Application of hsacirc0004662

Abstract

The invention belongs to the technical field of tumor molecular biology, and particularly relates to application of hsacirc0004662. It is found for the first time that hsacirc0004662 is significantly increased in a bladder cancer cis-platinum drug-resistant cell line and serum of a drug-resistant patient; besides, the expression of the hsacirc0004662 can be inhibited by using specific siRNA or shRNA, so that the cis-platinum drug resistance of bladder cancer cells can be treated in cell experiments and tumor-bearing mouse experiments, and hsacirc0004662 is prompted to be used as a diagnostic index and a potential therapeutic target of the cis-platinum drug resistance of bladder cancer. Therefore, the application of the hsacirc0004662 provided by the invention to the preparation of the preparation for diagnosing and treating the cis-platinum drug resistance of the bladder cancer has profound clinical significance and important popularization prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor molecular biology, and specifically relates to an application of circular RNA hsa_circ_0004662 in the preparation of a bladder cancer cisplatin-resistant diagnosis and treatment preparation and a corresponding preparation. Background Art

[0002] Bladder cancer is a malignant tumor that occurs in the bladder mucosal epithelium, mainly including urothelial carcinoma, squamous cell carcinoma and adenocarcinoma. According to the statistics of the International Agency for Research on Cancer in 2022, bladder cancer ranks ninth in incidence and thirteenth in mortality among malignant tumors worldwide. In recent years, due to the aging of the population, changes in lifestyle and the application of new diagnostic and treatment technologies, the incidence of bladder cancer in my country has also been increasing year by year.

[0003] Cisplatin is widely used in the treatment of bladder cancer and is considered the "gold standard" for bladder cancer chemotherapy. Cisplatin induces cell apoptosis by forming DNA adducts, significantly inhibiting tumor growth. However, although cisplatin has achieved certain efficacy in the treatment of bladder cancer, most patients will develop drug resistance after a period of cisplatin chemotherapy, which is manifested by factors such as enhanced DNA repair mechanism, increased expression of drug efflux pumps, dysregulation of apoptosis pathways, and microenvironmental immunosuppression, which greatly limits the treatment effect and prognosis. Therefore, in-depth exploration of the diagnostic and therapeutic targets of cisplatin resistance in bladder cancer is of great significance for early identification of cisplatin-resistant patients and optimization of treatment options.

[0004] In recent years, circular RNAs (circRNAs), as a type of non-coding RNA, have attracted extensive attention due to their special single-stranded endogenous characteristics and stable expression. CircRNAs are mainly formed by exons and / or introns, do not have a 5' cap structure and a 3' Poly A tail, and present a closed circular structure. Compared with linear RNA molecules, they have higher stability and are not easily degraded. CircRNAs are mainly located in the cytoplasm or extracellular fluid and are not easily affected by nucleases. Therefore, finding and identifying circRNAs that are specifically elevated in cisplatin-resistant bladder cancer cells is of great significance for subsequent clinical prognosis prediction and screening of therapeutic targets. Through these comprehensive measures, it is expected to improve the efficacy and prognosis of cisplatin-resistant bladder cancer and bring new treatment hope to patients. Summary of the invention

[0005] The first object of the present invention is to provide an agent for inhibiting the expression of hsa_circ_0004662 for use in preparing a cisplatin-resistant therapeutic preparation for bladder cancer, wherein the nucleotide sequence of hsa_circ_0004662 is shown in SEQ ID NO.1.

[0006] The above applications provide a new approach for the treatment of cisplatin-resistant bladder cancer.

[0007] Furthermore, the reagent for inhibiting the expression of hsa_circ_0004662 includes: a small interfering RNA or a short hairpin RNA designed based on the nucleotide sequence of the hsa_circ_0004662 circular splicing site.

[0008] The small interfering RNA sequence is shown in SEQ ID NO.4 or SEQ ID NO.5, and the short hairpin RNA sequence is the same as the small interfering RNA sequence.

[0009] Furthermore, the therapeutic reagent also includes a liposome transfection reagent (commercially available), a transfection-specific culture medium (commercially available) The above-mentioned reagents for inhibiting the expression of hsa_circ_0004662 include but are not limited to the above-mentioned small interfering RNA or short hairpin RNA designed based on the nucleotide sequence of the hsa_circ_0004662 circular splicing site.

[0010] The small interfering RNA designed based on the nucleotide sequence of the hsa_circ_0004662 circular splicing site includes but is not limited to the above-mentioned specific sequence.

