Role of circSEMA3C encoding SEMA-123aa in targeted therapy of bladder cancer lymph node metastasis

Abstract

The present invention discloses the role of SEMA-123aa, encoded by circSEMA3C, in the targeted treatment of bladder cancer lymphatic metastasis, belonging to the field of biomedicine technology. The present invention uses the circRNA encoding product that drives bladder cancer lymphatic metastasis as the starting point, screens out circRNA-circSEMA3C that is upregulated in bladder cancer tissue through high-throughput sequencing, and confirms that SEMA3C-123aa, encoded by circSEMA3C, is positively correlated with the occurrence of bladder cancer lymphatic metastasis and can promote bladder cancer lymphangiogenesis and lymphatic metastasis, thereby revealing the biological role of SEMA3C-123aa in bladder cancer lymphatic metastasis and providing a theoretical basis and scientific basis for SEMA3C-123aa 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 biomedicine technology, and particularly to the role of circSEMA3C encoding SEMA-123aa in the targeted treatment of bladder cancer lymph node metastasis. Background Art

[0002] Bladder cancer is one of the most common tumors of the urinary system. However, bladder cancer lymph node metastasis is not only difficult to diagnose early but also challenging to treat effectively. Research data indicate that treatment options, including radical surgical resection, radiotherapy, chemotherapy, and immunotherapy, have limited efficacy in improving the prognosis of patients with bladder cancer lymph node metastasis. Therefore, exploring the molecular mechanisms of bladder cancer lymph node metastasis, identifying effective therapeutic targets, and developing targeted therapeutic agents based on these mechanisms are currently challenging challenges in the clinical diagnosis and treatment of bladder cancer lymph node metastasis.

[0003] Circular RNA (circRNA) is a class of single-stranded, covalently closed RNA molecules, most of which are composed of exons within protein-coding genes. They are stable and impervious to RNA exonucleases. They are widely expressed in eukaryotes and participate in a variety of physiological and pathological processes. Studies have shown that circRNAs can act as molecular sponges in cells, adsorbing microRNAs (miRNAs), relieving their inhibitory effects on target genes, and thereby upregulating their expression. Furthermore, circRNAs can also exert regulatory functions through protein binding, such as molecular modification and molecular scaffolding. Some circRNAs have also been reported to promote or repress gene transcription within the cell nucleus. CircRNAs were initially thought to lack coding capacity, but recent studies have revealed that some circRNAs can also possess translational capacity. Although circRNAs lack key translational elements, such as the 5'-untranslated region (5'-UTR) and poly(A) tail, circRNAs with open reading frames (ORFs) can mediate translation in a cap-independent manner through internal ribosome entry sites (IRES) or N6-methyladenosine (M6A) modifications. At the same time, due to its unique closed structure, the open reading frame in circular RNA may bypass the splice site or even exceed its length, producing short peptides or proteins that do not exist in the protein translated from the linear transcript. These short peptides or proteins encoded by circular RNA have been reported to play an important role in the progression of various cancers. However, the biological role and molecular mechanism of short peptides encoded by circRNA in bladder cancer and bladder cancer lymph node metastasis have not yet been reported. Summary of the Invention

[0004] In response to the shortcomings of the existing technology, the present invention screened out circSEMA3C, which is highly expressed in the tissues of bladder cancer patients with positive lymphatic metastasis. It can encode SEMA-123aa and elucidate the molecular mechanism by which SEMA-123aa mediates bladder cancer lymphatic metastasis. It can serve as an early diagnostic marker and new treatment target for bladder cancer lymphatic metastasis.

[0005] In order to achieve the above purpose, the present invention solves the technical problem by adopting the following technical solutions:

[0006] On the one hand, the present invention provides the use of circSEMA3C encoding SEMA-123aa 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 expression level of SEMA-123aa.

[0009] Preferably, the amino acid sequence of SEMA-123aa is shown in SEQ ID NO:5.

[0010] Preferably, the patients with high SEMA-123aa expression have a poor prognosis, and the patients with low SEMA-123aa expression have a better prognosis; the patients with high SEMA-123aa expression have a shorter overall survival than those with low SEMA-123aa expression.

[0011] The present invention also provides a reagent for detecting the expression level of circSEMA3C, which is used to predict the prognosis of bladder cancer lymph node metastasis or diagnose bladder cancer.

