Use of glycosylated y5 rna as a diagnostic and prognostic marker for pancreatic ductal adenocarcinoma

Abstract

The application discloses application of glycosylation Y5 RNA as a pancreatic ductal adenocarcinoma diagnosis and prognosis marker. The application firstly discloses a key role of Glyco Y5 RNA in PDAC immune escape and clinical application value of the Glyco Y5 RNA as a PDAC prognosis marker, and experimentally proves that exosome Glyco Y5 produced under hypoxia induction can mold an immunosuppressive microenvironment by regulating immune cell functions, thereby providing a new mechanism explanation for malignant progression of PDAC.

Description

Technical Field

[0001] This invention relates to the field of biomarker technology, and in particular to the application of glycosylated Y5 RNA as a diagnostic and prognostic biomarker for pancreatic ductal adenocarcinoma. Background Technology

[0002] Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignant tumor of the digestive tract. Due to its atypical early symptoms and rapid progression, approximately 80% of patients are diagnosed at a locally advanced stage or with distant metastases, resulting in a five-year survival rate as low as 3% and an extremely poor prognosis. Furthermore, the current lack of effective early diagnostic methods and precise prognostic indicators for PDAC severely limits the effectiveness of clinical interventions. Therefore, there is an urgent need to identify new molecular biomarkers for PDAC to aid in early detection and improve patient survival outcomes.

[0003] The tumor microenvironment of PDAC is predominantly characterized by significant immunosuppression, and its malignant progression, drug resistance, and immune escape are closely related. Studies have found that the PDAC microenvironment is enriched with various immunosuppressive cells, such as tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs). These cells suppress anti-tumor immune responses by secreting immunosuppressive factors, thereby accelerating tumor progression. Furthermore, PDAC exhibits a pronounced hypoxic microenvironment. Hypoxia not only promotes metabolic reprogramming of tumor cells and enhances their invasiveness and drug resistance, but also induces tumor cells to secrete exosomes, further influencing microenvironment shaping and closely contributing to the poor prognosis of PDAC.

[0004] In recent years, the importance of exosomes in the immune regulation of PDAC has become increasingly prominent. As a key carrier of intercellular communication, exosomes can carry various bioactive molecules (such as proteins, non-coding RNAs, and modified RNAs) and regulate the functional state of recipient cells. Glycosylated RNA (GlycoRNA) is a newly discovered RNA structure that plays an important role in the regulation of the tumor immune microenvironment. However, no systematic studies have yet revealed the potential value of GlycoRNA in the prognosis of PDAC; therefore, whether it can be delivered via the exosome pathway and affect the immune function of recipient cells remains unknown.

[0005] Given the extremely poor prognosis of PDAC patients, if the feasibility of exosomal GlycoRNA as a poor prognostic biomarker for PDAC can be confirmed, it will provide new insights for the prognostic assessment and individualized treatment of PDAC. Summary of the Invention

[0006] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide an application of glycosylated Y5 RNA as a diagnostic marker for pancreatic ductal adenocarcinoma.

[0007] In this invention, the inventors discovered that there were significant differences in the content of glycosylated Y5 RNA (Glyco RNA) detected in RNA from hypoxic and normoxic control pancreatic cancer cell lines. Furthermore, the exosomal Glyco Y5 RNA fragments in hypoxic-treated exosomes were significantly increased compared to normoxic treatment (no glycosylated Y5 RNA was detected in normoxic-treated exosomes), suggesting that exosomal Glyco Y5 RNA may be a potential biomarker for poor prognosis in pancreatic cancer.

[0008] In this invention, the inventors isolated exosomes from cells after hypoxia and normoxic treatments and labeled them with rPAL. They found that no glycosylated Y5 RNA was detected in exosomes isolated from normoxic cells, while exosomes isolated from hypoxic cells showed a significant increase in glycosylated Y5 RNA (GlycoY5).

[0009] In this invention, the inventors, using a mouse model of pancreatic carcinoma in situ, discovered that Glyco Y5 RNA treatment can enhance the immunosuppressive state, demonstrating its key role in PDAC immune escape and poor prognosis. Furthermore, the inventors further confirmed through clinical samples that the expression levels of Glyco Y5 RNA in tissues and serum exosomes are positively correlated. Moreover, in tumor tissues with high Glyco Y5 RNA expression, CD8... + Decreased T cell infiltration, FoxP3+Tregs, and M2 macrophages (CD68) + The proportion of Glyco Y5 RNA increased significantly, which further supports the above-mentioned mechanism of action of Glyco Y5 RNA in immune escape.

[0010] In this invention, the inventors, through clinical data analysis, demonstrated that high levels of Glyco Y5 RNA were significantly associated with shorter overall survival (OS) and relapse-free survival (RFS) in patients, while non-glycosylated Y5 RNA showed no significant association with prognosis, suggesting that exosomal Glyco Y5 RNA can serve as a key biomarker for immunosuppression and poor prognosis. To further evaluate the prognostic differences between tissue-derived and exosomal Glyco Y5 RNA, we compared their predictive efficacy for OS / RFS in the same patient population using ROC curves (receiver operating characteristic curves) and AUC values ​​(area under the curve). The results showed that the predictive ability of exosomal Glyco Y5 RNA was not inferior to that of tissue-derived Glyco Y5 RNA, and its non-invasive detection characteristics were more suitable for dynamic clinical monitoring and prognostic assessment.

