Effect of circSEMA3C-coded SEMA-123aa in targeted therapy of lymphatic metastasis of bladder cancer

Abstract

The invention discloses an effect of circSEMA3C coding SEMA-123aa in targeted therapy of lymphatic metastasis of bladder cancer, and belongs to the technical field of biological medicine. According to the application disclosed by the invention, circRNA-circSEMA3C up-regulated in bladder cancer tissues is screened out through high-throughput sequencing by taking a circRNA coding product for driving bladder cancer lymphatic metastasis as an entry point, and it is proved that the circSEMA3C-123aa coded by the circSEMA3C is positively correlated with bladder cancer lymphatic metastasis, so that bladder cancer lymphatic neogenesis and lymphatic metastasis can be promoted, and bladder cancer lymphatic metastasis can be promoted. Therefore, the biological action of the SEMA3C-123aa in the lymphatic metastasis of the bladder cancer is disclosed, and a theoretical basis and a scientific basis are provided for the SEMA3C-123aa serving as an early diagnosis marker and a novel treatment target of the lymphatic metastasis of the bladder cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the role of circSEMA3C encoding SEMA-123aa in targeted therapy of bladder cancer lymph node metastasis. Background Art

[0002] Bladder cancer is one of the most common tumors of the urinary system. The average incidence of bladder cancer in my country is 5.80 / 100,000, and the average mortality rate of bladder cancer is 2.37 / 100,000. Its high incidence and mortality seriously threaten the lives and health of the people. Distant metastasis is an important factor in the death of malignant tumors. Lymphatic metastasis is the most common metastatic route of bladder cancer. Once a patient has lymphatic metastasis, the 5-year survival rate will drop from 77.6% to 18.6%. However, bladder cancer lymphatic metastasis is not only difficult to diagnose early, but also difficult to give effective treatment. Research data show that surgical radical resection, radiotherapy, chemotherapy and immunotherapy have limited effects on improving the prognosis of patients with bladder cancer lymphatic metastasis. Therefore, exploring the molecular mechanism of bladder cancer lymphatic metastasis, finding effective therapeutic targets and developing targeted therapeutic drugs based on this are currently difficult problems in the clinical diagnosis and treatment of bladder cancer lymphatic metastasis.

[0003] Circular RNA (circRNA) is a type of single-stranded RNA molecule with a covalently closed loop structure. Most of them are composed of exons of protein-coding genes. They are stable in molecular properties and are not affected by RNA exonucleases. They are widely expressed in eukaryotes and participate in a variety of physiological and pathological processes. Studies have shown that circular RNA can act as a molecular sponge in cells to adsorb miRNA, relieve the inhibitory effect of miRNA on target genes, and then upregulate their expression. In addition, circular RNA can also play a role in molecular modification and regulation and molecular skeleton by binding to proteins. Some circular RNAs have also been reported to promote or inhibit gene transcription in the nucleus. Generally, circular RNA was initially considered to have no coding ability, but recent studies have shown that some circular RNAs can also have translation ability. Although circRNA lacks some key translation elements, such as 5'-untranslated region (5'-UTR) and Poly (A) tail, circular RNA with open reading frames (ORFs) can mediate translation in a cap-independent manner through the mechanism of internal ribosome entry site (IRES) or N6-methyladenosine (M6A) modification. At the same time, due to its unique closed structure, the open reading frame in circular RNA may cross the splicing site or even exceed its length, producing short peptides or proteins that do not exist in the protein translated from the linear transcript, and these short peptides or proteins encoded by circular RNA have been reported to play an important role in the progression of various cancers. However, there has been no report on the biological role and molecular mechanism of short peptides encoded by circRNA in bladder cancer and bladder cancer lymph node metastasis. Summary of the invention

[0004] In view of the defects of the prior art, the present invention screened out circSEMA3C, which is highly expressed in the tissues of bladder cancer patients with positive lymphatic metastasis, and can encode SEMA-123aa. The molecular mechanism of SEMA-123aa-mediated bladder cancer lymphatic metastasis was also clarified, and it can be used 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: 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.