[0012] Preferably, the reagents include an upstream primer F1 as shown in SEQ ID NO: 1, a downstream primer R1 as shown in SEQ ID NO: 2, an upstream primer F2 as shown in SEQ ID NO: 3, and a downstream primer R2 as shown in SEQ ID NO: 4.

[0013] The present invention also provides use of a SEMA-123aa inhibitor in preparing a drug for treating bladder cancer.

[0014] The present invention also provides the use of an inhibitor of circSEMA3C 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 comprising an upstream primer F1 as shown in SEQ ID NO: 1, a downstream primer R1 as shown in SEQ ID NO: 2, an upstream primer F2 as shown in SEQ ID NO: 3, and a downstream primer R2 as shown in SEQ ID NO: 4.

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

[0018] The present invention takes the circRNA-encoded product that drives bladder cancer lymphatic metastasis as the starting point, screens out the circRNA-circSEMA3C that is upregulated in bladder cancer tissue through high-throughput sequencing, and confirms that SEMA3C-123aa encoded by circSEMA3C is positively correlated with the occurrence of bladder cancer lymphatic metastasis. It further confirms that SEMA3C-123aa promotes bladder cancer lymphangiogenesis and lymphatic metastasis, thereby revealing the biological role of SEMA3C-123aa in bladder cancer lymphatic metastasis, providing a theoretical basis and scientific basis for SEMA3C-123aa as an early diagnostic marker and new treatment target for bladder cancer lymphatic metastasis, filling the gap in the mechanism of action of short peptides encoded by bladder cancer circRNA in the progression of bladder cancer, providing new ideas for the research of new tumor targets, and also has broad clinical application prospects. 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 Schematic diagram of the screening process for screening common highly expressed circRNAs by high-throughput sequencing of paired bladder cancer and adjacent adjacent tissues and expression detection of bladder cancer cell lines in Example 1

[0021] Figure 2 : is the screening and circularity identification diagram of circSEMA3C in Example 1, in which: A is the statistical analysis diagram of the expression of circSEMA3C and SEMA3C linear mRNA after application of RNaseR, and the statistical analysis method used is two-tailed Student t test, Represents p < 0.01; B, C are statistical analysis diagrams of the relative expression levels of circSEMA3C and SEMA3C linear mRNA in T24 or UM-UC-3 cell lines after application of actinomycin D, respectively. The statistical analysis method used was two-tailed Student t test. Represents p < 0.01; D is the statistical analysis of circSEMA3C expression in qRT-PCR detection of random primers and Oligo-dT primer amplification experiments. The statistical analysis method used was two-tailed Student t test. Represents p < 0.01.

[0022] Figure 3 This is an analysis diagram showing the translational potential of circSEMA3C. In the figure: F is a schematic diagram showing the predicted open reading frame and IRES site of circSEMA3C (predicted using the circBANK website, using the IRES prediction program IRESfinder); G is a schematic diagram showing the construction of the circular plasmid;

[0023] Figure 4 These are Western blotting results and immunofluorescence detection images. In the figure: H is Western blotting detection of SEMA3C-123aa expression after transfection of a circular plasmid (the vector is constructed with the IRES+ORF sequence fragment of circSEMA3C, fused to express FLAG tag protein); Figure I is immunofluorescence detection of the cellular localization of the encoded short peptide.

[0024] Figure 5 The expression of SEMA3C-123aa in bladder cancer tissue and adjacent tissues is shown in Figure 1. J and K are Western blot experimental images; L is a histogram of expression levels. The statistical method used is Mann–Whitney U test. Represents p < 0.01.

[0025] Figure 6 This is a diagram of LYVE-1 staining of SEMA3C-123aa in bladder cancer tumors and adjacent tissues. In the figure: Figure M is a representative diagram of LYVE-1 and pan-CK staining in bladder cancer tumor tissues, Figure N is a representative diagram of LYVE-1 and pan-CK staining in bladder cancer adjacent tissues, and Figure O is a statistical analysis of LYVE-1 expression in each group. The statistical analysis method used was the chi-square test. Represents p < 0.01.