[0011] The second object of the present invention is to provide a cisplatin-resistant therapeutic preparation for bladder cancer, which is an agent for inhibiting the expression of hsa_circ_0004662; further comprising: a small interfering RNA or a short hairpin RNA designed based on the nucleotide sequence of the hsa_circ_0004662 circular splicing site.

[0012] The small interfering RNA sequence is shown as SEQ ID NO.4 or SEQ ID NO.5.

[0013] The above-mentioned reagents for inhibiting the expression of hsa_circ_0004662 include but are not limited to the above-mentioned small interfering RNA or short hairpin RNA designed based on the nucleotide sequence of the hsa_circ_0004662 circular splicing site.

[0014] The small interfering RNA designed based on the nucleotide sequence of the hsa_circ_0004662 circular splicing site includes but is not limited to the above-mentioned specific sequence.

[0015] The third object of the present invention is to provide a reagent for detecting a new circRNA, hsa_circ_0004662, for use in preparing a diagnostic preparation for cisplatin resistance in bladder cancer, wherein the nucleotide sequence of hsa_circ_0004662 is shown in SEQ ID NO.1.

[0016] The above applications provide a new approach for the diagnosis of cisplatin resistance in bladder cancer.

[0017] Furthermore, the reagent for detecting hsa_circ_0004662 includes a reagent for detecting the expression level of hsa_circ_0004662 in the serum of bladder cancer patients, bladder cancer tissue or bladder cancer cells.

[0018] Furthermore, the reagent for detecting the expression level of hsa_circ_0004662 in the serum of bladder cancer patients, bladder cancer tissue or bladder cancer cells includes a PCR detection reagent or an in situ hybridization detection reagent.

[0019] Preferably, the PCR detection reagent comprises: The upstream primer is shown in SEQ ID NO.2, The downstream primer is shown as SEQ ID NO.3.

[0020] Furthermore, the PCR detection reagent is a fluorescent quantitative PCR detection reagent, and also includes fluorescent quantitative PCR enzyme (commercially available) and enzyme-free water (commercially available).

[0021] The reagents for detecting the expression level of hsa_circ_0004662 in the serum of bladder cancer patients, bladder cancer tissue or bladder cancer cells of the present invention include but are not limited to the above-mentioned PCR detection reagents or in situ hybridization detection reagents.

[0022] The primers in the PCR detection reagent include but are not limited to the above-mentioned specific primer sequences.

[0023] The fourth object of the present invention is to provide a diagnostic preparation for cisplatin resistance in bladder cancer, comprising a reagent for detecting the expression level of hsa_circ_0004662 in the serum of bladder cancer patients, bladder cancer tissue or bladder cancer cells; the hsa_circ_0004662 nucleotide sequence is shown in SEQ ID NO.1.

[0024] The reagent can be used to efficiently detect cisplatin-resistant cells, tissues or patient serum of bladder cancer.

[0025] Furthermore, the reagent for detecting the expression level of hsa_circ_0004662 in the serum of bladder cancer patients, bladder cancer tissue or bladder cancer cells includes a PCR detection reagent or an in situ hybridization detection reagent.

[0026] Preferably, the PCR detection reagent comprises: The upstream primer is shown in SEQ ID NO.2, The downstream primer is shown as SEQ ID NO.3.

[0027] The above-mentioned reagents for detecting the expression level of hsa_circ_0004662 in the serum of bladder cancer patients, bladder cancer tissue or bladder cancer cells include but are not limited to the above-mentioned PCR detection reagents, or in situ hybridization detection reagents.

[0028] The primers in the PCR detection reagent include but are not limited to the above-mentioned specific primer sequences.

[0029] When the diagnostic reagent of the present invention is used for detecting tissues, serum, etc. of bladder cancer patients, when the expression level of hsa_circ_0004662 in the patient's serum or tissue is significantly increased, it indicates that the subject's sensitivity to cisplatin is significantly reduced.

[0030] In the present invention, bladder cancer cells T24 and 5637 (purchased from the cell bank of the Chinese Academy of Sciences) are treated with long-term low-dose cisplatin to establish cisplatin-resistant cells T24 / CR and 5637 / CR, and the corresponding cisplatin-sensitive bladder cancer cells are T24 and 5637.

[0031] The terms "expression level" and "expression amount" used in the present invention generally refer to the content of the sample to be tested in a biological sample, which can be detected by the corresponding absorbance or the intensity of the fluorescence signal and then converted by a specific formula.

[0032] In the accompanying drawings, cisplatin, the English name is Cisplatin.

