Isothermally Sensitive Detection of Serum Circulating miRNAs for

Nov 11, 2013 - Here, we develop a new method for isothermally sensitive detection of serum miRNAs using hairpin probe-based rolling circle amplificati...
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Isothermally Sensitive Detection of Serum Circulating miRNAs for Lung Cancer Diagnosis Ying Li,†,§ Li Liang,†,‡ and Chun-yang Zhang*,§ §

Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong 518055, China ‡ Department of Tumor Chemotherapy and Radiation Sickness, Peking University Third Hospital, Beijing 100191, China S Supporting Information *

ABSTRACT: Tumor-derived miRNAs in serum are emerging as the new noninvasive biomarkers for the diagnosis of human cancers, especially at their early stage. An ideal method with high sensitivity, excellent selectivity, a simple procedure, and small amounts of starting materials is imperative for the detection of clinic circulating miRNAs. Here, we develop a new method for isothermally sensitive detection of serum miRNAs using hairpin probe-based rolling circle amplification (HP-RCA). This method exhibits ultrahigh sensitivity toward lung cancer-related miR-486-5p with a detection limit of as low as 10 fM and a large dynamic range of 6 orders of magnitude, and it can even discriminate miR486-5p from both miRNAs with high sequence homology and its precursors (pre-miRNAs). More importantly, this method can directly and accurately distinguish the expression of serum miR-486-5p among six nonsmall-cell lung carcinoma (NSCLC) patients and six healthy persons, holding a great potential for further applications in the clinical diagnosis of lung cancers.

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and interpretation.16 Owing to its high sensitivity and specificity, qRT-PCR has become the most widely used approach for miRNA assay.15 Especially, the miR-specific TaqMan miRNA assay has become commercially obtainable without the involvement of sophisticated primer design and large amounts of RNA starting material. However, qRT-PCR generally requires the use of modified primers, such as Taqman and LNA (locked nucleic acid) primer, to ensure the detection specificity, inevitably increasing the experimental cost. In order to improve the sensitivity, specificity, and simplicity of miRNA assay, a variety of new strategies has been developed, such as electrochemical devices,17,18 molecular beacons,19,20 nanopore sensors,21,22 and sequence-based amplification.23 Among these methods, rolling circle amplification (RCA) has been employed for sensitive detection of miRNAs in combination with a second primer,24 Q-STAR reporters,25 a dumbbell-shaped DNA probe,26,27 and DNAzyme.28,29 However, a high temperature of 55−65 °C is usually required to denature the oligonucleotides before annealing between miRNAs and padlock probes.24,26,27,29,30 Such a high temperature, which is close to or above the melting temperature of most hairpinstructured precursor miRNAs (pre-miRNAs), might increase the risk of annealing between pre-miRNA and padlock probe and eventually result in low discrimination between mature miRNA and pre-miRNA. In addition, most of these methods have to work with the purified miRNAs,11−31 which requires a

icroRNAs (miRNAs) are endogenous small noncoding RNAs, which regulate the gene expression by either repressing translation or decreasing the stability of target mRNAs.1 miRNAs are predicted to target over 50% of all human protein-coding genes, enabling them to play crucial roles in the regulation of biological development, cellular proliferation, differentiation, and apoptosis.2 The dysregulation of miRNAs has been proved to be associated with cancer initiation and progression.3,4 Recent studies have shown that sufficiently stable miRNAs can be isolated from serum, plasma, and other body fluids.5,6 Moreover, the distinct levels of circulating miRNAs, particularly serum miRNAs, have a potential to become new biomarkers for cancer diagnosis and prognosis.7 Among all types of cancers, lung cancer is the leading cause of cancer mortality worldwide, and its major subtype, nonsmall-cell lung carcinoma (NSCLC), accounts for approximately 80−85% of all cases of lung cancer.8 More and more evidence indicates that the deregulated expression of miRNAs is an early event in tumorigenesis.9,10 Therefore, the measurement of circulating miRNA levels might become a new strategy for early diagnosis of NSCLC. Due to its small size, sequence similarity among various members, and the low level in body fluids, sensitive detection of miRNAs has remained a great challenge. Currently, the main technologies for miRNA detection include quantitative-reverse transcription PCR (qRT-PCR),11,12 microarrays,13 and nextgeneration sequencing.14 Microarrays and next-generation sequencing have shown their advantages in the discovery of new miRNAs,15 but they are restricted to centralized laboratories because of costly infrastructure for date analysis © 2013 American Chemical Society

