacon suspension (100 mM) and 10 mL of 10 PBS (pH 7.4), followed by incubation for 30 min at room temperature. Free molecular beacons were removed by centrifugation with a 300 K spin filter. Molecular beacons for the AIMP2-DX2 mutant was used as a positive control, and random molecular beacon was used as a negative control (Table S1). Size and zeta potential of the liposomes were measured using a Zetasizer Nano ZS (Malvern Instruments, UK). Morphology of liposomes was observed by transmission electron microscopy (JEOL, Tokyo, Japan) with negative staining using 2% phosphotungstic acid.

2.6. Analysis of tissue samples from lung cancer patients Tissue samples of 75 lung cancer patients were obtained from the Samsung Medical Center and analyzed to detect the AIMP2DX2 mutation by real-time PCR (Supplementary information).

3. Results 3.1. Design of molecular beacons for the AIMP2-DX2 mutant We first designed a molecular beacon for the AIMP2-DX2 mutant. A loop region of molecular beacons complementary to the AIMP2-DX2 mutant was designed to hybridize with DNA or mRNA of the AIMP2-DX2 mutant comprising twelve nucleotides of exon 1 and nine nucleotides of exon 3. The stem region of the molecular beacon has a GC-rich sequence of six base-pairs. To maintain the stem-loop structure, cytosine was inserted between the loops and the stem region, because the junction sequence between exon 1 and exon 3 was shown to give rise to a self-formed stem structure. The designed molecular beacon specifically recognizes the junction sequence between exon 1 and exon 3 of the AIMP2-DX2 mutant so that the molecular beacons can be opened (Scheme 1A). Nucleotide sequences of the designed molecular beacons for the AIMP2-DX2 mutant and their targets are shown in Table S1. To determine the optimal hybridization temperatures for the molecular beacon targeting the AIMP2-DX2 mutation (Supplementary information), we examined the thermal profiles of the designed molecular beacons as described in Supplementary information. The changes in fluorescence signals were measured at temperatures ranging from 95 1C to 15 1C in the presence and absence of the synthetic targets, namely a target template with a junction sequence between exon 1 and exon 3, wild-type 1 with a sequence between exon 1 and exon 2, and wild-type 2 with a sequence between exon 2 and exon 3 (Fig. S1). No difference in fluorescence intensity was observed at temperatures ranging from 95 1C to 75 1C. At these high temperatures, the stem-loop structure of the molecular beacons is hard to maintain, because DNA is denatured. At low temperatures between 60 1C and 15 1C, the fluorescence intensities in the presence of the target template and wild-type 2 were almost identical. This is mainly due to the fact that some nucleotides are conserved in both the target template and wild-type 1, and that the molecular beacons bind partially to wild-type 1. On the other hand, the fluorescence intensities in the presence of the target template were considerably higher than those of wild-type 1 and 2 at temperatures ranging from 72 1C to 60 1C. This result indicated that the specificity of the designed molecular beacons for the target mRNA and the hybridization at this temperature range. These temperatures are the ‘window of discrimination’. Based on these results, fluorescence intensity was monitored at 66 1C during real-time PCR in this study.

2.5. Cell imaging with dual-conjugated liposomes

3.2. Detection of the AIMP2-DX2 mutation with molecular beacons

HeLa (cervical cancer) cells were cultured on slides, followed by addition of 10 mL of dual-conjugated liposomes in 150 mL of folate-free and serum-free media. The cells were incubated with the media containing the liposome for 1 h at 37 1C, and washed three times using DPBS. The cells were further incubated with folate and serum free media for 3 h at 37 1C, and fixed with 4% paraformaldehyde for 15 min. The resulting cells were incubated at 60 1C for 40 min, followed by addition of mounting media containing DAPI. Fluorescence images were observed by confocal microscopy.