[0011] In a first aspect, the present invention provides a diagnostic and / or prognostic biomarker for assessing the risk and prognosis of pancreatic ductal adenocarcinoma.

[0012] In some embodiments of the present invention, the diagnostic and / or prognostic biomarkers include at least one of glycosylated Y5 RNA and exosomes containing glycosylated Y5 RNA.

[0013] In this invention, the term "prognosis" refers to the various outcomes of a disease. "Prognostic assessment" refers to the assessment of the probability of the occurrence of various outcomes of a disease. Specifically, "prognostic assessment" broadly refers to the actual clinical progress and outcome of a disease after its onset, and is the assessment of the various consequences and outcomes of the disease course, including improvement, cure, relapse, deterioration, disability, complications, and death, as well as the study of the influencing factors.

[0014] In some embodiments of the present invention, the diagnostic and / or prognostic markers are exosomes containing glycosylated Y5 RNA.

[0015] In some embodiments of the present invention, the sequence of the Y5 RNA is shown in SEQ ID NO: 1.

[0016] In some embodiments of the present invention, the glycosylated Y5 RNA and the exosomes containing glycosylated Y5 RNA are derived from a subject.

[0017] In some embodiments of the present invention, the subject includes a human.

[0018] In this invention, the location where glycosylation occurs is not limited.

[0019] In some embodiments of the present invention, the glycosylation is a naturally occurring glycosylation, rather than an artificially modified glycosylation.

[0020] A second aspect of the invention provides the use of products for detecting glycosylated Y5 RNA in the preparation of products for the diagnosis and / or prognostic risk assessment of pancreatic ductal adenocarcinoma.

[0021] A third aspect of the invention provides the use of products for detecting exosomes containing glycosylated Y5 RNA in the preparation of products for the diagnosis and / or prognostic risk assessment of pancreatic ductal adenocarcinoma.

[0022] In some embodiments of the present invention, the product includes a method for qualitative and / or quantitative detection of glycosylated Y5 RNA or exosomes containing glycosylated Y5 RNA based on at least one of the following methods:

[0023] PCR amplification, isothermal amplification, sequencing, nucleic acid hybridization, mass spectrometry, colorimetry, immunochromatography, ultraviolet absorption, enzyme-linked immunosorbent assay (ELISA), and Western blotting.

[0024] Of course, those skilled in the art may also use other methods besides those described above to perform qualitative and / or quantitative detection of glycosylated Y5 RNA or exosomes containing glycosylated Y5 RNA. Such detection methods are well known in the art and do not involve inventive effort based on glycosylated Y5 RNA or exosomes containing glycosylated Y5 RNA themselves.

[0025] In some embodiments of the present invention, the product includes at least one of reagents, reagent kits, test strips, chips, and detection systems.

[0026] In this invention, the type of product itself is not limited, and it can take different forms depending on the choice of detection method, including but not limited to the above-mentioned types.

[0027] In some embodiments of the present invention, the product includes a detection probe.

[0028] In some embodiments of the present invention, the detection probe includes a probe with a sequence as shown in SEQ ID NO: 16-17.

[0029] In some embodiments of the present invention, the product includes amplification primers.

[0030] In some embodiments of the present invention, the amplification primers include primers with sequences as shown in SEQ ID NO: 18-19.

[0031] In some embodiments of the present invention, the diagnostic and / or prognostic risk assessment criteria for pancreatic ductal adenocarcinoma are as follows:

[0032] When glycosylated Y5 RNA is detected, or its content is higher than that of the negative control, it is determined that there is a risk of pancreatic ductal adenocarcinoma or that the prognosis of pancreatic ductal adenocarcinoma is poor.

[0033] Conversely, it indicates a low risk of developing pancreatic ductal adenocarcinoma or a low prognostic risk after treatment of pancreatic ductal adenocarcinoma.

[0034] In some embodiments of the present invention, the diagnostic and / or prognostic risk assessment criteria for pancreatic ductal adenocarcinoma are as follows:

[0035] When exosomes containing glycosylated Y5 RNA are detected, or when their levels are higher than those in the negative control, it is determined that there is a risk of pancreatic ductal adenocarcinoma or a poor prognosis for pancreatic ductal adenocarcinoma.

[0036] Conversely, it indicates a low risk of developing pancreatic ductal adenocarcinoma or a low prognostic risk after treatment of pancreatic ductal adenocarcinoma.

[0037] In some embodiments of the present invention, the negative control is a healthy person or a patient who does not have pancreatic ductal adenocarcinoma.

[0038] In some embodiments of the present invention, the negative control is a healthy person.

