Combining Protein and miRNA Quantification for Bladder Cancer

Jun 21, 2017 - Department of Urology, Union Hospital, Tongji Medical College, Hubei ... signals that correspond to the quantities of telomerase and mi...
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Combining Protein and miRNA Quantification for Bladder Cancer Analysis Ruixue Duan,†,⊥ Zhenyu Zhang,†,⊥ Fuxin Zheng,†,⊥ Longwang Wang,‡ Ju Guo,§ Tianchi Zhang,† Xiaomeng Dai,‡ Shengwei Zhang,† Dong Yang,∥ Renrui Kuang,‡ Gongxian Wang,§ Chaohong He,∥ Abdul Hakeem,† Chang Shu,† Ping Yin,† Xiaoding Lou,† Fuqing Zeng,† Huageng Liang,*,† and Fan Xia*,† †

Department of Urology, Union Hospital, Tongji Medical College, Hubei Key Laboratory of Bioinorganic Chemistry & Materia Medica, School of Chemistry and Chemical Engineering and Department of Epidemiology and Biostatistics, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology (HUST), Wuhan 430074, China ‡ Department of Urology, The Second Affiliated Hospital of Nanchang University, Nanchang 330006, China § Department of Urology, The First Affiliated Hospital of Nanchang University, Nanchang 330006, China ∥ Department of Urology, The Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou 450008, China S Supporting Information *

ABSTRACT: We combine the telomerase extension reaction and microRNA (miRNA)-induced rolling circle amplification, followed by graphene oxide (GO) and nicking enzyme-assisted signal amplification as a method to analyze telomerase and miRNA-21 in urine samples with the following merits. First, it is a binary assay and can simultaneously output double signals that correspond to the quantities of telomerase and miRNA, respectively. Second, telomerase activity is enhanced by using a DNA molecular beacon probe to inhibit the formation of Gquadruplex. Third, background noise is decreased significantly via introduction of GO. Fourth, performance tests on about 258 urine samples demonstrate that this binary assay can distinguish between urine from bladder cancer patients, those with cystitis, and normal individuals. Finally, this strategy also shows great potential in distinguishing between muscle-invasive bladder cancers and non-muscle-invasive bladder cancers. The proposed strategy will greatly contribute to clinical decision-making and individualized treatments. KEYWORDS: binary assay, telomerase, microRNA, bladder cancer, signal amplification, muscle-invasive bladder cancers, non-muscle-invasive bladder cancers

1. INTRODUCTION Bladder cancer, a burdensome disease with significant morbidity, mortality, and cost, ranks second among the cancers of the urine system. Cystoscopy, medical imaging, and urine cytology are the methods generally used for the diagnosis of bladder cancer.1 Among these technologies, cystoscopy is considered to be the gold standard for bladder cancer diagnosis due to its high specificity. However, this invasive and costly method has low sensitivity for early-stage disease and is not suitable for cancer screening of large sample groups. Fortunately, analysis based on biomarkers, such as telomerase and microRNA (miRNA), provides a useful test for cancer monitoring because the aberrant expressions of biomarkers tend to highly correlate with human cancers. For example, accumulating evidence has shown that about 85% of human tumors contain overexpressed telomerase.2 Likewise, recent studies have found that aberrant expression of miRNA is associated with cancer and appears to be tissue-specific.3,4 More importantly, a number of approaches © 2017 American Chemical Society

have been reported for cancer diagnosis that involve analyzing miRNA and telomerase activity. For telomerase detection, telomeric repeat amplification protocol (TRAP) with high sensitivity is the most frequently used method.5 Although modified TRAP and polymerase chain reaction (PCR)-free assays for telomerase have been developed to overcome the numerous artifacts from PCR that restrict the wide application of TRAP, the limitation of G-quadruplex formation cannot be neglected.6−9 Telomerase does not require any folding of its primer. However, studies have demonstrated that human telomere with tandem TTAGGG repeats can fold into intramolecular G-quadruplex structures that can be further stabilized by cations such as K+ and Na+.10 Moreover, many experiments have proved that agents stabilized by GReceived: April 22, 2017 Accepted: June 21, 2017 Published: June 21, 2017 23420

