tRF5-22-sectca-1, tRF5-22-sectca-1 detection reagents, kits and uses thereof

Abstract

This invention belongs to the field of molecular biology technology, specifically involving tRF5-22-SeCTCA-1, tRF5-22-SeCTCA-1 detection reagents, kits, and their applications. This invention discovers and verifies that tRF5-22-SeCTCA-1 plays a key regulatory role in the process of 5-fluorouracil (5-FU) chemotherapy resistance in colorectal cancer, filling a gap in the research of tRNA-derived fragments (tRFs) in colorectal cancer chemotherapy resistance. It is the first tRF molecule reported to be associated with 5-FU chemotherapy resistance in colorectal cancer, providing a novel molecular target and research direction for predicting chemotherapy resistance in colorectal cancer. This invention also provides a therapeutic strategy targeting tRF5-22-SeCTCA-1 and establishes α-ketoglutarate as an independent reversal agent, providing multi-dimensional technical solutions for the precision diagnosis and treatment of colorectal cancer and the reversal of chemotherapy resistance.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to tRF5-22-SeCTCA-1, tRF5-22-SeCTCA-1 detection reagents, kits and their applications. Background Technology

[0002] Colorectal cancer (CRC) is the third most common malignant tumor worldwide and a leading cause of cancer-related death, with persistently high incidence and mortality rates, posing a serious threat to human health. Chemotherapy is one of the core methods of comprehensive treatment for colorectal cancer, and for patients with advanced and metastatic colorectal cancer, it is crucial for prolonging survival and improving quality of life. Among these treatments, 5-fluorouracil (5-FU), as a pyrimidine analogue, exerts its anti-tumor effects through mechanisms such as inhibiting thymidine synthase to block DNA synthesis and repair, and its metabolites incorporating into RNA to interfere with RNA function. Based on 5-FU, combination chemotherapy regimens such as FOLFOX and FOLFIRI have become the standard first-line treatment for advanced colorectal cancer.

[0003] However, current chemotherapy treatment systems for colorectal cancer have significant shortcomings. Chemotherapy resistance is widespread and lacks effective prediction and intervention methods, becoming a key bottleneck restricting treatment effectiveness. In clinical practice, some patients do not respond to initial 5-FU chemotherapy regimens or gradually develop resistance during treatment. This not only leads to treatment failure and delays in optimal treatment, but also causes severe toxic side effects from ineffective chemotherapy, increasing patients' physical and mental suffering and medical burden.

[0004] To overcome the bottlenecks in chemotherapy treatment and achieve precise individualized therapy, clinicians have attempted to guide treatment strategy selection using biomarkers. However, existing technologies still cannot meet the actual needs of optimizing and adjusting chemotherapy strategies. Specifically, KRAS, NRAS, and BRAF gene mutation detection are only used to screen patients for anti-EGFR targeted therapy and cannot provide a reference for chemotherapy regimen selection; microsatellite instability (MSI-H) and mismatch repair deficiency (dMMR) detection are primarily used for screening patients for immunotherapy and are unrelated to assessing the suitability of chemotherapy strategies; serum carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) indicators can only assist in assessing efficacy or warning of recurrence after treatment, but cannot predict patient response before chemotherapy regimen initiation, let alone guide clinicians to adjust treatment strategies for patients at risk of drug resistance in advance, leading to some patients still facing ineffective chemotherapy and delayed treatment. It is evident that current methods for addressing chemotherapy resistance in colorectal cancer lack effective means, and treatment strategy adjustments rely on postoperative efficacy assessment, which is a passive approach and difficult to intervene early. Existing technological limitations make it difficult to predict chemotherapy resistance and result in poor regimen suitability. There is an urgent need to overcome technological barriers and develop new and optimal treatment strategies.

[0005] In view of this, there is an urgent need to propose a new and better treatment strategy for colorectal cancer, in order to achieve accurate prediction of chemotherapy response and timely and effective intervention for drug-resistant patients, improve the level of individualized treatment, reduce the medical burden and patient suffering caused by ineffective chemotherapy, and ultimately improve the quality of life and long-term prognosis of colorectal cancer patients. Summary of the Invention

[0006] Based on this, the purpose of the present invention is to provide a tRF5-22-SeCTCA-1, tRF5-22-SeCTCA-1 detection reagent, kit and its application, in order to solve a series of clinical dilemmas in the existing technology in the chemotherapy treatment system for colorectal cancer, such as the difficulty in predicting drug resistance and the lack of accurate basis for adjusting treatment strategies, which leads to poor patient prognosis.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] The present invention provides a tRF5-22-SeCTCA-1, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0009] This invention also discloses the application of the tRF5-22-SeCTCA-1 detection reagent in the preparation of detection reagents / kits for diagnosing chemotherapy resistance in colorectal cancer.

[0010] In some embodiments of the present invention, the detection reagent includes a primer pair for amplifying the tRF, the nucleotide sequences of which are shown in SEQ ID No: 5 and SEQ ID No: 6.

[0011] The present invention also discloses a kit for detecting chemotherapy resistance in colorectal cancer, the kit comprising reagents for detecting the expression level of tRF5-22-SeCTCA-1, wherein the reagents for inhibiting the expression or activity of tRF5-22-SeCTCA-1 include the sequence shown in SEQ ID No: 10 or SEQ ID NO: 17.

[0012] The present invention also discloses the application of the tRF5-22-SeCTCA-1 detection reagent in colorectal cancer drug screening.

[0013] This invention also discloses the use of at least one of the following reagents in the preparation of medicaments for treating colorectal cancer and / or reversing chemotherapy resistance in colorectal cancer: a. A reagent that inhibits the expression or activity of tRF5-22-SeCTCA-1; b. α-Ketoglutarate.

[0014] In some embodiments of the present invention, the reagent for inhibiting the expression or activity of tRF5-22-SeCTCA-1 is selected from antisense oligonucleotides, small molecule compounds that inhibit the expression of tRF5-22-SeCTCA-1, and one or more.

[0015] In some embodiments of the present invention, the reagent for inhibiting the expression or activity of tRF5-22-SeCTCA-1 includes the sequence shown in SEQ ID No: 11 or SEQ ID NO: 17.

[0016] The present invention also discloses a combination drug composition of α-ketoglutarate, a reagent that inhibits the expression or activity of tRF5-22-SeCTCA-1, and its use in the preparation of a drug that reverses chemotherapy resistance in colorectal cancer and enhances the efficacy of chemotherapy drugs for colorectal cancer.

[0017] In some embodiments of the present invention, the chemotherapy resistance in colorectal cancer includes chemotherapy resistance in colorectal cancer caused by one or more of the following drugs: 5-fluorouracil, leucovorin, capecitabine, oxaliplatin, and irinotecan.

[0018] This invention also discloses a pharmaceutical composition for treating colorectal cancer, characterized in that the active ingredient of the pharmaceutical composition is selected from at least one of the following, and a pharmaceutically acceptable carrier: a. A reagent that inhibits the expression or activity of tRF5-22-SeCTCA-1; b. α-Ketoglutarate.

[0019] Based on the technical solution of the present invention, the present invention has the following beneficial effects compared with the prior art: This invention is the first to discover and verify that tRF5-22-SeCTCA-1 plays a key regulatory role in the resistance to 5-fluorouracil (5-FU) chemotherapy in colorectal cancer, filling a gap in the research on tRNA-derived fragments (tRFs) in colorectal cancer chemotherapy resistance. It is the first tRF molecule reported to be associated with 5-FU chemotherapy resistance in colorectal cancer, providing a novel molecular target and research direction for the prediction of chemotherapy resistance in colorectal cancer.

