Prostate cancer treatment target and application thereof

Abstract

The invention discloses a prostate cancer treatment target spot and application thereof, and belongs to the technical field of biological medicine, and the prostate cancer treatment target spot comprises that the treatment target spot is tRF-1: T16-Glu-TTC-2-2. The invention relates to an application of a prostate cancer treatment target, in particular to an effect of tRF-1: T16-Glu-TTC-2-2 in proliferation, migration and invasion of prostate cancer cells. According to the prostate cancer early screening kit based on serum tRF-1: T16-Glu-TTC-2-2 detection, prostate cancer is diagnosed in an early stage, and the response of a patient to treatment is tracked by monitoring the dynamic change of the serum tRF-1: T16-Glu-TTC-2-2 level, so that a treatment scheme is adjusted in time. Specific drugs are researched and developed according to a signal channel and a target gene related to tRF-1: T16-Glu-TTC-2-2, and the prostatic cancer treatment pertinence is favorably provided.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and specifically to a prostate cancer treatment target and its application. Background Art

[0002] Prostate cancer is one of the common malignant tumors in men, and its incidence and mortality rates remain high globally. Due to the insidious onset of prostate cancer, the best treatment opportunity is often missed when it is discovered. Although existing treatment methods (such as surgery, radiotherapy, hormone therapy, and chemotherapy) can control the condition to a certain extent, for advanced or metastatic prostate cancer, the treatment effect is still limited, and drug resistance is likely to occur.

[0003] Due to the advantages of convenience, economy, rapidity, and simplicity of serological tests, detecting prostate-specific antigen as a method for early diagnosis of prostate cancer has become the currently recognized best way. However, when a tumor occurs, the concentrations of multiple substances in the serum will change greatly, so the detection sensitivity and specificity of a single tumor marker are greatly limited, thus restricting the effect of early diagnosis of prostate cancer.

[0004] In recent years, tRF (tRNA-derived fragments), as a new type of non-coding RNA, the role of tRF in tumorigenesis and development has been gradually revealed. tRF can participate in tumor progression by regulating gene expression, affecting the cell cycle, and apoptosis. In prostate cancer, certain tRFs have been found to be abnormally expressed and are closely related to tumor progression, metastasis, and drug resistance. Therefore, in-depth study of the biological functions of tRFs and their mechanism of action in prostate cancer will provide new ideas and methods for the diagnosis and treatment of prostate cancer. For this reason, a prostate cancer treatment target and its application are proposed.

[0005] The above information disclosed in this background art is only used to increase the understanding of the background art of the present invention. Therefore, it may include prior art that is not known to those of ordinary skill in the art. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art. For this reason, an object of the present invention is to propose a prostate cancer treatment target and its application.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The application of tRF-1:T16-Glu-TTC-2-2 as a biomarker in the diagnosis of prostate cancer. This nucleic acid is a non-coding RNA with a length of 16 nt, which is generated after the degradation of the 3' tail sequence at the end of the precursor tRNA-Glu-TTC-2-2 located on chromosome 15. Its sequence is TGACTGGACCTTTCTT, and its expression level in the serum of prostate cancer patients is significantly higher than that of healthy individuals, and it can be used as a biomarker for the diagnosis of prostate cancer.

[0009] Furthermore, the above application includes detecting the expression level of tRF-1:T16-Glu-TTC-2-2 (sequence: TGACTGGACCTTTCTT) in the blood sample of the subject; comparing the expression level of tRF-1:T16-Glu-TTC-2-2 with a preset value. If it is higher than the preset value, the possibility of malignancy is high, otherwise the possibility of inflammation is high.

[0010] A prostate cancer treatment target, including: the treatment target is tRF-1:T16-Glu-TTC-2-2, and the sequence is TGACTGGACCTTTCTT.

[0011] The application of a prostate cancer treatment target, the role of tRF-1:T16-Glu-TTC-2-2 (sequence: TGACTGGACCTTTCTT) in the proliferation, migration and invasion of prostate cancer cells.

