Clinical diagnosis of EML4–ALK mutation in NSCLC by a gold nanoparticle beacon

Abstract

The existence of EML4–ALK, a mutated fusion gene in non-small-cell lung cancer (NSCLC), is an important consideration in the decision of the treatment options for NSCLC patients. However, there has been no standardized methods for EML4–ALK fusion gene detection up to now, which makes the development of novel detection probes particularly appealing. A molecular beacon is effective in detecting the level of a particular mRNA in living cells with easy signal visualization and convenient application. In this study, a molecular beacon composed of a gold nanoparticle core densely packed with FITC-labeled hairpin DNA sequences was synthesized and characterized. The presence of the EML4–ALK fusion gene will specifically open the hairpin structure, leading to the recovery of FITC fluorescence. This molecular beacon could precisely distinguish NSCLC cell lines with different levels of EML4–ALK mRNA expression, as indicated by obvious fluorescence in the EML4–ALK positive H2228 cell line and a negligible signal in the negative A549 cell line. The sensitivity and specificity of the molecular beacon were further verified in H2228 cells treated with small interfering RNAs (siRNAs) against ALK, which demonstrated decreased fluorescence after gene silencing. These results were simultaneously confirmed by flow cytometry and Q-PCR. More importantly, our molecular beacon successfully identified the EML4–ALK fusion gene in tissue specimens from 2 of 23 NSCLC patients. The results were consistent with Q-PCR quantification. Therefore, this molecular beacon holds great potential in clinical diagnosis of the EML4–ALK fusion gene with remarkable sensitivity and convenience.

---

Introduction

Lung cancer is one of the malignant tumors with the highest morbidity and mortality worldwide, among which NSCLC accounts for 80% of mortality of all lung cancer patients. According to statistics, the EML4–ALK fusion gene is positive in 3–8% patients with lung cancer, which means that about 70 000 of the newly diagnosed lung cancer patients worldwide each year may be associated with fusion gene EML4–ALK.¹ However, no standardized methods have been developed yet for EML4–ALK diagnosis to guide clinical treatment.

In 2007, Japanese scholar soda found a tumorigenic mutated gene fused by Anaplastic Lymphoma Kinase (ALK) gene and Echinoderm Microtubule-Associated Protein-Like 4 (EML4) in the lung adenocarcinoma tissues from a smoking patient.² EML4 and ALK are located respectively in p21 and p23 of human chromosome 2, and they are about 10 Mb apart. The fusion of the two genes results in the expression of the new fusion protein EML4–ALK.³ The in vitro cloning transformation experiment and in vivo genetic recombination test have proved that all the different EML4–ALK fusion mutants have the potential for malignant transformation and tumorigenicity. Currently, most of the clinical researches about EML4–ALK focus on Asian patients.² Martelli et al. have also carried out related clinical research in Europe, and the results are similar. These research data showed that the positive rate of EML4–ALK was related to patients' age, adenocarcinoma, epidermal growth factor receptor (EGFR) and kirsten rat sarcoma viral oncogene (KRAS) mutations and other factors.⁴

Clinical studies have indicated that EML4–ALK mutated fusion gene is highly exclusive. EML4–ALK fusion gene mutation rarely coexist with EGFR mutations. As a result, few NSCLC patients with EML4–ALK fusion gene respond to EGFR targeted therapy.⁵ As a result, without mutation gene screening, the EML4–ALK positive patients may be treated with the ineffective drug and suffer from the side effects and economic burden that could have been avoided. On the contrary, if a patient is diagnosed with EML4–ALK fusion gene mutation, better therapeutic efficacy could be achieved after treatment with crizotinib, an ALK protein inhibitor.⁶ So screening of EML4–ALK fusion gene for NSCLC patients is critical to guide clinical treatment.

