Evaluation of Serum-Paired miRNA Ratios for Early Diagnosis of Non-Small Cell Lung Cancer Using Quantum Dot-Based Suspension Array

Department of Respiration Medicine, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, China Departments of Oncology and Respiration Medicine, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, China School of Information Management and Engineering, Shanghai University of Finance and Economics, Shanghai 200433, China Department of Nuclear Medicine, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, China Central Laboratory, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai 200433, China Department of Oncology, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai 200433, China


Background
Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related deaths in China or throughout the world [1].At present, most NSCLC patients are diagnosed with advanced stages and less than 10% of them can survive more than 5 years [2], whereas the survival rate can reach up to 92% for patients with clinical stage I who undergo surgical resection within one month after diagnosis [3].The establishment of a noninvasive and effective detection of early NSCLC would greatly improve the survival of patients with lung cancer.
Low-dose spiral computerized tomography (LDCT) scanning is the most important method for the screening and noninvasive diagnosis of lung cancer.But it is difficult to identify the benign and malignant nodules whose diameters are less than 1 cm [4].Serum tumor markers such as carcinoembryonic antigen (CEA), cytokeratins21-1 (CYFRA21-1), neuron-specific enolase (NSE), and squamous cell carcinoma antigen (SCC) do not show high significant value because of low sensitivity and specificity [5].Circulating microRNAs (miRNAs) have been recently detected in extracellular body fluids and proved themselves as promising biomarkers for a broad spectrum of diseases [6,7].Circulating miRNA in peripheral blood are notably stable and resist degradation despite the presence of RNase, due to being chaperoned by Ago2 or encapsuled in exosomes [8,9].Real-time PCR have been widely used to detect the expression of circulating miRNAs.However, it is a lowthroughput technique which can only detect one kind of miRNA in a single tube.Therefore, new detection methods which can detect a set of serum miRNAs simultaneously are required.
Fluorescence microsphere-based suspension array provides a new strategy on multiplex miRNA expression in a noninvasive manner [10,11].Optically encoded beads or microspheres have been used as an efficient and costeffective platform for multiplex detection of proteins or RNAs in a single tube [12,13].Quantum dots (QDs) are tiny light-emitting particles on the nanometer scale and are emerging as a new class of fluorescent labels for biology and medicine with exceptional resistance to photodegradation [14].In this study, we evaluated the sensitivity and specificity of 9 circulating miRNAs and the paired miRNA ratios for the early diagnosis of NSCLC using fluorescence microsphere-based suspension array.

Methods
2.1.Sample Collection and Determination of Serum CEA and CYFRA21-1.All the 128 blood samples from patients diagnosed with stage I NSCLC and 79 samples from healthy donors in this study were collected from Shanghai Pulmonary Hospital.Written informed consents were obtained from all patients and volunteers before the study, and the study was approved by the ethics committee of Shanghai Pulmonary Hospital.The serum levels of tumor markers CEA and CYFRA21-1 were measured with an ELISA kit (CanAg Diagnostics, China) according to the instruction of the manufacturer.

miRNA Extraction.
Total miRNA was extracted from 200 μL of serum using the miRNeasy kit (QIAGEN, USA) according to the manufacturer's instruction.Before miRNA extraction, all serum samples were thawed completely on ice followed by centrifugation once at 20,000g for 15 minutes at 4 °C to remove remaining cell debris.miRNA was extracted as per the manufacturer's instruction.The concentration of miRNA was measured using the NanoDrop 1000 (Nano-Drop, Wilmington, USA) and stored at −80 °C.
2.3.Preparation of QD-Based DNA Nanoprobes.Core/shell CdSe/ZnSe/ZnSQDs were synthesized according to the reported paper with minor modifications [15,16].Briefly, CdSe nanocrystal cores were prepared using thermal decomposition at 280 °C.The shells were formed by deposition of ZnSe and ZnS on CdS nanocrystals using successive ion layer adsorption and reaction (SILAR) and then were surface modified with glutathione (GSH) to provide them with hydrophiling.GSH-coated QDs were coupled with a thiolmodified DNA probe using a heterobifunctional MAL-PEG-NHS as the cross-linker.The general DNA probe combined in the DNA-QD can be suitable for a variety of target detection.Its sequence is QDot-s-s-5 ′ -AAAAAA AAAAAAAAAAAAAAGTGGAGTGTGAGGTGG-3′.The coupling of the DNA probe was verified by AGE.
The absorption spectrum of the QD solution was measured by an ultraviolet-visible spectrophotometer (UV1750, Shimadzu, Japan); the fluorescence spectrum of the QD solution was measured by a fluorescence spectrophotometer (LS55, PerkinElmer Inc., USA).The electrophoresis characteristic of the nanoparticle was observed by agarose gel electrophoresis.A transmission electron microscope (TEM) was used to test the size and distribution of QDs (Tecnai G2 Spirit 200kV).
2.4.Fluorescence Microsphere-Based Suspension Array.Optically encoded beads or microspheres have been used as an efficient and cost-effective platform for multiplex detection of proteins or RNAs in a single tube.The encoded beads were customized from Toujing Bioengineering Corp. (Shanghai, China), and the linkage of the DNA probes to beads was performed by water-soluble carbodiimide method.
Nine kinds of DNA probes for miRNA testing are synthesized and fixated on the corresponding coding microspheres to become a suspension array.The 9 probe sequences were shown in Table 1.The upper part of the DNA probes is the complementary sequence of the miRNA, and the lower segment is the complementary sequence of QD.The miRNA extraction is used to wash the microsphere.miRNAs will be combined in the upper probe of the microchip.Then the single-exonuclease automotive service engineer ExoVII is used to digest the single-strand probe without combination of miRNAs.After the hybridization and digestion, the microchip was washed and the probes without combined miRNA were removed.On the contrary, the probes with combined miRNAs were still fixated on the solid matrix.Then general DNA-QDs were hybridized to this microchip.The fluorescence intensity of QDs is proportionate to the content of fixated miRNA on the solid matrix (Figure 1).
The fluorescence intensity of QDs was measured by the flow cytometry (FCM) instrument (BD Company Accuri C6).Software Flow Joe7.6 was used to analyze the data.300 μL of sample was performed to the FCM, and the relative fluorescence intensity of each miRNA coding beads was read as FL3 and FL4 double parameters.The FL2 was used to determine the content of each miRNA.Samples can be calculated to measure the content of miRNAs through the preset standard curve.