[0012] A kit for diagnosing prostate cancer, the kit includes a diagnostic kit for tRF-1:T16-Glu-TTC-2-2 (sequence: TGACTGGACCTTTCTT), which is used for the early diagnosis and prognosis evaluation of prostate cancer.

[0013] A method for diagnosing prostate cancer is provided, and the method includes the following steps:

[0014] S1: Obtain the serum sample of the subject;

[0015] S2: Detect the expression level of tRF-1:T16-Glu-TTC-2-2 in the serum sample of the subject;

[0016] S3: Compare the expression level of tRF-1:T16-Glu-TTC-2-2 with a preset value. If it is higher than the preset value, the possibility of malignancy is high; otherwise the possibility of inflammation is high.

[0017] Detecting the expression level of tRF-1:T16-Glu-TTC-2-2 in the serum sample of a subject includes extraction of serum RNA, preparation of cDNA, and amplification of tRF-1:T16-Glu-TTC-2-2. For the preparation and amplification of cDNA of tRF-1:T16-Glu-TTC-2-2, the RNA sample obtained by extraction is subjected to cDNA preparation and fluorescence quantitative PCR amplification using the GenStar StarScript III miRNA RT Kit (by Stem-loop).

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] The present invention reveals the abnormal expression of tRF-1:T16-Glu-TTC-2-2 (sequence: TGACTGGACCTTTCTT) in prostate cancer, and uses it as a potential early diagnosis marker. Moreover, the expression level of tRF-1:T16-Glu-TTC-2-2 (sequence: TGACTGGACCTTTCTT) can be used as an indicator for monitoring tumor progression and treatment effect.

[0020] The present invention provides an early screening kit for prostate cancer based on the detection of serum tRF-1:T16-Glu-TTC-2-2 (sequence: TGACTGGACCTTTCTT) to early diagnose prostate cancer and track the patient's response to treatment by monitoring the dynamic changes in the serum tRF-1:T16-Glu-TTC-2-2 (sequence: TGACTGGACCTTTCTT) level, so as to adjust the treatment plan in a timely manner. In addition, specific drugs are developed for the signal pathways and target genes related to tRF-1:T16-Glu-TTC-2-2 (sequence: TGACTGGACCTTTCTT), which helps to provide targeted treatment for prostate cancer.

[0021] The above summary is only for the purpose of the specification and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a flow chart for the identification and mechanism of action of tRF in the present invention.

[0023] Figure 2 It is a tRNA map from which the sequence of tRF-1:T16-Glu-TTC-2-2 in the GtRNAdb database is derived.

[0024] Figure 3Chromosome localization map of tRF-1:T16-Glu-TTC-2-2 in the UCSC database.

[0025] Figure 4 Amplification curve and melting curve of tRF-1:T16-Glu-TTC-2-2 detected by real-time fluorescence quantitative PCR provided in Example 1.

[0026] Figure 5 Schematic diagram of PCR product cloning and sequencing provided in Example 1.

[0027] Figure 6 Schematic diagram of experimental agarose gel electrophoresis of PCR products provided in Example 1.

[0028] Figure 7 Schematic diagram of the expression difference of tRF-1:T16-Glu-TTC-2-2 in the sera of prostate cancer patients and healthy individuals provided in Example 1.

[0029] Figure 8 ROC curve and area under the curve (AUC) graph for examining the diagnostic efficacy of tRF-1:T16-Glu-TTC-2-2 provided in Example 1. Detailed implementation manners

[0030] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0031] Please refer to Figures 1 - 3 , Example 1

[0032] For the identification of tRF, the specific steps are as follows:

[0033] Step 1: Extract total RNA from epithelial cells of patients with prostate cancer and normal prostate epithelial cells, and perform high-throughput sequencing.

[0034] Step 2: Through bioinformatics analysis, screen out tRF molecules that are significantly differentially expressed in prostate cancer (tRF-1:T16-Glu-TTC-2-2, whose sequence is TGACTGGACCTTTCTT).