To verify the performance of the designed molecular beacons, real-time PCR was carried out at varying amounts of target templates inserted in plasmids. As shown in Fig. 1A, changes in fluorescence intensity were dependent on the amount of initial template, and the threshold cycles were inversely proportional to the amount of initial templates (R2 ¼ 0.9905) (Fig. 1B). Moreover, the molecular beacons could detect the AIMP2-DX2 mutation as low as 0.3 pg of the mutant template. Amplicons of the wild-type AIMP2 and AIMP2-DX2 mutant were located on 326 bp and 119 bp, respectively (Fig. S2). Conditions for real-time PCR, such as optimal


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Fig. 2. Detection of cDNA from H460 cells harboring the AIMP2-DX2 mutation using molecular beacons. (A) Real-time PCR with varying amounts of cDNA synthesized from H460 cells. (B) Two-step real time PCR at different volumes of cDNA synthesis reaction. Reverse transcription PCR was performed using 200 ng of total RNA.

Fig. 1. PCR assay for detection the AIMP2-DX2 mutation using molecular beacons. (A) Real-time PCR using molecular beacons at varying amounts of target template inserted in plasmids. (B) Relationship between threshold cycle value and the target concentrations. (C) The fluorescence intensity from molecular beacons for AIMP2-DX2 mutant at different contents of the AIMP2-DX2 mutant in a mixture of the wild-type AIMP2 and mutant templates.

amount of molecular beacons and monitoring temperature, were optimized prior to every main assay (Fig. S3). To test the selectivity of molecular beacons, we measured the fluorescence intensity from molecular beacons for the AIMP2-DX2 mutation at different contents of the AIMP2-DX2 mutant in a mixture of the wild-type and mutant templates. As shown in Fig. 1C, the fluorescence intensity from molecular beacons for AIMP2-DX2 decreased with the decreasing content, but gave rise to distinct fluorescence signal even in the

presence of 1% AIMP2-DX2 mutant in the reaction mixture. It is therefore likely that our approach using molecular beacons is selective and sensitive for the AIMP2-DX2 mutant. To evaluate whether the AIMP2-DX2 mutation in cancer cells can be detected by molecular beacons, real-time PCR was carried out with varying amounts of cDNA extracted from H460 cells containing the AIMP2-DX2 mutation and derived from lung cancer cells. An increase in fluorescence intensity was evident during real-time PCR, and the AIMP2-DX2 mutation could be detected within the range from 0.5 ng to 5 ng of cDNA (Fig. 2A). Two-step PCR is generally used for cDNA-based real-time PCR. When this was done, the AIMP2-DX2 mutation could be detected from 1/10 to 1/100 volume of non-purified cDNA mixture at 45 cycles (Fig. 2B), but no signal from 1/1000 volume appeared despite prolonged (up to 65) PCR cycles (Fig. S4). Even though H460 cells contain the AIMP2-DX2 mutation, a significant portion of wild-type AIMP2 is likely to exist in the cells. Accordingly, amplification of wild-type AIMP2 template would interfere with amplification of the AIMP2-DX2 mutant, resulting in a prolonged threshold cycle. Nonetheless, our result demonstrates that molecular beacons can be effectively used for a rapid and sensitive detection of the AIMP2-DX2 mutation. 3.3. Fluorescence imaging of the cells using molecular beacons We attempted to detect the AIMP2-DX2 mutation in cells by in situ fluorescence imaging using molecular beacons. H460 cells

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Fig. 3. In situ fluorescence imaging of the cells using AIMP2-DX2 mutant specific molecular beacons. Images of H460 cells at FAM channel (A) and merged (B). Images of WI-26 cells at FAM channel (C) and merged (D). The fixed cells were incubated with 200 nM molecular beacons for 30 min at 60 1C. Magnification was 1000-fold. (For interpretation of the references to color in this figure caption, the reader is referred to the web version of this article.)