[0026] Figure 7Figure 1 is a graph of in vitro experimental results. In the figure: O is a representative image of lymphatic endothelial cell tube formation and transwell taken under a microscope; P on the left is a statistical graph of lymphatic endothelial cell tube formation in different groups. The three dots represent three replicates of the experiment. The statistical method was one-way ANOVA followed by Dunnett's tests; P on the right is a statistical graph of the difference in the number of lymphatic endothelial cell transwell cells in different groups. The three dots represent three replicates of the experiment. The statistical method was one-way ANOVA followed by Dunnett's tests. Represents p < 0.01.

[0027] Figure 8 This is the result of the in vivo experiment. In the figure: Q is the 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. Represents p < 0.01; R is a diagram showing the metastasis of tumors in the popliteal lymph nodes of nude mice detected by immunofluorescence; S is a statistical diagram showing the size of popliteal metastatic lymph nodes in nude mice between different groups, and the statistical method is the same as Q; T is a statistical table showing the number of popliteal metastatic lymph nodes between different groups, and the chi-square test was used for significant difference analysis, one of which Represents statistical significance P < 0.05. DETAILED DESCRIPTION

[0028] 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.

[0029] 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.

[0030] Example 1: Screening and identification of circSEMA3C

[0031] 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 Genomics Co., Ltd. for library construction and sequencing. Subsequently, a highly expressed circular RNA, circSEMA3C, was identified and its circularity was confirmed through random oligo dT reverse transcription, actinomycin D, and RNase R digestion.

[0032] 1. Primers for circSEMA3C and SEMA3C linear mRNA were designed (using Primer3 and the primer design tool website). Then, qRT-PCR was performed to detect the expression of SEMA3C linear mRNA and circSEMA3C in T24 and UM-UC-3 cell lines after the addition of RNaseR (which degrades linear transcripts).

[0033] 1. Total RNA extraction:

[0034] (1) Tissue RNA lysis: Grind the frozen fresh bladder cancer tissue into small particles in liquid nitrogen, add 1 mL of Trizol (TAKARA) lysis buffer, and gently pipette to mix to fully lyse the cells. Transfer the lysis buffer to a 1.5 mL EP tube and let it stand at room temperature for 5 minutes.

[0035] (2) Cell RNA lysis: remove the culture medium and wash twice with PBS. 6 Add 1 mL of Trizol lysis buffer to each cell and gently pipette to mix to fully lyse the cells. Transfer the lysate to a 1.5 mL EP tube and let it stand at room temperature for 5 minutes. Then, add 1 / 5 volume of chloroform and shake vigorously to mix. Let it stand at room temperature until stratification occurs. Then, centrifuge at 12,000 rpm and 4°C for 15 minutes. At this time, the solution in the EP tube will separate into three layers. Carefully transfer the top supernatant (about 400-500 μL) to a new RNase-free EP tube, taking care not to touch the interphase and precipitate.

[0036] (3) Precipitate RNA: Add an equal volume of isopropanol to the supernatant (about 400-500 μL), mix thoroughly, let stand at room temperature for 10 min, then centrifuge at 4°C, 12000 rpm for 10 min, discard the supernatant, and obtain RNA precipitate; add 1 mL of 75% ethanol to wash once, centrifuge at 4°C, 7500 rpm for 5 min, discard the ethanol, and air dry until the RNA becomes completely transparent; add an appropriate amount of DEPC-treated water according to the size of the precipitate, and shake thoroughly to obtain total RNA.

[0037] 2. Reverse transcription: qRT-PCR was performed using Novozymes' HiScript III RT SuperMix for qPCR. The expression of lncRNAs was detected using qRT-PCR. The amplification system and procedure are shown in Tables 1 and 2 below:

[0038] Table 1 qRT-PCR amplification system

[0039]

[0040] Table 2 Amplification program

[0041]

[0042] The primer sequences (designed and synthesized by Shanghai Sangon Biotechnology Co., Ltd.) are as follows:

[0043] circSEMA3C:

[0044] Upstream primer F1: 5′-ACCATGTCCTGTTTCTCGGA -3′ (SEQ ID NO. 1);

[0045] Downstream primer R1: 5′-CGGGTTATCAGTTTCCAGCAG -3′ (SEQ ID NO. 2);

[0046] linear SEMA3C:

[0047] Upstream primer F2: 5′-AAACTCAGAGGACCGGGAAG-3′ (SEQ ID NO. 3);

[0048] Downstream primer R2: 5′-AACACGCAAATTGTCCGGAA-3′ (SEQ ID NO. 4).