[0006] Preferably, the product comprises a chip, a kit or a reagent.

[0007] Preferably, the product is judged by the expression level of SEMA-123aa.

[0008] Preferably, the amino acid sequence of SEMA-123aa is as shown in SEQ ID NO:5.

[0009] Preferably, the patients with high expression of SEMA-123aa have a poor prognosis, and the patients with low expression of SEMA-123aa have a better prognosis; the patients with high expression of SEMA-123aa have a shorter overall survival period than the patients with low expression of SEMA-123aa.

[0010] The present invention also provides a reagent for detecting the expression amount of circSEMA3C, wherein the reagent is used for predicting the prognosis of bladder cancer lymph node metastasis or diagnosing bladder cancer.

[0011] 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.

[0012] The present invention also provides the use of the SEMA-123aa inhibitor in preparing a drug for treating bladder cancer.

[0013] The present invention also provides the use of an inhibitor of circSEMA3C 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 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.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention takes the circRNA encoding product that drives bladder cancer lymph node metastasis as the entry 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 lymph node metastasis, and further confirms that SEMA3C-123aa promotes bladder cancer lymphangiogenesis and lymph node metastasis, thereby revealing the biological role of SEMA3C-123aa in bladder cancer lymph node metastasis, providing a theoretical basis and scientific basis for SEMA3C-123aa as an early diagnostic marker and new therapeutic target for bladder cancer lymph node 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

[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 Schematic diagram of the screening process of screening the commonly highly expressed circRNA by high-throughput sequencing of paired cancer and adjacent tissues of 5 bladder cancers and expression detection of bladder cancer cell lines in Example 1 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 the application of RNaseR, and the statistical analysis method used is two-tailed Student t test, Represents p < 0.01; B and C are statistical analysis diagrams of the relative expression 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 ttest. Represents p<0.01.

[0019] Figure 3 This is an analysis diagram showing that circSEMA3C has translational potential, in which: F is a schematic diagram showing the prediction of the open reading frame and IRES site of circSEMA3C (predicted using the circBANK website, and the IRES prediction program used is IRESfinder); G is a schematic diagram showing the construction of a circular plasmid; Figure 4 These are Western blotting results and immunofluorescence detection images. In the images: H is Western blotting detection of the expression of SEMA3C-123aa after transfection of a circular plasmid (the vector was constructed with the IRES+ORF sequence fragment of circSEMA3C, and fused to express the FLAG tag protein); Figure I is immunofluorescence detection of the cellular localization of the encoded short peptide.

[0020] 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 bar chart of expression level analysis. The statistical method used is Mann–Whitney U test. Represents p<0.01.

[0021] Figure 6 Figure 1 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 adjacent tissues of bladder cancer; Figure O is a statistical analysis of the expression of LYVE-1 in each group. The statistical analysis method used is the chi-square test. Represents p<0.01.

[0022] Figure 7The figure shows the results of in vitro experiments. In the figure: O is a representative picture of lymphatic endothelial cell tube formation and transwell taken under a microscope; P on the left is a statistical chart of lymphatic endothelial cell tube formation in different groups. The three points represent that the experiment was repeated three times, and the statistical method is one-way ANOVA followed by Dunnett's tests; P on the right is a statistical chart of the difference in the number of cells passing through the lymphatic endothelial cell transwell in different groups. The three points represent that the experiment was repeated three times, and the statistical method is one-way ANOVA followed by Dunnett's tests. Represents p<0.01.

[0023] 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 in different groups detected by in vivo imaging. The statistical method is 2-tailed test. represents p < 0.01; R is a diagram of tumor metastasis in the popliteal lymph nodes of nude mice detected by immunofluorescence; S is a statistical diagram of the size of nude mouse popliteal lymph nodes between different groups, and the statistical method is the same as Q; T is a statistical table of the number of popliteal lymph nodes between different groups, and the chi-square test was used for significant difference analysis, one of which Represents statistical significance of P < 0.05. DETAILED DESCRIPTION

[0024] 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.

[0025] 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.