Immunohistochemistry (IHC) as a relatively simple and practical means is usually applied to detect disease-related proteins from biopsy samples and surgical resection specimens. However, this method is not appropriate to detect EML4–ALK, due to the low expression level and poor stability of EML4–ALK protein in NSCLC.⁷ It is well established that messenger RNA (mRNA) is a single-strand ribonucleic acid encoded with genetic information, which is the blueprint for the cellular production of proteins. The amount of mRNA is related to protein expression and mRNA measurement indirectly reflects protein expression level. Several techniques for detecting mRNA are available, including fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR).^8,9 FISH can detect ALK gene rearrangement through different fluorescence signals of different colors, but this method may cause damage to the tissue specimens. RT-PCR is a fast and sensitive method for the identification of ALK fusion gene, but it requires mRNA with high purity. In the clinical RT-PCR detection, false positive results often occur.⁴ Actually, IHC, FISH and RT-PCR techniques have already been employed clinically to detect EML4–ALK fusion gene. However, due to their respective shortcomings and limitations, it is difficult to confirm the presence of EML4–ALK mutations by just one of them. In practice, the methods mentioned above are often complementarily combined for precise detection, but the cycle and costs are increased.⁹ Recently, a number of emerging non-invasive detection technologies have attracted much attention. For example, gene analysis has already been realized in circulating tumor cells (CTCs). However, there are almost no CTCs in patients with non-metastatic tumor or those at the early stage of tumor metastasis. In the meantime, the insufficient CTCs in peripheral blood often require extremely high sensitivity of the detection method. Due to these limitations, CTC isolation is not appropriate for all patients. Also, it will be a high cost for sorting CTCs. After that, FISH or RT-PCR is needed for gene analysis in CTCs, which make the procedure more complex.^10,11 Moreover, these techniques are time-consuming and cannot be used to detect mRNA in living cells.

To address these challenges, a gold nanoparticle molecular beacon was developed in this study to detect EML4–ALK expression in living cells. This molecular beacon was composed a gold nanoparticle core densely packed with monolayer hairpin DNAs.¹² Without target mRNAs, the hairpin structure is maintained in the closed state, where the FITC fluorescence is quenched by gold. However, the presence of target mRNAs will stretch out the hairpin DNA and increase the distance between the fluorophore FITC and the quenching agent gold, leading to fluorescence recovery (schematic illustration in Fig. 1). The probes take advantage of the highly efficient fluorescence quenching properties of gold, easy cellular uptake of oligonucleotide—nanoparticle conjugates without the facilitation of transfection agents,^13,14 the convenient visible detection of mRNA expression, and the continuous observation of target mRNA in living cells. Living cell detection could be used not only for the diagnosis, but also the further research. For example, after gene detection, the living cells can be further used to study their responses to certain therapies to screen effective drugs.¹⁵ We have compared our molecular beacon with the detection systems based on other nanoparticles, including up-conversion nanoparticles, carbon nanotubes and graphene oxide. Compared to these detection systems, our gold nanoparticle beacon showed better stability attributed to its unique three-dimensional structure. Also, the large loading amount of sequences facilitates easy detection of fluorescence signal.^16–18

Fig. 1 Schematic diagram of gold nanoparticle beacon for detecting mRNA in living cells from clinical tumor tissue.

In the first step, the EML4–ALK molecular beacon (EA-MB) was prepared and characterized. Subsequently, the EA-MB was used to detect EML4–ALK fusion gene in A549 and H2228, two NSCLC cell lines cultured in lab with different levels of target mRNA. More importantly, this molecular beacon was further exploited to examine EML4–ALK fusion gene expression in cells obtained directly from clinical tissue samples. This method not only features the advantages of convenient operation, short experiment duration and real-time detection in living cells, but also the accuracy comparable to that of Q-PCR. Therefore, this molecular beacon has remarkable prospects for clinical application.

Experimental section

Chemical reagents

Chloroauric acid (HAuCl₄), sodium citrate (Na₃C₆H₅O₇), sodium borohydride (NaBH₄), sodium chloride (NaCl), magnesium chloride hexahydrate (MgCl₂·6H₂O), triethylamine, ethylacetate and Tween-20, dithiothreitol (DTT), glutathione (GSH), sodium dodecyl sulphate (SDS) and dimethyl sulphoxide (DMSO). All chemicals were purchased from Aladdin Chemistry Co., Ltd (Shanghai, People's Republic of China).

Hairpin DNA modified with fluorescein isothiocyanate (FITC) at the 5′ end and thiol at the 3′ end was purchased from Sangon Biotech Co., Ltd. (Shanghai, People's Republic of China). siRNA of fusion gene EML4–ALK was also purchased from Sangon Biotech Co., Ltd.

H2228 and A549 cells lines were obtained from Shanghai Institutes for Biological Sciences (cell bank), Chinese Academy of Sciences. Roswell Park Memorial Institute medium 1640 (RPMI 1640) and Dulbecco's Modified Eagle's Medium (DMEM) were purchased from Thermo Fisher Scientific China Co., Ltd. Trizol reagent and tetrazolium salt (MTT) were purchased from America Invitrogen. Bovine serum albumin and DNase I were purchased from Sangon Biotech (Shanghai) Co., Ltd.