[0020] This invention, through multi-dimensional systematic verification using cell experiments, animal models, and clinical samples, confirms that the expression level of tRF5-22-SeCTCA-1 is significantly positively correlated with 5-FU chemotherapy resistance in colorectal cancer. Furthermore, this molecule possesses good predictive power for chemotherapy resistance, enabling precise identification of high-risk individuals for chemotherapy resistance before colorectal cancer patients receive 5-FU chemotherapy. Positive expression (scored as + or ++) indicates a higher likelihood of developing chemotherapy resistance. Based on this, individualized chemotherapy regimens can be developed in clinical practice, effectively avoiding ineffective chemotherapy, reducing patient suffering, and minimizing unnecessary medical resource consumption.

[0021] This invention elucidates for the first time the molecular mechanism by which tRF-1 promotes glycolytic metabolism in tumor cells by regulating IDH3B, ultimately affecting 5-FU chemotherapy resistance in colorectal cancer. This discovery not only reveals a novel regulatory axis of tRFs-metabolic reprogramming-tumor chemotherapy resistance, but also provides a new perspective for understanding the intrinsic link between tumor metabolic reprogramming and chemotherapy resistance. At the same time, the clarification of this molecular mechanism also provides a potential intervention target for reversing chemotherapy resistance in colorectal cancer, laying an important theoretical and experimental foundation for the development of novel treatments to reverse chemotherapy resistance.

[0022] This invention, based on the association and regulatory mechanism between tRF5-22-SeCTCA-1 and 5-FU chemotherapy resistance in colorectal cancer, not only provides a detection method for predicting chemotherapy resistance in colorectal cancer, but also constructs a treatment strategy targeting tRF5-22-SeCTCA-1, and establishes α-ketoglutarate as an independent reversal agent. Experiments have demonstrated that inhibiting tRF-SeCTCA-1 expression significantly enhances the sensitivity of colorectal cancer to 5-FU chemotherapy; exogenous supplementation of α-ketoglutarate effectively reverses the chemotherapy resistance phenotype and metabolic reprogramming induced by tRF5-22-SeCTCA-1 overexpression, and its mechanism of action is salvage regulation of downstream metabolic pathways of tRF5-22-SeCTCA-1. These technical solutions collectively constitute a multi-dimensional precision diagnosis and treatment system covering "prediction-targeted intervention-downstream reversal," which is more targeted and valuable than single treatment methods, providing a new approach and feasible path for clinically addressing the challenge of 5-FU chemotherapy resistance in colorectal cancer. Attached Figure Description

[0023] Figure 1 This is a map showing the origin and distribution of tsRNA (based on tsRNA sequencing results).

[0024] Figure 2 This is a combination of differential expression volcano plots for the three groups and the composition ratios of each tsRNA subtype; among them... Figure 2 In the diagram, A represents a volcano plot where each of the three groups is compared pairwise. Figure 2The B in the diagram represents the composition ratio of each tsRNA isotype.

[0025] Figure 3 The image shows the expression level of tRF5-22-SeCTCA-1 in tissues of different patient groups detected using in situ hybridization technology; among them, Figure 3 In the image, A represents the staining results of tRF5-22-SeCTCA-1 expression in chemotherapy-sensitive and chemotherapy-resistant tissues. Figure 3 The figure shows the statistical results of tRF5-22-SeCTCA-1 expression levels in chemotherapy-sensitive and chemotherapy-resistant patients.

[0026] Figure 4 The results of the detection of drug-resistant cells; among them, Figure 4 In this context, A represents the IC50 of 5-FU after HCT8-R transfection with tRF-SeCTCA-1 inhibitor. 50 ; Figure 4 In this context, B indicates that after HCT8-R transfection with tRF-SeCTCA-1 inhibitor, IC50 is achieved. 50 The curve of change; Figure 4 In this context, C represents the IC50 value after HCT116-R transfection with tRF-SeCTCA-1 inhibitor. 50 The curve of change; Figure 4 The figure shows the statistical results of tRF-SeCTCA-1 expression levels in two types of drug-resistant cells after transfection with the inhibitor and the control group.

[0027] Figure 5 The figure shows the statistical results of tRF-SeCTCA-1 expression level and drug resistance level after transfection of cells overexpressing tRF-SeCTCA-1 with a tRF-SeCTCA-1 mimic; among them, Figure 5 In the figure, A represents the statistical results of tRF-SeCTCA-1 expression level after transfection with tRF-SeCTCA-1 mimic; Figure 5 In this context, B represents the IC50 value of HCT8 cells after transfection with tRF-SeCTCA-1 mimicry. 50 The curve of change; Figure 5 In this context, C represents the IC50 value of HCT116 cells after transfection with tRF-SeCTCA-1 mimicry. 50 The curve of change; Figure 5 After transfection of tRF-SeCTCA-1 mimicry into D cells, the IC50 of 5-FU was reduced. 50 The statistical results are shown in the figure.

[0028] Figure 6 The statistical results of cell proliferation after transfecting HCT8 and HCT116 cells with tRF-SeCTCA-1 mimicry are shown in the figure; among them, Figure 6 In the diagram, A represents the proliferation curve of HCT8 cells; Figure 6 In the figure, B represents the proliferation curve of HCT116 cells.

[0029] Figure 7 The image shows the results of a plate colony assay after transfecting HCT8 and HCT116 cells with tRF-SeCTCA-1 mimicry; among them, Figure 7 In the diagram, A represents the results of staining two cell plate clones. Figure 7 In the figure, B represents the bar chart of the statistical results of the number of clones on the two cell plates.

[0030] Figure 8 The image shows the results of flow cytometry culture of apoptotic cells after transfection with tRF-SeCTCA-1 mimicry in two cell lines.

[0031] Figure 9 The graph shows the ratio of PI+ and annexin V+ in two cell lines after transfection with tRF-SeCTCA-1 mimicry.

[0032] Figure 10 The graph shows the statistical results of tRF-SeCTCA-1 expression levels and drug resistance levels after transfection with a specific inhibitor of tRF5-SeCTCA-1 to inhibit HCT116-R and HCT8-R; among them, Figure 10 In the figure, A represents the statistical results of tRF5-SeCTCA-1 expression level after transfection with a specific inhibitor; Figure 10 In this context, B indicates that after HCT8-R cells are transfected with a specific inhibitor, the IC50 value is lowered. 50 The curve of change; Figure 10 In this context, C represents the IC50 value after HCT116-R cells are transfected with a specific inhibitor. 50 The curve of change; Figure 10 In this context, D represents the IC50 of 5-FU after transfection with a transfection-specific inhibitor in the two cell types. 50 The statistical results are shown in the figure.

[0033] Figure 11 The statistical results of cell proliferation after transfection with a specific inhibitor of tRF5-SeCTCA-1 to inhibit HCT116-R and HCT8-R are shown in the figure; among them, Figure 11 In the diagram, A represents the proliferation curve of HCT8-R; Figure 11 In the diagram, B represents the proliferation curve of HCT116-R.

[0034] Figure 12 The image shows the results of a plate cloning experiment after HCT116-R and HCT8-R were transfected with tRF-SeCTCA-1 mimics; among them, Figure 12 In the diagram, A represents the results of staining two cell plate clones. Figure 12In the figure, B represents the bar chart of the statistical results of the number of clones on the two cell plates.

