Peptides translated from circular RNA-circ MYCBP2 and their application in regulating bladder cancer

Abstract

The present invention discloses polypeptides translated from circular RNA-circMYCBP2 and their application in regulating bladder cancer, belonging to the field of tumor molecular biology. Taking the ability of circular RNA to encode short peptides or proteins as an entry point, the present invention screened out circMYCBP2, which is highly expressed in tissues of bladder cancer patients with positive lymphoma metastasis, and verified that its translation can translate the sequence MYCBP2-227aa shown in SEQ ID NO.1. At the same time, it also confirmed the promotion effect of MYCBP2-227aa on bladder cancer lymphatic metastasis, thereby revealing the biological role of MYCBP2-227aa in bladder cancer lymphatic metastasis, and providing a theoretical basis and scientific basis for MYCBP2-227aa as an early diagnostic marker and new treatment target for bladder cancer lymphatic metastasis.

Description

Technical Field

[0001] The present invention relates to the field of tumor molecular biology, and in particular to polypeptides translated from circular RNA-circMYCBP2 and their application in regulating bladder cancer. Background Art

[0002] Bladder cancer is one of the most common urinary tract malignancies worldwide, ranking ninth in incidence among malignant tumors. The incidence rate in men is significantly higher than in women, approximately 3-4 times higher. Bladder cancer can be divided into muscle-invasive and non-muscle-invasive bladder cancer based on whether the cancer invades the muscle layer. Muscle-invasive bladder cancer is more likely to metastasize and has a worse prognosis. Studies have shown that lymph node metastasis is the primary and primary mode of bladder cancer metastasis. Among bladder cancer patients, the incidence of lymph node metastasis is approximately 25% to 30%, while in patients with muscle-invasive bladder cancer, the incidence can be as high as 50%. Bladder cancer patients with lymph node metastasis require more aggressive treatment, including surgery, chemotherapy, and radiotherapy. However, these treatments have limited effect on improving patient prognosis, with the five-year survival rate dropping from 77.6% to 18.6%. With the continuous development and deepening of basic research, we have gained a certain understanding of the molecular mechanisms of bladder cancer lymph node metastasis. However, improving patient prognosis still faces challenges, mainly due to the lack of effective drug treatment targets and predictive biomarkers. Therefore, finding effective biomarkers and new drug treatment targets for bladder cancer lymph node metastasis is a key task in current clinical and basic research on bladder cancer.

[0003] Circular RNA (circRNA) is a special type of RNA with a closed loop structure. Its transcript length is not clearly limited, and its unique circular structure confers high stability. Due to its excellent stability, circRNA has attracted considerable attention for its role in tumor metastasis. Studies have shown that circRNA can act as a "sponge" for microRNA (miRNA), absorbing and inhibiting miRNA function, thereby relieving miRNA's inhibitory effect on downstream target genes. Aberrant expression of these target genes may contribute to tumor development and progression. Furthermore, circRNA can further promote tumor development and progression by encoding tumor-associated peptides or proteins or interacting with tumor-associated proteins. However, the biological role and molecular mechanism of circRNA-encoded peptides or proteins in bladder cancer lymphatic metastasis remain unclear.

[0004] Recent studies have revealed that circRNAs can encode tumor-associated short peptides or proteins. These peptides or proteins can promote tumor cell proliferation, migration, and invasion by influencing cell signaling pathways and regulating cell cycle and apoptosis. CircRNAs can also play a crucial role in regulating tumor metastasis by modulating gene expression and altering the tumor microenvironment, affecting gene stability and tumor-related inflammatory and immune responses. Furthermore, benefiting from the stability and conserved biological properties of circRNAs, the peptides or proteins encoded by circRNAs can be stably present in the nucleus and cytoplasm and widely detected. Therefore, new targets for tumor metastasis targeted therapy and new predictive markers based on circRNA-encoded peptides or proteins have broad clinical application prospects. Screening for bladder cancer lymph node metastasis and circRNAs with coding capabilities is conducive to the development of circular RNAs. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention screened out circMYCBP2, which is highly expressed in the tissues of bladder cancer patients with positive lymphatic metastasis. It can translate the polypeptide MYCBP2-227aa and elucidate its molecular mechanism for mediating bladder cancer lymphatic metastasis. It can serve as a more effective new diagnostic target and treatment target for bladder cancer lymphatic metastasis.