[0039] The beneficial effects of this invention are:

[0040] 1. This invention reveals for the first time the crucial role of Glyco Y5 RNA in PDAC immune escape, and experimentally confirms that Glyco Y5 exosomes produced under hypoxia-induced conditions can regulate immune cell functions (such as promoting Tregs expansion, M2 macrophage polarization, and inhibiting CD8). + T cell activity shapes an immunosuppressive microenvironment, providing a new mechanistic explanation for the malignant progression of PDAC.

[0041] 2. This invention also proposes for the first time the clinical application value of GlycoY5 RNA in exosomes as a prognostic biomarker for PDAC. Through clinical sample analysis, this invention found that serum exosomal GlycoY5 RNA levels were significantly negatively correlated with overall survival (OS) and recurrence-free survival (RFS) in patients. Furthermore, it was found that its predictive efficacy for PDAC was not inferior to that of tissue GlycoY5 RNA, providing a highly specific molecular target for liquid biopsy of PDAC. Attached Figure Description

[0042] Figure 1 Northern blot analysis of Glyco RNA extracted from exosomes of the Panc02 cell line after normoxic and hypoxic culture.

[0043] Figure 2 To generate RNA-Seq volcano plots of Glyco RNA extracted from exosomes after normoxic and hypoxic culture of the Panc02 cell line.

[0044] Figure 3 The study aimed to demonstrate the regulatory effect of exosomal Glyco Y5 RNA on immune cell function. A represents the RT-qPCR results of TNF-α, CD163, and ARG1 mRNA levels; B represents the ELISA results of TGF-β and IL-10 levels in the supernatant; C represents the RT-qPCR results of Granzyme B, IFN-γ, PDCD1, and HAVCR2 mRNA levels; and D represents the ELISA results of IL-2, IFN-γ, and Granzyme B levels in the supernatant.

[0045] Figure 4 The results are from flow cytometry analysis of apoptosis levels in Panc02 cells.

[0046] Figure 5 This is a schematic diagram of the experimental procedure for verifying the effects of Glyco Y5 RNA exosomes on tumor growth, prognosis, and the immune microenvironment in mice.

[0047] Figure 6 Tumor growth and overall survival (OS) in a mouse model of pancreatic carcinoma in situ treated with Glyco Y5RNA.

[0048] Figure 7 To validate the effects of Glyco Y5 RNA exosomes on different indicators in mice, A represents the flow cytometry results of CD8+ T cell content in pancreatic cancer tissue, B represents the flow cytometry results of Treg cell content in pancreatic cancer tissue, C represents the flow cytometry results of M2 macrophage content in pancreatic cancer tissue, and D represents the ELISA results of TGF-β, IL-10, Granzyme B, and IFN-γ levels in mouse plasma. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0049] Figure 8 The level of Glyco Y5 RNA in total RNA in 50 ng exosomes and tissues is expressed in 40-CT units.

[0050] Figure 9 The results of flow cytometry analysis of the content of CD8+ T cells (A), M2 macrophages (B), and Treg cells (C) in the tissue of a pancreatic cancer patient.

[0051] Figure 10 The results show the serum levels of TGF-β, IL-10, Granzyme B, and IFN-γ in patients. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0052] Figure 11 The results validate the effectiveness of Glyco Y5 RNA as a biomarker. A shows the ROC curves for predicting overall survival (OS) using EGY5 and TGY5 levels; B shows the Kaplan-Meier analysis of OS stratified by TGY5 level; C shows the Kaplan-Meier analysis of OS stratified by EGY5 level; D shows the ROC curves for predicting relapse-free survival (RFS) using EGY5 and TGY5 levels; E shows the Kaplan-Meier analysis of RFS stratified by TGY5 level; and F shows the Kaplan-Meier analysis of RFS stratified by EGY5 level. Detailed Implementation

[0053] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0054] Example 1

[0055] In this embodiment, using hypoxic and normoxic pancreatic cancer cell lines as subjects, the effects of different oxygen environments on GlycoRNA in exosomes of cancer cells were evaluated by extracting cell line RNA and detecting the content of glycosylated RNA (GlycoRNA).

[0056] (1) In vitro hypoxic culture of cells:

[0057] Pancreatic cancer cell line Panc02 was cultured in DMEM medium containing 10% FBS and 100 U / mL penicillin-streptomycin. Cell culture was validated using short tandem repeat analysis in MSKCC before use, and all cells were periodically checked for mycoplasma contamination (mycoplasma test results were required to be negative).

[0058] The cultured pancreatic cancer cell line Panc02 was subjected to in vitro hypoxic culture. The specific steps were as follows: 2 × 10⁶ cells / well were placed in a 10 cm diameter culture dish. 6 After seeding cells at a certain density, the cells were allowed to grow for 24 hours, and then cultured for 2 days in a hypoxic incubator (STEMCELL, 5% carbon dioxide, 1% oxygen). Meanwhile, pancreatic cancer cell lines Panc-1 and Panc02, grown in a constant temperature incubator (5% carbon dioxide, 21% oxygen), served as normoxic culture controls.