Received: August 15, 2013 Accepted: November 11, 2013 Published: November 11, 2013 11174

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In the hairpin probe, the hybridization region for miRNA is shown in bold. The hybridization region between hairpin probe and circular template is underlined.

performed in 50 μL of reaction mixture containing 1 nM annealed hairpin probes, 5 nM circlular probes, 5 μL of 10× RCA buffer, 0.1 mg/mL BSA, 500 μM of each dNTP, 5 U Phi29 DNA polymerase, and an indicated amount of miRNAs or serum lysate for 4 h at 35 °C. The reactions were terminated by incubation at 65 °C for 10 min. Gel Electrophoresis Analysis. After amplification reaction, 10 μL of reaction products stained with 1× SYBR Green II was loaded on 0.8% agarose gel and electrophoresed in 1× TAE buffer at 100 V for 60 min. The gel was analyzed by an Imaging station 4000MM. Measurement of Fluorescence Spectra. The 50 μL of amplification product was mixed with 5 μL of 50× SYBR Green II and diluted to a final volume of 400 μL with water. The excitation wavelength was 485 nm, and the spectra were recorded between 500 and 700 nm with a fixed exposure time and PMT gain. The maximum fluorescence emission was at 512 nm. The fluorescence intensity of reaction products obtained in the absence of annealed hairpin probe and miRNAs is defined as the background signal. For serum samples, the background signal is defined as the fluorescence intensity of reaction products obtained in the absence of Phi29 DNA polymerase. The value of (F − F0)/F0 is used to calculated the concentration of miRNAs based on the obtained regression equation, where F0 and F are the fluorescence intensity at 512 nm in the absence and presence of miRNAs with the subtraction of background, respectively. Sample Analysis. Circulating miRNAs in serum were extracted using the miRNeasy RNA isolation kit from Qiagen, with the miRNeasy spike-in control (C. elegans miR-39 miRNA mimic) as an internal control for miRNA recovery and reverse transcription efficiency. Reference values of miRNA were obtained by qPCR and normalized according to the parallel recovery of Cel_miR-39. For direct profiling of circulating miRNA in serum, each 10 μL of serum sample, obtained from six nonsmall-cell lung carcinoma (NSCLC) patients and six healthy persons, respectively, was diluted with PBS to a final volume of 50 μL (1:5 dilution), heated at 95 °C for 5 min, and then cooled rapidly on ice for 3 min.31 Then, the heatdenatured serum lysates were centrifuged at 17 000g for 15 min at 4 °C. Finally, 20 μL of the supernatant (equal to 4 μL of serum) was added to each 50 μL of the RCA reaction.

China). Whole blood samples were derived from adult NSCLC patients, recovered patients, and healthy volunteers at the Peking University Third Hospital (Beijing, China). All of the donors provided written informed consents. Whole blood was separated into serum within 2 h after blood was derived and was stored at −80 °C. Isothermal amplification reaction was performed on a 96 Well Thermal Cycler (Eppendorf, Hamburg, Germany). Quantitative PCR was performed on a CFX96 Touch realtime PCR detection system (Bio-Rad, CA, USA). Gel electrophoresis was conducted using a Sub-Cell GT horizontal electrophoresis system (Biorad, Hercules, CA, USA) and an Imaging station 4000MM (Carestream Health, Rochester, NY, USA). The fluorescent spectra were measured using a Hitachi F-4500 fluorometer (Tokyo, Japan). RCA Reactions. Before amplification reaction, 50 nM hairpin probes were incubated in a buffer containing 5 mM MgCl2 and 10 mM Tris−HCl (pH 8.0) at 95 °C for 5 min and then slowly cooled to room temperature over 60 min to make the probes fold into hairpin structures. The RCA reaction was