[0006] In order to achieve the above purpose, the present invention solves the technical problem by adopting the following technical solutions:

[0007] On the one hand, the present invention provides the use of a polypeptide translated from circ MYCBP2 as a molecular marker in screening or preparing products for diagnosing bladder cancer, predicting the risk of bladder cancer lymph node metastasis, and bladder cancer survival prognosis.

[0008] Preferably, the product comprises a chip, a kit or a reagent.

[0009] Preferably, the product is judged by the amount of polypeptide expression.

[0010] Preferably, the polypeptide is MYCBP2-227aa, and its amino acid sequence is shown in SEQ ID NO.1.

[0011] Preferably, the patients with high expression of MYCBP2-227aa have a poor prognosis, and the patients with low expression of MYCBP2-227aa have a better prognosis; the patients with high expression of MYCBP2-227aa have a shorter overall survival than those with low expression of MYCBP2-227aa.

[0012] The present invention also provides a reagent for detecting the expression level of circ MYCBP2, which is used to predict the prognosis of bladder cancer lymph node metastasis or diagnose bladder cancer.

[0013] Preferably, the reagent comprises an upstream primer as shown in SEQ ID NO.2 and a downstream primer as shown in SEQ ID NO.3.

[0014] The present invention also provides the use of an inhibitor of circ MYCBP2 translation in the preparation of a drug for treating bladder cancer.

[0015] Preferably, the inhibitor comprises an IRES-ATGmut circular plasmid.

[0016] The present invention also provides a kit for diagnosing bladder cancer or predicting the prognosis of bladder cancer lymph node metastasis, the kit comprising an upstream primer as shown in SEQ ID NO.2 and a downstream primer as shown in SEQ ID NO.3.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention takes the translation ability of circular RNA as the entry point, and screens out circ MYCBP2, which is highly expressed in the tissues of bladder cancer patients with positive lymph node metastasis, through high-throughput sequencing and bioinformatics analysis. Its translation ability is verified by predicting the open reading frame, constructing expression vector binding and synthesizing specific antibodies. It is confirmed that circ MYCBP2 can translate MYCBP2-227aa, and MYCBP2-227aa is positively correlated with the lymph node metastasis of bladder cancer and the poor prognosis of patients. It also promotes the regeneration of lymphatic vessels and lymph node metastasis of bladder cancer, revealing the biological role of MYCBP2-227aa in the lymph node metastasis of bladder cancer. It provides a theoretical basis and scientific basis for using MYCBP2-227aa as an early diagnostic marker and new treatment target for bladder cancer lymph node metastasis, and provides new ideas for the application of circular RNA in tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings required for use in the implementation. Obviously, the drawings described below are only some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 These are different experimental results of circMYCBP2 in Example 1, where: A is a Random, oligo dT reverse transcription experiment; B is an actinomycin D experiment; and C is an RNase R digestion experiment.

[0021] Figure 2This is a translation potential analysis diagram of circMYCBP2 in Example 2, in which: G is a schematic diagram of the predicted open reading frame (ORF) and internal ribosome entry site (IRES) of circMYCBP2; H is a schematic diagram of the construction of a circular plasmid, the upper half of which shows the IRES sequence and ORF of circMYCBP2 inserted at both ends of the start codon ATG, with a Flag tag protein inserted at the end of the ORF; the lower half shows a schematic diagram of the IRES sequence deletion of circMYCBP2, with a Flag tag protein inserted at the end of the ORF.

[0022] Figure 3 This is a diagram showing the expression of MYCBP2-227a after circular plasmid transfection detected by Western blot.

[0023] Figure 4 Fluorescence images of UM-UC-3 cells transfected with a MYCBP2-227aa overexpression plasmid carrying a Flag-tagged protein.

[0024] Figure 5 The figure shows the expression of MYCBP2-227aa. In the figure, K is a Western blot analysis of MYCBP2-227aa using a specific short peptide antibody; L is a statistical analysis of MYCBP2-227aa protein expression in cancer and adjacent tissues. The statistical method used is the Mann–Whitney U test. Represents p < 0.01.

