Bladder cancer molecular marker AP2S1 and application thereof

Abstract

The invention relates to the technical field of biological medicine and tumor diagnosis, in particular to a bladder cancer molecular marker and application thereof, and the bladder cancer molecular marker is a coding grid protein related linker compound AP2sigma 1 subunit AP2S1. Through bioinformatics analysis and clinical verification, the research proves that AP2S1 has good correlation with bladder cancer, can be used as a marker for diagnosis and prognosis of bladder cancer, can be used as a target for preparing medicines for treating bladder cancer, and has important clinical significance.

Description

Technical Field

[0001] The present invention belongs to the fields of biomedicine technology and tumor diagnosis technology, and specifically relates to AP2S1 as a bladder cancer biomarker and its application. Background Art

[0002] The incidence of bladder cancer ranks first among urinary tract malignancies in my country and the incidence is still increasing year by year. The main histological subtype of bladder cancer is urothelial carcinoma, which accounts for 90% of all bladder tumors, and the rest are squamous cell carcinoma, adenocarcinoma or neuroendocrine tumors. According to the degree of infiltration, bladder cancer is divided into muscle-invasive bladder cancer (MIBC) that has spread or passed through the detrusor muscle or non-muscle-invasive bladder cancer (NMIBC) that is limited to the mucosa or submucosal connective tissue, accounting for approximately 25% and 75% of newly diagnosed bladder cancer patients, respectively. Non-muscle-invasive bladder cancer has a high recurrence rate and is prone to progression. After transurethral bladder tumor resection and intravesical chemotherapy or BCG instillation, more than 50% of patients may relapse. Despite local treatment, 10-40% of high-risk non-muscle-invasive bladder cancers may eventually progress to muscle-invasive bladder cancer, resulting in a survival rate that is even lower than that of newly diagnosed muscle-invasive bladder cancer (5-year overall survival rates are 37% and 49%, respectively). Patients with muscle-invasive bladder cancer have a poor prognosis (5-year overall survival <50%), which has not improved substantially in the past few decades. Advanced bladder cancer that progresses to metastatic tumors and colonizes distant organs has limited treatment options and poor efficacy. Bladder cancer requires long-term intensive treatment and monitoring, which imposes a considerable economic burden on patients' health care systems, which is a major clinical challenge.

[0003] Early detection and treatment of bladder cancer is an effective means to improve the survival rate of patients. Therefore, finding a method for early diagnosis of bladder cancer is of great significance for the control and intervention of bladder cancer. However, existing bladder cancer tumor markers have problems such as sensitivity and specificity that cannot meet the expected values ​​and limited diagnosis of single markers. Therefore, finding a new marker with high specificity and good sensitivity that can be used for bladder cancer diagnosis and prognosis judgment has great clinical value and research significance.

[0004] AP2S1 is a gene encoding a subunit of the clathrin-associated adaptor complex AP2sigma 1, which is essential for clathrin-mediated endocytosis of plasma membrane components such as the calcium-sensing receptor. Calcium signaling has been reported to regulate cytokine release and tumor growth, but the role of AP2S1 in bladder cancer diagnosis has not been reported. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides the use of the AP2S1 gene as a target for the diagnosis and treatment of bladder cancer. The AP2S1 gene can be used as a molecular marker for diagnosing bladder cancer.

[0006] The specific technical solutions are as follows: The present invention provides a bladder cancer tumor marker AP2S1 and application thereof. The coding sequence of the AP2S1 gene is the DNA sequence shown in SEQ ID NO.1.

[0007] The present invention found through analysis of TCGA database and GEO database that AP2S1 was significantly upregulated in bladder cancer tissue, and its expression in cancer tissue of patients with higher pathological grades was further increased. Survival curve analysis found that the expression level of AP2S1 was negatively correlated with the patient's overall survival (OS) and disease-free survival (DFS).

[0008] The present invention finds through clinical specimen analysis that the AP2S1 mRNA and protein levels in bladder cancer tissues are significantly higher than those in the corresponding adjacent cancer tissues.

[0009] The reagent for specifically detecting the AP2S1 gene or its expression product of the present invention can be used to prepare a product for diagnosing early bladder cancer. The product can diagnose whether a person has bladder cancer by detecting the expression level of the AP2S1 gene.

[0010] Further, the reagents include reagents for detecting the expression level of AP2S1 gene or its expression product by RT-PCR, real-time quantitative PCR, immunoassay, in situ hybridization or chip. The reagents for detecting the expression level of AP2S1 gene or its expression product by RT-PCR or real-time quantitative PCR also include forward primers and reverse primers for specific amplification of AP2S1 gene, the sequence of the forward primer is shown in SEQ ID NO.2, and the sequence of the reverse primer is shown in SEQ ID NO.3. The product includes a detection chip or a kit.