[0026] Example 1: Screening and identification of circSEMA3C Bladder cancer tissue samples and paired adjacent tissues were obtained from 5 patients with bladder cancer at Sun Yat-sen Memorial Hospital through intraoperative sampling and sent to BGI for library construction and sequencing. Subsequently, the highly expressed circular RNA-circSEMA3C in bladder cancer was screened out, and its circular characteristics were identified through random and oligo dT reverse transcription experiments, actinomycin D experiments, and RNase R digestion experiments.

[0027] First, primers for circSEMA3C and SEMA3C linear mRNA were designed (designed by Primer3 and primer design tool website), and then the expression of SEMA3C linear mRNA and circSEMA3C in T24 and UM-UC-3 cell lines after the addition of RNaseR (degradation of linear transcripts) was detected by qRT-PCR.

[0028] 1. Total RNA extraction: (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, gently blow and 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 min.

[0029] (2) Cell RNA lysis: Aspirate the culture medium and wash twice with PBS for 10 min each. 6 Add 1 mL of Trizol lysis buffer to each cell, gently blow and 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 min; then add 1 / 5 volume of chloroform, shake vigorously to mix, and let it stand at room temperature until stratification occurs; then centrifuge at 12000 rpm and 4°C for 15 min. At this time, the solution in the EP tube is divided 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 middle phase and precipitate.

[0030] (3) Precipitate RNA: Add an equal volume of isopropanol (about 400-500 μL) to the supernatant, mix thoroughly, and let stand at room temperature for 10 min. Centrifuge at 4°C, 12,000 rpm for 10 min, and discard the supernatant to obtain RNA precipitate; add 1 mL of 75% ethanol to wash once, centrifuge at 4°C, 7,500 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.

[0031] 2. Reverse transcription: The expression of lncRNAs was detected by qRT-PCR using HiScript III RT SuperMix for qPCR from Novozymes. The amplification system and amplification procedure are shown in Tables 1 and 2 below: Table 1 qRT-PCR amplification system

[0032] Table 2 Amplification program

[0033] The primer sequences (designed and synthesized by Shanghai Sangon Biotechnology Co., Ltd.) are as follows: circSEMA3C: Upstream primer F1: 5′-ACCATGTCCTGTTTCTCGGA -3′ (SEQ ID NO. 1); Downstream primer R1: 5′-CGGGTTATCAGTTTCCAGCAG -3′ (SEQ ID NO. 2); Linear SEMA3C: Upstream primer F2: 5′-AAACTCAGAGGACCGGGAAG-3′ (SEQ ID NO. 3); Downstream primer R2: 5'-AACACGCAAATTGTCCGGAA-3' (SEQ ID NO. 4).

[0034] 2. Add 1 μM actinomycin D to T24 and UM-UC-3, extract cellular RNA at various time points of 0, 6, 12, 18, and 24 h, and use qRT-PCR to detect the expression of SEMA3C linear mRNA and circSEMA3C in T24 and UM-UC-3 cell lines after the addition of actinomycin D, and draw degradation curves.

[0035] like Figure 1-2 As shown, circSEMA3C is highly expressed in bladder cancer tissues, and circSEMA3C is identified as a covalently closed circRNA.

[0036] Random primers and Oligo-dT primers were designed. Since circRNA has no 3'polyA tail, Oligo-dT primers cannot amplify circRNA.

[0037] Example 2: Analysis of the coding capacity of circSEMA3C 1. Through circbank, IRESfinder (IRES prediction program), CPC analysis, Fickett_score and other analysis methods, it was found that circSEMA3C has IRES sequence and open reading frame (ORF), suggesting that circSEMA3C has translation potential.

[0038] Second, the IRES sequence and open reading frame of circSEMA3C were inserted into a circular plasmid and fused with the Flag tag protein (synthesized by Guangzhou Aiki Biotechnology Co., Ltd.), which was then transfected into the UM-UC-3 cell line to simulate the translation process of circSEMA3C in the cell. The short peptide was detected by Western blotting experiments and Flag tag antibodies.

[0039] Experimental method: UM-UC-3 cells were transfected with circular plasmid for 48 h at 1×10 5 The cells were seeded into confocal dishes at a density of 1000 cells / dish and cultured 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.