[0035] Figure 13 The image shows the results of flow cytometry culture of apoptotic cells after transfection with specific inhibitors of HCT116-R and HCT8-R.

[0036] Figure 14 The graph shows the ratio of PI+ and annexin V+ in cultured with HCT116-R and HCT8-R after transfection with specific inhibitors.

[0037] Figure 15 This is an image showing the results of in situ hybridization in clinical tumor tissues; among them, Figure 15 In the diagram, A represents the result of in situ hybridization staining; Figure 15 In the figure, B represents the statistical results of tRF5-SeCTCA-1 expression levels in different tissues.

[0038] Figure 16 The image shows the organoid growth detection results after transfection with two drug-resistant organoid inhibitors; among them, Figure 16 In the diagram, A represents two drug-resistant organoid phenotypes; Figure 16 In the figure, B represents the statistical results of the growth of drug-resistant organoid PDO1 in different treatment groups; Figure 16 The figure shows the statistical results of the growth of different treatment groups of drug-resistant organoid PDO2.

[0039] Figure 17 Phenotypic images showing tumor volume changes in different treatment groups of a mouse subcutaneous tumor model; among them... Figure 17 In the figure, A represents the phenotypic diagram of tumor volume change after treatment in mice injected with HCT116 cells. Figure 17 In the figure, B represents the phenotypic diagram of tumor volume change after treatment in mice injected with HCT116-R cells.

[0040] Figure 18 Tumor growth curves for different treatment groups in a mouse subcutaneous tumor model; among them, Figure 18 In the figure, A represents the tumor growth curve after HCT116 inoculation at different time points; where, Figure 18 In the figure, B represents the tumor growth curve at different times after HCT116-R inoculation.

[0041] Figure 19 Immunohistochemical staining results of Ki67 cells after injection of tRF-SeCTCA-1 inhibitor; among them, Figure 19 In the image, A represents the result of Ki67 immunohistochemical staining of HCT116 cells; Figure 19 The B in the image represents the result of Ki67 immunohistochemical staining of HCT116-R cells.

[0042] Figure 20 The growth curves of HCT8 and HCT116 with different concentrations of α-ketoglutarate are shown in the figure; Figure 20 In the figure, A represents the growth curve result of HCT116; Figure 20 In the figure, B represents the growth curve result of HCT8.

[0043] Figure 21 The results of the cellular energy metabolism analysis experiment after adding α-ketoglutarate to HCT8 and HCT116 cells are shown in the figure; Figure 21 In the figure, A represents the statistical results of ECAR for HCT116 at different treatment times; Figure 21 In the figure, B represents the statistical results of ECAR for HCT8 at different treatment times; Figure 21 In the figure, C represents the statistical results of OCR for HCT116 at different processing times; Figure 21 In the figure, D represents the statistical results of OCR for HCT8 at different processing times.

[0044] Figure 22 The image shows the results of detecting the sensitivity of exogenous α-ketoglutarate HCT8 and HCT116 cells to the chemotherapeutic drug 5-FU; among them, Figure 22 In the figure, A represents the cell viability change curve of HCT116 cells; Figure 22 In the figure, B represents the curve of HCT8 cell viability change. Detailed Implementation

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0046] The following description is based on specific embodiments.

[0047] Unless otherwise specified, all reagents used in the following examples are commercially available; and all methods used in the following examples are conventional methods unless otherwise specified.

[0048] Example 1: Study on tRF5-22-SeCTCA-1 and its effects 1. Sample collection and grouping (1) Sample size and grading criteria: Tumor samples collected from Sun Yat-sen Memorial Hospital of Sun Yat-sen University were evaluated according to the NCCN tumor regression grading criteria (as shown in Table 1), and 32 tumor samples were finally selected. The colorectal cancer tissue samples used in this embodiment were previously archived anonymized samples from Sun Yat-sen Memorial Hospital of Sun Yat-sen University. They could not identify specific individuals, the research risk was extremely small, informed consent was exempted in accordance with regulations, the sample source was legal and compliant, and the relevant research complied with relevant medical ethics and laws and regulations.

[0049] Table 1 NCCN Tumor Regression Grade (2) The specific groupings are as follows: 1) Non-Chemo group: 8 cases. This group consisted of colorectal cancer patients who did not receive preoperative chemotherapy.

[0050] 2) TRG1 and TRG2 groups (TRG1 / 2 group) who underwent preoperative chemotherapy: 10 patients. This group consisted of patients who received 5-FU-based chemotherapy preoperatively and obtained a tumor regression grade (TRG) of 1 or 2 after surgery. TRG1 and TRG2 indicate that the tumor responded to chemotherapy.

[0051] 3) TRG3 group (14 patients) who underwent preoperative chemotherapy. This group consisted of patients who received 5-FU-based chemotherapy preoperatively and achieved a tumor regression score (TRG) of 3 after surgery. TRG3 indicates that the tumor regression effect after chemotherapy was not significant.

[0052] (3) Inclusion criteria: a. Patients diagnosed with colorectal adenocarcinoma by histopathology; b. Has received standard neoadjuvant chemotherapy with oxaliplatin + 5-fluorouracil (such as FOLFOX or CapeOx regimen) or first-line chemotherapy for advanced disease; c. A clear TRG classification record with imaging assessment or surgical resection specimen; Patients sign informed consent forms before sampling.

[0053] (4) Exclusion criteria: a. Patients with concurrent primary malignant tumors; b. Patients with incomplete chemotherapy regimens or who were not administered the standard dose; Those with missing clinical data or whose sample quality (RNA integrity) does not meet the standards.

[0054] 2. Sample processing methods (1) Acquisition and preservation of clinical tissue samples 1) Collection process: Colorectal cancer tissue samples for each group were obtained from surgical resection specimens at the hospital. After the tissues were removed from the body, they were immediately infiltrated with RNA later solution to maintain RNA stability.

[0055] 2) Deep cryopreservation: The sample is first quickly placed in liquid nitrogen to freeze for 5 minutes, and then immediately transferred to an ultra-low temperature freezer at -80℃ for long-term storage to ensure that biomolecules such as RNA and proteins are not degraded until they are used in subsequent experiments.

[0056] (2) Extraction process of total RNA from tissues 1) Tissue homogenization: Take frozen colorectal cancer tissue (about the size of a soybean), place it in a 1.5 mL enzyme-free centrifuge tube, add 1 mL of TRIzol reagent and 2 small homogenizing beads. Use a tissue homogenizer to homogenize at a frequency of 70 Hz for 90 seconds, repeat 10 times to ensure complete lysis of tissue cells.

[0057] 2) Phase separation: Add 500µL of chloroform to the lysis buffer, shake vigorously to mix and form a homogeneous emulsion, and let stand at room temperature for 5 minutes to allow the nucleoprotein complex to completely dissociate.

[0058] 3) Centrifugation precipitation: Centrifuge at 4℃ and 12000 RPM for 15 minutes, and carefully aspirate the upper aqueous phase containing RNA into a new tube.

[0059] 4) Purification and washing: Add an equal volume of isopropanol to precipitate total RNA, discard the supernatant, and wash the precipitate with 75% ethanol.

[0060] 5) Dissolution and Quality Control: Dissolve the RNA precipitate using DEPC water (preparation: 1 mL DEPC added to 1 L deionized water, stirred for 30 min, allowed to stand overnight and autoclaved). Measure the A260 / A280 ratio using NanoDrop (should be between 1.8 and 2.1) to ensure purity.