RESULTS AND DISCUSSION Principle of miRNA Assay. The principle for circulating miRNA detection on the basis of hairpin probe-based rolling circle amplification (HP-RCA) is shown in Figure 1. The hairpin probe contains two sections: (1) 5′ strand of the stem and the loop are set as a complementary region for the target miRNA; (2) the 3′ strand of the stem with a short 3′ tail is set as a complementary region for the circular template (Figure 1A). The melting temperature (Tm) of hairpin probe is designed to be no less than 45 °C for keeping it stable under the reaction condition. The addition of a short 3′ tail in the hairpin probe is to balance the GC content (40−60%) and to keep the Tm between hairpin probe and circular template no less than 45 °C. Importantly, the Tm between the 3′ tail and circular template should be less than 15 °C to make their hybrid unstable under the reaction condition. In order to apply the HP-RCA for lung cancer diagnosis, miR-486-5p, one of the downregulated miRNAs in lung tumor tissues,9,32,33 is selected as the model. The first step of HP-RCA is initiated by the hybridization of miR-486-5p with the hairpin probe, resulting in

large amount of RNA sample from body fluids (several hundred microliters according to the manuals of commercial RNA extraction kits). Therefore, a new method with high sensitivity, excellent selectivity, simple procedure, and small amounts of starting materials is highly desirable for the detection of clinic circulating miRNAs. Here, we develop a new method for isothermally sensitive detection of serum circulating miRNAs using hairpin probe-based rolling circle amplification (HPRCA). Our method can selectively discriminate target miRNAs from both the related analogues and the miRNA precursors (pre-miRNAs), with a detection limit of as low as 10 fM and a large dynamic range of 6 orders of magnitude. Moreover, the method can directly and accurately distinguish the expression of serum miR-486-5p among six nonsmall-cell lung carcinoma (NSCLC) patients and six healthy persons, holding a great potential for further applications in the clinical diagnosis of lung cancers.

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EXPERIMENTAL SECTION Materials and Apparatus. The Phi29 DNA polymerase was purchased from New England Biolabs, Inc. (Ipswich, MA, USA). The deoxynucleotide solution mixture (dNTPs) and RNase inhibitor were purchased from TaKaRa Bio Inc. (Dalian, China). SYBR Green II, RNAase free water, fetal bovine serum (FBS), and miRNA qRT-PCR kit were purchased from Life Technologies (Carlsbad, CA, USA). The oligonucleotides (Table 1) were obtained from TaKaRa Bio Inc. (Dalian, Table 1. Sequences of the Oligonucleotidesa name

sequence (5′−3′)

hairpin probe

CTC GGG GCA GCT CAG TAC AGG AAG CTG CCC CGA GAT TAC ATT GGG GCA GAG CTT ACG ACC TCA ATG CTG CTG CTG TAC TAC TCT TCG AAC AAT GTA ATC TC UCC UGU ACU GAG CUG CCC CGA G AUU GGA CUG CUG AUG GCC CGU GCA UCC UGU ACU GAG CUG CCC CGA GGC CCU UCA UGC UGC CCA GCU CGG GGC AGC UCA GUA CAG GAU AC

circular probe miR-486-5p miR-4529-3p pre-miR-486 a

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Figure 1. (A) The structure of hairpin probe. The complementary region for target miRNA is colored in blue, and the complementary region for circular template is colored in brown. (B) Schematic illustration of miRNA detection with the hairpin probe-based rolling circle amplification (HPRCA).

the opening of hairpin probe and the exposure of the complementary part, which can anneal with the circular template and further function as a primer to initiate the RCA reaction in the presence of phi29 DNA polymerase. Notably, the whole reaction is maintained at a suitable low temperature, at which the free probe without hybridization with miR-486-5p remains a stable hairpin structure, unable to initiate the RCA reaction. The products of RCA reaction are large amounts of single-stranded DNAs with various lengths, which can be simply quantified by SYBR Green II dye, enabling the detection of miRNAs with high sensitivity and high selectivity (Figure 1B). Selectivity of miRNA Assay. We first investigated the selectivity of the proposed method using the synthesized miR486-5p. In the presence of miR-486-5p, multiple bands corresponding to the amplification products with various lengths are observed in the gel electrophoresis, but no distinguishable band is detected in the absence of miR-4865p (see Supporting Information, Figure S1A). According to the principle of HP-RCA, both the ratio of circular template to hairpin probe and the reaction temperature are two crucial factors for the detection sensitivity and selectivity. On the basis of Figure S1B (see Supporting Information), we determined that the optimal ratio of circular template to hairpin probe was 5:1. Sequence-specificity of miRNA assay is of great importance because different miRNAs often possess closely related sequences with a few bases difference. Moreover, pre-miRNAs containing an entire mature miRNA sequence, which could be released into the circulating compartments and bloodstream,34 may also interfere with the detection of mature circulating miRNAs. According to the latest miRBase Version 20, miR486-5p and miR-4529-3p have the highest sequence homology in all human miRNAs (Figure 2C). In this research, we challenged the proposed method with miR-486-5p, miR-45293p, and pre-miR-486. At the recommended working temperature of 30 °C for phi29 DNA polymerase, the fluorescence