[0033] Dimethyl sulfoxide, the English name is Dimethyl Sulfoxide, abbreviated as DMSO.

[0034] Advantages of the present invention: The present invention first discovered that hsa_circ_0004662 is significantly expressed in cisplatin-resistant bladder cancer cells and bladder cancer patient serum. By detecting the expression level of hsa_circ_0004662 in the subject's serum, tissue or cells, it can be determined whether the subject has the risk of cisplatin resistance, so as to diagnose cisplatin resistance in bladder cancer at an early stage.

[0035] In addition, in order to make up for the shortcomings of existing treatment methods, the present invention provides a therapeutic reagent related to cisplatin resistance of bladder cancer hsa_circ_0004662, which is a small interfering RNA (siRNA) or short hairpin RNA (shRNA) designed based on the nucleotide sequence of the hsa_circ_0004662 circular splicing site, which can effectively treat cisplatin chemotherapy resistance of bladder cancer by inhibiting the expression level of hsa_circ_0004662, and has far-reaching clinical significance and important prospects for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1The location, composition, and circularization pattern of hsa_circ_0004662 on chromosomes, as well as the nucleic acid sequence of the circular splicing site of hsa_circ_0004662 evaluated by Sanger sequencing; Figure 2 It is a DNA gel image of the expression of hsa_circ_0004662 specific primers (reverse primer pair) and non-specific primers (forward primer pair) in genomic DNA (genomeDNA, gDNA) and complementary DNA (complementary DNA, cDNA) detected by using the diagnostic kit of Example 1; Figure 3 This is a statistical result diagram of the difference in hsa_circ_0004662 expression in the serum of bladder cancer cisplatin-resistant patients (n=30) and sensitive patients (n=30) detected by the diagnostic kit of Example 1, * represents P<0.05; FIG4 is a diagram showing the statistical results of the difference in expression of hsa_circ_0004662 in bladder cancer cisplatin-resistant cell lines (T24 / CR and 5637 / CR) and sensitive cells (T24 and 5637) using the diagnostic kit of Example 1, * represents P<0.05; FIG5 shows the intracellular subcellular localization of hsa_circ_0004662 in bladder cancer cisplatin-resistant cells T24 / CR and sensitive cells T24 detected using fluorescent in situ hybridization reagents.

[0037] Figure 6 shows the small interfering RNA (siRNA) or short hairpin RNA (shRNA) sequence designed based on the nucleotide sequence of the hsa_circ_0004662 circular splicing site.

[0038] Figure 7 This is a statistical chart of the hsa_circ_0004662-specific siRNA inhibition efficiency detected in bladder cancer cisplatin-resistant cell lines T24 / CR and 5637 / CR cells using the hsa_circ_0004662 diagnostic kit. * represents P < 0.05.

[0039] Figure 8 This is a statistical chart of the effect of cisplatin on cytotoxicity, using CCK-8 kit to detect the inhibition of hsa_circ_0004662 by specific siRNA in cisplatin-resistant bladder cancer cell lines T24 / CR and 5637 / CR. * represents P < 0.05.

[0040] Fig. 9This is an experiment using tumor-bearing mice. The expression level of hsa_circ_0004662 in cisplatin-resistant bladder cancer cells T24 / CR was inhibited by specific shRNA. The mice were then intraperitoneally injected with cisplatin or saline to evaluate the responsiveness to cisplatin after inhibiting hsa_circ_0004662.

[0041] Fig.10 Specific shRNA inhibited the expression level of hsa_circ_0004662 in T24 / CR cells, and then mice were intraperitoneally injected with cisplatin or saline to evaluate the statistical results of the proliferation curves of tumor tissues in different groups; * represents P < 0.05.

[0042] Fig.11 is Fig.10 The final tumor size of each group was determined based on the results.

[0043] Fig.12 is Fig.11 Statistical graph of final tumor weight based on the data presented above; * represents P < 0.05.

[0044] Fig.13 The expression level of hsa_circ_0004662 in T24 / CR cells was inhibited by specific shRNA, and then the mice were intraperitoneally injected with cisplatin or normal saline. The malignancy of tumor tissues in different groups was evaluated by hematoxylin and eosin staining (HE) and immunohistochemical staining (Ki-67). DETAILED DESCRIPTION

[0045] The present invention is described in detail below in conjunction with the accompanying drawings and examples. The following examples are only illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the implementation are all recommended conventional molecular biology experimental conditions or the conditions recommended in the manufacturer's instructions.