Design of the oligonucleotides sequence

Specifically, the sequence of the EML4–ALK mRNA was obtained from the nucleotide database on the website of National Center for Biotechnology Information (NCBI). Several 20-base mRNA recognition sequences were chosen for target and evaluated the specificity as the cellular mRNA target. By extensive BLAST search analysis, sequence whose homology is less than 70% with other human sequences' was screened out and regarded as a specific sequence of the mRNA. According to the specificity of complementary base-pair, the loop region was designed to hybridize with the target mRNA. Through simulation of the probe's secondary structure by RNA structure software, specific sequence was selected, the nucleotide sequence of the stem section was designed. The number of the bases was adjusted to an optimal length. Subsequently, the hairpin DNA modified by fluorescein isothiocyanate (FITC) at the 5′ end and thiol at the 3′ end was designed and purchased from Sangon Biotech (Shanghai) Co. The final sequence is 5′-CTCGCTGGGTGACACTTGGTTGATGATGCGAGAAAAAAAAAA-3′.

Synthesis of EML4–ALK molecular beacon

The AuNPs were synthesized first according to the previous method. The above thiol-terminated fluorescently labeled EML4–ALK hairpin DNA were activated by 100 mM DTT firstly in phosphate buffer (PBS, pH = 8.0) and then added to the solution of AuNPs. The molar ratio of oligonucleotides to particles was 60 : 1. After 12 h incubation in the dark, 10% sodium dodecyl sulphate and 2 M sodium chloride was added to achieve final concentrations of 0.1% and 0.1 M, respectively. An additional aliquot of sodium chloride was gradually added to achieve a final concentration of 0.3 M and then kept at 4 °C in dark for 12 h. At last, the solution was centrifuged at 13 000 rpm for 20 min. The precipitated beacons were resuspended in PBS (pH = 7.0). The process of centrifugation and re-suspension was repeated three times. Meanwhile, the supernatant was collected for determining the efficacy of conjugation of hairpin DNA onto the AuNP surface.^19,20 Polyethylene glycol (PEG) modification was first completed by adding PEG solution to AuNPs to give a molar ratio between AuNPs and PEG of 1 : 20 and allowing the reaction for 6 h. For the functionalization of beacon with cell penetrating peptide (CPP), 10 nM of AuNPs@PEG-COOH were incubated in 0.5 mM EDC and NHS solution with 25 mM phosphate buffer of pH 6.0. Then, 500 nM CPP was added and incubated for 16 h, followed by centrifugation at 13 000 rpm for 20 minutes to remove the excess CPP.

Fluorescence quenching and recovery properties

EA-MB (1.5 nM) in PBS (pH = 7.0, 0.1% Tween-20) was used to determine the fluorescence quenching properties of the beacons. In brief, EA-MB was mixed with the cDNA (5′-TTTTTTTTTCTCGCATCATCAACCAAGTGTCACCCAGCGAG-3′) or single-base-mismatched DNA (5′-GAAACCCGGGTTTGTTATTT-GCGCCCGGGATAATGAACTA-3′) and incubated in the above beacon solution for 1 h at 70 °C for complete hybridization. After reaction, the solution was subsequently cooled gradually to 25 °C for 12 h in the dark, and fluorescent signals at the wavelength of 520 nm were measured to assess the target selectivity of the beacon. In addition, excess DTT (0.5 M) was added to achieve complete fluorescence recovery of EA-MB.

Cytotoxicity of the EML4–ALK molecular beacon

The cytotoxicity of the beacon on H2228 and A549 cell lines were investigated. Briefly, cells were seeded at a density of 3000 cells per well in 96-well culture plates in complete 1640 (500 mL) with serum until adherent. Cells were then incubated with the beacons at concentrations of 0.5 nM, 1 nM, 1.5 nM and 2 nM, cultured for 24 h at a 37 °C in a humidified atmosphere of 5% CO₂. Cell viability was calculated based on the colorimetric MTT assay. Absorption of the samples was measured with a microtiter plate reader at 570 nm. All experiments were carried out in triplicates.²¹

Qualitative imaging of EML4–ALK mRNA in H2228 and A549 cell lines

H2228 and A549 were used as testing and control cell lines, respectively, due to their inherent positive and negative EML4–ALK expression. These two kinds of cells were cultured in glass bottom wells to 50% confluence and then treated with media containing EA-MB (1 nM) for 18 h. The fluorescence signal was recovered after the complete hybridization of the beacon oligonucleotide with that of EML4–ALK in the living cells. The cells were washed three times with PBS (pH 7.4). The recovered fluorescence signals were imaged under a laser scanning confocal microscope (LSCM, Olympus FV1000) with excitation and emission wavelengths at 488 nm and 520 nm, respectively.²²

Quantitative measurement by flow cytometry

The recovered fluorescence was further quantified by flow cytometry. Cells were cultured in 6-well plate and treated with EA-MB for 4 h. After washing with PBS (pH 7.4), the cells were detached from the growth surface using the trypsin (0.25%), and collected. Flow cytometry was performed using a BD FACSCanto flow cytometer with 488 nm excitation. The signals were collected by FL1 channel.