[0040] 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 about 123aa, and its amino acid sequence is: MRTTKEFPDDVVTFIRNPLMYNSIYPIHKRPLIVRIGTDYKYTKIAVDRVNAADGRYHVLFLGTDRGTVQKVVVLPTNNSVSGELILEELEVFKNHAPITTMKISSKKRMCFCWKLITRGQH (SEQ ID NO. 5).

[0041] Example 3: Analysis of the relationship between SEMA3C-123aa and lymph node metastasis and poor prognosis in bladder cancer patients Tissues from patients with bladder cancer lymph node metastasis in Sun Yat-sen Memorial Hospital were collected and prepared into tissue sections by paraffin embedding. The tissues were quickly frozen in liquid nitrogen and then ground. Protein was then extracted using a protein extraction kit (Biosharp) and detected by Western blotting.

[0042] Subsequently, SEMA3C-123aa was detected in bladder cancer tissues with positive and negative lymph node metastasis by using specific antibody detection. 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.

[0043] In addition, Western blotting analysis of the expression of SEMA3C-123aa in 30 bladder cancer patient tissues and adjacent tissues indicated that SEMA3C-123aa was upregulated in bladder cancer patient tissues, such as Figure 5 shown.

[0044] Tissue section immunofluorescence (Ruchuang Biotech Four-color Multiple Immunofluorescence Kit plus): 1. Place the tissue in 4% paraformaldehyde and fix for 3-4 hours. The time can be adjusted according to the size of the specimen.

[0045] 2. Take a 0.5×0.5×0.5cm 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.

[0046] 3. Turn on the embedding machine, and turn on the cold stage, paraffin tank, and tissue tank respectively. Place the paraffin block in the paraffin tank of the embedding machine and heat to dissolve. Place the dehydrated tissue together with the embedding box into the machine tissue tank for wax dipping. Select a mold of the right size, drip liquid paraffin into the mold, and then use tweezers to take the tissue out of the embedding box and put it into the mold. Finally, cover the mold with the other half of the embedding box, drip a little paraffin, and place it on the cold stage to cool.

[0047] 4. Turn on the slicer, fix the wax block, and adjust the distance between the blade and the wax block. Adjust the slice thickness, make a rough cut first, and switch to the target thickness after the ideal tissue section is cut. After slicing, use a brush and tweezers to lift the slice or strip slice, put it in a water tank and spread it. The water temperature is about 37 degrees. Finally, use an anti-shedding slide to pick up the slice.

[0048] 5. Bake the slides in a 65°C oven for 30-60 minutes. Dewax and hydrate 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.

[0049] 6. Prepare antigen repair solution and microwave on medium-high heat for 7 minutes. Then put the slices in it and heat on medium-low heat for 15 minutes, and finally cool naturally to room temperature. Wash twice with PBS for 5 minutes.

[0050] 7. Use an immunohistochemistry pen to circle the tissue, place the slices in a wet box, add a small amount of distilled water, add 3% hydrogen peroxide, incubate at room temperature for 10 minutes, and wash twice with PBS for 5 minutes.

[0051] 8. Shake off the water, add goat serum blocking solution, place in a wet box, and incubate at 37℃ for 1 hour.

[0052] 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.

[0053] 10. Take out the wet box from the refrigerator at 4 degrees, warm it up to room temperature for 30 minutes, and wash it twice with PBS for 5 minutes. Shake off the water, add 50 microliters of secondary antibody, incubate it at room temperature for 30 minutes, and wash it twice with PBS for 5 minutes.

[0054] 11. Add fluorescent signal amplification solution to each slide, incubate at room temperature for 15 minutes, and wash twice with PBS for 5 minutes.

[0055] 12. Add 50 μl of DAB color developing solution to each slide and observe under a microscope for 3-10 minutes. Stop staining when staining is appropriate and rinse with tap water.

[0056] 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.

[0057] 14. After the slide is dried, add an appropriate amount of anti-fluorescence decay quencher to seal the slide, cover with a cover glass, and be careful of bubbles. Store away from light. Take pictures under a microscope.