[0061] 3. tRF microarray sequencing and differential screening Thirty-two colorectal cancer samples were collected and sent to Kangcheng Biotechnology for tsRNA sequencing. The general steps are as follows: RNA was extracted from tissue samples; a total of 100 ng of RNA was extracted from each colorectal cancer sample. T4 polynucleotide kinase was added to dephosphorylate the RNA, removing residual phosphate and cyclic phosphate groups at the 3' end to expose the 3'-OH terminus. The small RNA containing the 3'-OH terminus was then denatured in dimethyl sulfoxide (DMSO) and fluorescently labeled using a Cy3-labeled enzyme via a ligation reaction. The Cy3-labeled tsRNA was hybridized to an Arraystar human small RNA matrix chip in an Agilent microarray hybridization oven. Sections were washed, scanned using an Agilent Scanner G2505C, and data were processed for tsRNA quantification. Differentially expressed tsRNA sequences are shown in Table 2.

[0062] Differential analysis used a threshold of 1.5 for the absolute value of the fold change, with fold change > 1.5 indicating upregulation and fold change < -1.5 indicating downregulation, to screen for differentially expressed tsRNAs.

[0063] 4. Quantitative detection of differentially expressed tsRNAs (reverse transcription + qPCR) (1) Specific reverse transcription of tRF (preparation of cDNA) Because the tRF fragment is short (sequence shown in Table 2), containing only about 20 nucleic acids, it needs to be lengthened before qPCR detection. The systems and methods used for tRF reverse transcription and mRNA reverse transcription differ and need to be prepared separately. For ordinary mRNA genes, the reverse transcription reaction system is prepared according to Table 3. After mixing, vortex and centrifuge for 5 seconds. The prepared reaction system is then placed in a PCR instrument and the following reaction program is executed: incubation at 37°C for 15 minutes, treatment at 85°C for 5 seconds, and storage at 4°C (until subsequent experiments). For tRF, the reverse transcription reaction system is prepared according to Table 4. After mixing, vortex and centrifuge for 5 seconds. The prepared reaction system is then placed in a PCR instrument and the following reaction program is executed: incubation at 16°C for 10 minutes, incubation at 42°C for 20 minutes, treatment at 85°C for 10 minutes, and storage at 4°C (until subsequent experiments). The sequences of reverse transcription primers and internal reference gene primers are shown in Table 5. All reverse transcription reaction systems are prepared under ice bath conditions.

[0064] Table 2 tRF sequences Table 3. mRNA reverse transcription reaction system (10µL) Table 4. tsRNA reverse transcription reaction system (10µL) Table 5 Primer sequences for internal reference genes (2) Real-time quantitative PCR (qPCR) Because tRF has been lengthened, tRF qPCR also requires specific primers (sequences are shown in Table 6). Therefore, the systems used for tRF and mRNA qPCR experiments are different and need to be prepared separately. The specific operations are as follows: cDNA template pretreatment: Take out the cDNA product obtained from reverse transcription, add 90µL of DEPC water to each cDNA sample, dilute the cDNA 10 times, and use it as a template for qPCR.

[0065] mRNA-qPCR reaction system (10µL): Prepare the reaction system under ice bath conditions according to the following components and volumes: 2×ChamQ Universal SYBR qPCR Master Mix: 5 µL, upstream primer (Primer-F): 0.5 µL, downstream primer (Primer-R): 0.5 µL, DEPC water: 2 µL, diluted cRNA template: 2 µL.

[0066] tRF-qPCR reaction system preparation (10µL): Prepare the reaction system under ice bath conditions according to the following components and volumes: 2×RT Master Mix: 5 µL, PCR Primer: 0.5 µL, DEPC water: 2.5 µL, diluted cDNA template: 2µL.

[0067] Table 6 Primers After preparing the reaction mixture, load 10 µL of sample into each well of the qPCR plate. Gently shake the plate after loading to allow the liquid to settle to the bottom. Carefully attach the sealing film and centrifuge to further collect the liquid at the bottom of the wells. Then, place the qPCR plate into a Roche LightCycler 480 II real-time quantitative PCR instrument and run the qPCR reaction according to the following parameters: Pre-denaturation stage: 95℃ for 5 minutes, 1 cycle; Amplification cycle stage: 40 cycles in total, each cycle including 95℃ denaturation for 5 seconds and 60℃ annealing / extension for 30 seconds; Melting curve analysis stage: temperature programmed to change from 95℃ to 60℃ to 95℃, continuously acquiring fluorescence data during this process, 1 cycle.

[0068] 5. In situ hybridization to verify tissue expression of Rtf In another study, 30 patients who received preoperative chemotherapy underwent postoperative tumor tissue sections. The expression level of tRF5-SeCTCA-1 was detected by in situ hybridization. The patients were divided into a sensitive group (TRG1 / 2) and a resistant group (TRG3) according to the chemotherapy response. The expression difference of tRF5-SeCTCA-1 between the two groups was compared.

[0069] In situ hybridization was performed as follows: Paraffin sections were baked in a hybridization oven at 70°C for 60 minutes to fix the tissue onto the sections. The sections were then dewaxed by sequentially immersing them in three solutions of xylene, 10 minutes in each solution. After dewaxing, the sections were then sequentially immersed in a gradient of 100%, 95%, and 75% ethanol solutions, 5 minutes each time, followed by rehydration in tap water. An immunohistochemical pen was used to circle the tissue at a distance of 0.5 mm, forming a closed loop.

[0070] Add peroxidase solution to the tissue, completely covering it for 30 minutes. Then, insert the staining rack into tap water to wash away the peroxidase solution. Repeat this process three times, for three minutes each time.

[0071] Dilute the pepsin stock solution with 2 drops of pepsin per milliliter of 3% citric acid, vortex to mix, and obtain the diluted pepsin working solution. Add freshly diluted pepsin working solution to the tissue sections, cover the tissue, and digest the tissue at room temperature for 10 minutes to expose RNA fragments. Then, wash the tissue sections with PBS four times, for 5 minutes each time, to remove the pepsin working solution.

[0072] 6. Data Analysis and Statistical Methods In this study, all data analyses were performed using R4.2.0 software, and statistical charts were created using GraphPad Prism 9 software. Independent samples t-tests were used to compare continuous variables between groups, and the Mann-Whitney U rank-sum test was used to compare ordinal data from two independent samples. All experiments were repeated at least three times. A p-value less than 0.05 was considered statistically significant. (Figure labels are provided.) This indicates that P < 0.05. This indicates that P < 0.01. ns indicates that P is less than 0.001, and ns indicates that P is greater than 0.05.

[0073] Independent samples t-test was used for comparisons of continuous variables between groups, and Mann-Whitney U rank-sum test was used for comparisons of ordinal data.

[0074] Ct value acquisition: Extract the Ct values ​​of the target gene and the internal reference gene actin from the real-time PCR instrument. Three technical replicates are required for each experiment, and the average value is used as the measurement data. If the Ct value deviation between replicate tubes is >0.5, the sample needs to be retested.

[0075] Relative expression level was 2 -ΔΔCt The formula for calculating ΔCt is: ΔCt = Ct (target gene) - Ct (Beta-actin). ΔCt is calculated using the chemotherapy-sensitive group as a baseline, ultimately yielding the relative expression level (Fold Change = 2).-ΔΔCt ).

[0076] Using chemotherapy-sensitive (TRG grade 1-2) samples as the baseline (calibrator), calculate ΔCt = ΔCt(test sample) - ΔCt(baseline sample).