[0025] Figure 6 The expression of MYCBP2-227aa in bladder cancer cells or normal cells is shown in Figure 1. D is a representative image taken under a microscope; E is a bar graph for statistical analysis, which is a χ 2 test, Represents p < 0.01.

[0026] Figure 7 This is a diagram showing the expression of MYCBP2-227aa in bladder cancer adjacent tissues, lymph node metastasis-positive and -negative bladder cancer tissues. In the figure: M on the left is an immunofluorescence staining diagram; M on the right is a statistical analysis diagram. The statistical analysis method used is the chi-square test. Represents p < 0.01.

[0027] Figure 8 The left picture is a UM-UC-3 cell adhesion experiment. The right picture is a statistical analysis of the differences in the number of UM-UC-3 cell adhesions in different groups. The three dots represent three replicates of the experiment. The statistical method was one-way ANOVA followed by Dunnett's tests, in which two The statistical comparison between the two groups was P < 0.01.

[0028] Figure 9 The left picture shows the transwell migration and invasion experiment of UM-UC-3 cells. The right picture shows the statistical analysis of the difference in the number of UM-UC-3 cells passing through the transwell in different groups. The three dots represent three replicates of the experiment. The statistical method was one-way ANOVA followed by Dunnett's tests, in which two The statistical comparison between the two groups was P < 0.01.

[0029] Figure 10 This is an analysis of MYCBP2-227aa promoting lymph node metastasis in bladder cancer. In the figure: Figure A is a statistical analysis of the nude mouse popliteal lymph node metastasis between different groups detected by in vivo imaging. The statistical method is 2-tailed test; Figure B is a statistical graph of the size of nude mouse popliteal lymph node metastasis between different groups. The statistical method is the same as A; Figure C is a statistical table analyzing the number of popliteal lymph node metastasis between different groups, and the chi-square test was used to analyze the significance of the difference. Represents statistical significance P < 0.05.

[0030] Figure 11 This is a diagram showing tumor metastasis in the popliteal lymph nodes of nude mice. DETAILED DESCRIPTION

[0031] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0033] Example 1: Screening of circ MYCBP2

[0034] Bladder cancer tissue samples and paired adjacent adjacent tissues were obtained intraoperatively from five bladder cancer patients at Sun Yat-sen Memorial Hospital and sent to BGI for library construction and sequencing. The sequencing results identified a highly expressed circular RNA, circ MYCBP2, in bladder cancer. Its circularity was confirmed through random oligo dT reverse transcription, actinomycin D, and RNase R digestion assays. The specific procedures are as follows:

[0035] 1. Oligo dT, Random Reverse Transcription Experiment:

[0036] Total RNA was extracted from tissues using the trizol method and reverse-transcribed into cDNA using Invitrogen™ Oligo (dT) primers (Cat. No. AM5730G) and random primers. CircFOCAD expression was detected by RT-qPCR and analyzed using the nonparametric Mann-Whitney U test. The RT-qPCR primer sequences (designed and synthesized by Shanghai Sangon Biotech Co., Ltd.) are shown below:

[0037] Upstream primer F1: 5′-CCTAGCACACAAGTCACTGC-3′ (SEQ ID NO. 2);

[0038] Downstream primer R1: 5′-TGTCTTGCCTCTTGACCATCT -3′ (SEQ ID NO. 3);

[0039] Table 1 RT-qPCR amplification system

[0040]

[0041] The above system was incubated at 44°C for 1 h and then incubated at 92°C for 10 min for subsequent PCR experiments.

[0042] Table 2 qPCR amplification system

[0043]

[0044] Table 3 Amplification program

[0045]

[0046] 2. Actinomycin D experiment:

[0047] Tumor cells were seeded in six-well plates 24 hours in advance. The next day, actinomycin D powder was prepared to a concentration of 2 mM using DMSO solution. The concentration of actinomycin D was then diluted to 0.1 μM using cell culture medium and 2 mL was added to each well of the six-well plate. Normal cell culture medium without actinomycin D was also added to the control group. Total RNA was extracted from the experimental and control groups using an EZ-press RNA purification kit (Cat. No. B0004D) at 0, 6, 12, 18, and 24 hours. After reverse transcription into cDNA using HiScript III RT SuperMix for qPCR, the expression of circFOCAD was detected by RT-qPCR and analyzed using the nonparametric Mann-Whitney U test.