[0011] The present invention also provides the use of AP2S1 as a target in the preparation of a drug for treating bladder cancer.

[0012] Furthermore, the drug for treating bladder cancer includes an inhibitor of the AP2S1 gene and / or its expression product; the inhibitor is an agent that inhibits the expression of the AP2S1 gene, or an agent that inhibits the stability of the AP2S1 gene expression product, or an agent that reduces the activity of the AP2S1 gene expression product, or an agent that inhibits the function of the AP2S1 gene expression product; the agent that inhibits the expression of the AP2S1 gene is an agent that inhibits the transcription of the AP2S1 gene, or an agent that inhibits the translation of the AP2S1 gene, or an agent that inhibits the content of the AP2S1 protein.

[0013] Beneficial effects of the present invention: The present invention has discovered a molecular marker AP2S1 for diagnosing and judging the prognosis of bladder cancer, which has high specificity and high sensitivity. The use of this molecular marker can be used to judge the occurrence and prognosis of bladder cancer, and combined with other tumor markers, it can provide a new strategy for the diagnosis and treatment of bladder cancer. In addition, the present invention provides a basis for the development of new drugs or new technologies for treating bladder cancer with the AP2S1 gene and its expression products as diagnostic and therapeutic targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is an analysis of the expression of AP2S1 in bladder cancer in the TCGA database; the expression of AP2S1 in bladder tissue (Figure A), the expression of AP2S1 in cancer tissue of patients with higher pathological grades (Figure B), the influence of gender (Figure C), race (Figure D), smoking status (Figure E), and molecular typing (Figure F) on the high expression of AP2S1 in bladder cancer tissue.

[0015] Figure 2 This is the expression analysis diagram of AP2S1 in bladder cancer in the GEO database.

[0016] Figure 3 The effect of high expression of AP2S1 on the prognosis of bladder cancer.

[0017] Figure 4 The results of AP2S1 mRNA level analysis in cancer tissues and adjacent tissues in clinical samples.

[0018] Figure 5 These are the results of analysis of AP2S1 protein levels in cancer tissues and adjacent tissues in clinical samples. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the embodiments. The equipment and reagents used in each embodiment and test example can be obtained from commercial sources unless otherwise specified. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0020] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0021] Example 1: Expression of AP2S1 in the TCGA database The ualcan tool (https: / / ualcan.path.uab.edu / ) was used to analyze the expression level of AP2S1 in TCGA database samples, and the relationship between AP2S1 expression level and pathological grade, gender, race, smoking status, and molecular typing was further analyzed, and statistical analysis was performed.

[0022] Figure 1This is an analysis chart of AP2S1 expression in bladder cancer in the TCGA database; ualcan data analysis showed that the expression of AP2S1 in bladder tissue was significantly upregulated (Figure A), and the expression in cancer tissues of patients with higher pathological grades was further increased (Figure B), and the high expression of AP2S1 in bladder cancer tissue was not affected by gender (Figure C), race (Figure D), smoking status (Figure E), or molecular typing (Figure F).

[0023] Example 2: Expression of AP2S1 in the GEO database The Oncomine tool (https: / / www.oncomine.org / ) was used to analyze the clinical specimens of bladder cancer in the GEO database and the data set of normal patients, and the expression level of the AP2S1 gene in several breast cancer data sets was analyzed. To reduce the error rate, P < 0.0001, Fold Change > 2 and the top 10% of the gene ranking were selected as the screening thresholds, and the results were further analyzed.

[0024] Figure 2 This is the expression analysis diagram of AP2S1 in bladder cancer in the GEO database; Oncomine data analysis showed that the expression of AP2S1 in bladder cancer tissue was significantly higher than that in adjacent cancer tissues.

[0025] Example 3: Analysis of the effect of high expression of AP2S1 on the prognosis of bladder cancer (1) The GEPIA tool (http: / / gepia.cancer-pku.cn / ) was used to analyze the impact of high AP2S1 expression on OS and DFS in bladder cancer patients in TCGA database samples. The AP2S1 expression cutoff value was set to the median; (2) The PrognoScan tool (http: / / www.abren.net / PrognoScan / ) was used to analyze the effect of high AP2S1 expression on OS and DFS in bladder cancer patients in GSE13507 database samples.

[0026] Figure 3 The effect of high expression of AP2S1 on the prognosis of bladder cancer. In the GSE13507 and TCGA bladder cancer databases, the OS and DFS of patients with high expression of AP2S1 were worse than those of the low expression group.