Primary cell capture and culture

Fresh sterile tissues of primary lung tumors larger than 3 cm³ were obtained as surgical waste from patients newly diagnosed for lung cancer at the China–Japan Union Hospital, Jilin University (Table 1) immediately after surgical resection and maintained in organ transportation medium (sterile RPMI-1640 containing 25 mM N-2-hydroxyethylpiperazine N′-2-ethane-sulfonic acid buffer and no added serum or antibiotics; GIBCO/Invitrogen Life Technologies) on ice for preservation. On arrival, the tumor was minced into small pieces using sterile scalpels or scissors, and the pieces were placed in a cell culture dish for initial cell outgrowth. Cells were grown in the RPMI-1640 medium with serum at 37 °C in an atmosphere of 5% CO₂. Over time, cells moved out of the tumor pieces and formed a monolayer at the bottom of the dish.²³ Subsequently, LSCM imaging and flow cytometry analysis were experimented on primary cell lines we captured. All patients were informed and agreed to use postoperative tumor tissue for further study in this article.

Table 1 Characteristics of 23 NSCLC patients^a

Characteristic	Number (%)
a Our study included 23 NSCLC patients referred to China–Japan Union Hospital, Jilin University from March 2014 to April 2016 for the diagnostic workup of NSCLC. The data collected included age, gender, smoking history, EML4–ALK mutation and EGFR mutation.
Age (mean age 58.3 years)
≤50 years	9 (39.1)
>50 years	14 (60.9)
Gender
Male	17 (73.9)
Female	6 (26.1)
Smoking history
Yes	15 (65.2)
No	8 (34.8)
EML4–ALK mutation
Yes	2 (87.0)
No	21 (13.0)
EGFR mutation
Yes	21 (13.0)
No	2 (87.0)

---

Assessment of EML4–ALK expression in H2228 and A549 by Q-PCR

Q-PCR was used to correlate the expression of EML4–ALK with the recovered fluorescence signal after hybridization. Briefly, total RNA was extracted from freshly isolated H2228 and A549 cells using Trizol reagent. RNA (3 µg) from each cell line was converted into cDNA with Superscript III reverse transcriptase. Subsequently, 1.0 µL cDNA was used for Q-PCR amplification with EML4–ALK specific primers: sense, 5′-TACCAGTGCTGTCTCAATTGCAGG-3′, and antisense, 5′-TCTTGCCAGCAAAGCAGTAGTTGG-3′. The 1.0 µL cDNA was used for Q-PCR amplification using GAPDH-specific primers: sense, 5′-AAGGTCGGAGTCAACGGATT-3′, and antisense, 5′-CTGGAAGATGGTGATGGGATT-3′. All of the samples were run on the ABI 7500 Real-Time PCR system (ABI, Foster City, CA, USA) as follows: 30 s at 95 °C, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. The relative abundance of EML4–ALK mRNA transcript was normalized to GAPDH expression. The standard deviation for these data was calculated from three independent experiments. As for tumor tissues, homogenization should be performed before using Trizol reagent, then operations were proceeded as above.

Small RNA interfering targeting ALK gene in H2228 cells

H2228 cells were plated at 50–60% confluence in 6-well plates or 25 cm² flasks and then incubated for 24 hours before transient transfection for the indicated times with siRNAs mixed with the Lipofectamine reagent (Invitrogen). The siRNAs specific for EML4–ALK mRNA, (ALK-1,5′-ACACCCAAAUUAAUACCAA-3′; ALK-2,5′-UCAGCAAAUUCAACCACCA-3′), a nonspecific siRNA, (5′-GUUGAGAGAUAUUAGAGUU-3′). The cells were then subjected to confocal laser scanning microscope, flow cytometry and RT-PCR assay.