DOI: 10.1021/acsami.7b05639 ACS Appl. Mater. Interfaces 2017, 9, 23420−23427

Research Article

ACS Applied Materials & Interfaces

was firstly incubated at 37 °C for 15 min. Then, 5 μL of the product of the extended reaction and 10 U Nt.CviPII nicking endonuclease were added to the above solution. The mixture was incubated at 37 °C for another 50 min and the fluorescence intensities were recorded. The telomerase extractions in the control experiments were heated at 95 °C for about 10 min. For detection of the urine samples, the volume of product from the first step was 25 μL. 2.5. MiRNA Extraction from Urine Samples. MiRNA was extracted using a Urine microRNA Purification Kit. Lysis buffer A (1.5 mL) containing β-mercaptoethanol was directly added into 1 mL of urine sample and vortexed for 15 s. Then, 1.5 mL of 100% ethanol was added and vortexed for 10 s. The resulting mixture was applied onto the column provided by the kit and centrifuged for 1 min at 8000 rpm. Then 400 μL of Wash Solution A was applied to the column and centrifuged at 14 000 rpm for 1 min. After that, the above column was centrifuged at 14 000 rpm for 2 min. Elution Solution A (50 μL) provided by the kit was added to the column, which was placed into a fresh tube, followed by centrifuging at 2000 rpm for 2 min and then an additional 2 min at 14 000 rpm. Finally, the resulting purified miRNA samples were stored at −70 °C. 2.6. MiRNA Detection. Experiments were performed in 50 μL of solution consisting of phi29 DNA polymerase reaction buffer, 1 mM dNTP, 1 μM loop probe, 1 μM padlock beacon, 0.2 mg/mL BSA, 40 U phi29 DNA polymerase, 0.4 U/μL RNase inhibitor, and an appropriate amount of target. The mixture was firstly incubated at 37 °C for 180 min. For the nicking enzyme-assisted signal amplification, a solution consisting of CutSmart buffer, 1 μM padlock beacon, and 5 μL of 0.5 mg/mL GO was firstly incubated at 37 °C for 15 min. Then, 25 μL of the product of the extended reaction and 10 U Nt.CviPII nicking endonuclease were added to the above solution. The mixture was incubated at 37 °C for another 50 min and the fluorescence intensities were recorded. 2.7. Simultaneous Detection of Telomerase and miRNA. Firstly, a reaction mixture consisting of a mixture of NEB buffer 2 and phi29 DNA polymerase reaction buffer, 0.4 U/μL RNase inhibitor, 2 mM NTP, 200 μg/mL BSA, 1 μM MB, 1 μM loop probe, 1 μM padlock beacon, 40 U phi29 DNA polymerase, 4 μM primer, an appropriate amount of target telomerase, and an appropriate amount of target miRNA was incubated at 37 °C for 180 min. Then, a solution consisting of CutSmart buffer, 1 μM MB, 1 μM padlock beacon, and 0.5 mg/mL GO was firstly incubated at 37 °C for 15 min. After that, 25 μL of the product of the extended reaction and 10 U Nt.CviPII nicking endonuclease were added to the above solution. The mixture was incubated at 37 °C for another 50 min and the fluorescence intensities were recorded. Fluorescence measurements for telomerase detection were carried out on an F-4500 fluorometer (Hitachi, Japan) with the following settings: λex: 490 nm, λem: 515 nm, bandwidth: 5 nm. Fluorescence measurements for miRNA detection were carried out on an F-4500 fluorometer (Hitachi, Japan) with the following settings: λex: 585 nm, λem: 608 nm, bandwidth: 5 nm.

