Aptamer for specifically targeting extracellular vesicles derived from prostatic cancer and application of aptamer as biological detection marker for diagnosing prostatic cancer

Abstract

The invention relates to a targeting nucleic acid molecule, in particular to an aptamer specifically targeting a prostatic cancer source extracellular vesicle and application of the aptamer as a biological detection marker for PCa diagnosis. The invention comprises a DNA molecule with a base sequence as shown in SEQ ID NO: 1, and application of the DNA molecule as a biological detection marker for prostatic cancer diagnosis. The kit can be directly and selectively combined with prostatic cancer source EVs, can effectively distinguish EVs from body fluid samples of prostatic cancer patients and BPH patients, and is convenient to operate.

Description

Technical Field

[0001] This invention relates to a targeted nucleic acid molecule, specifically to an aptamer that specifically targets extracellular vesicles derived from prostate cancer and its application as a biomarker for the diagnosis of prostate cancer. Background Technology

[0002] Prostate cancer (PCa) is a common malignant tumor in men, posing a huge health burden on the world (see reference Bergengren O, Pekala KR, Matsoukas K, Fainberg J, Mungovan SF, Bratt O, Bray F, Brawley O, Luckenbaugh AN, Mucci L et al: 2022 Update on Prostate Cancer Epidemiology and Risk Factors-A Systematic Review. Eur Urol 2023, 84(2):191-206.).

[0003] Currently, prostate-specific antigen (PSA) has become the most commonly used serum biomarker for screening high-risk groups for prostate cancer, favored for its cost-effectiveness and availability (see reference Loeb S, Bjurlin MA, Nicholson J, Tammela TL, Penson DF, Carter HB, Carroll P, Etzioni R: Overdiagnosis and overtreatment of prostate cancer. EurUrol 2014, 65(6):1046-1055.). However, patients with acute / chronic prostatitis or benign prostatic hyperplasia (BPH) may also have elevated serum PSA levels. Therefore, the clinical application of PSA is limited by its inability to distinguish between BPH and malignant tumors, as well as its inability to distinguish between low-risk and high-risk prostate cancer (see reference Louie KS, Seigneurin A, Cathcart P, Sasieni P: Do prostate cancer risk models improve the predictive accuracy of PSA screening Ameta-analysis. Ann Oncol 2015, 26(5):848-864.). Thus, the development of new prostate cancer biomarkers has become an urgent clinical need.

[0004] Liquid biopsy is a cutting-edge technology in the field of tumor detection, and the diagnostic value of extracellular vesicles (EVs) has been widely recognized (see reference Batool SM, Yekula A, Khanna P, Hsia T, Gamblin AS, Ekanayake E, Escobedo AK, You DG, Castro CM, Im H et al: The Liquid Biopsy Consortium: Challenges and opportunities for early cancer detection and monitoring. Cell Rep Med 2023,4(10):101198.). However, the presence of numerous extracellular vesicles derived from normal cells in the body fluids significantly dilutes tumor-derived signals, making the specific isolation and detection of tumor-derived extracellular vesicles a critical challenge (see reference Weng J, Xiang X, Ding L, Wong AL, Zeng Q, Sethi G, Wang L, Lee SC, Goh BC: Extracellular vesicles, the cornerstone of next-generation cancer diagnosis Semin Cancer Biol 2021, 74:105-120.). Although numerous studies have explored the application of extracellular vesicles in prostate cancer diagnosis (see reference Wang X, Zhang L, Cheng L, Wang Y, Li M, Yu J, Ma Z, Ho PC, Sethi G, Chen X et al: Extracellular vesicle-derived biomarkers in prostate cancer care: Opportunities and challenges. Cancer Lett 2024, 601:217-184.), most methods require complex pretreatment steps for extracellular vesicles and their contents, which severely limits their practical application in clinical diagnosis. Aptamers, as functional nucleic acid molecules, have become ideal candidates for constructing various biosensors due to their high stability, strong affinity, ease of modification, and low cost (see reference Zhu C, Li L, Wang Z, Irfan M, Qu F: Recent advances of aptasensors for exosomes detection. Biosensors and Bioelectronics 2020, 160.).This provides a potential solution for the specific isolation and detection of tumor-derived extracellular vesicles. Summary of the Invention

[0005] The purpose of this invention is to address the problems of low accuracy or complex operation of existing methods for diagnosing prostate cancer, and to provide an aptamer that specifically targets extracellular vesicles derived from prostate cancer and its application as a biomarker for the diagnosis of prostate cancer.

[0006] To achieve the above objectives, the technical solution provided by this invention is:

[0007] An aptamer that specifically targets extracellular vesicles derived from prostate cancer cells, characterized by:

[0008] This includes DNA molecules with base sequences as shown in SEQ ID NO: 1.

[0009] Furthermore, the 3' or 5' end of the DNA molecule is modified with a detectable labeling group.