[0048] 3. RNase R digestion experiment:

[0049] Total RNA was extracted from cells using an EZ-press RNA purification kit (Cat. No. B0004D). The RNase R digestion reaction system was prepared using a GSPure RNase R kit (Cat. No. R0300). The reaction system was then added to the cells and incubated at 37°C for 15 min. The digested RNA was reverse transcribed into cDNA using HiScript III RT SuperMix for qPCR. RT-qPCR was then used to detect the expression of circFOCAD and FOCAD mRNA.

[0050] Table 4 GSPure RNase R reaction system

[0051]

[0052] like Figure 1 As shown, circ MYCBP2 was identified as a covalently closed circRNA.

[0053] Example 2: Verification that circ MYCBP2 encodes a short peptide

[0054] 1. Through circ bank, IRES finder, ORF finder, and CPC analysis, it was found that circ MYCBP2 (sequence shown in SEQ ID NO.4) has an IRES sequence, an open reading frame (ORF), and an initiation codon ATG in the ORF, suggesting that circ MYCBP2 has translation potential.

[0055] (SEQ ID NO.4).

[0056] Second, the IRES sequence (sequence shown in SEQ ID NO.5) and ORF (sequence shown in SEQ ID NO.6) of circ MYCBP2 were inserted into a circular plasmid and fused with the Flag-tagged protein (constructed by Guangzhou Aiki Biotechnology Co., Ltd.). The UM-UC-3 cell line was transfected to simulate the translation process of circ MYCBP2 in the cells. The short peptide was detected by Western blot and Flag-tag antibody.

[0057] IRES:ATTCTTGATGCAGTGAAAGAAATGATACCTTTAGATCTTCTTTTAGCTGTCCCAGTGCCCGGGGTTAACATTGAAGAACACCTTCAGTTACGACAAGAAGAAAAACGGCAACGTGTAATCAGAAGGCACAGATTAGAGGAAGGAAGAGGCCCCCTTGTATTTGCTGGTCCTATT(SEQ ID NO.5)。

[0058] ORF: TGGAAAATGGAGATGTCTATACATTTGGTTATGGGCAGCATGGGCAGCTAGGACATGGAGATGTCAACTCCAGGGGATGTCCCACTCTTGTTCAAGCATTGCCAGGCCCTAGCACACAAGTCACTGCAGGCAGCAACCATACGGCAGTACTTTTAATGGATGGACAGGTCTTCACATTTGGAAGTTTTTCTGATCTGTGGTTGTGGTTCCGGAGAATCTGGTTGTGCTGTGTGTGGATGTTGCAAGGCCTGTGCAAGAGAGTTAGATGGTCAAGAGGCAAGACAAAGAGGAATTCTTGATGCAGTGAAAGAAATGATACCTTTAGATCTTCTTTTAGCTGTCCCAGTGCCCGGGGTTAACATTGAAGAACACCTTCAGTTACGACAAGAAGAAAAACGGCAACGTGTAATCAGAAGGCACAGATTAGAGGAAGGAAGAGGCCCCCTTGTATTTGCTGGTCCTATTTTTATGAACCATCGAGAACAGGCTCTAGCCAGACTCAGATCCCATCCAGCACAGCTAAAGCATAAACGGGACAAGCACAAAGATGGAAGTGGAGAAAGAGGCGAAAAGGATGCAAGCAAAATCACAACATACCCTCCAGGCTCTGTGCGATTTGACTGTGAGCTCCGGGCAGTCCAAGTCAGCTGTGGATTTCACCATTCAGTGGTTTTAATGGAAAATGGAGATGTCTATACATTTGGTTATGGGCAGCATGGGCAGCTAGGACATGGAGATGTCAACTCCAGGGGATGTCCCACTCTTGTTCAAGCATTGCCAGGCC (SEQ ID NO.6).

[0059] The results are as Figure 2-4 shown. circ MYCBP2 can translate a short peptide with a molecular weight of approximately 227 aa (the sequence is shown in SEQ ID NO.1) in cells, named MYCBP2-227aa.