quadruplexes have the potential to inhibit telomerase activity.11 On the other hand, various sensitive approaches for miRNA detection have also been reported based on RT-PCR,12 modified invader assay,13 ribozyme amplification,14 and isothermal enzymatic amplification.15−22 Among these methods, isothermal enzymatic amplification has attracted much attention due to its simple operation. Nevertheless, the sensitivity is always limited by fluorescent signals from background noise from the intact fluorescent probe. Up to now there have been few reports on the simultaneous detection of telomerase and miRNA for cancer analysis. Because the technical ability to analyze nucleic acids is far more advanced than that for other biomarkers, herein, by skillful sequence design, graphene oxide (GO) and nicking enzyme-assisted amplification is used to overcome the abovementioned drawbacks and fill the gap in telomerase and miRNA detection. We apply this proposed strategy in bladder cancer analysis and divide it into two steps. The first step is to extend the TS primer (telomerase as template) and miRNA (loop probe as template). The extended TS primer and miRNA can hybridize with multiple telomerase molecular beacons (TMBs) and miRNA molecular beacons (M-MBs), respectively, resulting in the detection of emission peaks at about 520 and 610 nm. The second step is a nicking enzyme-assisted recycle, generating products from the first step and enhancing the fluorescence signals. GO is employed to adsorb the redundant T-MBs and M-MBs to decrease signal noise. In this study, we have demonstrated a promising method based on analysis of a combination of biomarkers with high sensitivity and accuracy, providing a platform for the diagnosis of bladder cancer.

2. EXPERIMENTAL SECTION 2.1. Reagents and Materials. Bovine serum albumin (BSA), deoxyribonucleotide triphosphates (dNTPs), RNase inhibitor, miRNA, DNA, and diethylpyrocarbonate-treated water were purchased from TaKaRa Bio Inc. (Dalian, China). Phi29 DNA polymerase, T4 RNA ligase 2, Nt.CviPII nicking endonuclease, Cutsmart buffer, and NEB buffer 2 were purchased from New England Biolabs, Inc. Au nanoparticles (Au) with 10 and 20 nm diameters, and SYBR Green I were purchased from Sigma-Aldrich. GO was obtained from Nanjing XFNANO Materials Tech Co., Ltd. Patient urine samples were obtained from union hospital, Wuhan, China. 2.2. Telomerase Extraction from MCF-7 Cells. In total, 1 million MCF-7 cells were collected into microcentrifuge tubes. Cells were pelleted at 1000g for 5 min and washed with phosphate-buffered saline (PBS) buffer (pH 7.4). The cells were centrifuged again and resuspended in 200 μL of ice 1× CHAPS lysis buffer. The resuspended cells were incubated on ice for 30 min followed by centrifuging at 10 000g for 20 min at 4 °C. After that, the supernatant was collected into a clean tube, then stored at −80 °C for further use. 2.3. Telomerase Extraction from Patient Urine Samples. Patient urine samples were centrifuged for 10 min (850g, 4 °C) followed by washing with PBS. The above samples were centrifuged for 5 min (2300g, 4 °C) and dispersed in 200 μL of ice lysis buffer. The resulting mixtures were incubated for 30 min on ice and then centrifuged for 20 min (10 000g, 4 °C). After that, the supernatant was collected into a clean tube, then stored at −80 °C for further use. 2.4. Telomerase Detection. For the telomerase extended reaction, the reaction mixture consisted of NEB buffer 2, 1 mM dNTP, 0.4 U/μL ribonuclease inhibitor, 4 μM primer, 1 μM MB, and an appropriate amount of target telomerase in a reaction volume of 50 mL. The solution was incubated at 37 °C for 60 min, and then heated at 95 °C for 10 min to deactivate the telomerase. For the nicking enzyme-assisted signal amplification, a solution consisting of CutSmart buffer, 1 μM MB, and 5 μL of 0.5 mg/mL GO

3. RESULTS AND DISCUSSION 3.1. Design of Binary Assay. An outline of the design principle is shown in Figure 1. Specifically, in the presence of telomerase, the TS primer is extended. The reaction products containing tandem TTAGGG can hybridize with multiple TMBs, forming nicking enzyme recognition sites and emitting maximum fluorescence signals at 520 nm. Following the nicking reaction by Nt.CviPII, the T-MBs are cleaved and dissociated from the extended products, permitting hybridization between tandem TTAGGG repeats and intact T-MBs and forming new nicking enzyme recognition sites. With each cycle, the extended product is regenerated, and multiple T-MBs are nicked and released. For miRNA detection, the loop probe acts as the template and target miRNA serves as the primer to initiate RCA reaction 23421