[0046] Example 1 Design and verification of primer pairs for hsa_circ_0004662 1. Design of specific primer pairs for specific amplification of hsa_circ_0004662: Download the fasta format sequence of hsa_circ_0004662 from the circBase database. The full length is 462 bp and the sequence is shown in SEQ ID NO.1. Connect the sequence end to end to form a continuous base sequence to form a reverse splicing site. Figure 1As shown in the figure, the chromosomal location, exon composition and specific reverse splicing site sequence of hsa_circ_0004662. Specific primers are designed for the site, and the upstream sequence or downstream sequence of the primer must span the reverse splicing site to amplify a special sequence containing the splicing site bases, which is called the upstream primer of the reverse primer pair for specific amplification of hsa_circ_0004662 as shown in SEQ ID NO.2; the downstream primer of the reverse primer pair is shown in SEQ ID NO.3.

[0047] 2. Preparation of genomic DNA (gDNA) and complementary DNA (cDNA) from bladder cancer cells 1) Extraction and preparation of gDNA from bladder cancer cells Take bladder cancer T24 cells (commercially available, purchased from the cell bank of the Chinese Academy of Sciences) and prepare a tumor cell suspension. Take the cell DNA extraction kit (Cat. DP304-03) provided by Tiangen Biochemical Technology Co., Ltd. as an example. Add buffer GA to the above cells and shake and mix, add proteinase K and mix, then add buffer GB and mix and heat in a 70°C water bath. Then add anhydrous ethanol and shake and mix, add the above liquid to the adsorption column CB3, rinse thoroughly with rinse solution PW, and finally elute with elution buffer to obtain gDNA.

[0048] 2) Extraction and purification of total RNA from bladder cancer cells Take bladder cancer T24 cells and prepare tumor cell suspension. Use Trizol reagent (Cat. 15596018) provided by Invitrogen to extract total RNA from tissue cancer cells. Put 0.5g of cancer tissue into 1mL liquid nitrogen (commercially available) for grinding, then add 1mL of Trizol to dissolve and lyse on ice, then extract with 200μL of chloroform, precipitate with 400μL of isopropanol, stand on ice for 20 minutes and centrifuge at high speed. Then resuspend with 200μL of 75% ethanol and centrifuge at high speed again. After drying the product at room temperature, add 50μL of enzyme-free water to dissolve it, which is the total RNA.

[0049] 3) Preparation of cell cDNA Taking RNase R (Cat. R0301) provided by Gisai Biotechnology as an example, prepare 10× Reaction Buffer, RNase R (20U / μL), and total RNA of 1μg, 1U, and 1μg respectively, and finally dilute to 20μL system with enzyme-free water. Incubate the above system at 37°C for 30 minutes. Use phenol: chloroform: isoamyl alcohol (25:24:1) solution to extract RNA, use ethanol precipitation, and keep it at 70°C for 10 minutes to inactivate the enzyme so that the subsequent reverse transcription can be directly carried out. Taking the circRNA pseudo-transcription kit (Cat. GS0201) provided by Gisai Biotechnology as an example, use the random primers and reverse transcriptase provided in the kit for reverse transcription, and the final product is cDNA.

[0050] 3. Quantitative PCR Reaction and DNA Gel Electrophoresis 1) Preparation of quantitative PCR mixture: Quantitative PCR reaction mixture: 2×Hieff PCR Master Mix (for example, Yeasen, Cat.10102ES03) 10μL, upstream primer SEQ ID NO.2 0.5μL and downstream primer SEQ ID NO.3 0.5μL for specific amplification of hsa_circ_0004662 (or upstream primer SEQ ID NO.6 0.5μL and downstream primer SEQ ID NO.7 0.5μL for internal reference β-actin), cDNA or gDNA extracted above 1μL, enzyme-free water (commercially available) 8μL, a total volume of 20μL of the mixture.

[0051] 2) PCR reaction Pre-denaturation: Place the above mixture into a fluorescent quantitative PCR instrument (commercially available) and perform the following reaction: Step 1: pre-denaturation at 94°C for 5 minutes; Step 2: denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, and the above reaction is carried out for 35 cycles; Step 3: termination extension at 72°C for 10 minutes.

[0052] 3) DNA gel electrophoresis: Weigh 0.5 g of agarose (commercially available), add 50 mL of 1×TAE buffer (commercially available), and prepare an agarose gel. Use 1×DNA loading buffer (commercially available) to mix 10 μL of the above PCR product and perform electrophoresis in an electrophoresis tank (commercially available). The final results were observed and photographed under ultraviolet light. The intensity of the bands in different groups can reflect the expression level of hsa_circ_0004662.