[0059] (2) Isolation of exosomes:

[0060] Panc02 cells were cultured again according to the method in step (1) and seeded in 100 mm culture dishes (2 × 10⁻⁶ cells per cell). 6 (each piece / plate). After 90% fusion, incubate in serum-free medium for 24 hours. Then, under hypoxic conditions (1% O2) or normal oxygen levels (21% O2), replace the medium with serum medium and incubate for another 48 hours.

[0061] Exosomes were collected from cells in each group using a gradient ultracentrifugation method. The specific steps were as follows: centrifugation at 500×g for 10 minutes to remove cell contamination from the cell slurry; centrifugation at 3000×g for 20 minutes to remove apoptotic bodies and larger cell debris; and centrifugation at 12000×g for 20 minutes to remove larger microvesicles. Then, exosomes were collected by centrifugation at 100,000×g for 70 minutes (Beckman Ti70 rotor). The obtained exosomes were resuspended in 20 mL of phosphate-buffered saline (PBS) and purified by ultracentrifugation at 100,000×g for 70 minutes. The obtained exosomes were validated using transmission electron microscopy (TEM). Simultaneously, the exosome particle size was measured using dynamic light scattering (DLS) analysis with a Zetasizer Nano ZSE analyzer (Malvern Panalytical). Exosomes were characterized using Western blotting of CD63, CD81, TSG101, HSP70, and calnexin in both cell and exosome samples.

[0062] (3) RNA extraction:

[0063] Panc02 cancer cell line and exosomes derived from Panc02, cultured under hypoxic and normoxic conditions obtained in steps (1) and (2), were collected. Total RNA was extracted using the RNAzolRT method with a commercially available kit (Molecular Research Center, Inc.). The extracted total RNA precipitate was purified using a Zymo RNA purification column (hereinafter referred to as Zymo column) according to the manufacturer's instructions, and the obtained RNA product was quantified using a NanoDrop micro-spectrophotometer. Based on this, the purified RNA was further fragmented into small RNA (17-200 nt) and large RNA (>200 nt) using the Zymo column. The details are as follows:

[0064] RNA binding buffer (Zymo Research) and an equal volume of anhydrous ethanol were mixed to obtain a diluted RNA binding buffer solution. Two volumes of this buffer were added to the total RNA, and the mixture was thoroughly vortexed before passing through a column. At this point, large RNA was bound to the Zymo column, and the eluent contained small RNA, which needed to be stored. One volume of anhydrous ethanol was added to the small RNA eluent and thoroughly mixed before binding to a new Zymo column. After both large and small RNAs were bound to the column, they were washed separately with 2 × 400 μL of 80% ethanol, centrifuged for 30 seconds during the second wash. Finally, elution was performed with 2 × 50 μL of water. The obtained small and large RNAs were lyophilized.

[0065] (4) rPAL labeling and glycoRNA visualization:

[0066] The small RNA and large RNA obtained in step (2) were labeled with rPAL. The specific steps are as follows:

[0067] Preparation of blocking buffer: Mix 1 μL of 16 mM mPEG3-Ald (BP-23750, BroadPharm), 15 μL of 1 M MgSO4, and 12 μL of 1 M NH4OAc (pH 5, adjusted with HCl) (final concentration: 570 μM mPEG3-Ald, 500 mM MgSO4, 450 mM NH4OAc, pH 5). Then add 28 μL of aldehyde reaction blocking buffer to the small RNA and large RNA powder obtained in step (2) (maximum 3 μg of RNA powder), vortex to mix thoroughly, and incubate at 35 °C for 45 min to block all molecules that are reactive to aldehyde groups.

[0068] After a brief (2-3 minutes) cooling to room temperature, 1 μL of a 30 mM aldehyde reactive probe (ARP / AminooxyBiotin, Cayman Biotin, purchased from Cayman Chemicals, catalog number 10009350) and 2 μL of 7.5 mM periodic acid (NaIO4) were rapidly added. The RNA was oxidized at room temperature in the dark for 10 minutes to expose the aldehyde group (–CHO) at the terminal sugar residues. Then, 3 μL of 22 mm sodium sulfite was added to terminate the oxidation reaction (25°C, 5 minutes) to obtain the stock solutions for small and large RNA testing. The reaction system was then subjected to a ligation reaction at 35°C for 90 minutes to allow Aminooxy Biotin to undergo a stable covalent condensation reaction with the aldehyde group at the RNA terminal. 19 μL of water was then added to the reaction system to bring the total volume to 50 μL, and the system was purified using a Zymo column as described in step (2). To further analyze the samples in agarose gel to visualize glycoRNA, 6.2 μL of water was added twice to elute the corresponding RNA from the column.