[0049] 2. Actinomycin D at a concentration of 1 μM was added to T24 and UM-UC-3 cells. Cell RNA was extracted at various time points of 0, 6, 12, 18, and 24 h. The expression of SEMA3C linear mRNA and circSEMA3C in the T24 and UM-UC-3 cell lines after the addition of actinomycin D was detected by qRT-PCR, and the degradation curves were drawn.

[0050] like Figure 1-2 As shown, circSEMA3C is highly expressed in bladder cancer tissues, and circSEMA3C was identified as a covalently closed circRNA.

[0051] Random primers and Oligo-dT primers were designed. Since circRNA does not have a 3'polyA tail, Oligo-dT primers cannot amplify circRNA.

[0052] Example 2: Analysis of the coding capacity of circSEMA3C

[0053] 1. Through circbank, IRESfinder (IRES prediction program), CPC analysis, Fickett_score and other analysis methods, it was found that circSEMA3C has an IRES sequence and an open reading frame (ORF), suggesting that circSEMA3C has translation potential.

[0054] Second, by inserting the IRES sequence and open reading frame of circSEMA3C into a circular plasmid and fusion-expressing Flag-tagged protein (synthesized by Guangzhou Aiki Biotechnology Co., Ltd.), the UM-UC-3 cell line was transfected to simulate the translation process of circSEMA3C in the cells. The short peptide was detected by Western blotting experiments and Flag-tagged antibodies.

[0055] Experimental method: UM-UC-3 cells were transfected with circular plasmid for 48 h at a concentration of 1×10 5 The cells were seeded into confocal dishes at a density of 1000 cells / dish and plated overnight. The next day, the cells were washed with PBS, permeabilized with Tridon, blocked with BSA, and incubated with primary antibody (FLAG) and fluorescent secondary antibody before sealing the slides. Finally, the cells were photographed using a confocal microscope.

[0056] The results are as follows Figure 3-4 As shown, circSEMA3C has translational potential and can encode a short peptide SEMA3C-123aa with a molecular weight of approximately 123aa, and its amino acid sequence is:

[0057] MRTTKEFPDDVVTFIRNPLMYNSIYPIHKRPLIVRIGTDYKYTKIAVDRVNAADGRYHVLFLGTDRGTVQKVVVLPTNNSVSGELILEELEVFKNHAPITTMKISSKKRMCFCWKLITRGQH (SEQ ID NO. 5).

[0058] Example 3: Analysis of the relationship between SEMA3C-123aa and lymph node metastasis and poor prognosis in bladder cancer patients

[0059] Tissues of patients with bladder cancer lymph node metastasis from Sun Yat-sen Memorial Hospital were collected and paraffin-embedded for preparation of tissue sections. The tissues were quickly frozen in liquid nitrogen and then ground. Protein was extracted using a protein extraction kit (Biosharp) and detected by Western blotting.

[0060] Subsequently, SEMA3C-123aa was detected in bladder cancer tissues with positive and negative lymph node metastasis by using specific antibodies. It was found that the expression of SEMA3C-123aa in bladder cancer tissues was significantly higher than that in bladder cancer adjacent tissues; the expression level in bladder cancer tissues with lymph node metastasis was significantly higher than that in bladder cancer tissues with negative lymph node metastasis.

[0061] In addition, Western blotting analysis of the expression of SEMA3C-123aa in 30 bladder cancer tissues and adjacent tissues suggested that SEMA3C-123aa was upregulated in bladder cancer tissues. Figure 5 shown.

[0062] Tissue section immunofluorescence (Ruchuang Biotech Four-color Multiplex Immunofluorescence Kit plus):

[0063] 1. Place the tissue in 4% paraformaldehyde and fix for 3-4 hours. The time should be adjusted according to the size of the specimen.

[0064] 2. Take a 0.5×0.5×0.5 cm tissue and dehydrate it in 75% alcohol for 1.5 hours, 95% alcohol for 1.5 hours, 95% alcohol for 1 hour, anhydrous ethanol for 1.5 hours, anhydrous ethanol for 1 hour, xylene No. 1 for 0.5 hours, and xylene No. 2 for 0.5 hours.