Statistical Analysis. Statistical comparison of the demographic features between the NSCLC cases and control samples or between the NSCLC cases was performed by using
Student's t-test.The differences were considered statistically significant at p < 0 05.Risk score analysis was performed to evaluate the associations between NSCLC and the expression levels of the serum miRNAs.All the statistical analyses were performed with Statistical Product and Service Solutions19.0 (Spss 19.0).

Synthesis and Characterization of QD-Based DNA
Nanoprobes.The core CdSe QDs were prepared and several layers of ZnSe and ZnS shells were coated on the surface of the CdSe QDs by successive ionic layer adsorption and reaction (SILAR).Thus the oil-soluble CdSe/ZnSe/Zns core/shell QD was obtained.In order to transfer the oilsoluble QDs to water-soluble QDs, the surface of QDs was modified with functional PEG spaced by GSH.As showed in Figure 2(a), the TEM image showed the homogenous distribution of GSH-QDs with uniform size.Figure 2(b) showed that with the increased ratio of coupling reagent of NHS-PEG-Mal, the electrophoresis migration rate of GSH-QDs in the AGE was significantly decreased, which demonstrated the coupling of functional PEG with QDs increasing the particle size of QDs.The migration rate of QDs no longer changed when the ratio of QD to PEG decreased to 1 : 2000, suggesting saturated modification of PEG on the surface of QDs.
Figure 2(c) showed the AEG images of PEG-GSH-QDs coupled with DNA probe.The terminal sulfhydrylmodified DNA probe was used to couple with the PEG-GSH-QDs.The electrophoresis migration obviously increased after being coupled with DNA probe because of the negative charge of DNA, indicating successful linkage of DNA and QDs. Figure 2(d) showed the ultraviolet-visible absorption and emission spectrum of DNA-PEG-GSH-QDs.The emission peak was narrow and symmetric with a wavelength of around 560 nm.