[0069] To visualize the eluted RNA, it needs to be run on a denaturing agarose gel, transferred to a nitrocellulose (NC) membrane, and stained with streptavidin. The specific steps are as follows:

[0070] The eluted RNA was mixed with 12 μL of Gel Loading Buffer II (GLBII, containing 95% formamide, 18 mM EDTA, and 0.025% SDS), followed by the addition of 2x concentration of SybrGold (ThermoFisher Scientific) dye, and denatured at 55°C for 10 minutes. Immediately after denaturation, the sample was placed on ice to cool for at least 2 minutes. The sample was then loaded into a denaturing gel containing 1% agarose, 0.75% formaldehyde, and 1.5×MOPS buffer (Sigma-Aldrich). RNA was electrophoresed in 1×MOPS buffer at 115V for 34 to 45 minutes, depending on the gel length. After electrophoresis, the RNA bands were observed under UV imaging, and excess gel was removed. Then, the membrane transfer was performed, following the instructions of the Northern Max kit: Transfer using a 0.45 μm nitrocellulose membrane (NC membrane, GE Life Sciences) at 25°C for 90 minutes (transfer buffer: 3M NaCl solution at pH 1, adjusted acidity with HCl). After transfer, the membrane was exposed to UV light (0.18 J / cm²). 2 RNA was cross-linked onto the NC membrane. The transferred NC membrane was dried and then blocked in Intercept Protein-Free Blocking Buffer (TBS) at 25°C for 30 minutes. Streptavidin-IR800 (Li-Cor Biosciences) diluted 1:5000 was added and stained at 25°C for 30 minutes. The stained membrane was washed three times with a mixture of 0.1% Tween-20 and 1×PBS for 3 minutes each time, maintaining the temperature at 25°C. The membrane surface was then rapidly rinsed with PBS to remove Tween-20, and the membrane was scanned on a Li-Cor Odyssey CLx imaging system.

[0071] (5) Glyco RNA sequencing and analysis:

[0072] use The Mini Kit was used to extract total RNA from the exosomes obtained in step (4) according to the procedure in the kit instructions, and the RNA was evaluated using the high-sensitivity RNA kit of the Qsep100 fully automated nucleic acid analysis system. The total RNA from the exosomes was then labeled with rPAL using the procedure in step (3). 250 μg of RNA was used as the test sample for each replicate experiment. The rPAL labeling procedure was performed as described in the previous example, with the reagents added proportionally to the sample volume.

[0073] After rPAL labeling and purification using the Zymo kit, biotinylated RNA was captured using 250 μL Neutravidin magnetic beads (ThermoFisher Scientific), with each experiment performed once. The capture reaction was incubated at 4°C in 5×PBS with a reaction volume of 1000 μL for 2 hours by rotation. Subsequently, the magnetic beads were washed sequentially with preheated buffers to 45°C for 30 seconds each: 1) twice with 1000 μL 4M NaCl and 100 mM HEPES buffer; 2) twice with 1000 μL 2M GuHCl and 100 mM HEPES buffer; 3) twice with 1×PBS at room temperature.

[0074] After washing, the magnetic beads were subjected to nuclease treatment in the following enzyme mixture: 1) 250 μg RNase A (Sigma, 10109169001), 5 units of Phosphodiesterase (PDE1, Sigma, P3243-1VL), and 0.5 mM MgCl2 were added to a 300 μL reaction system and prepared in 0.5×PBS; 2) The mixture was incubated at 37°C for 4 hours with a shaker set to 10 seconds every 60 seconds at a speed of 1100 rpm. After the reaction, the supernatant was collected, stored in a new tube, and the magnetic beads were washed with 1000 μL of LC-MS grade water (Thermo Fisher Scientific). The washing solution was combined with the supernatant released by the nuclease. The final combined sample was frozen and lyophilized for pandora sequencing (sequencing and cDNA library construction were performed by Guangzhou Epigenetics Biotechnology Co., Ltd.).

[0075] The results are as follows Figure 1 As shown.

[0076] It was found that the number of GlycoRNA fragments in exosomal tissues under hypoxia treatment was significantly higher than that under normoxic conditions. Further analysis revealed that the RNA types with significantly increased glycosylation levels in hypoxic-treated exosomal tissues (logFC≥2, p<0.05) were significantly upregulated in hypoxic-treated exosomal tissues. Figure 2 ).

[0077] Example 2

[0078] In this embodiment, to further investigate the role of exosomal Glyco Y5 RNA in the immune regulation of pancreatic cancer, exosomes were extracted from pancreatic cancer cells that had been stably overexpressing Y5 RNA after hypoxia and normoxic treatment, and then used to treat immune cells such as T cells and macrophages to explore the regulatory effect of Glyco Y5 RNA on immune cell function.

[0079] The specific experimental steps are as follows:

[0080] (1) Construction of a pancreatic cancer cell model with stable Y5 RNA overexpression:

[0081] Using Y5 RNA (purchased from Dharmacon) at a final concentration of 50 nM or an empty vector as a control, pancreatic cancer cells Panc-1 and Panc02 were transfected according to the manufacturer's protocol. Stable lines of pancreatic cancer cells that stably overexpress Y5 RNA were obtained through antibiotic screening.