[0053] 4. Interpretation of results: The results are as follows Figure 2As shown in the figure, the specific primer (reverse primer) of hsa_circ_0004662 can only amplify cDNA products, but not gDNA products (gDNA does not contain sheared circRNA), while the non-specific forward primer (forward) can amplify gDNA and cDNA products and form specific bands. The above results suggest that hsa_circ_0004662 has the characteristics of circular RNA, and the reverse primer can specifically amplify the circular splicing site of hsa_circ_0004662.

[0054] Example 2 Application of hsa_circ_0004662 detection kit 1. Total RNA Extraction and cDNA Preparation from Cisplatin-Resistant Bladder Cancer Cell Lines (T24 / CR and 5637 / CR) and Sensitive Cell Lines (T24 and 5637) 1) Establishment of cisplatin-resistant cell line for bladder cancer: According to the standard "acquired resistance method", cisplatin-resistant cell line for bladder cancer was established. The brief description is as follows: First, T24 cells were treated with 0.05μg / mL initial concentration of cisplatin (commercially available, English name Cisplatin) for 3 days, and then replaced with normal complete culture medium. After 5-7 days, most sensitive cells died, and then replaced with the same concentration of cisplatin for another 3 days and then replaced with medium. Repeated 3-5 cycles until the cells can survive more than 80-90% at this concentration. By analogy, the concentration of cisplatin was gradually increased to 0.05, 0.1, 0.2, 0.5, and 1.0μg / mL. The whole process took about 9-10 months, and finally the resistant cell lines (marked as T24 / CR and 5637 / CR cells) were screened out; at the same time, the parental cells of the above control group should be exposed to the corresponding dimethyl sulfoxide (commercially available, English name DimethylSulfoxide, referred to as DMSO) solution during the same period. The CCK-8 experimental method was used to detect the survival ability of resistant cells and control cells, confirming that the above-mentioned resistant cell lines had a resistance effect to cisplatin.

[0055] 2) Extract total RNA from cells and prepare corresponding cDNA (the method is the same as in Example 1).

[0056] 2. Extraction of total RNA from serum of patients with bladder cancer and preparation of cDNA 1) Extract serum from cisplatin-resistant patients (patients with metastatic bladder cancer) and sensitive patients Serum specimens were collected from patients with metastatic bladder cancer before cisplatin chemotherapy. The specific steps are as follows: 5 ml of whole blood was collected using a non-anticoagulated vacuum blood collection tube (commercially available), which was left to coagulate at room temperature for about half an hour, followed by centrifugation and the supernatant was taken. The supernatant was immediately frozen in liquid nitrogen after being aliquoted, and then stored in a -80°C refrigerator (commercially available) for standby use. According to the patient's responsiveness to cisplatin chemotherapy, the specific clinical evaluation index was the progression of imaging (abdominal MRI or PET-CT, etc.), and the patients were divided into a cisplatin-sensitive group (30 cases) and a cisplatin-resistant group (30 cases). Among them, the diagnostic criteria for cisplatin chemotherapy sensitivity were: patients had at least 3 cycles of cisplatin chemotherapy, and the disease showed partial or complete remission (such as a reduction in imaging metastatic load, etc.); the diagnostic criteria for cisplatin chemotherapy resistance were: patients had at least 3 cycles of cisplatin chemotherapy, and the disease progressed (such as imaging progression).

[0057] 2) Extraction and reverse transcription of serum total RNA Taking the serum plasma free RNA extraction kit (Cat. BTN130978) provided by Biolabs as an example, add solution A and shake to mix, then add it to the centrifugal adsorption column and let it stand at room temperature, and then centrifuge it at high speed. Then add the washing solution to the centrifuge column and centrifuge it again. Finally, add the eluent again, collect the eluent after high-speed centrifugation, and it is the RNA sample. Taking the circRNA pseudo-transcription kit (Cat. GS0201) provided by Gisai Biotechnology as an example, the reverse transcription process is performed using the random primers and reverse transcriptase provided in the kit, and the final product is cDNA.

[0058] 3. Amplification and detection of hsa_circ_0004662 1) This part requires the hsa_circ_0004662 diagnostic reagent used in the present invention, which includes: a primer pair for specific amplification of hsa_circ_0004662, a primer pair for the internal reference gene β-actin (commercially available), a fluorescent quantitative PCR enzyme (commercially available), and enzyme-free water (commercially available); suitable for detecting the expression difference of hsa_circ_0004662 in the cDNA of cells, tissues or serum.