DOI: 10.1021/acsami.7b05639 ACS Appl. Mater. Interfaces 2017, 9, 23420−23427

Research Article

ACS Applied Materials & Interfaces

by phi29 DNA polymerase. The RCA product, single-strand DNA, has many repeated sequences that could hybridize with the M-MBs. When the M-MBs bind to the RCA product, nicking enzyme recognition sites form and a maximum fluorescent signal at 610 nm is detected. In the presence of nicking enzyme, M-MBs can be nicked and then separated from the single-stranded DNA. Then, the nicking enzyme-assisted signal amplification is triggered. If telomerase and miRNA coexist in the system, telomerase extension and RCA production occur at the same time, producing two kinds of single-stranded DNA with multiple repeats. Then, the two kinds of repeats produced from telomerase extension and RCA hybridize with the T-MBs and M-MBs, respectively, producing two emission peaks at 520 and 610 nm. After that, Nt.CviPII nicks and frees the hybridized TMBs and M-MBs simultaneously, which allows the intact TMBs and M-MBs to bind to the repeats again. Therefore, the nicking enzyme unites the oligonucleotide and protein detection and enhances their detection signals. 3.2. Experimental Demonstration and Optimization of the Proposed Principle. To investigate the feasibility of nicking enzyme-assisted amplification, we first chose synthesized DNA (S-DNA) with TTAGGG repeats as the target and used polyacrylamide gel electrophoresis (PAGE) to test the principle (Figure 2a). As we can see from lane 3, the S-DNA can hybridize with the T-MBs. The reason for the ladder in lane 3 is that the number of T-MBs hybridized to each S-DNA is random and different. Comparison between lanes 1 and 4 demonstrates that the hybridized T-MBs are nicked by Nt.CviPII endonuclease and dissociate from S-DNA. The principle is also confirmed by fluorescence. Figure 2b shows that when adding nicking enzyme, the fluorescent signal is

Figure 1. Schematic outline of the binary assay for telomerase and miRNA. In step 1, telomerase extends its primer and miRNA triggers RCA reaction, respectively, producing two kinds of single-strand DNA with repeats. Both kinds of linear products can hybridize with many copies of T-MBs and M-MBs, respectively. In step 2, the resulting hybridized molecular beacons can be recognized and nicked by Nt.CviPII, and then the linear products regenerate, resulting in GO and nicking enzyme-assisted recycling.

Figure 2. (a) Analysis results from PAGE. Lane 1: the sample containing only beacon; lane 2: the sample containing only S-DNA; lane 3: the sample containing beacon and S-DNA; lane 4: the sample containing beacon, S-DNA, and nicking enzyme. (b) Analysis results from fluorescence. When adding nicking enzyme (green), the fluorescent signal is enhanced by about three times. The reaction time is 50 min. (c) Effects of GO, 10 nm Au, and 20 nm Au on the background signals. (d) Effects of GO and 10 nm Au on the proposed method. The final concentrations of GO, 10 nm Au, and 20 nm Au were 0.05 mg/mL, 1 nM, and 1 nM, respectively. Error bars were calculated from three independent experiments. Telomerase reaction time was 60 min; 5 μL of telomerase product participated in the nicking enzyme-assisted amplification and the amplification time was 50 min. 23422

DOI: 10.1021/acsami.7b05639 ACS Appl. Mater. Interfaces 2017, 9, 23420−23427

Research Article

ACS Applied Materials & Interfaces

Figure 3. Schematic illustration of the effect of T-MBs on the telomerase extension reaction (a, b). (a) With the TS primer extension triggered by telomerase, the tandem TTAGGG repeats will fold into an intramolecular G-quadruplex structure, which inhibits telomerase activity. (b) If T-MBs are added at the extension step, hybridization between the T-MBs and telomerase products will hinder the formation of G-quadruplex and maintain telomerase activity. Analysis results from fluorescence study (c, d). (c) The detection signals for telomerase can be enhanced by introducing T-MBs at the telomerase extension step. Telomerase concentration is equivalent to 1.6 cells/μL in lysis buffer. The reaction time is 60 min, 5 μL of telomerase product participates in the nicking enzyme-assisted amplification, and the amplification time is 50 min. (d) The detection signals for miRNA detection will not be affected by adding M-MBs at the RCA step. The concentration of miRNA is 10 pM. RCA reaction time is 120 min, 25 μL of RCA product participates in the nicking enzyme-assisted amplification, and the amplification time is 50 min.