harboring the AIMP2-DX2 mutation were used. As a negative control, WI-26 cells, which originated from normal lung fibroblast, were used (Choi et al., 2011; Won and Lee, 2012). Fig. 3 shows the fluorescence images of the cells harboring the AIMP2DX2 mutation, indicating that molecular beacons could bind specifically to mRNA of the mutant target. In the case of H460 cells, strong green fluorescence signals were observed from the cytoplasm where mRNA of the AIMP2-DX2 mutant exists (Fig. 3A and B). Blue spheres indicate cell nucleus stained by DAPI. On the other hand, WI-26 cells gave rise to negligible fluorescence signals (Fig. 3C and D). This result demonstrates that existence of the AIMP2-DX2 mutation in the cells can be simply detected by in situ fluorescence imaging using molecular beacons. 3.4. Imaging by dual-conjugated liposomes with folate and molecular beacons The fluorescence imaging by in situ hybridization using molecular beacons has a limited resolution, because both the wild-type and mutant cells are exposed to the same amount of the molecular beacons. Thus, the resolution is mainly determined by the binding specificity of the molecular beacons to a target nucleotide. In an approach to more sensitive detection of the AIMP2-DX2 mutation

by the fluorescence imaging, we attempted a folate receptormediated delivery of molecular beacons via liposomes. Folate receptors are overexpressed in cancer cells, and this receptor has been broadly used for targeted delivery of various reagents inside the cells (Zhao et al., 2000; Byrne et al., 2008). Our dual-conjugated liposomes with folate and molecular beacons (Scheme 1B) would efficiently deliver molecular beacons into only cancer cells, and the transported molecular beacons would hybridize with mRNA of the AIMP2-DX2 mutant in the cytoplasm, resulting in a fluorescence signal (Scheme 1C). We used HeLa cells harboring the AIMP2-DX2 mutation and overexpressing folate receptors (Lee and Low, 1995; Saul et al., 2003; Weng et al., 2011). We fabricated folateconjugated liposomes composed of DOPE and DC-Chol. Liposomes are vesicles of phospholipids bilayers that have been widely used for delivery of a variety of compounds. Especially, the DOPE/DCChol liposomes have excellent performance as nucleotide carriers due to their cationic property (Li et al., 1996; Gao et al., 2010). The resulting liposomes were further conjugated with molecular beacons via electrostatic interaction and characterized in terms of size, zeta potential, and morphological properties (Fig. S5 and Table S2). The size of the liposomes was slightly increased by conjugation with molecular beacons, and the zeta potential was shifted to a weak negative charge. This result verified the synthesis of dual-conjugated


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Fig. 4. Fluorescence imaging of the cells using molecular beacon-conjugated liposomes. (A) Image of HeLa cells using dual-conjugated liposomes with folate and molecular beacons for AIMP2-DX2 mutant. (B) Image of HeLa cells using liposomes conjugated only with molecular beacons for AIMP2-DX2 mutant. (C) Image of HeLa cells using liposomes conjugated with folate and random molecular beacons. (D) Image of HeLa cells using liposomes conjugated only with random molecular beacons. Molecular beacons were delivered into living cells using 10 mg liposomes for 1 h and further kept for 3 h after a washing. The resulting cells were fixed and incubated for 40 min at 60 1C. Magnification is 1000-fold. (For interpretation of the references to color in this figure caption, the reader is referred to the web version of this article.)

liposomes with folate and molecular beacons. Furthermore, bilayer structures of liposomes could be observed before and after conjugation with molecular beacons. It seems that conjugation of molecular beacons have no deleterious effect on liposome structure. We performed fluorescence imaging of the cells containing the AIMP2-DX2 mutation using dual-conjugated liposomes. As shown in Fig. 4A, strong green fluorescence signals was observed in the cytoplasm of HeLa cells when hybridized at 60 1C. On the other hand, very low green signals were obtained when liposomes without folate were employed (Fig. 4B). This result indicates that a large amount of molecular beacons were effectively delivered inside the cells via folate receptor-mediated endocytosis pathway, resulting in high fluorescence signals compared with that obtained in the absence of folate on liposomes. At the same time, molecular beacons were easily released from the delivered liposomes and hybridized with mRNA of the AIMP2-DX2 mutant. Previous studies have shown that cationic carriers such as DOPE/DC-Chol liposomes could escape from late-endosome by ‘proton sponge effect’ with loaded nucleotide chains (Boussif et al., 1995; Varkouhi et al., 2011). Non-specific cellular uptake was observed in case of liposomes with no PEGylation and 2.5% PEGylated liposomes (data not