[0035] Step 3: Collect serum samples from prostate cancer patients and healthy controls, and detect the expression level of tRF-1:T16-Glu-TTC-2-2 (sequence is TGACTGGACCTTTCTT) by qPCR.

[0036] To provide the detection, identification and clinical diagnostic value of serum tRF-1:T16-Glu-TTC-2-2 in prostate cancer patients. The patients are from the First Affiliated Hospital of Jinan University, and the specific steps are as follows:

[0037] S1, Extraction of serum RNA. The specific steps are as follows:

[0038] (1) Serum preparation: Collect 10 mL of blood from each subject and place it in an EATA anticoagulant tube. After blood collection, gently invert the blood collection tube 4 - 5 times, and then place it upright at room temperature until the blood completely coagulates (about 1 h). Separate the serum at a centrifugation speed of 1000 rpm for 10 min. Transfer the collected serum to a separate cryopreservation tube, and aliquot 200 - 300 μL per tube. Samples for short-term storage will be placed in a -20°C or -80°C refrigerator, and samples for long-term storage will be placed in liquid nitrogen waiting for the next experiment.

[0039] (2) Extract serum RNA: Use Trizol Reagent (Ambion, USA, catalog number 15596018) to extract total RNA from the sample serum. Take out the serum specimen from the liquid nitrogen tank and thaw it at room temperature. Add 250 μL of serum to 750 μL of TRIzol reagent and let it stand at room temperature for 5 min. Then add 200 μL of chloroform, seal the tube cap, and shake vigorously for 30 s. Let it stand at room temperature for 5 min. Centrifuge at 12000 rpm for 15 min at 4°C. After centrifugation, the mixture is divided into 3 layers. Transfer the upper aqueous phase to a new 1.5 mL Eppendorf tube and label it. Then add an equal volume of isopropanol to precipitate RNA. Let it stand at room temperature for 15 min. Centrifuge at 12000 rpm for 10 min at 4°C and discard the supernatant. Add 1 mL of 75% ethanol solution to wash the RNA precipitate and shake gently. Centrifuge at 7500 rpm for 5 min at 4°C. Discard the supernatant, centrifuge the empty tube at 12000 rpm for 3 min at 4°C, and air dry at room temperature for 3 min. Finally, add 25 μL of DEPC water to dissolve the RNA precipitate and aspirate about 20 times.

[0040] (3) RNA concentration determination: From the extracted 10 μL of total RNA, accurately pipette 1 μL of RNA using a pipette and detect it using a Nano-drop instrument to measure the OD260 and OD280 values. The selected RNA should have high purity, and the A260 / A280 ratio should be between 1.8 and 2.1.

[0041] S2, Detection, identification and clinical diagnostic value of serum tRF-1:T16-Glu-TTC-2-24 in prostate cancer patients. The specific steps are as follows:

[0042] (1) Preparation of cDNA: Using the StarScript III miRNA RT Kit (by Stem-loop) (purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd., product number A237-02), the RNA samples extracted were used for cDNA preparation. The reaction system was prepared according to Table 1 for cDNA preparation.

[0043] Component 20 μL Reverse Transcription Reaction System RNA Sample 10 pg - 1 μg Total RNA Stem-loop RT primer 1 μL 2×StarScript III miRNA-L Buffer 10 μL StarScript III miRNA-L Enzyme Mix 1 μL DEPC Water Make up to 20 μL Total 20 μL

[0044] Table 1 Reaction system for cDNA preparation

[0045] Note: The amount of Stem-loop RT primer can be adjusted between 2 - 5 μM according to experimental requirements. The reverse transcription reaction conditions are as follows: 25°C for 5 min, 50°C for 15 min, 85°C for 5 min. The cDNA obtained after the reaction can be continued for experiments on ice or stored in a -20°C or -80°C refrigerator after proper labeling.