G-quadruplex folded by tandem TTAGGG repeats can appear in human cells and has been proved to be an ineffective substrate for telomerase, inhibiting telomerase activity.28 To solve this problem, we added T-MBs into the process of telomerase reaction (Figure 3a,b). In theory, with extension of the telomerase primer, the loops of T-MBs will bind to the TTAGGG repeats, hindering the fold of telomerase primer and maintaining telomerase activity. As expected, Figure 3c shows that when we add T-MBs into the telomerase extension step, the detection signal is enhanced by about 2 times. This gap will become wider if more telomerase products participate in the following nicking amplification (Figure S2). This phenomenon can only be observed in telomerase detection because the RCA product cannot fold into G-quadruplex. As we can see from Figure 3d, the addition of M-MBs into the RCA reaction does not increase the fluorescence signals. 3.3. Investigation of the Sensitivity and Specificity of the Proposed Method. The telomerase extracted from MCF7 cell lines and synthesized miR-21 were selected as targets to demonstrate the sensitivity and selectivity of the proposed

almost 3 times higher than that of the control, which also demonstrates that the amplification reaction is effective. In addition, the feasibility of this strategy is also demonstrated by the result from using different concentrations of tissue telomerase (Figure S1). An interesting property of Au nanoparticles and GO is that they can strongly bind single-strand DNA and protect the adsorbed DNA from nuclease cleavage. Another useful character of Au nanoparticles and GO is that the fluorescence of fluorophores bound with adsorbed DNA can be quenched.23−27 Therefore, in step 2, to suppress the level of background signals, we incubated Au nanoparticles or GO with the T-MBs firstly before triggering nicking enzyme reaction. Compared with the 10 nm Au nanoparticles or 20 nm Au nanoparticles, GO can greatly reduce background noise (Figure 2c). Additionally, the detection signals were not affected by GO or the Au nanoparticles (Figure 2d). Optimally, the assay used GO to ensure low levels of background signals while maintaining high levels of detection signals. 23423

DOI: 10.1021/acsami.7b05639 ACS Appl. Mater. Interfaces 2017, 9, 23420−23427

Research Article

ACS Applied Materials & Interfaces

Figure 4. Investigation of the sensitivity and specificity of the analysis method toward telomerase (a, b), miRNA (c, d), and telomerase and miRNA (e, f). Telomerase reaction time was 60 min, 25 μL of telomerase product participated in the nicking enzyme-assisted amplification, and the amplification time was 50 min. RCA reaction time was 120 min, 25 μL of RCA product participated in the nicking enzyme-assisted amplification, and the amplification time was 50 min. In the binary assay, the reaction time for step 1 was 180 min, and for step 2 it was 50 min.

method (Figure 4). For telomerase analysis, as we can see from Figure 4a, the fluorescence intensity is enhanced as the number of MCF-7 cells increased from 0 to 375 cells/μL. A detection limit of 0.4 cells/μL is obtained. The mean value of the controls added to 3 times the standard deviation was set as the threshold line. To interrogate the specificity of our strategy, BSA and thrombin were used as negative controls. As expected, the signals from BSA and thrombin are similar to those from the inactive samples, whereas the signal from telomerase is almost 14 times higher than that from the control (Figure 4b). To investigate the sensitivity for miRNA detection, the fluorescence intensity was measured by adding different concentrations of miR-21 (Figure 4c). A detection limit of 1 pM was obtained according to the 3σ method. To evaluate the specificity for miRNA, we challenged the system with miR155 and 100 fM miR-21. Figure 4d shows that the signal from miR-21 can be clearly discriminated from the others. The binary assay also shows great sensitivity and selectivity. Figure 4e shows that when telomerase extracted from 250 cells/ μL and 10 nM miR-21 coexist in the system, fluorescence emission peaks at 520 and 610 nm can be detected respectively. With decreasing biomarker concentration, the relative detection signals decrease. For example, the fluorescence intensities for