Figure 2. Selectivity of the proposed method. (A) Fluorescence emission spectra in response to miR-486-5p (red), miR-4529-3p (green), and pre-miR-486 (blue). (B) The value of (F − F0)/F0 in response to miR-486-5p (red), miR-4529-3p (green), and pre-miR486 (blue). Error bars show the standard deviation of three experiments. (C) Sequence alignment among miR-486-5p, miR4529-3p, and pre-miR-486. The consensus bases are marked in yellow.

signal produced by miR-4529-3p is 16.2 ± 1.4% of that produced by miR-486-5p (see Supporting Information, Figure S1C). To further improve the detection specificity, we moderately increased the reaction temperature by 5 °C to destabilize the hybridization between miR-4529-3p and the hairpin probe. As expected, the fluorescence signal produced by 11176

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miR-486-5p. The fluorescence intensity increases with the increase of miR-486-5p concentration from 0 to 1 nM (Figure 3A). In the logarithmic scales (Figure 3B), the value of (F − F0)/F0 has a linear correlation with the concentration of miR486-5p over the range from 0.2 fM to 1 nM. The regression equation is log10 Y = 3.545 + 0.2884 log10 C with a correlation coefficient of 0.9927, where Y is the value of (F − F0)/F0, C is the concentration of miR-486-5p, and F0 and F are the fluorescence intensity in the absence and presence of miRNA, respectively. The limit of detection is estimated to be 10 fM based on the 3σ method. Notably, the sensitivity of the proposed method has improved by as much as 5 orders of magnitude as compared with that of molecular beacon-based methods,19 4 orders of magnitude as compared with that of the Q-STAR probe-based rolling circle amplification assay,25 and 2 orders of magnitude as compared with that of molecular beacon-based isothermal amplification assay23 and is comparable to the branched rolling-circle amplification assay but with a more simple procedure.24 The improved sensitivity of the proposed method can be attributed to both the unique design of hairpin probe (Figure 1A) and the extremely high amplification efficiency of RCA. Moreover, the large dynamic range of the proposed method with 6 orders of magnitude enables the accurate quantification of miRNAs under various experimental conditions. In addition, the proposed method has a good reproducibility with a relative standard deviation (RSD) of 5.0% for five repetitive measurements of 100 fM miR-486-5p. Detection of miRNAs in Serum. Recent research demonstrated that expression levels of miR-486-5p in plasma might provide a new diagnostic approach for NSCLC.32 In this research, we investigated the feasibility of the proposed method for direct detection of miRNAs in serum. Circulating miRNAs are associated with protein complexes and exosomes,34 which are conducive to their high stability but also are deterrent to the hybridization between miRNA and hairpin probe. We first released miRNAs from the enclosure of protein and exosome using heat treatment and then detected miR-486-5p in the supernatant of serum lysate. As shown in Figure S2 (see Supporting Information), the value of (F − F0)/F0 obtained from the healthy persons is significantly (unpaired t test, P < 0.0001) higher than that obtained from the NSCLC patients. As a negative control, the values of (F − F0)/F0 obtained from fetal bovine serum (FBS) are negligible (see Supporting Information, Figure S2). The concentrations of miR-486-5p in serum were further calculated according to the calibration curve in Figure 3B. As shown in Figure 4 and Table S1 (see Supporting Information), the results obtained by HP-RCA are in good agreement with those obtained by qPCR (see Supporting Information, Figure S3A,B) and are comparable to the reported concentration range of miR-486-5p in plasma (mainly 106−107 copies/μL),35 further confirming the accuracy of the HP-RCA assay. The median concentration of serum miR-486-5p from six NSCLC patients (2.3 pM) is significantly lower than that of serum miR-486-5p from six healthy persons (13 pM), which is consistent with the function of miR-486-5p as a tumor-suppressor to inhibit the progression and metastasis of lung cancer.33 In addition, both inter (between-days) and intra (within-day)-measurements of miR-486-5p from the serum lysates of one patient and one healthy person show a good reproducibility (see Supporting Information, Figure S3C). Even though the dilution of serum lysates may result in the concomitant decrease in the value of (F − F0)/F0, the obtained initial concentrations of miR-486-5p in serum remain