[0059] The upstream primer of the primer pair for specific amplification of hsa_circ_0004662 is shown in SEQ ID NO.2, and the downstream primer is shown in SEQ ID NO.3; 2) Steps of Fluorescence Quantitative PCR ①Configure according to the following components Fluorescence quantitative PCR reaction mixture: 2×qPCR SYBR Green Master Mix 10μL, upstream primer SEQ ID NO.2 0.5μL and downstream primer SEQ ID NO.3 0.5μL for specific amplification of hsa_circ_0004662 (or upstream primer 0.5μL and downstream primer 0.5μL for internal reference β-actin), cDNA 1μL, enzyme-free water 8μL, to make a total volume of 20μL of the mixture.

[0060] ②Place the above mixed solution into the fluorescent quantitative PCR instrument (commercially available) Carry out the following reaction: Step 1: pre-denaturation at 95°C for 5 minutes; Step 2: 95°C, 10 seconds, 60°C, 30 seconds, 72°C, 10 seconds, and the above reaction is carried out for 40 cycles; Step 3: Melting curve (set according to the requirements of the instrument).

[0061] ③ Result calculation: The CT value of hsa_circ_0004662 detected by the instrument was compared with β-actin as the internal reference. The final result was calculated by 2 -△△CT The index can be calculated by this method, and the size of this index can reflect the expression difference of hsa_circ_0004662.

[0062] 4. Interpretation of results: Figure 3 As shown in the figure, after real-time fluorescence quantitative PCR reaction, the results showed that the relative expression level of hsa_circ_0004662 in the serum of cisplatin-resistant bladder cancer patients (n=30) was significantly higher than that of cisplatin-sensitive patients (n=30). Figure 4 As shown in the figure, the relative expression level of hsa_circ_0004662 in cisplatin-resistant bladder cancer cells T24 / CR and 5637 / CR was significantly higher than that in cisplatin-sensitive bladder cancer cells T24 and 5637.

[0063] Example 3 In situ hybridization experiment of hsa_circ_0004662 in cisplatin-resistant and sensitive cells of bladder cancer 1. Kit and probe preparation: Taking the fluorescent in situ hybridization kit (Cat. C10910) provided by Ruibo Bio as an example, the hsa_circ_0004662-specific Cy3-labeled probe Probe is designed and provided by Ruibo Bio.

[0064] 2. Preparation of cell slides, cell fixation, and permeabilization: Take cisplatin-resistant T24 / CR cells and cisplatin-sensitive T24 cells in the logarithmic growth phase, inoculate them into cell slides or confocal dishes for continued culture, and conduct subsequent experiments after the cells are fully expanded. Use PBS buffer to wash thoroughly, fix with 4% paraformaldehyde, permeabilize with pre-cooled PBS buffer containing 0.5% Triton X-100, and finally wash thoroughly with PBS buffer again.

[0065] 3. Probe incubation and DAPI staining: First, incubate with pre-hybridization solution for half an hour and then discard it. Then, drop the hybridization solution mixed with FISH probe onto the cell surface and incubate at 37°C overnight. Then, wash with hybridization washing solution I, washing solution II, washing solution II and PBS buffer respectively. Finally, use 1×DAPI staining solution to stain the cell nucleus.

[0066] 4. Observation and analysis: A laser confocal microscope was used for observation, photography and analysis, with a maximum excitation wavelength of 555 nm and a maximum emission wavelength of 570 nm. ImageJ software was used for cell localization and quantitative analysis.

[0067] 5. Interpretation of results: Figure 5 As shown, the expression of hsa_circ_0004662 in cisplatin-resistant T24 / CR cells was significantly higher than that in sensitive T24 cells; in addition, in cisplatin-resistant T24 / CR cells, hsa_circ_0004662 mainly existed in the cytoplasm and exerted its biological function.

[0068] Example 4 Preparation and verification of siRNA molecules designed based on the nucleotide sequence of the hsa_circ_0004662 circular splicing site 1. Design of hsa_circ_0004662-specific small interfering RNA (siRNA) molecules: siRNA molecules are a type of double-stranded RNA molecules with a length of 21-25 nucleotides, which can specifically degrade the target RNA by complementary binding to homologous RNA, thereby inhibiting its expression. Figure 6 As shown, according to the principle of base complementary pairing, an siRNA molecule designed based on the nucleotide sequence of the hsa_circ_0004662 circular splicing site is designed, as shown in SEQ ID NO.4 or SEQ ID NO.5.

