Cyclic RNA-circ MYCBP2 translated polypeptide and application thereof in regulation and control of bladder cancer

Abstract

The invention discloses a polypeptide translated by circular RNA-circMYCBP2 and application of the polypeptide in regulation and control of bladder cancer, and belongs to the field of tumor molecular biology. According to the application disclosed by the invention, circMYCBP2 which is highly expressed in tissues of a lymph cancer metastasis positive bladder cancer patient is screened by taking the capability of coding short peptides or proteins by circular RNA as an entry point, the translation of circMYCBP2 is verified, MYCBP2-227aa with a translation sequence as shown in SEQ ID NO.1, meanwhile, the promotion effect of MYCBP2-227aa on bladder cancer lymphatic metastasis is also verified, and the circMYCBP2 can be used for preparing the bladder cancer lymphatic metastasis. Therefore, the biological effect of the MYCBP2-227aa in the lymphatic metastasis of the bladder cancer is disclosed, and a theoretical basis and a scientific basis are provided for taking the MYCBP2-227aa as an early diagnosis marker and a new treatment target of the lymphatic metastasis of the bladder cancer.

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 applications thereof in regulating bladder cancer. Background Art

[0002] Bladder cancer is one of the most common malignant tumors of the urinary system in the world, and its incidence rate ranks ninth among malignant tumors. The incidence rate in men is significantly higher than that in women, about 3-4 times. Bladder cancer can be divided into muscle-invasive bladder cancer and non-muscle-invasive bladder cancer according to whether the muscle layer is invaded or not. Muscle-invasive bladder cancer is more likely to metastasize and the patient's prognosis is worse. Studies have shown that lymph node metastasis is the most important and first metastasis mode of bladder cancer. In patients with bladder cancer, the incidence of lymph node metastasis is about 25% to 30%, while in patients with bladder cancer that invades the muscle layer, the incidence of lymph node metastasis can be as high as 50%. For patients with bladder cancer with lymph node metastasis, more active treatment is required, including surgery, chemotherapy and radiotherapy, but these treatments have limited effect on improving the patient's prognosis. The five-year survival rate of patients has dropped from 77.6% to 18.6%. With the continuous development and deepening of basic research, we have a certain understanding of the molecular mechanism of bladder cancer lymph node metastasis, but we still face challenges in improving the prognosis of patients, mainly because we lack effective drug treatment targets and predictive biomarkers. Therefore, finding effective biomarkers and new drug treatment targets for bladder cancer lymph node metastasis is an important task in the current clinical and basic research of bladder cancer.

[0003] Circular RNA (circRNA) is a special type of RNA with a closed loop structure. There is no clear limit on the transcription length, and its unique circular structure makes it highly stable. Due to its good stability, the role of circRNA in tumor metastasis has attracted the attention of many scholars. Studies have found that on the one hand, circRNA can act as a "sponge" for microRNA (miRNA), adsorbing and inhibiting the function of miRNA, thereby relieving the inhibitory effect of miRNA on downstream target genes. The abnormal expression of these target genes may lead to the occurrence and development of tumors; on the other hand, circRNA can further promote the occurrence and development of tumors by encoding tumor-related peptides or proteins, or interacting with tumor-related proteins. However, in current studies, the biological role and molecular mechanism of circRNA-encoded peptides or proteins in bladder cancer lymph node metastasis are still unclear.

[0004] In recent years, studies have found that circRNA can encode tumor-related short peptides or proteins, which can promote the proliferation, migration and invasion of tumor cells by affecting cell signaling pathways and regulating cell cycle and apoptosis; circRNA can also regulate gene expression and change the tumor microenvironment, affecting gene stability and tumor-related inflammation and immune response, thereby playing an important regulatory role in tumor metastasis. And benefiting from the stability and conservative biological characteristics of circRNA, the short peptides or proteins encoded by circRNA can be stably present in the nucleus and cytoplasm and be widely detected. Therefore, new targets and new predictive indicators for tumor metastasis targeted therapy based on circRNA-encoded short peptides or proteins have broad clinical application prospects. Screening for bladder cancer lymph node metastasis and circRNA with coding ability is conducive to the development of circular RNA. Summary of the invention

[0005] In view of the defects of the prior art, the present invention screened out circMYCBP2, which is highly expressed in the tissues of bladder cancer patients with positive lymphatic metastasis, and can translate the polypeptide MYCBP2-227aa. The molecular mechanism by which circMYCBP2 mediates bladder cancer lymphatic metastasis was clarified, and it can serve as a more effective new diagnostic target and new therapeutic 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: 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.