[0060] MIPLDLLLAVPVPGVNIEEHLQLRQEEKRQRVIRRHRLEEGRGPLVFAGPIFMNHREQALARLRSHPAQLKHKRDKHKDGSGERGEKDASKITTYPPGSVRFDCELRAVQVS CGFHHSVVLMENGDVYTFGYGQHGQLGHGDVNSRGCPTLVQALPGPSTQVTAGSNHTAVLLMDGQVFTFGSFSDLWLWFRRIWLCCVWMLQGLCKRVRWSRGKTKRNS (SEQ ID NO.1).

[0061] Example 3: Analysis of the relationship between MYCBP2-227aa and lymph node metastasis and poor prognosis in bladder cancer patients

[0062] Bladder cancer tissues and adjacent tissues were collected from 30 patients with bladder cancer and lymph node metastasis at Sun Yat-sen Memorial Hospital. Tissue sections were prepared by paraffin embedding, quick-frozen in liquid nitrogen, and then ground. Protein was then extracted using a protein extraction kit (Cornwell, Micro BCA Protein Quantification Kit CW2011S). Western blot analysis of MYCBP2-227aa expression in both cancer and adjacent tissues from these 30 bladder cancer patients revealed that MYCBP2-227aa was upregulated in bladder cancer tissue. MYCBP2-227aa was then detected in both lymph node metastasis-positive and lymph node metastasis-negative bladder cancer tissues using a specific antibody designed by Huaan Biotechnology based on the translated short peptide MYCBP2-227aa. The specific antibody was then used for immunohistochemistry (IHC) staining of sections.

[0063] The results are as follows Figure 5-7 As shown in the figure, the expression of MYCBP2-227aa in bladder cancer tissue was significantly higher than that in bladder cancer adjacent tissue; the expression level in lymph node metastasis bladder cancer tissue was significantly higher than that in lymph node metastasis negative bladder cancer tissue.

[0064] Example 4: In vitro experiments

[0065] First, a circular plasmid expressing MYCBP2-227aa was transfected into the UM-UC-3 cell line as an experimental group, while an empty plasmid transfection group served as a control group. An IRES-ATGmut circular plasmid (which inhibits the production of MYCBP2-227aa) was constructed by Guangzhou Aiki Biotechnology Co., Ltd. and transfected into the UM-UC-3 cell line to investigate the role of circ MYCBP2 itself. Subsequently, the UM-UC-3 cell lines in each group were trypsinized and centrifuged at 1000 g for 5 minutes. The culture medium was then collected and removed for subsequent tumor cell adhesion, transwell migration, and invasion assays.

[0066] 2. Adhesion test:

[0067] Resuspend the cells in normal complete medium containing 5% serum and count them. 4 Gently add 100 cells / well to a 24-well plate pre-plated with human lymphatic endothelial cells. Gently shake to evenly spread the cells. Incubate in a 5% CO2, 37°C cell culture incubator. Observe cell adhesion every 2 hours. Once adhered, photograph the cells under an inverted microscope and count the adhered cells using Image J software. Statistical analysis will be used to compare differences in adhesion between the different induced groups.

[0068] The experimental process of 24-well plate human lymphatic endothelial cell seeding is as follows: seed the plate one day in advance. When seeding the plate, prepare human lymphatic endothelial cells and centrifuge tubes in advance. Digest the lymphatic endothelial cells in the cell culture dish with trypsin and add 1×10 5 The cells were prepared into a cell suspension with a total volume of 700 μL and added to a 24-well plate. The suspension was shaken and leveled. The 24-well plate was then placed in an incubator. The plate was ready for use when the lymphatic endothelial cells covered the bottom of the 24-well plate.

[0069] like Figure 8 As shown in the results, it was found that overexpression of MYCBP2-227aa significantly enhanced the ability of bladder cancer cells to adhere to lymphatic endothelial cells compared with the control group, while the mutant ATG, IRES-ATGmut, weakened this effect.