[0077] Calculate the relative expression number (Fold Change): Relative Expression = 2 -ΔΔCt .

[0078] 7. Experimental Results (1) Microarray sequencing results A total of 4107 tsRNAs were detected in this sequencing study. They were classified according to the codons of the upstream tRNAs from which they originated, such as... Figure 1 and Figure 2 As shown in A and B, the proportions of different tRNA-derived tsRNA types and tsRNA subtypes vary. In this sequencing result, the tsRNA with the highest proportion was tRF5, reaching 34%, while the lowest was tRF1, at only 3.1%.

[0079] As shown in Tables 7 and 8, two genes, tRF5-22-SeCTCA-1 (hereinafter referred to as tRF-SeCTCA-1) and tRF5-22-SeCTCA-2 (hereinafter referred to as tRF-SeCTCA-2), were significantly upregulated in both chemotherapy groups. At the same time, tRF5-SeCTCA-1 was always the most significant. Compared with tissues that did not receive chemotherapy, tRF-SeCTCA-1 was upregulated 6-fold in the TRG3 group and 7-fold in the TRG1 / 2 group.

[0080] Table 7 Top 10 Differences Between TRG3 Group and Non-Chemo Group Table 8. Top 10 Differences Between TRG 1 / 2 Group vs. Non-Chemo Group (2) tRF-SeCTCA-1 is associated with chemotherapy resistance in colorectal cancer The results are as follows Figure 3As shown in Figures A and B, tRF-SeCTCA-1 expression was negative in the chemosensitive group and positive in the chemoresistant group. Furthermore, the expression level of tRF-SeCTCA-1 in the chemoresistant group was significantly higher than that in the chemosensitive group. These results indicate that high expression of tRF-SeCTCA-1 is closely related to resistance to 5-FU chemotherapy in colorectal cancer, suggesting that it may play an important role in the resistance mechanism and warrants further investigation.

[0081] Example 2: Regulation and clinical validation of tRF5-22-SeCTCA-1 on 5-FU chemotherapy resistance in colorectal cancer cells. This embodiment explores the regulatory effect of tRF5-22-SeCTCA-1 (tRF5-SeCTCA-1) on chemoresistance in colorectal cancer cells by constructing a tRF5-22-SeCTCA-1 overexpressing cell line, inhibiting tRF5-SeCTCA-1 expression in drug-resistant cells, and combining 5-fluorouracil (5-FU) related functional experiments. Its expression characteristics and clinical significance were verified using clinical tissue samples. The specific methods are described below: I. Cellular Level Validation 1. Source and preparation method of experimental cells The original cell line HCT116 was purchased from Guangzhou Saiku Biotechnology, cell number CC0506; HCT8 cells were purchased from Guangzhou Saiku Biotechnology, cell number CC0510.

[0082] 2. Experimental Methods (1) Construction and detection of drug-resistant cells: 5-FU-resistant cell lines HCT116-R and HCT8-R were established using a concentration gradient escalation method. HCT116 and HCT8 cells in the logarithmic growth phase were seeded into complete medium containing 5-FU for induction culture. At the start of induction, the concentration of 5-FU in the medium was set to the parental cell's IC50 concentration. 50 The cells were cultured at 20% concentration at 37°C with 5% CO2, and their condition was observed daily. After the cells stabilized, the drug concentration was increased by 20%, the medium was changed, and the cells were passaged and cultured again, repeating the concentration escalation process. After induction to the target concentration, the cells were cultured for 3 more passages to consolidate drug resistance, then transferred to drug-free medium and cultured for 2 weeks, with IC50 measured periodically. 50 After verifying stability, the cells were frozen for later use to complete the construction of drug-resistant cell lines.

[0083] The complete culture medium is prepared by mixing the following reagents in a clean bench at a volume ratio of 89:10:1: Basic culture medium: RPMI 1640 (Shanghai Yuanpei Biotechnology, catalog number: L210KJ) or high glucose DMEM (Shanghai Yuanpei Biotechnology, catalog number: L110KJ). In this example, RPMI 1640 is used as the basic culture medium. Fetal bovine serum (SERANA GmbH, Germany, catalog number: FBS-AS500); Penicillin-streptomycin bispecific antibody (Shanghai Yuanpei Biotechnology, catalog number: S110JV).

[0084] (2) tRF5-SeCTCA-1 overexpressing cells, the specific construction method is as follows: 1) Cell seeding and pretreatment: 24 hours in advance, healthy cells in the logarithmic growth phase (HCT116 cells and HCT8 cells, respectively) were seeded at a density of approximately 3 × 10⁶ cells per well. 5 Seeds were placed at a density of [number] cells per well in 6-well cell culture plates and incubated overnight to allow for full cell adhesion and proliferation. During transfection, cell confluence should be ensured to reach 60%–80%.

[0085] 2) Preparation of transfection complex: Take a 1.5 mL centrifuge tube, add 200 µL of jetPrime buffer and 10 µL of target RNA to each well (as shown in Table 9), vortex for 10 s to mix thoroughly, and then briefly centrifuge to allow the liquid to settle to the bottom of the tube. Add 10 µL of jetPrime transfection reagent to each well of the centrifuge tube, vortex again for 10 s to mix thoroughly, and incubate at room temperature for 10 minutes to form a stable RNA-jetPrime liposome transfection complex.

[0086] 3) Cell transfection: Gently aspirate the existing culture medium from each well of the 6-well plate and add 1.8 mL of complete culture medium to each well. Add 10 µL of the prepared transfection complex directly to the corresponding cell culture well, and gently shake the plate to distribute the transfection complex evenly. Return the plate to a 37°C, 5% CO2 incubator and continue culturing. Replace with complete culture medium after 4 hours.

[0087] Cells were harvested 24 hours after transfection to extract RNA and assess transfection efficiency. Cells were harvested 48 hours after transfection to extract protein and assess protein expression efficiency. Cells were cultured with gradient concentrations of 5-FU, with three parallel wells for each concentration. Dose-response curves were plotted, and IC50 was calculated using nonlinear regression. 50 Cells obtained using the above construction method were then used, and 5-FU was added to a complete culture medium at a final concentration of 5 µM. Cell proliferation assays, plate colony assays, and flow cytometry double staining apoptosis assays were performed to analyze the regulatory effect of tRF5-SeCTCA-1 overexpression on cell proliferation, colony formation, and apoptosis under the action of 5-FU.

[0088] Table 9 Transfected RNA Sequences (3) Cells that inhibit tRF-SeCTCA-1 expression are constructed as follows: Inhibition experiments of tRF5-22-SeCTCA-1 were performed on drug-resistant cell lines HCT116-R and HCT8-R. The specific method is as follows: In the experimental group, drug-resistant cells were transfected with a specific inhibitor of tRF5-SeCTCA-1 (tRF inhibitor, specific sequence shown in Table 10) to reduce the expression level of tRF5-SeCTCA-1; in the control group, drug-resistant cells were transfected with a negative control sequence of the inhibitor. The transfection procedure was performed according to the procedure for transfecting tRF5-22-SeCTCA-1 overexpressing cell lines.

[0089] After transfection, the inhibitory effect was verified by real-time quantitative PCR. Tumor cells were cultured in gradient concentrations of 5-FU, with three parallel wells for each concentration. Dose-response curves were plotted, and the IC50 was calculated using nonlinear regression. 50 Cells obtained using the above construction method were then used, and 5-FU was added to a complete culture medium at a final concentration of 5 µM. Cell proliferation assays, plate colony assays, and flow cytometry double staining apoptosis assays were performed to analyze the regulatory effect of tRF5-SeCTCA-1 overexpression on cell proliferation, colony formation, and apoptosis under the action of 5-FU.