[0065] 3. Turn on the embedding machine and open the cold stage, paraffin tank, and tissue tank. Place the paraffin block in the embedding machine's paraffin tank and heat to dissolve. Place the dehydrated tissue and the embedding cassette into the machine's tissue tank for wax dipping. Select a mold of appropriate size and drip liquid paraffin into it. Use tweezers to remove the tissue from the embedding cassette and place it into the mold. Finally, cover the mold with the other half of the embedding cassette, drip a small amount of paraffin, and place it on the cold stage to cool.

[0066] 4. Turn on the microtome, secure the wax block, and adjust the distance between the blade and the wax block. Adjust the slice thickness, starting with a rough cut. Once you have the desired tissue section, adjust the desired thickness. After slicing, use a brush and tweezers to lift the slice or strip. Place it in a sink with water at approximately 37°C for spreading. Finally, use a slide to remove the slice.

[0067] 5. Bake the slides in a 65°C oven for 30-60 minutes. Dewax and rehydrate at room temperature: xylene No. 1 for 10 minutes, xylene No. 2 for 10 minutes, anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes, 75% ethanol for 5 minutes, and PBS for 5 minutes twice.

[0068] 6. Prepare antigen retrieval solution and microwave on medium-high for 7 minutes. Then place the sections in the solution and heat on medium-low for 15 minutes. Finally, cool to room temperature. Wash twice with PBS for 5 minutes.

[0069] 7. Use an immunohistochemistry pen to circle the tissue, place the slices in a wet box, add a small amount of distilled water to the box, add 3% hydrogen peroxide, incubate at room temperature for 10 minutes, and wash twice with PBS for 5 minutes.

[0070] 8. Shake off the water, add goat serum blocking solution, place in a wet box, and incubate at 37℃ for 1 hour.

[0071] 9. Shake off the blocking solution, add primary antibody to cover the tissue, and then place it in a humidified box at 4°C overnight.

[0072] 10. Remove the wet chamber from the refrigerator at 4°C and warm to room temperature for 30 minutes. Wash twice with PBS for 5 minutes each. Shake dry and add 50 μL of secondary antibody. Incubate at room temperature for 30 minutes. Wash twice with PBS for 5 minutes each.

[0073] 11. Add fluorescent signal amplification solution to each slide, incubate at room temperature for 15 minutes, and wash twice with PBS for 5 minutes.

[0074] 12. Add 50 μl of DAB colorant to each slide and observe under a microscope for 3-10 minutes. Stop staining when staining is appropriate and rinse with tap water.

[0075] 13. Dehydrate the sections using 70% alcohol for 1 minute, 80% alcohol for 1 minute, 95% alcohol for 2 minutes, anhydrous ethanol for 4 minutes, xylene No. 1 for 3 minutes, and xylene No. 2 for 3 minutes.

[0076] 14. After the slides have dried, add an appropriate amount of anti-fluorescence decay quencher to seal the slides. Cover with a coverslip, being careful of air bubbles. Store in a dark place. Photograph under a microscope.

[0077] The results are as follows Figure 6 As shown in the results, the density of LYVE-1-labeled lymphatic vessels in bladder cancer tissues with high SEMA3C-123aa expression was significantly higher than that in bladder cancer tissues with low SEMA3C-123aa expression.

[0078] Example 4: In vitro experiments

[0079] First, UM-UC-3 cells were transfected with the circular plasmid circSEMA3C-OV, which expresses SEMA3C-123aa, as the experimental group. A group transfected with an empty plasmid served as the control group. A circular plasmid circSEMA3C-ATG-mut (synthesized by Agen Biotechnology, which inhibits the production of SEMA3C-123aa) was also constructed and transfected into UM-UC-3 cells to investigate the role of SEMA3C-123aa itself.

[0080] Subsequently, each group of UM-UC-3 cell lines was incubated with human lymphatic endothelial cells for 48 h. After 48 h of incubation and induction, the human lymphatic endothelial cells were digested with trypsin and centrifuged, and the culture medium was collected and removed for subsequent tube formation and transwell experiments.