[0058] The results are as follows Figure 6 As shown, 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.

[0059] Example 4: In vitro experiments 1. The UM-UC-3 cell line transfected with the circular plasmid circSEMA3C-OV expressing SEMA3C-123aa was used as the experimental group, and the empty plasmid transfection group was set as the control group. The circSEMA3C-ATG-mut circular plasmid (synthesized by Aigi Bio, which inhibits the production of SEMA3C-123aa) was also constructed and transfected into the UM-UC-3 cell line to explore the role of SEMA3C-123aa itself.

[0060] Subsequently, each group of UM-UC-3 cell lines was incubated with human lymphatic endothelial cells for 48 h. After 48 h of incubation, 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.

[0061] 2. Tube Formation Experiment: One day in advance, matrix gel was seeded into the 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 and mixed at a ratio of 1:2. 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. The matrix gel was solidified overnight and then used. The next day, the human lymphatic endothelial cells were resuspended in a normal complete medium containing 5% serum and the human lymphatic endothelial cells were cultured at 7×10 4 The number of cells / well was seeded into the matrix gel, gently shaken, placed in a 37° incubator and waited for 3-5 hours. After the cells formed tubes, they were photographed under an inverted fluorescence microscope and measured with imageJ. Subsequently, statistical analysis was performed to compare the differences between the two groups.

[0062] The results are as follows Figure 7 As shown, SEMA3C-123aa significantly promoted the ability of UM-UC-3 to induce lymphatic endothelial cells to form tubes.

[0063] 3. Transwell Experiment Take 3×10 4 Cells / well The induced human lymphatic endothelial cells in the above groups were diluted to a volume of 300 μL, and 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. After the entire system was placed in an incubator for 5 hours, the chamber was taken out, the residual supernatant was removed, and it was immersed in PBS for 1 minute, and then immersed in 4% paraformaldehyde to fix the cells for 15 minutes, and then gently washed three times with PBS, and then the cells were stained with crystal violet stain for 15 minutes, and the excess crystal violet stain was washed with PBS. Then, the cells inside the chamber were gently wiped with a cotton swab, and then observed and photographed under a microscope, and random fields of view were taken for counting using Image J, and finally the differences in the migration ability of lymphatic endothelial cells in each group were statistically analyzed.

[0064] 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.

[0065] Example 5: In vivo experiments The UM-UC-3 bladder cancer cell line with GFP fluorescence labeling was constructed by transfecting UM-UC-3 with lentiviral vector plasmid labeled with GFP in vitro; the UM-UC-3 bladder cancer cell line with GFP fluorescence labeling and stable overexpression of SEMA3C-123aa was constructed by transfecting UM-UC-3 with lentiviral overexpression plasmid of SEMA3C-123aa gene fragment in vitro; the 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 5 The footpad tumor model was constructed. 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 Or nude mice died. Popliteal lymph nodes of nude mice were isolated, their volumes were measured, and the metastasis of tumors was analyzed by immunohistochemistry and immunofluorescence. The differences in the metastasis rate of popliteal lymph nodes of nude mice between different groups were recorded and analyzed.

[0066] like Figure 8As shown in RT, it was found that bladder cancer cells overexpressing SEMA3C-123aa could significantly promote the lymph node metastasis of bladder cancer.

[0067] 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 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.

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 expression level of SEMA-123aa.

4. The use according to claim 1, characterized in that: The amino acid sequence of SEMA-123aa is shown in SEQ ID NO:

5.

5. The use according to claim 4, characterized in that The patients with high expression of SEMA-123aa have a poor prognosis, and the patients with low expression of SEMA-123aa have a good prognosis; the patients with high expression of SEMA-123aa have a shorter overall survival period than those with low expression of SEMA-123aa.

6. A reagent for detecting the expression level of circSEMA3C, characterized in that: The reagent is used to predict the prognosis of bladder cancer lymph node metastasis or diagnose bladder cancer, and the reagent includes 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.

7. Application of SEMA-123aa inhibitors in the preparation of drugs for treating bladder cancer.

8. Application of inhibitors of circSEMA3C 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 comprises 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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