[0007] Preferably, the product comprises a chip, a kit or a reagent.

[0008] Preferably, the product is judged by the amount of polypeptide expression.

[0009] Preferably, the polypeptide is MYCBP2-227aa, and its amino acid sequence is shown in SEQ ID NO.1.

[0010] 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.

[0011] The present invention also provides a reagent for detecting the expression amount of circ MYCBP2, wherein the reagent is used to predict the prognosis of bladder cancer lymph node metastasis or diagnose bladder cancer.

[0012] 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.

[0013] The present invention also provides the use of an inhibitor of circ MYCBP2 translation in the preparation of a drug for treating bladder cancer.

[0014] Preferably, the inhibitor comprises an IRES-ATGmut circular plasmid.

[0015] 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.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention takes the translation ability of circular RNA as the entry point, 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, and verifies its translation ability by predicting the open reading frame, constructing expression vector binding and synthesizing specific antibodies, confirming that circ MYCBP2 can translate MYCBP2-227aa, and that MYCBP2-227aa is positively correlated with the lymph node metastasis of bladder cancer and the poor prognosis of patients, and promotes the lymphangiogenesis and lymph node metastasis of bladder cancer, revealing the biological role of MYCBP2-227aa in the lymph node metastasis of bladder cancer, providing 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 providing new ideas for the application of circular RNA in tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solution of the present invention, the drawings required for use in the implementation mode will be briefly introduced below. Obviously, the drawings described below are only some implementation modes of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 These are different experimental results of circMYCBP2 in Example 1, wherein: A is a Random, oligo dT reverse transcription experiment; B is an actinomycin D experiment; and C is an RNase R digestion experiment.

[0019] Figure 2This is a translation potential analysis diagram of circMYCBP2 in Example 2, in which: G is a schematic diagram of the prediction of the 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 is a schematic diagram of the IRES sequence and ORF of circMYCBP2 inserted at both ends of the start codon ATG, and a Flag tag protein inserted at the end of the ORF, and the lower half is a schematic diagram of the IRES sequence deletion of circMYCBP2, and a Flag tag protein inserted at the end of the ORF.

[0020] Figure 3 This is a diagram showing the expression of MYCBP2-227a detected by Western blot after circular plasmid transfection.

[0021] Figure 4 Fluorescence images of UM-UC-3 cells transfected with MYCBP2-227aa overexpression plasmid carrying Flag-tagged protein.

[0022] Figure 5 The expression of MYCBP2-227aa is shown in Figure 1. K is a detection diagram of MYCBP2-227aa by Western blot using a specific short peptide antibody; L is a statistical analysis diagram of MYCBP2-227aa protein expression in cancer and adjacent tissues, and the statistical method used is Mann–Whitney U test. Represents p<0.01.

[0023] 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 chart of statistical analysis, which is a χ 2 test, Represents p<0.01.

[0024] Figure 7 This is 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 the immunofluorescence staining diagram; M on the right is the statistical analysis diagram. The statistical analysis method used is the chi-square test. Represents p<0.01.

[0025] Figure 8 The left picture is a picture of UM-UC-3 cell adhesion experiment. The right picture is a statistical analysis of the difference in the number of UM-UC-3 cell adhesion in different groups. The three dots represent three replicates of the experiment. The statistical method is one-way ANOVA followed by Dunnett's tests, in which two The comparison between the two groups was statistically analyzed with P < 0.01.

[0026] Fig. 9 The left picture shows the UM-UC-3 cell transwell invasion and migration under a microscope. The right picture shows the statistical analysis of the differences in the number of UM-UC-3 cells that passed 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 comparison between the two groups was statistically analyzed with P < 0.01.

[0027] Fig.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 nude mouse popliteal lymph node metastasis between different groups detected by nude mouse live imaging. The statistical method is 2-tailed test; Figure B is a statistical chart 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 using chi-square test for significant difference analysis. One of them is Represents statistical significance of P < 0.05.