Results and discussion

Characterization of EML4–ALK molecular beacon

The absorption spectra exhibited that the EA-MB shared the same absorption peak at 520 nm as the bare gold nanoparticles (Fig. 2B). When the hairpin DNAs were coated on the surface of nanoparticles, the UV-vis absorption of beacon has an obvious shoulder peak at 260 nm, which did not exist in the absorption spectrum of bare gold nanoparticles (Fig. 2B). Dynamic light scattering was used to measure the diameter of beacon and bare gold particles (Fig. 2C and D). Compared with the bare Au particles, the beacon exhibited increased diameter. In addition, the hydrodynamic diameters of the molecular beacons covered a broad size distribution, which further proved that the hairpin DNAs were successfully coated on the surface of nanoparticles. In addition, the broad size distribution of hydrodynamic diameters of beacons possibly attributed to the difference in the number of DNA sequences modified on gold nanoparticles.

Fig. 2 (A) TEM image of EA-MB. (B) UV-visible absorption spectra of gold nanoparticles and EA-MB. (C) The hydrodynamic diameter of bare gold particles. (D) The hydrodynamic diameter of EA-MB.

Transmission electron microscope (TEM) images (Fig. 2A) showed a good dispersibility of EA-MB, and the diameter of the gold core was 15 nm on average.²⁴ The image of TEM also suggested that the conjugation of the hairpin DNA did not induce AuNPs aggregation. In addition, nanoparticles with moderate size have high absorption, large specific surface area, and are easy to be uptaken by cells. The oligonucleotide-to-particle ratio was calculated to be 60 strands in average per EA-MB. In addition, PEG modification was used to improve the stability and ion interference resistance of beacons, and make the particles escape from the serum protein adherence in the medium.²⁵

Stability, specificity, cytotoxicity assay of EML4–ALK molecular beacons

Stability assay of EML4–ALK molecular beacons. It is necessary to investigate the stability of EA-MB because the DNA sequence and the thiol-gold bond were easily to be destroyed by enzyme or other reductive substances in living cells, which would induce false positive signals.^26,27 The stability of EA-MB against degradation by DNase I and damage by DTT and GSH were examined using a fluorescence assay (Fig. 3A). DTT was used as a kind of reducing agent that can break Au–S bond through thio-exchange reaction. Deoxyribonuclease (DNase I) and GSH are two main biodegradation- and damage-causing substances in the cytoplasm. The addition of DTT resulted in the rapid increase in the fluorescence, due to the release of FITC from the surface of AuNPs through thiol-exchange reaction. However, the fluorescence scarcely increased after the addition of DNase I. Also, no obvious fluorescence was found in EA-MB solution treated with 10 mM GSH, a concentration resembling intracellular conditions. These results confirmed the stability of our EA-MB against the biological components in living cells, ensuring the reliability of the subsequent fluorescence detection.

Fig. 3 (A) Fluorescence spectra of EA-MB before and after adding GSH, Dnase I and excess DTT. (B) Fluorescence spectra of EA-MB before and after addition of complementary DNA or mismatched DNA. (C) Cytotoxicity of the bare AuNP and EA-MB in H2228 and A549 cells by MTT assay.

Specificity assay of EML4–ALK molecular beacons. To characterize the target specificity of the beacons, fluorescence spectrum was measured before and after addition of complementary or mismatched DNAs (Fig. 3B). In the mismatched group, the solution of beacons exhibited a low fluorescence signal similar to that of the bare AuNP solution. The addition of the complementary DNA sequence resulted in obvious increase in the EA-MB fluorescence. Therefore, the EA-MB only responded to target sequence, which verified the specificity of our molecular beacons and excluded possible false results caused by non-specific binding in cells. In this study, the connection part of EML4–ALK mRNA containing the characteristics sections of both EML4 and ALK mRNAs was selected as the target sequence. This design ensured the specific identification of fusion EML4–ALK mRNA sequence rather than individual EML4 or ALK.

Cytotoxicity of EML4–ALK molecular beacons. The cytotoxicity of EA-MB was tested on NSCLC H2228 and A549 cell lines (Fig. 3C). Cell viability was evaluated by MTT assay following 24 h incubation with different concentrations of beacons or bare AuNPs. The results indicated that EA-MB did not cause evident loss of cell viability in both cell lines at a concentration up to 2 nM. High cell survival rates of 85% or more were observed in cells exposed to either EA-MB or bare AuNPs. Gold nanoparticles are widely used in biomedical imaging and diagnostic tests. Based on their unique optical properties and the chemical stability of Au, gold nanoparticles are widely applied in the fields of biomedicine. In this study, the 15 nm spherical gold nanoparticle was chosen. The spherical structure has the larger surface area which could aggregate more sequences to the surface of nanoparticles.²⁸ The stereo structure which consists of spherical nanoparticles and hairpin sequence could improve the efficiency of hybridization with target mRNA.²⁹ Furthermore, the dense nanostructure was proved to be internalized easily into cells which could enhance the detection signal. It is known that absorption peak of different size nanoparticle differs from each other because of the diversity of the surface plasmon resonance. 15 nm gold nanoparticle has a strong absorption peak at 520 nm wavelength which precisely fits for the fluorescence emission peak of FITC dye.