[0010] Furthermore, the detectable labeling group is a fluorescent group such as FAM, VIC, Cy5, HEX, or ROX.

[0011] Furthermore, the detectable labeling group is HRP.

[0012] Furthermore, the detectable labeling group is a fluorescent group FAM, and it is modified at the 5' end of the DNA molecule.

[0013] Furthermore, this invention also provides the application of the above-mentioned aptamers specifically targeting extracellular vesicles derived from prostate cancer as biomarkers for prostate cancer diagnosis.

[0014] Compared with the prior art, the beneficial effects of the present invention are:

[0015] 1. The aptamer specifically targeting extracellular vesicles derived from prostate cancer provided by this invention can directly and selectively bind to EVs derived from prostate cancer, and can effectively distinguish EVs from bodily fluid samples from prostate cancer patients and BPH patients, and is easy to operate.

[0016] 2. The aptamer specifically targeting extracellular vesicles derived from prostate cancer provided by this invention can be used for disease detection using non-invasive urine samples, and has a wide range of applications. Attached Figure Description

[0017] Figure 1 A schematic diagram of the DNA molecular structure (represented in base sequence) of an embodiment of the present invention that specifically targets extracellular vesicles derived from prostate cancer.

[0018] Figure 2 This is a comparison of the affinity detection results of the aptamer examples of the present invention that specifically targets extracellular vesicles derived from prostate cancer and other aptamers with positive screening, where the vertical axis represents fluorescence intensity and the horizontal axis represents time.

[0019] Figure 3 This is a graph showing the affinity detection results of the aptamer embodiment of the present invention, which specifically targets extracellular vesicles derived from prostate cancer, with positive screening at different concentrations. The vertical axis represents fluorescence intensity and the horizontal axis represents time.

[0020] Figure 4 The graph shows the percentage of positive EVs after incubation with positive screening at different concentrations of the aptamer examples that specifically target extracellular vesicles derived from prostate cancer according to the present invention.

[0021] Figure 5 This diagram shows the results of incubation of aptamers specifically targeting extracellular vesicles derived from prostate cancer and the percentage of positive vesicles after incubation with EVs samples from different cell sources. In the diagram, a is a representative scatter plot of the prostate cancer cell line DU145, b is a representative scatter plot of the prostate cancer cell line LnCap, c is a representative scatter plot of the immortalized normal prostate cell line RWPE-1, and d is a comparison of the percentage of positive vesicles from EVs derived from the prostate cancer cell lines DU145, LnCap, and RWPE-1.

[0022] Figure 6 This is a graph showing the percentage of positive EVs after incubation with urinary EVs from PCa and BPH patients, respectively, in an embodiment of the aptamer that specifically targets extracellular vesicles derived from prostate cancer according to the present invention.

[0023] Explanation of reference numerals in the attached figures:

[0024] 1-Adenine deoxyribonucleotide, 2-cytosine deoxyribonucleotide, 3-guanine deoxyribonucleotide, 4-thymine deoxyribonucleotide. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0026] An aptamer that specifically targets extracellular vesicles derived from prostate cancer cells, see [link to aptamer]. Figure 1The DNA molecule includes a base sequence as shown in SEQ ID NO: 1, and the 5' end of the DNA molecule is modified with a detectable labeling group. In this embodiment, the detectable labeling group used is the fluorescent group FAM. The function of the fluorescent group is to facilitate the quantification of the percentage and / or number of positive EVs (prostate cancer-derived EVs bound to aptamers). In practical applications, the fluorescent group can also be VIC, Cy5, HEX, or ROX, or even without modification, enzyme modification can be used. By adding the corresponding substrate and measuring the amount of substrate consumed, the percentage and / or number of positive EVs can be quantified. Theoretically, the detectable labeling group can also be modified at the 3' end.

[0027] If enzyme modification is used, the commonly used group is HRP (horseradish peroxidase). HRP is covalently linked to the 5' end of the aptamer, and then the binding ratio of the aptamer is determined by adding an HRP substrate (such as TMB, 3,3',5,5'-tetramethylbenzidine) and the HRP-catalyzed reaction producing color or light.

[0028] The bond-line formula of the fluorescent group FAM used in this embodiment is as follows:

[0029]

[0030] 1) The affinity of the aptamer (seq25) for prostate cancer-derived EVs was detected using the surface plasmon resonance (SPR) method. This method includes the following steps:

[0031] Step a: Dilute the PCa cell-derived EVs sample to a concentration of 50 μg / mL;

[0032] Step b: Dilute with 10 mM sodium carboxylate buffer (pH = 4.0), inject into CM5 chip (Guy Healthcare, USA) at a flow rate of 5 μL / min and fix for 600 s to fix it on CM5 chip;

[0033] Step c: Block the CM5 chip containing PCa cell-derived EVs with ethanolamine solution at a flow rate of 5 μL / min for 10 min.