Diagnostic Value Evaluation of Circulating miRNAs for NSCLC.
In order to determine the values of these 9 miRNAs in diagnosis of NSCLC, we analyzed them by receiver operating characteristic (ROC) (Figure 4).The expression of hsa-miR-15b-5p and hsa-miR-17-5p was significantly increased in the serum of NSCLC cases.Under ROC curves, the area under curve (AUC) for them was 0.776 and 0.685, respectively.However, the expression of hsa-miR-20a-5p, hsa-miR-16-5p, and hsa-miR-19a-3p was downregulated in patients with NSCLC compared with the controls.The AUC for them were 0.  22,354, YI is −0.344.We defined the values of hsa-miR-17-5p and hsa-miR-15b-5p to be higher than the best cut-off value as positive, while the values of hsa-miR-20a-5p, hsa-miR-16-5p, and hsa-miR-19a-3p are lower than the best cut-off value as positive.According to each cut-off value, the specificity and sensitivity of 5 miRNAs for diagnosis of NSCLC was calculated and compared with pathological diagnosis, which is the golden standard for NSCLC diagnosis.The sensitivity and specificity of has-miR-20a-5p, hsa-miR-15b-5p, hsa-miR-17-5p, hsa-miR16-5p, and hsa-miRN-19a-3p were 0.776 and 0.836, 0.838 and 0.695, 0.759 and 0.659, 0.797 and 0.679, and 0.723 and 0.679, respectively (Table S1).It demonstrated that these 5 circulating miRNAs have high sensitivity and specificity for early diagnosis of NSCLC, separately.liquid QD microchip hybridization method can simultaneously detect a variety of miRNAs in a single tube.However, this problem can be solved by using the paired miRNA ratio (content of lowest expressed miRNA versus content of higher expressed miRNA) to evaluate the diagnosis value for NSCLC.We analyzed the paired differentially expressed miRNA values of hsa-miR-15b-5p/hsa-miR-16-5p, hsa-miR-15b-5p/hsa-miR-20a-5p, hsa-miR-17-5p/hsa-miR-20a-5p, and hsa-miR-17-5p/hsa-miR-16-5p by ROC (Figure 5).The AUC for them were 0.831, 0.842, 0.784, and 0.808, respectively.Then we chose two miRNA pairs with higher AUC for further study.The best cut-off value for hsa-miR-15b-5p/hsa-miR-20a-5p is 0.2599 and YI is 0.577.The best cut-off value for miR-15b-5p/hsa-miR-16-5p is 0.122 and YI is 0.567.We defined the values of hsa-miR-15b-5p/hsa-miR-20a-5p and hsa-miR-15b-5p/hsa-miR-16-5p as higher than the best cut-off value as positive.According to each cut-off value, the specificity and sensitivity of these two paired miRNAs for diagnosis of NSCLC can also be calculated and compared with the golden standard (biopsy) (Table S2).The results showed that evaluation of specificity and sensitivity of NSCLC can be further improved by using paired miRNA ratio as diagnostic index.The sensitivity and specificity for diagnostic has-miR-15b-5p/hsa-miR-20a-5p and has-miR-15b-5p/hsa-miR-16-5p were 0.792 and 0.868 and 0.832 and 0.743, respectively.

Discussion
Lung cancer is still the leading cause of cancer-related mortality both worldwide and in China, especially NSCLC.The main obstacle is that the diagnosis of NSCLC is usually made at advanced stages.In the recent years, researchers have found that there are a large number of stable existing miR-NAs in serum, named as circulating miRNAs.The circulating miRNA can be combined with Ago2 to form Ago2-miRNA protein complex so as to escape the RNase degradation in serum.Hence, circulating miRNA as a noninvasive detection method satisfies all the requirements for the ideal tumor The quantum dot-based suspension array provides a miRNA detection method with high throughput.This serum miRNA detection method may enhance the sensitivity and specificity of diagnosis for early NSCLC.Semiconductor quantum dot can produce fluorescence by absorbing exciting light [18].Comparing with organic fluorescent dyes, the advantages of QD include continuous excitation spectrum, narrow emission spectrum, high fluorescence efficiency, high resistance to photobleaching, and high photostability [19].These excellent optical properties make QD a great potential in the application of immunofluorescence diagnosis, cell imaging, and living imaging and in other biological medicine areas [20].In our study, the QD-based suspension array was able to detect low abundant miRNAs.Meanwhile, the QD with multiple shells has advantages such as high fluorescence and stable luminescence; thus, the sensitivity is greatly improved.For the high specificity, exonuclease ExoVII was used to digest nonhybrid probe, which could eliminate the influence of nonspecific adsorption on the determination results at maximum.

FCM:
Flow cytometry ROC: Receiver operating characteristic AUC: Area under the curve YI: Youden's index.

Figure 1 :
Figure 1: The diagram of the QD-based suspension arrays.

Figure 2 :
Figure 2: (a) TEM image of QDs.(b) The agarose gel electrophoresis imaging of QDs and PEG cross-linker-modified QDs.(c) The agarose gel electrophoresis imaging of QD-based DNA nanoprobes, prepared by different reaction conditions.(d) The spectroscopic properties of QD-based DNA nanoprobes.The absorbance and emission spectra were denoted by a dash line and a solid line, respectively.

3. 4 .
Diagnostic Value Evaluation of Paired miRNAs for NSCLC.One important problem is that the circulating serum miRNA detection lacks a reliable inner reference, since this

Figure 3 :
Figure 3: Flow cytometry instrument to detect fluorescent microsphere, coding, and quantum dot fluorescent signal.

3. 5 .
Comparison of Paired miRNA Ratios with Serum CEA and CYFRA21-1 in Early Diagnosis of Early NSCLC.CEA and CYFRA21-1 are commonly used for the serum diagnosis of NSCLC.We compared the sensitivity and specificity of paired miRNA ratios with serum CEA and CYFRA21-1 in the 128 cases of stage I NSCLC (Figure6).It demonstrated that serum CEA and CYFRA21-1 have little discriminatory power for diagnosis of early-stage NSCLC with AUC 0.559 and 0.508, respectively.The has-miR-15b-5p/hsa-miR-20a-5p and has-miR-15b-5p/hsa-miR-16-5p ratios were much better than those of CEA and CYFRA21-1 for the diagnosis of stage I NSCLC.

Table 2 :
Clinical features of NSCLC patients.

Table 3 :
Comparison of expression of 9 miRNAs between the NSCLC group and the healthy group.