[0046] (2) Real-time fluorescence quantitative qPCR detection: For amplification, the GenStar 2×RealStar Fast SYBR qPCR Mix (purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd., product number Cat#A301) was used to prepare the real-time fluorescence quantitative PCR reaction system. Using the reverse-transcribed cDNA as a template for fluorescence quantitative PCR amplification, the ABI7500 system was applied for qRT-PCR experiments on the cDNA prepared in step 1. The upstream and downstream primers of tRF-Val-CAC-024 were commissioned by Suzhou GenePharma Co., Ltd. to be synthesized. A qRT-PCR reaction system containing tRF-1:T16-Glu-TTC-2-2 was configured (as shown in Table 2).

[0047] Component Volume cDNA 1 μL 2×RealStar FastSYBR qPCR Mix 10 μL PCR Forward Primer 0.5 μL PCR Reverse Primer 0.5 μL DEPC Water Make up to 20 μL Total 20 μL

[0048] Table 2 qRT-PCR reaction system

[0049] Temperature Time Number of Cycles Pre-denaturation at 95°C 2 min Amplification at 95°C 15s 40 Annealing and Extension at 60°C 40s

[0050] Table 3 RT-qPCR reaction conditions

[0051] Analysis of PCR results: The amplification curves and melting curves shown by the qPCR results are as attached Figure 4 shown. The left figure is the amplification curve, showing an S shape and the CT value difference of each replicate well in each group is less than 0.5 CT values, meeting the standard; the right figure is the melting curve. The melting curve of the tRF-1:T16-Glu-TTC-2-2 primer is a single peak, indicating that the amplification product is single. The Tm value represents the melting temperature of the primer itself. The Tm value of tRF-1:T16-Glu-TTC-2-2 is 79°C, meeting the primer design requirements.

[0052] (3) Cloning, sequencing and agarose gel electrophoresis of PCR experimental products. The results of cloning and sequencing of PCR experimental products are shown in the appendix Figure 5 as follows. tRF-1:T16-Glu-TTC-2-2 (sequence: TGACTGGACCTTTCTT). The results of cloning and sequencing showed that the PCR products contained the complete tRF-1:T16-Glu-TTC-2-2 sequence, which confirmed the accuracy of PCR amplification and ensured the reliability of the experimental results.

[0053] Agarose gel electrophoresis experiment: Diluting 50×TAE buffer 10-fold can obtain 1×TAE buffer. The specific operation is as follows:

[0054] Take 10 ml of the stock solution and add 490 ml of distilled water to finally obtain 500 ml of the working solution. When preparing 1% agarose gel, first weigh 0.5 g of agarose powder and pour it into a conical flask. Then add 50 mL of 1×TAE buffer for dissolution. Seal the conical flask with tin foil and shake it manually to make the mixed solution uniform. Then heat it in the microwave oven at high power for 3 - 4 min until the liquid becomes clear. After heating, rinse the outer wall of the conical flask with tap water to cool it down to 45℃ - 55℃ (the standard is that it doesn't feel hot to the hand). Then, add 2.5 μL of EB dye and gently shake the bottle to make the nucleic acid dye evenly distributed. Finally, pour the shaken liquid into the gel casting tank, insert the comb teeth, and wait for 30 min for the gel to solidify. After loading the samples, perform electrophoresis in 1×TAE buffer under the conditions of: 150 V, 25 min.

[0055] The agarose gel electrophoresis results of PCR experimental products are shown in the appendix Figure 5 as follows. The leftmost in the figure is the DNA Marker (StarMarker 50bp Ladder, purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd., product number M018 - 01), and its bands indicate the sizes of DNA fragments. What is shown in the lane is the PCR product of tRF-1:T16-Glu-TTC-2-2, and its size is approximately 50 bp, which is consistent with the primer design and PCR experimental standards.

[0056] (3) Comparison of tRF expression differences in patient sera and ROC curve analysis

[0057] Appendix Figure 6The differential expression of tRF-1:T16-Glu-TTC-2-2 in the sera of prostate cancer patients and healthy individuals detected by real-time quantitative PCR is presented. The results show that the expression of tRF-1:T16-Glu-TTC-2-2 in the sera of prostate cancer patients is significantly higher than that in healthy individuals, and the difference is statistically significant (P<0.01). This result indicates that tRF-1:T16-Glu-TTC-2-2 can be used as a serum biomarker for the diagnosis of prostate cancer.