both telomerase from 25 cells/μL and 100 pM miR-21 reduced significantly. To demonstrate the selectivity of the binary assay, the combinations thrombin−SM miR-21, inactive telomerase− miR-155, and BSA−TM miR-21 were used as negative controls. As shown in Figure 4f, the signals from the negative controls are similar to those of the blank controls, which are distinctly lower than those of the samples, telomerase−miR-21. These results demonstrate that the selectivity and specificity for simultaneously detecting both protein and oligonucleotide remain attractive in a complex environment. 3.4. Determination of Telomerase and miRNA in Urine Samples from Normal Individuals, Those with Cystitis, and Bladder Cancer Patients. Cystitis and bladder cancer are the general diseases observed in clinical urine detection. Furthermore, recent evidence has demonstrated that telomerase activity in urine is a potential biomarker for the early detection of bladder cancer, and miR-21 is one of the most commonly overexpressed miRNAs in bladder cancers.29,30 Herein, we compared three methods for bladder cancer diagnosis based on analysis of telomerase, miRNA, and telomerase and miRNA, respectively (Figure 5). We firstly analyzed the telomerase levels using variance analysis in 20 normal, 36 cystitis, and 36 bladder cancer cases. Table 1 in 23424

DOI: 10.1021/acsami.7b05639 ACS Appl. Mater. Interfaces 2017, 9, 23420−23427

Research Article

ACS Applied Materials & Interfaces

Figure 5. Variance analysis of three strategies for diagnosis of patients with bladder cancer, those with cystitis, and normal individuals. Analysis based on telomerase (Table 1), miRNA (Table 2), and telomerase and miRNA (Table 3). The institutional Ethical Committee approved the experimental protocol, and the protocol was explained to each participant and informed consent was obtained.

and can require cystectomy, radiotherapy, or chemotherapy. A technique with the ability to distinguish between MIBC and NMIBC would contribute to clinical decision-making and individualized treatment.31,32 In this context, tests based on reliable urine biomarkers have been developed. Although these tests have a higher sensitivity, lower cost, and are more comfortable than cytology, their specificity is not satisfactory. Here, our binary assay based on dual biomarker detection was also investigated in distinguishing between MIBC and NMIBC (Figure 6a−c). Figure 6d shows that for the detection system based on telomerase, the fluorescence intensity from MIBC is obviously higher than that from NMIBC (P < 0.0001). For the detection system based on miRNA, there is no significant difference between MIBC and NMIBC (P = 0.788). However, in the combination assay, the fluorescence intensities of both telomerase and miRNA from MIBC are higher than those from NMIBC (P = 0.001 and 0.003, respectively). It is obvious that the detection signal for miRNA in the combination assay is enhanced compared to that in the assay based on only miRNA. The reason for this may be that the telomerase extracts contain some amount of miRNA. The potential of the proposed method to differentiate between MIBC from NMIBC will contribute to clinical decision-making and individualized treatment.

Figure 5 shows that there are significant differences between the normal and cancer group, and between the normal and cystitis group. However, there is no difference between the cystitis and cancer group. The results demonstrate that the detection method based on telomerase has the ability to distinguish between normal individuals and cancer patients, or between normal individuals and those with cystitis. Nevertheless, it cannot differentiate between those with cystitis and the cancer patients. We also analyzed miRNA expression using variance analysis in 19 normal, 21 cystitis, and 53 bladder cancer cases. As with telomerase detection, Table 2 in Figure 5 indicates that the detection method based on miRNA-21 can only distinguish between normal individuals and cancer patients. There is no significant difference observed between the normal and cystitis group, and the cystitis and cancer group. The quantities of telomerase and miRNA in the binary assay were measured using variance analysis in 19 normal, 20 cystitis, and 34 bladder cancer cases. Table 3 in Figure 5 shows that there are significant differences among the normal, cystitis, and cancer groups. These results prove that only the binary assay can be used to diagnosis normal individuals, those with cystitis, and cancer patients, respectively. 3.5. Investigation of the Ability of the Three Detection Modes To Distinguish between MuscleInvasive Bladder Cancer (MIBC) and Non-MuscleInvasive Bladder Cancer (NMIBC). Urothelial carcinoma is the most common type of bladder cancer (representing >90% of cases) and can be divided into two subgroups: non-muscleinvasive and muscle-invasive. NMIBC infrequently progresses to invasion with a five-year survival rate of >90% and can require transurethral resection of the bladder tumor. MIBC is a life-threatening disease with a five-year survival rate of