The detection of EML4–ALK mRNA in lab cell lines

Our previous study gave the evidence that the EA-MB could be applied for living cell imaging.²⁷ Here, we aimed to visualize EML4–ALK mRNA expression in cell lines cultured in lab. To accomplish this goal, cells were cultured on glass microscope cover slips, incubated with EA-MB, and imaged by LSCM (Fig. 4A). H2228 cells treated with the EA-MB showed strong fluorescence signal in comparison to A549 cells with no fluorescence, because A549 cell line does not contain the human EML4–ALK transcript.

Fig. 4 (A) Fluorescence images of EML4–ALK positive cell line H2228 and EML4–ALK negative cell line A549, treated with EA-MB. (B) Flow cytometry data of H2228 and A549 treated with EA-MB to qualify the results of LSCM. (C) Relative expression of EML4–ALK in H2228 and A549 cell lines by Q-PCR.

To quantify the intracellular fluorescent signals of EA-MB, flow cytometry analysis was further carried out. Flow cytometry measurements revealed that H2228 cells treated with EA-MB were highly fluorescent (mean fluorescence intensity: 15.62) but A549 cell models showed only marginal signal (mean fluorescence intensity: 3.34). These flow cytometry results were consistent with the confocal imaging.

To evaluate the accuracy of the above living cell mRNA detection strategy, the relative amount of EML4–ALK mRNA was measured by quantitative polymerase chain reaction (Q-PCR), as shown in Fig. 4C. EML4–ALK mRNA level was normalized to GAPDH mRNA. The level of EML4–ALK mRNA in H2228 cell line was significantly higher than that in A549 cell line. The expression difference between the two types of cells coincided with the result of the fluorescence imaging in living cells. Although Q-PCR method was used widely, the complex operation and high demands on the environment limited its application in clinical detection.

The assay of response to gene silence by EML4–ALK molecular beacon in H2228 cells

In order to further illustrate the sensitivity and specificity of the combination between EA-MB and target mRNA EML4–ALK, small interfering RNAs were transfected into EML4–ALK positive cell line H2228. EA-MB was used to detect the expression of EML4–ALK mRNA in H2228 cells with or without siRNA transfection respectively. The result was displayed by LSCM image in Fig. 5A. It was found that fluorescence of cells with siRNA transfection (mean fluorescence intensity: 5.01) was much lower than the original H2228 cells (mean fluorescence intensity: 15.03). This result indicated that the expression of fusion gene EML4–ALK was down-regulated, and the phenomena could be observed visually. At the same time, the flow cytometry analysis and Q-PCR results also exhibited the decrease of EML4–ALK expression in H2228 cells after gene silencing, which verified the results of LSCM (Fig. 5B and C). These results confirmed that EA-MB could combine with target mRNA specifically in cells, which promised the subsequent detection in clinical tumor tissues.³⁰

Fig. 5 (A) Fluorescence images of H2228 cells treated with EML4–ALK beacon before and after adding siRNA of EML4–ALK. (B) Flow cytometry data of H2228 treated with EA-MB before and after adding siRNA of EML4–ALK to qualify the results of LSCM. (C) Relative expression of EML4–ALK in original H2228 cells and H2228 treated with siRNA of EML4–ALK detected by Q-PCR.

The detection of EML4–ALK gene in clinical tumor tissue

The same methods as above were also used for analysis of EML4–ALK mRNA expression in 23 lung tumor tissues. The primary cells from tumor tissues were captured and cultured in flasks and then 10⁴ cells were seeded in glass cover slips, incubated with EA-MB, and imaged using scanning confocal microscopy. Fig. 6 showed the representative data of a positive sample and a negative sample (both 2 positive groups showed significant signals, other 21 groups revealed negative results). The EML4–ALK positive group treated with the EA-MB showed sharp fluorescence while there was almost no signal in the negative group (Fig. 6A). Furthermore, flow cytometry results were consistent with the confocal imaging and demonstrated the uniform cellular internalization and intracellular fluorescent signal of EA-MB (Fig. 6B). To evaluate the above result from the level of mRNA, the relative amount of EML4–ALK mRNA was measured by Q-PCR, as shown in (Fig. 6C). EML4–ALK mRNA level was normalized to GAPDH mRNA. The level of EML4–ALK mRNA in 2 of the 23 tissues was higher than that in the rest 21 tissues. The results above suggested that the EA-MB could be applied both in the lab-cultured cells and clinical tissue-extracted cells. Notably, the accuracy of EA-MB detection strategy was consistent with that of Q-PCR.