[0069] 2. Transfection of siRNA molecules into cisplatin-resistant cell lines Using a liposome transfection kit (commercially available), siRNA molecules that specifically silence hsa_circRNA or negative control siRNA molecules (commercially available from Rebo Biotech) were transfected into T24 / CR and 5637 / CR cells, and the cells were collected after 48 hours of culture. Total RNA was extracted using the Trizol method, and cDNA was generated using a circRNA reverse transcription kit.

[0070] 3. Detection of siRNA silencing efficiency and interpretation of results The expression level of hsa_circ_0004662 in the above cells was detected by the fluorescent quantitative kit designed by the present invention. Figure 7 As shown, the siRNA molecules designed based on the hsa_circ_0004662 circular splicing site can specifically inhibit the expression of hsa_circ_0004662 in T24 / CR and 5637 / CR cells.

[0071] Example 5 Effect of siRNA silencing hsa_circ_0004662 expression on cell proliferation and cisplatin resistance 1. CCK-8 detection of cell proliferation ability and cisplatin toxicity: 1) Cell preparation and transfection: 1000 cells / 100 μL of cell suspension were inoculated into a clean 96-well plate, and the 96-well plate was placed in a 37°C cell culture incubator for overnight culture. Subsequent experiments were performed only after the cells were completely attached to the wall. Lipofectamine transfection reagent (commercially available) was used to transfect specific hsa_circ_0004662 siRNA or negative control siRNA (Ribo Bio, commercially available); 24 hours after transfection, the culture medium of the above two groups of cells was replaced with a culture medium containing different concentrations of cisplatin (0, 0.5, 1.0, 2.0, 4.0, 8.0 μg / mL), and culture was continued for 48 hours.

[0072] 2) CCK-8 detection: Add 100 μL of CCK-8 solution (Tongren Biotechnology, Cat. CK04) to the wells to be tested and centrifuge briefly to eliminate bubbles; continue to culture the above cells for 2 hours, and finally use a microplate reader to measure the OD value at an absorbance of 450 nm to detect cell activity.

[0073] 3) Calculation and interpretation of results: Cell survival rate = (OD value of experimental well - OD value of blank well) / (OD value of control well - OD value of blank well) × 100%. Figure 8 As shown in the results, after inhibiting the expression of hsa_circ_0004662 using siRNA, the sensitivity of cisplatin-resistant T24 / CR and 5637 / CR cells to cisplatin was enhanced, suggesting that inhibiting hsa_circ_0004662 can treat cisplatin resistance.

[0074] Example 6 Effect of inhibiting the expression of hsa_circ_0004662 on tumor growth and cisplatin responsiveness in tumor-bearing mice 1. Use short hairpin RNA (shRNA #1) to establish bladder cancer cisplatin-resistant T24 / CR cells with stable silencing of hsa_circ_0004662, marked as stable silencing hsa_circ_0004662 (shRNA #1) group and control (shNC) group: 2. Establishment of a subcutaneous tumor-bearing mouse model: 5-6 week-old male Balb / c nude mice (purchased from Beijing Huafukang Biological Company) were kept in a constant temperature, constant humidity and pathogen-free SPF-level experimental animal room. All the above animal experimental methods and processes were in compliance with the regulations of the Animal Ethics Committee of Tianjin Medical University. After the BALB / c nude mice were randomly divided into cages and adapted to sexual life for 1 week, subsequent experiments were started. Prepare the above-mentioned logarithmic growth phase, stably transfected bladder cancer resistant cells, and prepare a cell concentration of 5×10 per 100 μL of pre-cooled PBS. 6 Then add an equal volume of Matrigel to a final concentration of 2.5×10 6 Use a microsyringe to slowly implant 100 μL of the drug subcutaneously in the right inguinal region of the mouse and continue feeding the mouse.

[0075] 3. Preparation of cisplatin-containing saline and control saline: First, calculate the required cisplatin mass and saline volume according to the concentration formula c=m / V. For example, to prepare 10mL of a solution with a concentration of 2.5mg / mL, 25mg of cisplatin is required. Then use a precision balance to accurately weigh cisplatin. Because cisplatin is toxic, be sure to take precautions when weighing to prevent contact with the skin and inhalation. After that, place the cisplatin powder in a container such as a volumetric flask, first add a small amount of saline and stir or oscillate thoroughly to completely dissolve it. Cisplatin has a certain solubility in water. Finally, continue to add saline until the solution volume reaches 10mL, so as to obtain a cisplatin saline solution with the target concentration. The entire preparation process must strictly follow laboratory operating procedures to ensure safe operation and accurate solution concentration.