[0028] Fig.11 This is a diagram showing tumor metastasis in the popliteal lymph nodes of nude mice. DETAILED DESCRIPTION

[0029] 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 creative work are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0031] Example 1: Screening of circ MYCBP2 Bladder cancer tissue samples and paired adjacent tissues were obtained from 5 bladder cancer patients at Sun Yat-sen Memorial Hospital by intraoperative sampling and sent to BGI for library construction and sequencing. The highly expressed circular RNA-circ MYCBP2 in bladder cancer was screened out from the sequencing results, and its circular characteristics were identified by random, oligo dT reverse transcription experiments, actinomycin D experiments, RNase R digestion experiments, etc. The specific operations are as follows: 1. Oligo dT, Random reverse transcription experiment: Total RNA from tissues was extracted using the trizol method and reverse transcribed into cDNA using Invitrogen™ Oligo (dT) primer (Cat. No.: AM5730G) and Random primers. The expression of circFOCAD 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 as follows: Upstream primer F1: 5′-CCTAGCACACAAGTCACTGC-3′ (SEQ ID NO. 2); Downstream primer R1: 5′-TGTCTTGCCTCTTGACCATCT -3′ (SEQ ID NO. 3); Table 1 RT-qPCR amplification system

[0032] The above system was incubated at 44°C for 1 h and then at 92°C for 10 min for subsequent PCR experiments.

[0033] Table 2 qPCR amplification system

[0034] Table 3 Amplification program

[0035] 2. Actinomycin D experiment: Tumor cells were seeded in six-well plates 24 h in advance. The next day, DMSO solution was used to prepare actinomycin D powder to a concentration of 2 mM. Then, the concentration of actinomycin D was diluted to 0.1 μM using cell culture medium and added to the 6-well plate at 2 mL per well. At the same time, normal cell culture medium without actinomycin D was added to the control group. The total RNA in the experimental group and the control group was extracted using an RNA purification kit (EZ-press RNA purification kit, catalog number: B0004D) at 0 h, 6 h, 12 h, 18 h, and 24 h, respectively. 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.

[0036] 3. RNase R digestion experiment: Total RNA in the cells was extracted using an EZ-press RNA purification kit (Cat. No. B0004D), and the reaction system required for RNase R digestion was prepared using the GSPureRNase R kit (Cat. No. R0300, Gisai Biotechnology). 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, and RT-qPCR was used to detect the expression of circFOCAD and FOCAD mRNA.

[0037] Table 4 GSPure RNase R reaction system

[0038] like Figure 1 As shown, circ MYCBP2 was identified as a covalently closed circRNA.

[0039] Example 2: Verification that circ MYCBP2 encodes a short peptide 1. Through circ bank, IRES finder, ORF finder and CPC analysis, it was found that circ MYCBP2 (sequence as shown in SEQ ID NO.4) has an IRES sequence, an open reading frame (ORF) and a start codon ATG in the ORF, suggesting that circ MYCBP2 has translation potential.

[0040] (SEQ ID NO.4).

[0041] Second, the IRES sequence (sequence as shown in SEQ ID NO.5) and ORF (sequence as shown in SEQ ID NO.6) of circ MYCBP2 were inserted into the circular plasmid and fused with the Flag tag protein (constructed by Guangzhou Aiki Biotechnology Co., Ltd.), and transfected into the UM-UC-3 cell line to simulate the translation process of circ MYCBP2 in the cell. The short peptide was detected by Western blot experiment and Flag tag antibody.

[0042] IRES:ATTCTTGATGCAGTGAAAGAAATGATACCTTTAGATCTTCTTTTAGCTGTCCCAGTGCCCGGGGTTAACATTGAAGAACACCTTCAGTTACGACAAGAAGAAAAACGGCAACGTGTAATCAGAAGGCACAGATTAGAGGAAGGAAGAGGCCCCCTTGTATTTGCTGGTCCTATT(SEQ ID NO.5)。