Fig. 6 (A) Fluorescence images of EML4–ALK positive and negative cells from clinical tumor tissues, treated with EA-MB. (B) Flow cytometry data of EML4–ALK positive and negative cells from clinical tumor tissues treated with EA-MB to qualify the results of LSCM. (C) Relative expression of EML4–ALK in its positive and negative cell lines detected by Q-PCR.

NSCLC accounts for 80% morbidity of all lung cancer. At the same time, EML4–ALK fusion gene is a common mutation in NSCLC and plays an important role on the determination of clinical regimen. But until now there has been no ideal method for clinical EML4–ALK detection. Because of the poor stability of fusion gene, IHC method could not effectively detect the protein expression of EML4–ALK. Q-PCR was used as a verification experiment to test the accuracy of beacon detection. However, the disadvantages of Q-PCR mentioned above limit its clinical application in large scale. In comparison, the method with EA-MB demonstrates simpler operation and shorter detection cycle. The designed molecular beacon could be prospectively applied in clinical EML4–ALK detection with the merit of simple operation and real-time observation in living cells.

Conclusions

In this study, we firstly synthesized and characterized EA-MB based on the previous study.²⁷ Then the expression of EML4–ALK fusing gene was detected by EA-MB in both lab-cultured cell lines and cells obtained from clinical tumor specimens. EML4–ALK positive and negative cells could be differentiated simply and accurately. Flow cytometry was used to quantify the result and Q-PCR was carried out for verification. The detection accuracy of EA-MB was comparable to Q-PCR, and even more convenient and intuitive. Next, by means of RNA interference, EA-MB was confirmed to accurately reflect the expression level of EML4–ALK fusion gene, implying the specific hybridization of EA-MB to the target gene. Compared with the common methods in clinical diagnosis of EML4–ALK fusion gene, the detection by molecular beacon features short experiment circle and convenient operation. More importantly, the accuracy of beacon was comparable to that of Q-PCR method, which was widely reported to examine gene expression.⁹ The present study provides insight into the development of new strategies to detect EML4–ALK mRNA expression. The molecular beacon is expected to further the development of clinical diagnostic strategies for the detection of EML4–ALK fusion gene.

Acknowledgements

The authors are grateful to Natural Science Foundation Committee of China (NSFC 81220108012, 61335007, 81371684, 81000666, 81171395 and 81328012), the 973 project (Grant No. 2015CB755504) for their financial support.