[0090] Table 10. Specific inhibitor sequences of tRF5-SeCTCA-1 3. Experimental Results (1) Results of drug-resistant cell detection like Figure 4 As shown in A, the HCT116 original cell line IC 50 It is 5.672µM, and the IC50 of the HCT8 original cell line is... 50 It is 3.752µM; while the HCT116-R drug-resistant cell line IC 50 The IC50 of the HCT8-R drug-resistant cell line was 27.58 µM. 50 It is 11.07µM. The resistance curve is as follows: Figure 4 As shown in B and C, the expression of tRF-SeCTCA-1 was compared between the drug-resistant and original cell lines. qPCR results showed ( Figure 4As shown in Figure D), the expression level of tRF-SeCTCA-1 in both cell lines was significantly higher than that in their corresponding original cell lines (HCT116-R and HCT8-R), increasing by 8-fold and 10-fold, respectively. This finding suggests that high expression of tRF5-22-SeCTCA-1 may be closely related to the resistance of colorectal cancer cells to 5-FU.

[0091] (2) Detection results of cells overexpressing tRF-SeCTCA-1 The results are as follows Figure 5 As shown in Figure A, compared with the NC mimic group, the expression level of tRF-SeCTCA-1 in HCT116 and HCT8 cells in the mimic group was significantly upregulated, indicating that the tRF-SeCTCA-1 mimic transfection was successful.

[0092] HCT8 and HCT116 cells followed by tRF-SeCTCA-1 mimicry IC 50 Changes such as Figure 5 As shown in B~D, HCT116 cells, NC mimic group IC 50 The value was 5.597 μM; IC of the tRF-SeCTCA-1 mimic group 50 The value significantly increased to 16.82 μM (P<0.01). Similarly, in HCT8 cells, the IC50 value in the tRF5-22-SeCTCA-1 mimic group was significantly higher. 50 The IC value of the NC mimic group increased significantly compared to the NC mimic group. 50 The value was 3.725 μM, IC of the tRF5-22-SeCTCA-1 mimic group. 50 The value increased to 10.93 μM (P < 0.01). Using IC... 50 (mimic group) / IC 50 (Control group) The drug resistance index RI was calculated and found to be 3 (HCT116) and 2.91 (HCT8), indicating that overexpression of tRF-SeCTCA-1 increased the drug resistance of cells by more than 2 times.

[0093] Cell proliferation results showed ( Figure 6 Cells transfected with the mimic tRF-SeCTCA-1 mimic (A and B in the original text) significantly resisted the inhibitory effect of fluorouracil on cell proliferation in the presence of the drug, and the proliferation capacity of the overexpression group (mimic) was significantly higher than that of the control group (NC mimic). Similarly, the results of the plate colony assay showed that ( Figure 7In the cases of A and B), under complete culture medium containing 5 µM fluorouracil, the HCT116 and HCT8 cells overexpressing tRF-SeCTCA-1 formed significantly more cell colonies than the control group. Cells were cultured in 5 µM 5-FU medium for 48 hours, and then flow cytometry was used for double staining apoptosis detection. The results showed that (…). Figure 8 and Figure 9 In the case of drug-treated culture, the number of apoptotic cells in the control group was significantly higher than that in the tRF-SeCTCA-1 overexpression group. These results indicate that upregulation of tRF-SeCTCA-1 expression can enhance the tolerance of colorectal cancer cells to 5-FU and reduce the inhibitory effect of 5-FU on colorectal cancer cells.

[0094] (3) Detection results of tRF-SeCTCA-1 inhibited cells Compared with the control group (NC inhibitor), the expression level of tRF-SeCTCA-1 was reduced by 30% in cells transfected with tRF-SeCTCA-1 inhibitor, demonstrating that the inhibitory effect was effective. Figure 10 (As shown in A in the diagram).

[0095] Drug IC 50 The results show ( Figure 10 (As shown in B~D), HCT116-R cells, IC50 of the NC inhibitor group 50 The value was 27.58 μM; the IC50 value of the tRF-SeCTCA-1 inhibitor group was 27.58 μM. 50 The value decreased significantly to 8.251 μM. Similarly, in HCT8-R cells, the IC50 value in the tRF-SeCTCA-1 inhibitor group was significantly lower. 50 The IC50 value was also significantly lower than that of the NC inhibitor group. 50 The value was 11.02 μM, IC50 of the inhibitor group 50 The value decreased to 6.154 μM, indicating that inhibition of tRF-SeCTCA-1 significantly reversed cellular drug resistance and restored cellular sensitivity to 5-FU.

[0096] Cell proliferation results showed ( Figure 11 As shown in A and B in the figure), cells transfected with tRF-SeCTCA-1 inhibitor exhibited significantly lower proliferation capacity than the control group in the presence of 5-FU, suggesting that inhibiting tRF-SeCTCA-1 expression significantly reduced the drug resistance of colorectal cancer cells compared to the control group (NC inhibitor). Plate colony assay results also showed… Figure 12As shown in A and B), under conditions of culture containing 5 µM 5-FU, the cell colonies formed by the tRF-SeCTCA-1 inhibitor group of drug-resistant HCT116-R and HCT8-R cell lines were significantly fewer than those formed by the NC inhibitor group. Flow cytometry double staining apoptosis detection results showed that ( Figure 13 and Figure 14 In the case of drug-treated culture, the number of apoptotic cells in the tRF-SeCTCA-1 inhibitor group was significantly higher than that in the NC mimic group.

[0097] It can be seen that by inhibiting the expression of tRF-SeCTCA-1, the sensitivity of drug-resistant cells to 5-FU can be restored, which again suggests that overexpression of tRF-SeCTCA-1 can promote the tolerance of colorectal cancer cells to 5-FU.

[0098] II. Clinical validation experiments Following the inclusion criteria in Example 1, 228 colorectal cancer patients from Sun Yat-sen Memorial Hospital of Sun Yat-sen University were selected. In situ hybridization was used to detect the expression level of tRF5-SeCTCA-1 in clinical tissue samples (postoperative tumor tissue and corresponding adjacent normal tissue samples) of colorectal cancer patients. The expression differences between tumor tissue and corresponding adjacent normal tissue were compared and analyzed. Furthermore, combined with clinical case data, the correlation between tRF5-SeCTCA-1 expression level and patient clinical characteristics (such as tumor stage) was analyzed, and its correlation with prognosis was evaluated.

[0099] The results showed that tRF5-SeCTCA-1 expression in tumor tissues was significantly higher than that in the corresponding adjacent normal tissues. Figure 15 (As shown in A and B in Table 11). Further analysis with clinical characteristics (see Table 11) revealed a difference in tRF5-SeCTCA-1 expression between M0 and M1 stages (P<0.01), suggesting that tRF5-SeCTCA-1 is associated with poor prognosis. These results suggest that tRF5-SeCTCA-1 plays an important role in the development and progression of colorectal cancer, and its high expression may promote tumor progression and chemotherapy resistance, thus possessing potential clinical application value.

[0100] Table 11 Relationship between tRF5-SeCTCA-1 expression level and clinical characteristics Example 3: Organoid model validation of inhibiting tRF-SeCTCA-1 and reversing 5-FU resistance Two colorectal cancer tissues that met the inclusion criteria in Example 1 and had undergone 5-FU-based chemotherapy before surgery and showed drug resistance were collected from Sun Yat-sen Memorial Hospital of Sun Yat-sen University. Organoid models (PDOs) were constructed using these tissues and designated as PDO1 and PDO2, respectively, to further verify the role of tRF-SeCTCA-1 in chemotherapy resistance in colorectal cancer.