[0043] ORF: TGGAAAATGGAGATGTCTATACATTTGGTTATGGGCAGCATGGGCAGCTAGGACATGGAGATGTCAACTCCAGGGGATGTCCCACTCTTGTTCAAGCATTGCCAGGCCCTAGCACACAAGTCACTGCAGGCAGCAACCATACGGCAGTACTTTTAATGGATGGACAGGTCTTCACATTTGGAAGTTTTTCTGATCTGTGGTTGTGGTTCCGGAGAATCTGGTTGTGCTGTGTGTGGATGTTGCAAGGCCTGTGCAAGAGAGTTAGATGGTCAAGAGGCAAGACAAAGAGGAATTCTTGATGCAGTGAAAGAAATGATACCTTTAGATCTTCTTTTAGCTGTCCCAGTGCCCGGGGTTAACATTGAAGAACACCTTCAGTTACGACAAGAAGAAAAACGGCAACGTGTAATCAGAAGGCACAGATTAGAGGAAGGAAGAGGCCCCCTTGTATTTGCTGGTCCTATTTTTATGAACCATCGAGAACAGGCTCTAGCCAGACTCAGATCCCATCCAGCACAGCTAAAGCATAAACGGGACAAGCACAAAGATGGAAGTGGAGAAAGAGGCGAAAAGGATGCAAGCAAAATCACAACATACCCTCCAGGCTCTGTGCGATTTGACTGTGAGCTCCGGGCAGTCCAAGTCAGCTGTGGATTTCACCATTCAGTGGTTTTAATGGAAAATGGAGATGTCTATACATTTGGTTATGGGCAGCATGGGCAGCTAGGACATGGAGATGTCAACTCCAGGGGATGTCCCACTCTTGTTCAAGCATTGCCAGGCC (SEQ ID NO.6).

[0044] The results are as Figure 2-4 shown. circMYCBP2 can translate a short peptide with a molecular weight of approximately 227 aa in cells (the sequence is shown in SEQ ID NO.1), named MYCBP2-227aa.

[0045] MIPLDLLLAVPVPGVNIEEHLQLRQEEKRQRVIRRHRLEEGRGPLVFAGPIFMNHREQALARLRSHPAQLKHKRDKHKDGSGERGEKDASKITTYPPGSVRFDCELRAVQVS CGFHHSVVLMENGDVYTFGYGQHGQLGHGDVNSRGCPTLVQALPGPSTQVTAGSNHTAVLLMDGQVFTFGSFSDLWLWFRRIWLCCVWMLQGLCKRVRWSRGKTKRNS (SEQ ID NO.1).

[0046] Example 3: Analysis of the relationship between MYCBP2-227aa and lymph node metastasis and poor prognosis in bladder cancer patients Bladder cancer tissues and paracancerous tissues from 30 patients with bladder cancer lymph node metastasis from Sun Yat-sen Memorial Hospital were collected, tissue sections were prepared by paraffin embedding, the tissues were quickly frozen in liquid nitrogen and ground, and then extracted using a protein extraction kit (Conway Century, Micro BCA Protein Quantification Kit CW2011S). Western blot analysis of the expression of MYCBP2-227aa in the cancer and paracancerous tissues of 30 bladder cancer patients indicated that MYCBP2-227aa was upregulated in the cancer tissues of bladder cancer patients. Subsequently, MYCBP2-227aa was detected in bladder cancer tissues with positive and negative lymph node metastasis by using specific antibody detection (Hua'an Bio designed specific antibodies based on the translated short peptide MYCBP2-227aa, and then used for IHC section staining).

[0047] The results are as follows Figure 5-7 As shown, 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 metastatic bladder cancer tissue was significantly higher than that in lymph node metastasis negative bladder cancer tissue.

[0048] Example 4: In vitro experiments 1. The circular plasmid expressing MYCBP2-227aa was transfected into the UM-UC-3 cell line as the experimental group, and the empty plasmid transfection group was set as the control group. The IRES-ATGmut circular plasmid (which inhibited the production of MYCBP2-227aa) was constructed by Guangzhou Aiki Biotechnology Co., Ltd. and transfected into the UM-UC-3 cell line to explore the role of circ MYCBP2 itself. Subsequently, each group of UM-UC-3 cell lines was digested with trypsin and centrifuged at 1000 g for 5 minutes, and the culture medium was collected and removed for subsequent tumor cell adhesion experiments, transwell migration and invasion experiments.

[0049] 2. Adhesion experiment: Resuspend the cells in normal complete medium containing 5% serum and count them. 4 cells / well, gently add them to the 24-well plate with human lymphatic endothelial cells laid in advance, gently shake and evenly spread the cells. Place in a 5% CO2, 37℃ cell culture incubator for culture, observe the cell adhesion every 2 hours, take pictures under an inverted microscope after the cells adhere, and use Image J software to measure the adhered cells. Through statistical analysis, compare the differences in adhesion between different induced groups.