Notes and references

1. K. Inamura, K. Takeuchi, Y. Togashi, K. Nomura, H. Ninomiya, M. Okui, Y. Satoh, S. Okumura, K. Nakagawa, M. Soda, Y. L. Choi, T. Niki, H. Mano and Y. Ishikawa, J. Thorac. Oncol., 2008, 3, 13–17.
2. M. Soda, Y. L. Choi, M. Enomoto, S. Takada, Y. Yamashita, S. Ishikawa, S. Fujiwara, H. Watanabe, K. Kurashina, H. Hatanaka, M. Bando, S. Ohno, Y. Ishikawa, H. Aburatani, T. Niki, Y. Sohara, Y. Sugiyama and H. Mano, Nature, 2007, 448, 561–566.
3. L. Horn and W. Pao, J. Clin. Oncol., 2009, 27, 4232–4235.
4. M. P. Martelli, G. Sozzi, L. Hernandez, V. Pettirossi, A. Navarro, D. Conte, P. Gasparini, F. Perrone, P. Modena, U. Pastorino, A. Carbone, A. Fabbri, A. Sidoni, S. Nakamura, M. Gambacorta, P. L. Fernandez, J. Ramirez, J. K. Chan, W. F. Grigioni, E. Campo, S. A. Pileri and B. Falini, Am. J. Pathol., 2009, 174, 661–670.
5. T. Fan, Y. J. Song and X. L. Liu, Mol. Clin. Oncol., 2016, 4, 203–205.
6. M. Wen, X. Wang, Y. Sun, J. Xia, L. Fan, H. Xing, Z. Zhang and X. Li, OncoTargets Ther., 2016, 9, 1989–1995.
7. M. von Laffert, P. Schirmacher, A. Warth, W. Weichert, R. Buttner, R. M. Huber, J. Wolf, F. Griesinger, M. Dietel and C. Grohe, Pneumologie, 2016, 70, 277–281.
8. Y. C. Wu, I. C. Chang, C. L. Wang, T. D. Chen, Y. T. Chen, H. P. Liu, Y. Chu, Y. T. Chiu, T. H. Wu, L. H. Chou, Y. R. Chen and S. F. Huang, PLoS One, 2013, 8, e70839.
9. L. Liu, P. Zhan, X. Zhou, Y. Song, X. Zhou, L. Yu and J. Wang, PLoS One, 2015, 10, e117032.
10. E. Pailler, J. Adam, A. Barthelemy, M. Oulhen, N. Auger, A. Valent, I. Borget, D. Planchard, M. Taylor, F. Andre, J. C. Soria, P. Vielh, B. Besse and F. Farace, J. Clin. Oncol., 2013, 31, 2273–2281.
11. W. Wu, H. Hu, F. Li, L. Wang, J. Gao, J. Lu and C. Fan, Chem. Commun., 2011, 47, 1201–1203.
12. S. Ghosh, S. Mishra and R. Mukhopadhyay, Langmuir, 2013, 29, 11982–11990.
13. A. Jayagopal, K. C. Halfpenny, J. W. Perez and D. W. Wright, J. Am. Chem. Soc., 2010, 132, 9789–9796.
14. S. R. Harry, D. J. Hicks, K. I. Amiri and D. W. Wright, Chem. Commun., 2010, 46, 5557–5559.
15. Y. B. Kuo, J. S. Chen, C. W. Fan, Y. S. Li and E. C. Chan, Clin. Chim. Acta, 2014, 433, 284–289.
16. Q. Ding, Q. Zhan, X. Zhou, T. Zhang and D. Xing, Small, 2016 DOI:10.1002/smll.201601724.
17. W. Wu, H. Hu, F. Li, L. Wang, J. Gao, J. Lu and C. Fan, Chem. Commun., 2011, 47, 1201–1203.
18. J. Chen, Y. Huang, M. Shi, S. Zhao and Y. Zhao, Talanta, 2013, 109, 160–166.
19. J. Frens, Lab. Anim., 1973, 7, 287–288.
20. D. Deng, D. Zhang, Y. Li, S. Achilefu and Y. Gu, Biosens. Bioelectron., 2013, 49, 216–221.
21. Y. Yang, J. Nan, J. Hou, B. Yu, T. Zhao, S. Xu, S. Lv and H. Zhang, Int. J. Nanomed., 2014, 9, 5441–5448.
22. W. P. Kloosterman, E. Wienholds, E. de Bruijn, S. Kauppinen and R. H. Plasterk, Nat. Methods, 2006, 3, 27–29.
23. K. Kolostova, J. Spicka, R. Matkowski and V. Bobek, Am. J. Transl. Res., 2015, 7, 1203–1213.
24. H. Ji, H. Dong, F. Yan, J. Lei, L. Ding, W. Gao and H. Ju, Chemistry, 2011, 17, 11344–11349.
25. W. Wang, Q. Q. Wei, J. Wang, B. C. Wang, S. H. Zhang and Z. Yuan, J. Colloid Interface Sci., 2013, 404, 223–229.
26. J. Hoypierres, V. Dulong, C. Rihouey, S. Alexandre, L. Picton and P. Thebault, Langmuir, 2015, 31, 254–261.
27. J. Xue, L. Shan, H. Chen, Y. Li, H. Zhu, D. Deng, Z. Qian, S. Achilefu and Y. Gu, Biosens. Bioelectron., 2013, 41, 71–77.
28. K. Sato, M. Onoguchi, Y. Sato, K. Hosokawa and M. Maeda, Anal. Biochem., 2006, 350, 162–164.
29. G. A. Barhate, S. M. Gaikwad, S. S. Jadhav and V. B. Pokharkar, Int. J. Pharm., 2014, 471, 439–448.
30. Y. Pan, S. Neuss, A. Leifert, M. Fischler, F. Wen, U. Simon, G. Schmid, W. Brandau and W. Jahnen-Dechent, Small, 2007, 3, 1941–1949.

---

Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra21230a

‡ These authors contributed to the work equally and should be regarded as co-first authors.

---