shown). Thus, we used the 5% PEGylated liposomes in this study. HeLa cells with random molecular beacons-conjugated liposomes displayed negligible signals regardless of the presence of folate on liposomes (Fig. 4C and D). In these cases, even though molecular beacon-conjugated liposomes were delivered inside the cells via a folate receptor-mediated endocytosis, they gave rise to no fluorescence signal because molecular beacons kept closed forms due to no target mRNA existed. It was reported that molecular beacons are susceptible to degradation by nucleases or nonspecific binding by proteins (Santangelo et al., 2004). However, our experiments using random molecular beacons gave rise to negligible fluorescence signals as shown in Fig. 4C and D, which indicates that molecular beacons remain intact during experiments and degradation by nucleases or non-specific binding by proteins is negligible. 3.5. Quantitative analysis of fluorescence images To quantify the fluorescence images, we calculated the fluorescence intensities form the confocal images by ZEN 2011 image analyzing software. We chose a single cell without bias in each case, and set the compartments in proper size in a single cell. All pixels in territory were counted and classified according to their

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fluorescence intensities. Fig. S6A displays the quantified results of images obtained from H460 cells and WI-26 cells by in situ hybridization, respectively. Much stronger green fluorescence intensities were obtained from H460 cells, whereas negligible green signals were observed from WI-26 cells. Most of pixels showing high fluorescence intensity were observed from H460 cells (cancer cells), whereas WI-26 cells, non-tumorigenic lung fibroblast harboring normal levels of AIMP2-DX2 mutation, generated few pixels exhibiting the intensity above 15. Therefore, the threshold intensity distinguishing the tumorigenic cells from normal cells was determined to be 15. This result is coincident with transcription levels of mRNA in respective cell lines as observed in PCR band (Choi et al., 2011; Won and Lee, 2012). The use of dual-conjugated liposomes for the fluorescence imaging of cells also led to stronger fluorescence intensities compared with that of only molecular beacon-conjugated liposomes (Fig. S6B). Imaging by dual-conjugated liposomes with folate and molecular beacons only generated high intensity pixels. This result indicates that folate receptor-mediated delivery of molecular beacons via liposomes is very effective for the fluorescence imaging of the cells containing specific mutations.

3.6. Detection of the AIMP2-DX2 mutation in tissue samples from lung cancer patients To get some insight into a potential of the AIMP2-DX2 mutation as a biomarker for lung cancer, we attempted to detect this mutation in tissue samples from 75 lung cancer patients by real-time PCR with molecular beacons (Supplementary information). By using 200 ng of total RNA as an initial template, two-step PCR was performed under the same conditions described above, and the results were compared with end-point PCR. As shown in Table 1, the AIMP2-DX2 mutation was detected in 38 samples, and 26 samples had only wild-type AIMP2 (1st trial). Three samples displayed discrepancy between PCR band and molecular beacon signals, and eight samples resulted in failure of PCR assay. As a result, around 60% of lung cancer patients was estimated to harbor the AIMP2-DX2 mutation. Additional experiment using another primer set (2nd trial) also displayed a similar result, indicating that approximately 66.0% of the patients harbored the AIMP2-DX2 mutation. Correlation between first trial and second trials was 77.1% in overlapped 48 valid cases. Small amount of the AIMP2-DX2 mRNA might be present in cancer cells, and the purity of clinical samples would be considerably low. In addition, mRNAs from the wild-type AIMP2 and AIMP2-DX2 mutant will compete for the same primers, because their binding regions are exactly same. In other words, amplification of wild-type AIMP2 mRNA is more dominant than the AIMP2-DX2 mutant. These considerations could explain the 22.9% discrepancy between the first and second trials, and this discrepancy seemed not to be caused by malfunction of the Table 1 Real-time PCR of tissue samples from lung cancer patients using molecular beacons. Characteristic

1st Trial (N ¼75)

2nd Trial (N ¼75)

Valid cases AIMP2-DX2 positive AIMP2-DX2 negative Overlapped Correlation between 1st and 2nd trial Inconsistence between band and signal Failed PCR

64 38 (59.4%) 26 (40.6%)

56 37 (66.1%) 19 (33.9%)

48 37 (77.1%) 3 8

6 13

molecular beacons. Our result indicates that the AIMP2-DX2 mutation occurred at considerable rate in lung cancer patients, implying that this mutation has a great potential to be a lung cancer biomarker.