[0070] 3. Transwell invasion and migration assay:

[0071] Resuspend the UM-UC-3 cells in each group with fresh serum-free medium and count the cells. Take 1×10 5 Cells were diluted to a total volume of 300 μL with serum-free culture medium. The cell suspension was then added to the upper chamber of a transwell chamber, and 700 μL of culture medium containing 5% serum was added to the lower chamber. The entire system was placed in an incubator and cultured for 6 hours. The chamber was then removed and the cells were fixed with 4% paraformaldehyde for 15 minutes. The cells were then gently washed three times with PBS and stained with crystal violet for 15 minutes. The excess crystal violet stain was then washed away with PBS. The cells inside the chamber were gently wiped clean with a cotton swab and observed and photographed under a microscope. Random fields of view were counted using Image J, and differences in cell migration ability between different bladder cancer cell groups were statistically analyzed.

[0072] The results are as follows Figure 9As shown in the results, compared with the control group, overexpression of MYCBP2-227aa significantly enhanced the migration ability of bladder cancer cells, while the mutant ATG, IRES-ATGmut, weakened this effect.

[0073] Example 5: In vivo experiments

[0074] 1. Construct a UM-UC-3 bladder cancer cell line with GFP fluorescence labeling; construct a UM-UC-3 bladder cancer cell line with GFP fluorescence labeling that stably overexpresses MYCBP2-227aa. The specific steps are as follows:

[0075] An expression vector containing the GFP gene and an antibiotic resistance gene as a selection marker was constructed. The GFP gene was inserted into the multiple cloning site of the plasmid using recombinant DNA technology to ensure proper expression of the GFP gene. UM-UC-3 cells were seeded in six-well plates 24 hours in advance. The GFP-tagged plasmid was transfected into UM-UC-3 bladder cancer cells using Lipofectamine 3000 and p3000 (Life Technologies, California, USA). Forty-eight hours after transfection, the culture medium was replaced with fresh medium containing the selective antibiotic (G418) to kill untransfected cells. After approximately two weeks of selection, GFP fluorescence expression was observed using a fluorescence microscope, and single clones with strong fluorescence and good growth were selected for use in subsequent experiments. A plasmid stably overexpressing the GFP-tagged MYCBP2-227aa, constructed by Guangzhou Aiji Biotechnology Co., Ltd., was transfected into UM-UC-3 bladder cancer cells according to the above method and selected for use in subsequent experiments.

[0076] 2. Purchase 24 healthy female nude mice aged 4-5 weeks and inject 5×10 UM-UC-3 cells labeled with GFP and UM-UC-3 cells overexpressing MYCBP2-227aa with GFP fluorescence into their right footpads. 5 To establish a footpad tumor model, mice were treated with 100 mg / day of nasopharyngeal carcinoma (100 mg / day) ...

[0077] The results are as follows Figure 10-11 As shown in the results, it was found that bladder cancer cells overexpressing MYCBP2-227aa could significantly promote the lymph node metastasis of bladder cancer.

[0078] In summary, the present invention screened out circMYCBP2, which is highly expressed in the tissues of bladder cancer patients, and verified its coding ability by predicting the open reading frame, constructing an expression vector and synthesizing specific antibodies. At the same time, it was confirmed that the expression of the short peptide MYCBP2-227aa encoded by it in bladder cancer tissue was significantly higher than that in bladder cancer adjacent tissues. Further in vitro and in vivo experiments confirmed the promoting effect of MYCBP2-227aa on bladder cancer lymphatic metastasis, thereby revealing the biological role of MYCBP2-227aa in bladder cancer lymphatic metastasis, and providing a theoretical basis and scientific basis for using MYCBP2-227aa as an early diagnostic marker and new treatment target for bladder cancer lymphatic metastasis.

[0079] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. Use of a reagent for detecting the expression of the polypeptide MYCBP2-227aa translated from circular RNA-circMYCBP2 in the preparation of a product for predicting the risk of bladder cancer lymph node metastasis, characterized in that: The polypeptide is MYCBP2-227aa, and its amino acid sequence is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The product includes a chip, a kit or a reagent.

3. The use according to claim 1, characterized in that The product is judged by the amount of polypeptide expression.

4. Use of a reagent for detecting the expression of circular RNA-circMYCBP2 in the preparation of a kit for predicting the risk of lymph node metastasis of bladder cancer, characterized in that: The reagents include an upstream primer as shown in SEQ ID NO.2 and a downstream primer as shown in SEQ ID NO.3.

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