[0034] Step d: Dilute aptamers seq1-seq30 with PBST buffer to 50-500 nM (preferably 100 nM), then incubate at 90-99℃ (preferably 95℃) for 5-15 min (preferably 10 min), followed by rapid cooling in an ice-water bath for 5-20 min (preferably 5 min). The purpose is to transform the spatial conformation of the aptamers from the initial inactive conformation to the specific functional conformation, thus obtaining the activated aptamers.

[0035] Step e: Analyze aptamer seq1-seq30 using an SPR instrument (Guy Healthcare, USA). Each injection contact time is 120 s. "nM" represents nmol / L. "PBST buffer" is a PBS buffer with Tween 20. The PBS buffer is a phosphate buffer containing sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4), and NaCl. The purpose of adding Tween 20 is to improve washing ability.

[0036] Step e: Dilute the target aptamer seq25 to different desired concentrations (including 1.95 nM, 15.63 nM, 31.25 nM, 62.50 nM, 125.00 nM and 1000.00 nM) in PBST buffer and analyze them using an SPR instrument (Guy Healthcare, Inc., USA).

[0037] 2) The affinity of the nanofluidic aptamer (seq25) for prostate cancer-derived EVs was then determined. This method includes the following steps:

[0038] Step a: Incubate the initial aptamer seq25 at 90-99℃ (preferably 95℃) for 5-15 min (preferably 10 min), and then rapidly cool it in an ice-water bath for 5-20 min (preferably 5 min). The purpose is to change the spatial conformation of aptamer seq25 from the initial inactive conformation to the specific functional conformation, so as to obtain the activated aptamer seq25.

[0039] Step b: Adjust the concentration of PCa cell-derived EVs samples to 1×10⁻⁶. 7 ~1×10 9 particles / mL (preferably 1×10⁻⁶) 8 (particles / mL);

[0040] Step c: Dilute the activated aptamer seq25 to different desired concentrations (concentration values ​​include 15.63 nM, 31.25 nM, 62.50 nM, 125.00 nM, 250.00 nM, and 500.00 nM), and then incubate it with PCa cell-derived EVs in the dark at room temperature for 20–60 min (preferably 30 min).

[0041] Step d: Use a 0.5 mL (or larger, preferably 0.5 mL) 100 kDa (or 30 kDa) ultrafiltration tube (model: Millipore, USA) to remove free activated aptamers seq25 from the stained EVs sample by centrifugation.

[0042] Step e: Quantify the percentage of positive EVs using microflow cytometry. Complete.

[0043] See results Figure 4 ,Depend on Figure 4 It can be seen that there is a good dose-dependent interaction between aptamer seq25 and PCa cell-derived EVs samples, as shown by the R curve in the figure. 2 The value is 0.976, indicating a high degree of fit.

[0044] Depend on Figure 2 It can be seen that ① represents the fluorescence intensity detection result of aptamer seq25 binding to PCa cell-derived EVs (positive screen), and ② represents the fluorescence intensity detection result of other aptamers binding to the positive screen. It is evident that aptamer seq25 has a significantly higher affinity for PCa cell-derived EVs than other aptamers. Figure 3 It can be seen that its K D The value (equilibrium dissociation constant) is 24.02 nM.

[0045] In addition, this embodiment also provides the application of aptamers that specifically target extracellular vesicles derived from prostate cancer as biomarkers for the diagnosis of prostate cancer, specifically in cell sample detection and urine sample detection.

[0046] When the aptamer seq25 is used for cell sample detection, the procedure is as follows:

[0047] Step A1: Incubate the initial aptamer seq25 at 50-500 nM (preferably 100 nM) at 90-99℃ (preferably 95℃) for 5-15 min (preferably 10 min), and then rapidly cool it in an ice-water bath for 5-20 min (preferably 5 min). The purpose is to change the spatial conformation of aptamer seq25 from the initial inactive conformation to the specific functional conformation, so as to obtain the activated aptamer seq25.

[0048] Step A2: Adjust the concentration of EVs from two prostate cancer cell lines, DU145 and LnCap, and EVs from the normal immortalized prostate cell line RWPE-1, to 1×10⁻⁶. 7 ~1×10 9 particles / mL (preferably 1×10⁻⁶) 8 (particles / mL); the prostate cancer cell line here can also be selected from PC3, VCap, C4-2B and other cell lines, and the normal immortalized prostate cell line can also be selected from RWPE-2, WPMY-1 and other cell lines;

[0049] Step A3: Incubate the sample obtained in step A2 with the activated aptamer seq25 in the dark at room temperature for 20-60 min (preferably 30 min) to allow the seq25 aptamer to selectively bind to prostate cancer cell lines DU145 and LnCap-derived EVs.