[0101] 1. Methods for constructing tumor organoids: (1) Sampling and cleaning: Fresh tumors were removed and transported in ice-cold PBS; in a clean bench, the tumors were randomly transferred to PBS containing penicillin-streptomycin antibiotics, and the tumors were repeatedly inverted and shaken 5 times to further clean them and remove necrotic tissue.

[0102] (2) Mechanical shearing: cut the tissue into small pieces of 1-2 mm to increase the surface area.

[0103] (3) Enzymatic digestion: Depending on the size of the tissue, add type I collagenase to fully cover the tumor tissue, and incubate at 37°C by rotation until it is separated into single cells or small clumps; add complete culture medium containing fetal bovine serum to terminate digestion.

[0104] (4) Filtration and collection: Pass the digestion product through a 70µm filter, collect the filtrate, centrifuge at 1000 RPM for 5 minutes, discard the supernatant, resuspend in ice-cold PBS and count.

[0105] (5) Plate culture: Mix cell suspension and matrix gel at a volume ratio of 1:4 on ice; seed into 24-well plates (50µL / well); incubate at 37℃ for solidification (15min upright + 15min upside down); add organoid culture medium (manufacturer: Zhongke Puruisheng; catalog number: PRS-ICM-3D).

[0106] 2. Organoid passage After aspirating the culture medium, add 500 µL / well of frozen StemPro. TM Accutase TM Cell dissociation reagents were used to mechanically dissociate organoids and separate the matrix gel. Serum-containing culture medium and a large amount of ice-cold PBS were then added to terminate the dissociation reaction and melt the residual matrix gel. The mixture was centrifuged at 1300 RPM for 5 minutes. If there was still residual matrix gel after centrifugation, the PBS washing and centrifugation steps were repeated. The cell pellet was then resuspended in PBS and mixed with matrix gel at a ratio of 1:4 before it could be plated for culture or cryopreserved.

[0107] 3. Organoid RNA transfection After organoids regained viability 24 hours after passage, transfection experiments were performed. 100 µL of jetPRIME buffer and 10 µL of RNA solution (from Table 10) were added to each well of a 1.5 mL centrifuge tube, mixed by pipetting and vortexing for 5 seconds, then centrifuged. Next, 10 µL of jetPRIME transfection reagent was added to each well, mixed by pipetting and vortexing for 10 seconds, centrifuged, and allowed to stand for 10 minutes. The original organoid culture medium was carefully aspirated, and 400 µL / well of organoid culture medium was added, followed by 100 µL of transfection mix. Subsequent experiments were performed 48 hours after transfection. The half-maximal inhibitory concentration (IC50) of the drug was determined after 48 hours of continuous culture.

[0108] 4. Half-maximal inhibitory concentration (IC50) of the drug 50 ) Measurement Organoids were digested and dissociated into single cells, centrifuged at 1300 RPM, and the final cell concentration was adjusted to 2.5 × 10⁻⁶. 5 Cell suspensions were prepared at a concentration of 1 cell / mL (including Matrigel) and seeded into 96-well plates (20µL / well). A blank control, gradient concentrations, and a negative control group were set up, with three parallel wells for each group. 20µL of cell suspension was added to each well. After 30 minutes, the Matrigel solidified, and organoid culture medium was added. After 24 hours, organoid viability was observed under a microscope. The original organoid culture medium was then replaced with organoid culture medium containing 5-FU. After 96 hours of drug-containing culture, 100µL of CCK8 working solution was added to each well of the culture medium. The plates were incubated at 37°C, 5% CO2, in the dark for 3 hours. The absorbance at 450nm was measured using a multi-mode microplate reader, and the IC50 of the organoids was calculated. 50 .

[0109] 5. Test Results like Figure 16 As shown in A~C, after successful organoid construction, its drug IC was measured. 50 PDO1 IC 50 The IC of PDO2 is 25.48 μM. 50The concentration was 29.17 μM. The same negative control oligonucleotide (NC inhibitor) and tRF-SeCTCA-1 specific inhibitor (tRF-SeCTCA-1 inhibitor) as in Example 2 were transfected into the control and inhibition group organoids, respectively. After transfection, the organoids were passaged, and 15 μM 5-FU was added to the culture medium. The cultures were incubated at 37°C in a 5% CO2 incubator, and observed under a microscope daily. After 5 days of culture, photographs were taken. The control group organoids (NC inhibitor) showed slower growth after the addition of 5-FU, but still maintained a certain degree of proliferative capacity. However, the growth of the inhibition group (tRF-SeCTCA-1 inhibitor) organoids was significantly inhibited, with some organoids showing atrophy and disintegration. This indicates that inhibiting tRF-SeCTCA-1 expression can significantly enhance the sensitivity of patient-derived organoids (PDO) models from colorectal cancer to 5-FU chemotherapy.

[0110] Example 4: Animal model validation of inhibiting tRF-SeCTCA-1 and reversing 5-FU resistance I. Laboratory Animals Male BALB / c-nu nude mice aged 4-6 weeks were purchased from Area C (large and small mouse experiments) of the Experimental Animal Center, East Campus, Sun Yat-sen University, with Experimental Animal Use License No.: SYXK (Guangdong) 2023-0112. They were acclimatized for one week to ensure their health.

[0111] II. Animal Model Construction In this embodiment, a mouse subcutaneous tumor model was used to verify the inhibition of tRF-SeCTCA-1 and reversal of 5-FU resistance. Specifically, male BALB / c-nu nude mice aged 4-6 weeks, weighing 18-22g, were selected. HCT116 tumor cells and the drug-resistant cell line HCT116-R were cultured to the logarithmic growth phase to ensure cell viability. Cells were then digested with trypsin, collected, and counted. The tumor cells were resuspended in sterile, ice-cold PBS, and the cell concentration was adjusted to 1×10⁻⁶. 7 Cells / mL, placed in an ice box, and transported to the animal facility as soon as possible to maintain viability.

[0112] Before inoculation, pipette the cells to ensure even distribution. Use a 1 mL sterile syringe to take 100 µL of cell suspension (approximately 1 × 10⁻⁶ cells). 6 HCT116 cells (1 × 10⁻⁶ cells) were injected subcutaneously into the axilla of one nude mouse, and the mice were randomly divided into four groups: agomir group, NCagomir group, antagomir group, and NC antagomir group. The agomir group and NC agomir group were injected with the original HCT116 cell line at a dose of 1 × 10⁻⁶ cells. 6Cells per mouse; the antagomir group and the NC antagomir group were injected with the HCT116 resistant cell line, injection dose: 1×10⁻⁶ cells / mouse; 6 Cells per mouse. Tumor growth was observed and measured every 3 days after injection, and tumor volume was calculated. When the tumor reached a diameter of 5 mm, the first intratumoral injection of agomir / NC agomir / antagomir / NC antagomir was administered at a dose of 5 mol per tumor per injection. The miRNA mimic sequences are shown in Table 12. A second injection was administered one week later. After the first intratumoral RNA injection, 5-FU was administered intraperitoneally every 3 days. Data from each measurement was recorded, and a tumor growth curve was plotted. Tumor volume calculation formula: Volume = 1 / 2 × Length × Width 2 .