[0076] 4. Establishment of cisplatin treatment model in tumor-bearing mice: When the tumor volume reaches 50 mm 3 At 14:00, the patients were given intraperitoneal injections of saline containing cisplatin (at a concentration of 2.5 mg / kg) or an equal volume of saline in the control group, once every 3 days for a total of 4 times. Fig. 9 shown.

[0077] 5. Evaluation of tumor growth: Monitor the growth of tumor-bearing mice every 3 days, change bedding and food, and use a vernier caliper to measure the short diameter and diameter (in cm) of the mouse tumor during the growth period; when the volume of the inoculated tumor is greater than 1 cm 3 At the same time, the mice were dislocated by cervical dislocation, and the tumors were completely removed, then weighed and photographed.

[0078] 6. Interpretation of results: Figure 10-12 As shown in the figure, compared with the control group (shNC), the growth rate of tumors in the hsa_circ_0004662 (shRNA) silencing group was significantly reduced, and the tumor volume was also significantly reduced; compared with the treatment with normal saline containing dimethyl sulfoxide (DMSO), the sensitivity of tumors to cisplatin (DTX) was significantly enhanced after silencing hsa_circ_0004662 (shRNA). Fig.13 As shown, HE staining and Ki-67 immunohistochemical staining results showed that the morphology of tumor cells in the hsa_circ_0004662 (shRNA) silencing group changed significantly and the malignancy was significantly reduced; compared with the treatment with normal saline containing dimethyl sulfoxide (DMSO), cisplatin (DTX) treatment after silencing hsa_circ_0004662 (shRNA) could significantly inhibit the malignancy of the tumor.

[0079] The above implementation description is only used to explain the method and design ideas of the present invention. It should be pointed out that for ordinary technicians in this field, without departing from the principle of the present invention, the present invention can be appropriately modified or improved, and these modifications are also within the scope of protection required by the present invention.

Claims

1. Use of an agent for inhibiting the expression of hsa_circ_0004662 in the preparation of a preparation for treating cisplatin-resistant bladder cancer, wherein the nucleotide sequence of hsa_circ_0004662 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The reagent for inhibiting the expression of hsa_circ_0004662 includes: a small interfering RNA or a short hairpin RNA designed based on the nucleotide sequence of the hsa_circ_0004662 circular splicing site.

3. The use according to claim 2, characterized in that The small interfering RNA sequence is shown as SEQ ID NO.4 or SEQ ID NO.

5.

4. A preparation for treating cisplatin-resistant bladder cancer, characterized in that: An agent for inhibiting the expression of hsa_circ_0004662; further comprising: a small interfering RNA or a short hairpin RNA designed based on the nucleotide sequence of the hsa_circ_0004662 circular splicing site.

5. The preparation for treating cisplatin-resistant bladder cancer according to claim 4, characterized in that: The small interfering RNA sequence is shown as SEQ ID NO.4 or SEQ ID NO.

5.

6. Use of a reagent for detecting hsa_circ_0004662 in the preparation of a diagnostic preparation for cisplatin resistance in bladder cancer, wherein the nucleotide sequence of hsa_circ_0004662 is shown in SEQ ID NO.

1.

7. The use according to claim 6, characterized in that The reagents for detecting hsa_circ_0004662 include reagents for detecting the expression level of hsa_circ_0004662 in the serum of bladder cancer patients, bladder cancer tissue or bladder cancer cells.

8. The use according to claim 7, characterized in that The reagent for detecting the expression level of hsa_circ_0004662 in bladder cancer patient serum, bladder cancer tissue or bladder cancer cells includes a PCR detection reagent or an in situ hybridization detection reagent; preferably, the PCR detection reagent includes: The upstream primer is shown in SEQ ID NO.2, The downstream primer is shown as SEQ ID NO.

3.

9. A preparation for diagnosing cisplatin resistance in bladder cancer, characterized in that: It includes a reagent for detecting the expression level of hsa_circ_0004662 in the serum of bladder cancer patients, bladder cancer tissue or bladder cancer cells; the nucleotide sequence of hsa_circ_0004662 is shown in SEQ ID NO.

1.

10. The diagnostic preparation for cisplatin resistance of bladder cancer according to claim 9, characterized in that The reagent for detecting the expression level of hsa_circ_0004662 in bladder cancer patient serum, bladder cancer tissue or bladder cancer cells includes a PCR detection reagent or an in situ hybridization detection reagent; preferably, the PCR detection reagent includes: The upstream primer is shown in SEQ ID NO.2, The downstream primer is shown as SEQ ID NO.3.

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