miR-4529-3p is reduced to 2.1 ± 1.1% of that produced by miR-486-5p (see Supporting Information, Figure S1C), suggesting the better performance to distinguish the perfectmatched miRNA from the mismatched one at 35 °C than at 30 °C. Moreover, the signal ratios of pre-miR-486 to miR-486-5p are both ca. 2.7% at the reaction temperature of either 30 or 35 °C (see Supporting Information, Figure S1C), suggesting similar performance to distinguish the miRNA from its premiRNA at these two temperatures. Taken together, 35 °C is selected as the optimal temperature in the subsequent research. Figure 2A shows the comparison of fluorescence spectra in response to different kinds of miRNAs targets under the optimal conditions. Notably, the value of (F − F0)/F0 in response to miR-486-5p is approximately 50-fold higher than that in response to miR-4529-3p and approximately 36-fold higher than that in response to pre-miR-486 (Figure 2B). These results demonstrate the high selectivity of the proposed method to discriminate miR-486-5p from both analogous miRNA and pre-miRNA. Sensitivity of miRNA Assay. We further investigated the sensitivity of the proposed method using different concentrations of miR-486-5p. Figure 3A shows the emission spectra of reaction products in the presence of different-concentration

Figure 3. (A) Variance of fluorescence intensity with the concentration of miR-486-5p. The concentration of miR-486-5p is 0, 2 fM, 20 fM, 200 fM, 2 pM, 20 pM, 200 pM, and 1 nM from bottom to top. (B) The log−linear correlation between the value of (F − F0)/ F0 and the concentration of miR-486-5p, where F0 and F are the fluorescence intensity in the absence and the presence of miR-486-5p, respectively. The concentration of hairpin probe is 1 nM, and the concentration of circulating templates is 5 nM. 11177

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program 973 (Grant Nos. 2011CB933600 and 2010CB732600), the National Natural Science Foundation of China (Grant Nos. 21325523 and 21075129), the Award for the Hundred Talent Program of the Chinese Academy of Science, the funds from State Ministry of Health of China (Grant No. 201002009) and Peking University Third Hospital of China (Grant No. 2013-BYSY-CAS-007), the Guangdong Innovation Research Team Fund for Low-cost Healthcare Technologies, the Natural Science Foundation of Shenzhen City (Grant No. JCYJ20130401170306879), and the Funds for both Guangdong Key Laboratory of Nanomedicine and Shenzhen Engineering Laboratory of Single-molecule Detection and Instrument Development (Grant No. (2012) 433).

Figure 4. Detection of lung cancer-related miR-486-5p in serum samples. Bars represent the concentrations of miR-486-5p in serum from six NSCLC patients and six healthy persons detected with qRTPCR (green bars) and HP-RCA (red bars), respectively. Error bars show the standard deviation of three experiments.

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CONCLUSION In summary, we have developed a hairpin probe-based rolling circle amplification (HP-RCA) method to quantify miRNA expression and further applied it for sensitive and selective measurement of lung cancer-related miRNAs (miR-486-5p) in serum. The proposed method is an isothermally homogeneous assay, which can be performed by just simply mixing hairpin probes, circular templates, target miRNAs, and Phi29 DNA polymerase at 35 °C, without the involvement of either miRNA purification or the complicated experimental procedures. The proposed method allows for sensitive detection of miRNAs with a detection limit of as low as 10 fM and a large dynamic range of 6 orders of magnitude, and it can even discriminate target miRNAs from both related analogues and miRNA precursors. More importantly, the proposed method can directly and accurately distinguish the expression of serum miR-486-5p among six nonsmall-cell lung carcinoma (NSCLC) patients and six healthy persons, holding a great potential for further applications in the clinical diagnosis of lung cancers. ASSOCIATED CONTENT

S Supporting Information *

Supplementary figures and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.

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unchanged (see Supporting Information, Figure S3D). These results demonstrate that the proposed method can directly quantify miRNA in serum with great reliability, thus holding a great potential for further applications in the clinic diagnosis of lung cancers.

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Letter

AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 755 86392211. Fax: +86 755 86392299. E-mail: [email protected]. Author Contributions †

Y.L. and L.L. contributed equally.

Notes

The authors declare no competing financial interest. 11178

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