4. Discussion We have demonstrated that molecular beacon-based approaches are very effective for detecting the AIMP2-DX2 mutation, and that this mutation is closely associated with lung cancer. The AIMP2-DX2 mutation was shown to compromise the tumor suppressive ability of the wild type AIMP2, and this mutation occurs at high rates in lung and ovarian cancers (Choi et al., 2011, 2012; Kim et al., 2011). In this regard, a simple and fast detection of the AIMP2-DX2 mutation can be useful for prognostics of lung and ovarian cancers. Molecular beacons are versatile fluorescent probes, which can be used for detection of mutations in a target nucleotide instead of direct sequencing. We used FAM dye and DABCYL as a report dye and a fluorescence quencher, respectively, to construct molecular beacons. FAM dye has been widely used for many biological assays such as real-time PCR and in situ hybridization although its quantum yield was shown to decrease at high temperatures (Marras, 2006). In this study, however, no significant decrease in the fluorescence intensity was observed, which implies that thermal decay of FAM dye is negligible during a short assay period. By using real-time PCR and fluorescence imaging approaches, we could detect the AIMP2-DX2 mutation in different types of cancer cells. Detection sensitivity of the AIMP2-DX2 mutation was about 0.3 pg of the mutant template. Starting from cDNA, which corresponds to 2 ng of total RNA, we could identify the existence of the AIMP2-DX2 mutation. The amount of RNA in a single cell was estimated to range from 10 to 50 pg (Livesey, 2003; White et al., 2011). Thus, the molecular beacon approach might allow the detection of the AIMP2-DX2 mutation from around 100 cells. Real-time PCR is facile, sensitive, and widely used in the general laboratory, and molecular beacon-based realtime PCR could be useful for detecting mutations clinically. Fluorescent imaging techniques could eventually lead to the identification of the AIMP2-DX2 mutation in a single cell without an amplification step. Therefore, both approaches can be applied to the detection of the mutations from cell-free nucleic acids and circulating tumor cells due to their high sensitivity and requirement of small amount of sample (Schwarzenbach et al., 2011). The ratio of mRNA between AIMP2-DX2 mutant and wild-type AIMP2 would provide more insight into the development and progression of related cancers. Folate receptor-mediated delivery of molecular beacons via folate-conjugated liposomes was more effective for detecting the AIMP2-DX2 mutation in the cells by fluorescence imaging. Our results show that the AIMP2-DX2 mutation can be developed as a potential biomarker for lung cancer, because the occurrence rate of the mutation was above 60% in a patient group. Previous study showed that 44% of non-small-cell lung cancer patients have high occurrence rate of the AIMP2-DX2 mutation in tumor tissues compared with normal tissues (Choi et al., 2011). Therefore, a high rate of the AIMP2-DX2 mutation is likely to be closely associated with lung cancer. In addition, the AIMP2-DX2 mutation was suggested to play important roles in tumor survival with poor prognosis. Thus, existence of the AIMP2-DX2 offers an important guide for treating cancer patients. Expression of the AIMP2-DX2 mutant could be suppressed by specific small interfering RNA that blocks transcription of the target mRNAs (Choi et al., 2011). Moreover, a recent study revealed that chemosensitivities for paclitaxel and cisplatin are significantly increased


when expression of the AIMP2-DX2 mutant is suppressed (Choi et al., 2012).

5. Conclusion In summary, we have demonstrated that the AIMP2-DX2 mutation can be effectively detected by molecular beacons. Fluorescent imaging using dual-conjugated liposomes clearly identified the AIMP2-DX2 mutation in a single cell without an amplification step. Therefore, both approaches can be applied to the detection of the mutations from cell-free nucleic acids and single cells. We believe that molecular beacon-based approach will provide the information regarding the patient status and therapeutic guideline.

Acknowledgments This research was supported by the Korea Health 21C R&D Project (A040041) of Ministry for Health, Welfare and Family Affairs, and the Pioneer Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (20082000218).

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.02.037.

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