[0113] Table 12 miRNA mimic sequences Note: In the table, Agomir (miRNA agomir) is a miRNA mimic with special chemical modifications. It is modified on the antisense strand, with cholesterol at the 3' end, two thiocarbon backbones at the 5' end, four thiocarbon backbones at the 3' end, and full-chain methoxyl modification. NC agomir is the Agomir control mimic and has no function. Antagomir is a miRNA antagonist with cholesterol at the 3' end, two thiocarbon backbones at the 5' end, four thiocarbon backbones at the 3' end, and full-chain methoxyl modification.

[0114] III. Experimental Results The results are as follows Figure 17 As shown in Figures A and B, in the drug-resistant cell group, tumor growth in mice injected with antagomir was significantly inhibited, with tumor volume reduced by approximately 60% compared to the control group (P<0.01). After harvesting the tumors, we embedded the tumor tissues in paraffin and prepared tissue sections for further analysis. First, in situ hybridization (ISH) was used to detect the expression of tRF-SeCTCA-1 in the tumor tissues. The results are shown in Figures A and B. Figure 18 As shown in A and B, the expression signal of tRF-SeCTCA-1 in the tumor tissue of the drug-resistant cell group after injection of antagomir was significantly weakened, and the in situ hybridization staining showed weak positive or even negative results, proving that the expression of this molecule was successfully inhibited.

[0115] Subsequently, we performed Ki67 immunohistochemical staining to assess the proliferative activity of tumor cells. The results are as follows: Figure 19As shown in A and B, the proportion of Ki67-positive cells was significantly reduced in tumor tissues derived from drug-resistant cells that inhibited tRF-SeCTCA-1 (antagomir group), indicating that cell proliferation was inhibited. This histologically validated the key role of tRF-SeCTCA-1 in regulating the malignant proliferation of chemotherapy-resistant colorectal cancer cells, providing direct in vivo experimental evidence for targeting tRF-SeCTCA-1 to reverse chemotherapy resistance in colorectal cancer and inhibit tumor growth.

[0116] Example 5: α-Ketoglutarate reverses tRF5-SeCTCA-1-induced metabolic reprogramming and chemotherapy resistance I. Cells HCT116 was purchased from Guangzhou Saiku Biotechnology, cell number CC0506; HCT8 cells were purchased from Guangzhou Saiku Biotechnology, cell number CC0510.

[0117] II. Experimental Methods: Three concentration gradients of α-ketoglutarate (α-KG) (1 mM, 5 mM, and 10 mM) were added to the complete culture medium (the same formulation as in Example 2), and 5-FU was added to a final concentration of 5 μM. Colorectal cancer HCT116 and HCT8 cells overexpressing tRF5-SeCTCA-1 were cultured for 5 days at 37°C under 5% CO2 and 95% air conditions. During culture, cell viability was assessed using the CCK-8 assay, dose-response curves were plotted, and IC50 was calculated. 50 The drug resistance of two types of colorectal cancer cells was tested, and the effect of α-ketoglutarate on the 5-FU resistance of cells was analyzed.

[0118] III. Experimental Results In the tricarboxylic acid cycle, IDH3B catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate. α-ketoglutarate (α-KG) is not only an intermediate product of the TCA cycle, but can also participate in epigenetic regulation (such as histone and DNA demethylation) as a signaling molecule.

[0119] By supplementing with α-ketoglutarate, we found that as the concentration of α-ketoglutarate increased, the chemotherapy resistance induced by overexpression of tRF5-SeCTCA-1 gradually decreased. Figure 20 (A and B in the text).

[0120] To avoid direct tumor cell death due to excessively high α-ketoglutarate concentrations, we supplemented the culture medium with 5 mM α-ketoglutarate for our experiments. By supplementing α-ketoglutarate in cells overexpressing tRF5-SeCTCA-1, we observed its effects on cellular metabolic function and chemosensitivity. The results showed that α-ketoglutarate partially reversed metabolic reprogramming and chemosensitivity induced by tRF5-SeCTCA-1 overexpression. The results indicated ( Figure 21 As shown in Figures A-D), in colorectal cancer cells overexpressing tRF5-SeCTCA-1, the addition of α-ketoglutarate significantly reduced glycolytic activity and extracellular acidification rate (ECAR). However, mitochondrial oxidative phosphorylation function was also significantly altered.

[0121] More importantly, exogenous supplementation of α-ketoglutarate increased the sensitivity of cells to the chemotherapeutic drug 5-FU. Figure 22 (A and B in the text). Cells overexpressing tRF5-SeCTCA-1 showed an IC50 of 5-FU after α-ketoglutarate supplementation. 50 The levels decreased by approximately 50%. These results indicate that tRF5-SeCTCA-1 induces chemotherapy resistance by downregulating IDH3B expression, leading to decreased α-ketoglutarate levels, inhibiting mitochondrial function, and promoting glycolysis. Supplementation with α-ketoglutarate can partially compensate for this metabolic defect, inhibiting glycolysis, reversing metabolic reprogramming, and enhancing cellular sensitivity to chemotherapeutic drugs. Therefore, our results show that α-ketoglutarate supplementation provides a new approach to overcoming chemotherapy resistance in colorectal cancer, suggesting that regulating metabolic pathways may be an effective strategy for improving chemotherapy efficacy.

[0122] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0123] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A tRF5-22-SeCTCA-1, characterized in that, The nucleotide sequence of tRF5-22-SeCTCA-1 is shown in SEQ ID NO:

1.

2. The use of the detection reagent of tRF5-22-SeCTCA-1 as described in claim 1 in the preparation of reagents or kits for diagnosing chemotherapy resistance in colorectal cancer.

3. A kit for detecting chemotherapy resistance in colorectal cancer, characterized in that, The kit contains reagents for detecting the expression level of tRF5-22-SeCTCA-1 as described in claim 1.

4. The kit according to claim 3, characterized in that, The detection reagent includes a primer pair for amplifying the tRF5-22-SeCTCA-1; the nucleotide sequences of the primer pair are shown in SEQ ID No: 5 and SEQ ID No:

6.

5. The application of the detection reagent of tRF5-22-SeCTCA-1 as described in claim 1 in the screening of colorectal cancer drugs.

6. The use of at least one of the following reagents in the preparation of drugs for treating colorectal cancer and / or reversing chemotherapy resistance in colorectal cancer: a. A reagent that inhibits the expression or activity of tRF5-22-SeCTCA-1 as described in claim 1; b. α-Ketoglutarate.

7. The application as described in claim 6, characterized in that, The reagent that inhibits the expression or activity of tRF5-22-SeCTCA-1 is selected from antisense oligonucleotides, small molecule compounds that inhibit the expression of tRF5-22-SeCTCA-1, and one or more.

8. The application as described in claim 6, characterized in that, The reagents that inhibit the expression or activity of tRF5-22-SeCTCA-1 include the sequences shown in SEQ ID No: 11 or SEQ ID NO:

17.

9. The application as described in claim 8, characterized in that, The chemotherapy resistance in colorectal cancer includes chemotherapy resistance caused by one or more of the following drugs: 5-fluorouracil, leucovorin, capecitabine, oxaliplatin, and irinotecan.

10. A pharmaceutical composition for treating colorectal cancer, characterized in that, The active ingredient of the pharmaceutical composition is selected from at least one of the following, and a pharmaceutically acceptable carrier: a. A reagent that inhibits the expression or activity of tRF5-22-SeCTCA-1 as described in claim 1; b. α-Ketoglutarate.

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