[0082] The nucleotide sequence of the Y5 RNA is: 5'-AGUUGGUCCGAGUGUUGUGGGUUAUUGUUA AGUUGAUUUAACAUUGUCUCCCCCCACAACCGCGCUUGACUAGCU-3' (SEQ ID NO: 1).

[0083] Then, following the method described in Example 1 above, cells were subjected to hypoxia and normoxic treatments for 48 hours. Cells were collected and non-Glyco Y5RNA exosomes (i.e., exosomes derived from Y5 RNA-overexpressing pancreatic cancer cells under normoxic treatment) and Glyco Y5RNA exosomes (exosomes derived from Y5 RNA-overexpressing pancreatic cancer cells under hypoxia treatment) were separated for later use.

[0084] (2) In vitro verification of the effects of Glyco Y5 RNA exosome treatment on immune cell function and cytokine expression:

[0085] Glyco Y5 RNA exosomes derived from the pancreatic cancer cell line obtained in step (1) (final concentration after addition: 5 μg / mL) were added to the culture medium for macrophages obtained in the above steps and co-cultured for 24 hours. After centrifugation, the culture supernatant and cell pellet were collected separately. qPCR was used to detect the M1 marker (TNF-α) and M2 markers (CD163, Arg1) in the cells. The levels of TGF-β and IL-10 in the macrophage culture supernatant were detected using an ELISA kit (ELISA Kit for IL-10, TNF-β, BD Pharmingen).

[0086] Peripheral blood from healthy individuals was collected, and human peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll density gradient centrifugation. The PBMCs were then cultured in RPMI 1640 medium with 10 ng / mL M-CSF (PeproTech) and 10% FBS for 3–5 days. RoboSep was used concurrently. TM Human initial type CD8 + T-cell isolation kit (Stemcell) was used according to the instructions to isolate naïve CD8 cells from human peripheral blood.+ T cells were isolated and enriched to obtain naïve CD8. + The T cells were then cultured in RPMI 1640 medium containing 10% FBS.

[0087] At the same time, the initial CD8 obtained from the above steps + T cells were added to T cell culture medium and cultured. Then, Glyco Y5 RNA exosomes (5 μg / mL) were added and incubated for 24 or 48 hours (non-Glyco Y5 RNA exosomes). After centrifugation, the culture supernatant and cell pellet were collected separately. The concentrations of Granzyme B, IL-2, and IFN-γ in the culture supernatant were determined using ELISA kits: Human Granzyme B ELISA Kit (Beijing 4ABiotech Co., Ltd.), Human IFN-γ ELISA Kit (Beijing 4ABiotech Co., Ltd.), and IL-2 ELISA Kit (BD Pharmingen). CD8 was detected using RT-qPCR. + Activation and exhaustion phenotypic markers in T cells (Granzyme B, IFN-γ, PDCD1, and HAVCR2).

[0088] The primer information for the relevant markers is shown in the table below.

[0089] Table 1 Primer information for relevant biomarkers

[0090]

[0091]

[0092] The specific method for RT-qPCR is as follows: TRIzol RT method was used to extract macrophages and CD8+ cells from the treated cells. + Total RNA from T cells was collected. 1 μL of total RNA was added to 2 μL of gDNARemover, and the mixture was pipetted 10 times. After reacting at room temperature for 5 min, the mixture was placed on ice. Reverse transcription was performed using a commercially available reverse transcription kit to obtain cDNA. Then, real-time quantitative PCR was performed using the ChamQ SYBR qPCR Master Mix kit according to the manufacturer's instructions. The RT-qPCR reaction program was: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 10 s, annealing at 60°C for 30 s, extension at 72°C for 7 min, repeated for 40 cycles. 2... -ΔΔCT The results were calculated using the method, and all experiments were repeated three times.

[0093] The results showed that exosomal Glyco Y5 RNA can regulate immune cell function, including promoting M2 macrophage polarization and inhibiting CD8. + T cell activity ( Figure 3 A-3D), thereby creating an immunosuppressive microenvironment.

[0094] Furthermore, the inventors verified the effectiveness of initial CD8+ treatment with Glyco Y5 RNA exosomes derived from pancreatic cancer cell lines. + The cytotoxic activity of T cells. The specific experimental steps are as follows:

[0095] Panc02 cells were mixed with untreated, naïve CD8 cells. + T cells were co-cultured in exosome-free medium with an effector cell to target cell (E:T) ratio of 1:5. Prior to co-culture, Panc02 cells were labeled with Calcein AM dye for tracking. Glyco Y5 RNA exosomes obtained in the above-described example were then added to the co-culture system at a final concentration of 10 μg / mL, and the cells were cultured for another 24 hours. Cells were collected, co-stained with Annexin V-FITC and PI dyes, and the percentage of cell death was analyzed by flow cytometry.

[0096] The results are as follows Figure 4 As shown.