[0050] Step A4: Use a 0.5 mL (or larger, preferably 0.5 mL) 100 kDa (or 30 kDa) ultrafiltration tube (model: Millipore, USA) to remove free activated aptamers seq25 from the stained EVs sample by centrifugation.

[0051] Step A5: Quantify the percentage of positive vesicles using nanoflow cytometry (a nanoflow cytometer can be used). This is the first result; see [link to previous step]. Figure 5 .

[0052] Depend on Figure 5 It can be seen that the proportion of positive vesicles in EVs derived from prostate cancer cell lines DU145 and LnCap is significantly higher than that in EVs derived from RWPE-1, indicating that the aptamer seq25 can selectively bind to prostate cancer cell lines, while having a lower binding affinity to normal EVs.

[0053] When the aptamer seq25 is used for testing body fluid samples, the procedure is as follows:

[0054] Step B1: Incubate 50-500 nM (preferably 100 nM) of seq25 aptamer at 90-99℃ (preferably 95℃) for 5-15 min (preferably 10 min), and then rapidly cool it in an ice-water bath for 5-20 min (preferably 5 min). The purpose is to restore the specific functional conformation of aptamer seq25 and obtain activated aptamer seq25.

[0055] Step B2: Select body fluid samples (blood or urine, urine in this example) from 10 PCa patients and 10 BPH patients respectively, and adjust the concentration of the separated EVs samples to 1×10⁻⁶. 7 ~1×10 9 particles / mL (preferably 1×10⁻⁶) 8 (particles / mL);

[0056] Step B3: Incubate the sample obtained in step B2 with the activated aptamer seq25 in the dark at room temperature for 20-60 min (preferably 30 min) to allow the seq25 aptamer to selectively bind to the EVs sample.

[0057] Step B4: Use a 0.5 mL (or larger, preferably 0.5 mL) 100 kDa (or 30 kDa) ultrafiltration tube (model: Millipore, USA) to remove the free activated aptamer seq25 from the stained EVs sample by centrifugation.

[0058] Step B5: Quantify the percentage of positive vesicles using nanoflow cytometry (a nanoflow cytometer can be used). This is the second result; see [link to relevant documentation]. Figure 6 .

[0059] Figure 6 This is a comparison of the percentage of positive EVs in urine samples from patients with prostate cancer (PCa) and benign prostatic hyperplasia (BPH), representing an aptamer embodiment of the present invention specifically targeting prostate cancer-derived extracellular vesicles. Figure 6 It was found that the percentage of positive vesicles (EVs) from PCa patients was significantly higher than that from BPH patients; the asterisk (*) indicates that the difference was statistically significant (P < 0.05). While BPH and prostate cancer share similar symptoms, both potentially causing lower urinary tract symptoms (LUTS), their pathogenesis differs significantly. BPH is generally caused by a combination of aging and dihydrotestosterone (DHT), leading to an enlarged prostate. Treatment typically involves alpha-blockers (such as doxazosin, terazosin, alfuzosin, tamsulosin, and celodoxine) and / or 5α-reductase inhibitors (such as finasteride and dutasteride), and sometimes surgery is required. Prostate cancer, on the other hand, has more complex causes, and treatment typically involves androgen receptor inhibitors (such as bicalutamide), gonadotropin-releasing hormone (GnRH) agonists (such as leuprorelin), chemotherapy, or surgery. The treatment methods and prognoses for BPH and PCa differ significantly. This embodiment uses a specifically selected aptamer to identify extracellular vesicles derived from prostate cancer cells. Using the differential SELEX method (EV-SELEX), the high-affinity aptamer seq25 was successfully screened, which can rapidly distinguish between these two different diseases and has important guiding significance for clinical diagnosis and subsequent treatment selection.

[0060] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. An aptamer that specifically targets extracellular vesicles derived from prostate cancer cells, characterized in that: This includes DNA molecules with base sequences as shown in SEQ ID NO:

1.

2. The aptamer that specifically targets extracellular vesicles derived from prostate cancer according to claim 1, characterized in that: The DNA molecule is modified with a detectable labeling group at its 3' or 5' end.

3. The aptamer specifically targeting extracellular vesicles derived from prostate cancer according to claim 2, characterized in that: The detectable labeling group is a fluorescent group such as FAM, VIC, Cy5, HEX, or ROX.

4. The aptamer specifically targeting extracellular vesicles derived from prostate cancer according to claim 2, characterized in that: The detectable labeling group is HRP.

5. The aptamer specifically targeting extracellular vesicles derived from prostate cancer according to claim 3, characterized in that: The detectable labeling group is the fluorescent group FAM, and it is modified at the 5' end of the DNA molecule.

6. The application of the aptamer of any one of claims 1 to 5 that specifically targets extracellular vesicles derived from prostate cancer as a biomarker for the diagnosis of prostate cancer.

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