[0058] Appendix Figure 7 The diagnostic efficacy of tRF-1:T16-Glu-TTC-2-2 for prostate cancer patients evaluated by ROC curve analysis is presented. The results show that the AUC value is 0.8962 (the closer the AUC value is to 1, the better the diagnostic efficacy), indicating that detecting the abundance of tRF-1:T16-Glu-TTC-2-2 in the plasma of patients has good diagnostic efficacy for differentiating prostate cancer patients.

[0059] Example 2

[0060] For the functional study of tRF, the specific steps are as follows:

[0061] Step 1: Construct prostate cancer cell lines with overexpression and knockdown of tRF.

[0062] Step 2: Detect the effects of tRF-1:T16-Glu-TTC-2-2 on cell proliferation, migration, invasion, and apoptosis through cck8, scratch assay, Transwell assay, and cell cycle and apoptosis assays, etc.

[0063] Step 3: Establish a mouse model of prostate cancer to verify the role of tRF-1:T16-Glu-TTC-2-2 in vivo.

[0064] Example 3

[0065] For the study of the mechanism of action of tRF, the specific steps are as follows:

[0066] Step 1: Use bioinformatics analysis and experimental verification (such as RNA immunoprecipitation, luciferase reporter assay) to study the mechanism of action of tRF-1:T16-Glu-TTC-2-2.

[0067] Step 2: Construct a regulatory network of tRF-target gene-signaling pathway to reveal the molecular mechanism of tRF in prostate cancer.

[0068] Example 4

[0069] For the development of tRF as a diagnostic biomarker, the specific steps are as follows:

[0070] Step 1: Collect blood, urine or tissue samples from prostate cancer patients and healthy controls.

[0071] Step 2: Use qPCR to detect the expression level of tRF-1:T16-Glu-TTC-2-2 and verify its feasibility as a diagnostic marker.

[0072] Step 3: Develop a diagnostic kit based on tRF-1:T16-Glu-TTC-2-2 for early diagnosis and prognosis evaluation of prostate cancer.

[0073] Example 5

[0074] For verifying the application of tRF as a therapeutic target, the specific steps are as follows:

[0075] Step 1: Design and synthesize tRF-1:T16-Glu-TTC-2-2 inhibitors or mimics.

[0076] Step 2: Evaluate the therapeutic effect of tRF-1:T16-Glu-TTC-2-2 inhibitors or mimics on prostate cancer through in vitro and in vivo experiments.

[0077] Step 3: Conduct clinical trials to verify the safety and effectiveness of tRF inhibitors or mimics.

[0078] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example" or "some examples", etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example.

[0079] The parts not involved in the present invention are the same as the prior art or can be implemented by the prior art. Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A prostate cancer treatment target, characterized in that, Comprising: The treatment target is tRF-1:T16-Glu-TTC-2-2, with the sequence TGACTGGACCTTTCTT.

2. Use of the nucleic acid tRF-1: T16-Glu-TTC-2-2 as a biomarker according to claim 1 in the diagnosis of prostate cancer, characterized in that, Detect the expression level of tRF-1:T16-Glu-TTC-2-2 in the serum sample of the subject; compare the expression level of tRF-1:T16-Glu-TTC-2-2 with a preset value. If it is higher than the preset value, the possibility of being diagnosed as malignant is high, otherwise the possibility of being inflammation is high.

3. Use of a prostate cancer treatment target, characterized in that: The role of tRF-1:T16-Glu-TTC-2-2, with the sequence TGACTGGACCTTTCTT, in the proliferation, migration and invasion of prostate cancer cells.

4. A kit for diagnosing prostate cancer, characterized in that: The kit includes a diagnostic kit for tRF-1:T16-Glu-TTC-2-2, with the sequence TGACTGGACCTTTCTT, for the early diagnosis and prognosis evaluation of prostate cancer.

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