[0097] The results showed that the naïve CD8 cells treated with Glyco Y5 RNA exosomes... + T cells have weaker cytotoxic activity.

[0098] (3) In vivo experiments to verify the effect of Glyco Y5 RNA on immune escape

[0099] Furthermore, the inventors verified the effects of Glyco Y5 RNA exosomes on tumor growth, prognosis, and the immune microenvironment in mice.

[0100] The specific steps are as follows: After anesthetizing the mice with isoflurane, a 1cm incision is made in the skin approximately 1.5cm to the left of the abdominal midline using sterile surgical instruments, followed by a 1cm incision in the abdominal muscle. The spleen is located using forceps and gently pulled out of the abdominal cavity. The tail of the pancreas is positioned near the spleen, and 25μL of Panc02 cell suspension (containing 1×10⁻⁶ cells) is injected into the pancreas using a 29-gauge needle and a 0.3mL insulin syringe. 6(One pancreatic cancer cell), and then the spleen and pancreas were repositioned into the abdominal cavity. The abdominal muscle layer and external skin of the mice were then sutured using absorbable Bard 4-0 sutures with a continuous suture technique. Starting from day 7, exosomes extracted from hypoxic or normoxic Glyco Y5 RNA-overexpressing pancreatic cancer cells or control exosomes were injected twice weekly via the tail vein. Specifically, the dosage of exosomes was 5 μg twice weekly, with purified exosomes injected into the tail vein in 100 μL of PBS. Tumor growth was observed from day 7 to day 28. On day 28, half of the mice (n=8) were sacrificed for further testing, and the total survival time of the remaining mice was continuously recorded. The following tests were performed during and after the experiment.

[0101] (1) During the experiment, tumor volume was monitored regularly using the small animal IVIS Lumina imaging system (Caliper Life Sciences) to assess tumor growth and metastasis, and the volume of pancreatic cancer tumors in mice was measured.

[0102] (2) Time-of-flight flow cytometry (CyTOF) was used to detect tumor-associated immune cells. The specific experimental steps were as follows: pancreatic cancer tissue from experimental mice was collected, digested with collagenase P to obtain a single-cell suspension, stained with 5 μM Cell-ID Cisplatin (purchased from Fluidigm Corp.), incubated with Fc block (BD Biosciences), and incubated for a period of time after adding a surface antibody mixture. The cells were then washed and fixed in Maxpar Fix I buffer (Fluidigm Corp.). Cell-ID... TM Barcodes were created using a 20-Plex Pd barcode kit (Fluidigm Corp.). Incubation was then performed with an intracellular antibody cocktail, followed by washing and staining with 1.25 μM Cell-ID Intercalator-Ir (FluidigmCorp.). Samples were collected on a Helios mass spectrometer (Fluidigm Corp.) and analyzed using FlowJoversion 10 software (FlowJo LLC).

[0103] The antibody information used in step (2) is shown in the table below.

[0104] Table 2 Antibody Information

[0105]

[0106]

[0107] The experimental procedure is as follows Figure 5 As shown.

[0108] The results are as follows Figures 6-7 As shown.

[0109] In vivo experiments revealed that Glyco Y5 RNA can significantly inhibit tumor immunity, promote pancreatic cancer growth, and shorten overall survival (OS) in mice. Figure 6 Specifically, this manifests as CD8. + The proportion of T cells is reduced, and FoxP3+Tregs and M2 macrophages (CD68) are less active. + The proportion of cells increased significantly. Figure 7 A-7C) and increased secretion of inhibitory cytokines ( Figure 7 D). These findings provide important experimental evidence for elucidating the mechanism of action of GlycoY5 RNA in PDAC immune escape.

[0110] Example 3

[0111] The effectiveness of Glyco Y5 RNA for pancreatic cancer diagnosis was validated using real samples.

[0112] Surgical resection specimens and paired serum samples were collected from patients with early-stage pancreatic cancer. A total of 60 cases were included, and all patients had completed at least three years of follow-up.

[0113] Inclusion criteria were: age > 18 years; all patients underwent computed tomography (CT), magnetic resonance imaging (MRI), and serological examinations, and met the diagnostic criteria for pancreatic cancer as outlined in the Guidelines for the Diagnosis and Treatment of Pancreatic Cancer; surgical resection and pathological diagnosis of pancreatic ductal adenocarcinoma (PDAC); TNM stage I or II; no distant metastasis or peritoneal seeding metastasis preoperatively; underwent radical pancreatic resection (such as pancreaticoduodenectomy, distal pancreatectomy, or total pancreatectomy) and did not receive neoadjuvant radiotherapy or chemotherapy postoperatively; complete postoperative follow-up data was available for at least three years; and patients and their families were fully informed and signed informed consent forms, indicating their willingness to participate in this study.

[0114] Exclusion criteria were: patients with a history of other malignant tumors; patients with severe liver or kidney dysfunction, active infection or hematological disease; patients who died or were lost to follow-up after surgery due to other reasons, or whose follow-up data were incomplete; and patients who had received radiotherapy, chemotherapy or targeted therapy before surgery.