[0050] 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 plate was shaken and flattened, and then the 24-well plate was placed in an incubator. The plate was ready for use when the lymphatic endothelial cells covered the bottom of the 24-well plate.

[0051] 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, i.e. IRES-ATGmut, weakened this effect.

[0052] 3. Transwell invasion and migration assay: 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, and then the cell suspension was added to the upper chamber of the 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 for 6 hours, and then the chamber was taken out, and the cells were fixed with 4% paraformaldehyde for 15 minutes, and then gently washed three times with PBS, and then stained with crystal violet stain for 15 minutes, and the excess crystal violet stain was washed away with PBS. The cells inside the chamber were gently wiped with a cotton swab, and then observed and photographed under a microscope. Random fields of view were taken and counted using Image J, and the differences in cell migration ability between different bladder cancer cell groups were statistically analyzed.

[0053] The results are as follows Fig. 9 As shown, compared with the control group, overexpression of MYCBP2-227aa significantly enhanced the migration ability of bladder cancer cells, while the mutant ATG, i.e., IRES-ATGmut, weakened this effect.

[0054] Example 5: In vivo experiments 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 operations are as follows: An expression vector containing the GFP gene was constructed with an antibiotic resistance gene as a selection marker. The GFP gene was inserted into the multiple cloning site of the plasmid using recombinant DNA technology to ensure the correct expression of the GFP gene. UM-UC-3 cells were seeded in a 6-well plate 24 hours in advance, and the plasmid with GFP fluorescence marker was transfected into UM-UC-3 bladder cancer cells using Lipofectamine 3000 and p3000 (Life Technologies, California, USA). After 48 hours of transfection, fresh culture medium containing selective antibiotics (G418) was replaced to kill unsuccessfully transfected cells. After about 2 weeks of screening, the fluorescence expression of GFP was observed using a fluorescence microscope, and monoclonal cells with strong fluorescence and good growth status were selected as cells for subsequent experiments; the MYCBP2-227aa plasmid stably overexpressing GFP fluorescence marker constructed by Guangzhou Aiji Biotechnology Co., Ltd. was transfected into UM-UC-3 bladder cancer cells according to the above method and screened for subsequent experiments.

[0055] 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 foot pads. 5 Each mouse was treated with a footpad tumor model. Then, live imaging was performed once a week to observe the metastasis of the popliteal lymph nodes in the nude mouse footpad until the tumor volume of the nude mouse footpad was greater than 200 mm3 or the nude mouse died. The popliteal lymph nodes of the nude mice were separated, their volumes were measured, and the metastasis of the tumors was analyzed by immunohistochemistry. The differences in the metastasis rates of the popliteal lymph nodes of nude mice between different groups were recorded and analyzed. The antibody used for immunofluorescence was anti-GFP.

[0056] The results are as follows Figure 10-11 As shown, it was found that bladder cancer cells overexpressing MYCBP2-227aa could significantly promote the lymph node metastasis of bladder cancer.

[0057] In summary, the present invention screened out circMYCBP2 that 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 combination 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 tissues was significantly higher than that in bladder cancer adjacent tissues, and further in vivo and in vitro 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.

[0058] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description 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 may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. Application of circ MYCBP2-translated polypeptides as molecular markers in screening or preparing products for diagnosing bladder cancer, predicting the risk of bladder cancer lymph node metastasis, and bladder cancer survival prognosis.

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. The use according to claim 1, characterized in that: The polypeptide is MYCBP2-227aa, and its amino acid sequence is shown in SEQ ID NO.

1.

5. The use according to claim 4, characterized in that The patients with high expression of MYCBP2-227aa have a poor prognosis, while the patients with low expression of MYCBP2-227aa have a good prognosis; the patients with high expression of MYCBP2-227aa have a shorter overall survival than those with low expression of MYCBP2-227aa.

6. A reagent for detecting the expression level of circ MYCBP2, characterized in that: The reagent is used for predicting the prognosis of bladder cancer lymph node metastasis or diagnosing bladder cancer.

7. The reagent for detecting the expression level of circ MYCBP2 according to claim 6, 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.

8. Application of inhibitors of circ MYCBP2 translation in the preparation of drugs for the treatment of bladder cancer.

9. The use according to claim 8, wherein the inhibitor comprises an IRES-ATGmut circular plasmid.

10. A kit for diagnosing bladder cancer or predicting the prognosis of bladder cancer lymph node metastasis, characterized in that: The kit includes 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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