[0081] 2. Tube Formation Experiment:

[0082] One day in advance, matrix gel was seeded into a 24-well plate. When spreading the gel, the 24-well plate and centrifuge tube were pre-cooled in an ice bath. The matrix gel and serum-free medium were prepared at a ratio of 1:2 and mixed. 700ul of the diluted and mixed matrix gel was added to each well of the 24-well plate, shaken and spread flat, and then the 24-well plate was placed in an incubator. It can be used after the matrix gel solidifies overnight. The next day, the human lymphatic endothelial cells were resuspended in normal complete medium containing 5% serum and the human lymphatic endothelial cells were cultured at a density of 7×10 4 The number of cells / well was seeded into the matrix gel, gently shaken, and placed in a 37° incubator for 3-5 hours. After the cells formed tubes, they were photographed under an inverted fluorescence microscope and measured using ImageJ. Statistical analysis was then performed to compare the differences between the two groups.

[0083] The results are as follows Figure 7 As shown, SEMA3C-123aa significantly promoted the ability of UM-UC-3 to induce lymphatic endothelial cell tube formation.

[0084] transwell experiment

[0085] Take 3×10 4 The human lymphatic endothelial cells induced in each group were diluted to a volume of 300 μL. The diluted cell suspension was added to the upper chamber of the transwell chamber, and 700 μL of 5% serum culture medium was added to the lower chamber. The entire system was placed in an incubator and cultured for 5 hours. The chamber was removed, the residual supernatant was removed, and the cells were immersed in PBS for 1 minute. Then, the cells were fixed with 4% paraformaldehyde for 15 minutes, and then gently washed three times with PBS. The cells were then stained with crystal violet solution for 15 minutes, and the excess crystal violet solution was washed away with PBS. The cells inside the chamber were then 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. Finally, the differences in the migration ability of lymphatic endothelial cells in each group were statistically analyzed.

[0086] The results are as follows Figure 8 As shown in Q, SEMA3C-123aa significantly promoted the ability of UM-UC-3 to induce lymphatic endothelial cell migration.

[0087] Example 5: In vivo experiments

[0088] A GFP-labeled UM-UC-3 bladder cancer cell line was constructed by transfecting UM-UC-3 cells with a lentiviral vector plasmid (synthesized by Agi) in vitro. A GFP-labeled UM-UC-3 bladder cancer cell line stably overexpressing SEMA3C-123aa was constructed by transfecting UM-UC-3 cells with a lentiviral overexpression plasmid (synthesized by Agi) in vitro. A nude mouse popliteal lymph node metastasis model was used to evaluate the ability of SEMA3C-123aa to promote bladder cancer lymph node metastasis in vivo. Twenty-four healthy female nude mice aged 4-5 weeks were prepared and UM-UC-3 cells (5×10 overexpressing SEMA3C-123aa and control) were injected into the right footpad of nude mice. 5 The footpad tumor model was established. Then, live imaging was performed once a week to observe the metastasis of the popliteal lymph nodes in the footpad of nude mice until the tumor volume of the footpad of nude mice was greater than 200 mm. 3 Popliteal lymph nodes of nude mice were isolated and their volumes were measured. Immunohistochemistry and immunofluorescence were used to analyze tumor metastasis. Differences in popliteal lymph node metastasis rates among different groups were recorded and analyzed.

[0089] like Figure 8 As shown in RT, it was found that bladder cancer cells overexpressing SEMA3C-123aa could significantly promote the lymph node metastasis of bladder cancer.

[0090] 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 level of SEMA-123aa encoded by circSEMA3C in the preparation of a product for diagnosing bladder cancer, predicting the risk of bladder cancer lymph node metastasis, or predicting the survival prognosis of bladder cancer; the amino acid sequence of SEMA-123aa is shown in SEQ ID NO:5, and the product is judged by the expression level of SEMA-123aa.

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 patients with high SEMA-123aa expression have a poor prognosis, while the patients with low SEMA-123aa expression have a good prognosis; the patients with high SEMA-123aa expression have a shorter overall survival than those with low SEMA-123aa expression.

4. Use of a reagent for detecting circSEMA3C expression in the preparation of a kit for predicting the prognosis of bladder cancer lymph node metastasis, characterized in that: The reagents include an upstream primer F1 as shown in SEQ ID NO: 1, a downstream primer R1 as shown in SEQ ID NO: 2, an upstream primer F2 as shown in SEQ ID NO: 3, and a downstream primer R2 as shown in SEQ ID NO: 4.

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