[0115] The collected blood sample was transferred into a 5 mL vacuum blood collection tube (containing a coagulant and separating gel). Within 2 hours of sampling, the sample was centrifuged at 1800 × g for 10 minutes at room temperature to obtain serum. The serum was then centrifuged at 3000 × g for 10 minutes at 4°C to remove cell debris and apoptotic bodies. The precipitate was discarded, and the supernatant serum was extracted. Serum exosomes were obtained by ultracentrifugation following the method described in the previous example.

[0116] Exosomal RNA was extracted and rPAL-labeled according to the methods described in the above embodiments. The total amount of Y5 RNA and the content of Glyco Y5 RNA exosomes in serum exosomes of pancreatic cancer patients were detected using Northern Blot and RT-qPCR, and linear regression curves of Glyco Y5 RNA in tissue and serum exosomes were plotted accordingly.

[0117] In the Northern Blot method, Y5 RNA probes were used to quantify the total amount of Y5 RNA. The nucleotide sequences are as follows: 3′ wild-type probe: 5′-AGCTAGTCAAGCGCGGTTGTGGGGG-3′ (SEQ ID NO: 16); 5′ probe: 5′-TAACCCACAACACTCGGACCAACT-3′ (SEQ ID NO: 17).

[0118] The RT-qPCR primers are: upstream primer: 5'-AATACTAGTGAAGATCCATGGAGGTACATC-3' (SEQ ID NO: 18); downstream primer: 5'-GTAAACGTTGTCTACTACTGTTATTAGTGC-3' (SEQ ID NO: 19).

[0119] The results are as follows Figure 8 As shown.

[0120] It was found that the levels of exosomal GlycoY5 RNA (TGY5) in pancreatic cancer tissue were positively correlated with the levels of serum exosomal GlycoY5 RNA (EGY5).

[0121] Furthermore, the collected tissue and serum samples from pancreatic cancer patients were divided into a high-EGY5 expression group and a low-EGY5 expression group according to the Youden index of the receiver operating curve (ROC) of exosome EGY5. Flow cytometry was used to detect the level of immune cell infiltration in pancreatic cancer tissues, and ELISA kits were used to detect the cytokine content in the patient serum (the steps are the same as in the above embodiments).

[0122] The results are as follows Figure 9 and Figure 10 As shown.

[0123] The results showed that in tumor tissues with high EGY5 expression, CD8... + Decreased T cell infiltration, FoxP3+Tregs, and M2 macrophages (CD68) + The proportion of cells increased significantly. Figure 9This further supports the role of Glyco Y5 RNA in immune escape. Patients with high Glyco Y5 RNA expression showed upregulated levels of immunosuppressive factors such as IL-10 and TGF-β in their serum, while levels of IFN-γ and Granzyme B were decreased. Figure 10 ).

[0124] Example 4

[0125] To further determine the effectiveness of Glyco Y5 RNA in tissues and exosomes as biomarkers, the inventors used the Kaplan-Meier survival method to assess their availability and evaluated the prognostic accuracy and sensitivity of the model based on receiver operating characteristic (ROC) and area under the curve (AUC).

[0126] The specific steps are as follows: The Youden index of TGY5 is calculated from the collected clinical data according to the method in Example 3, and the data are divided into high and low groups. OS and RFS survival curves are then plotted for each group. Finally, the predictive efficacy of both methods for OS / RFS in the same patient population is compared using ROC curves and AUC values.

[0127] The results are as follows Figure 11 As shown.

[0128] The sensitivity of tissue Glyco Y5 RNA as a marker relative to OS was 0.4727, and its specificity was 0.8750. The sensitivity relative to RFS was 0.4833, and its specificity was 1.000. The sensitivity of exosomal Glyco Y5 RNA as a marker relative to OS was 0.7091, and its specificity was 0.8333. The sensitivity relative to RFS was 0.7167, and its specificity was 1.000.

[0129] It was found that high levels of TGY5 in both tissues and exosomes were significantly associated with shorter overall survival (OS) and remission rate (RFS) in patients, and the predictive ability of exosomal GlycoY5 RNA was not inferior to that of tissue-derived GlycoY5 RNA.

[0130] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of products that detect the expression level of glycosylated Y5 RNA in the preparation of products for prognostic risk assessment of pancreatic ductal adenocarcinoma.

2. Use according to claim 1, characterized in that, The product achieves quantitative detection of glycosylated Y5 RNA based on at least one of the following methods: PCR amplification or nucleic acid hybridization.

3. The use according to claim 2, characterized in that, The product includes reagents.

4. The use according to claim 2, characterized in that, The product includes a reagent kit.

5. The use according to claim 2, characterized in that, The product includes a testing system.

6. The use according to claim 2, characterized in that, The product includes detection probes.

7. The use according to claim 6, characterized in that, The detection probes include probes with sequences as shown in SEQ ID NO: 16-17.

8. The use according to claim 2, characterized in that, The product includes amplification primers.

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