[0027] Example 4: Analysis of AP2S1 mRNA levels in cancerous tissues and adjacent tissues in clinical samples (1) Sample acquisition: The bladder cancer tissues and paired adjacent cancer tissues used in the present invention were provided by the Urology Research Institute of the Second Hospital of Lanzhou University and stored in a liquid nitrogen tank; (2) RNA extraction: ① Prepare tumor tissue samples: Use scissors to cut samples from tumor tissue and immediately place them into RNase-free polyvinyl alcohol tubes; ② Tissue grinding: Take out the frozen tumor tissue sample and quickly cut it into small pieces, then grind the sample into fine powder in liquid nitrogen; ③ RNA extraction: Use TRIzol reagent to extract RNA, mix the ground tissue sample with TRIzol reagent, and add chloroform, ethanol and other reagents in stages for precipitation and washing; ④ RNA precipitation and washing: After adding chloroform, gently shake the sample to mix and allow the phases to separate. Collect the RNA supernatant and transfer it to a new RNase-free polyacryl alcohol tube, and wash the RNA with ethanol respectively; ⑤ RNA dissolution: dissolve RNA precipitate in RNase-free water; ⑥ RNA quality and concentration detection: Use RNA quality and concentration measuring instruments (such as NanoDrop) to detect the quality and concentration of the extracted RNA; (3) RNA reverse transcription: mRNA reverse transcription was performed using the reaction system provided by the PrimeScript RT reagent Kit. 0.5 μg of RNA was taken for reverse transcription, and the total system was 20 μL. The entire preparation process needed to be performed on ice. The specific system is as shown in the table below. Reaction conditions: 37°C for 15 minutes, 85°C for 5 seconds; (4) qRT-PCR detection of AP2S1 expression level: ①The reaction system of qRT-PCR is as described in the following table. Prepare the reaction system on ice, mix well after preparation, add into 8-tube strips in 3 portions, 15μl / tube, mark well, centrifuge quickly, and then react and detect on CFX96 real-time fluorescence quantitative PCR instrument; ②Reaction conditions Step 1: Pre-denaturation: 95 °C for 3 minutes; Step 2: PCR reaction at 95°C for 10 seconds, 55°C for 30 seconds, for a total of 40 cycles; Step 3: Dissolution curve ③The main primer sequences are as follows: Figure 4 The results of the analysis of AP2S1 mRNA levels in cancer tissues and adjacent tissues in clinical samples show that the AP2S1 mRNA level in clinical samples is significantly higher than that in adjacent tissues.

[0028] Example 5: Analysis of AP2S1 protein levels in cancer tissues and adjacent tissues in clinical samples (1) Extracting tissue cell proteins: For tissues, add liquid nitrogen to freeze-dry the tissues, grind them into powder immediately, add 300 uL of strong RIPA cell lysis buffer (containing 1 μM PMSF), and place on ice for 30 minutes. Gently blow and swirl every 10 minutes, and be careful to avoid vigorous blowing to produce too many bubbles. After centrifugation at 12000g in a 4℃ centrifuge for 20 minutes, aspirate the upper liquid as the protein solution; (2) BCA method to measure protein concentration: Add different concentrations of BSA to set up standard wells in a 96-well plate. Add the protein sample to be tested, and the final volume of the liquid in each well is 20μL. Then, prepare the reaction solution (liquid A: liquid B = 50:1), 200μL per well, and place it in a 37℃ carbon dioxide-free drying oven for 30 minutes. Finally, read the absorbance at 562nm in a microplate reader. In addition, a standard curve was made in Excel based on the concentration and absorbance of the standard, and the concentration of each group of protein samples was calculated. The sample concentration was diluted to 1.5μg / μL with cell lysis buffer. The protein sample was mixed with 5× loading buffer and boiled for 3-5 minutes; (3) Protein gel electrophoresis: Select 10% polyacrylamide gel and add 20 μL protein sample to each well. ① Set the voltage to 60V first, and adjust the voltage to 100V after the protein band runs into the separation gel. Stop the electrophoresis when the methylene blue band approaches the bottom of the gel. Before protein transfer, prepare 1× electrophoresis transfer solution, soak the PVDF membrane in methanol for 30 seconds, and then soak it in the transfer solution together with the filter paper. ② Next, according to the black gel and white membrane principle, transfer the gel and PVDF into the wet transfer electrophoresis tank, set the voltage to 110V and transfer for 120 minutes. ③ After the transfer is completed, place the PVDF membrane in 1× TBST solution and shake it slowly on a shaker for 10 minutes. ④ Transfer the PVDF membrane to 5% BSA blocking solution and continue to shake it slowly on a shaker for 30-60 minutes. ⑤ According to the size of the protein marker, cut the target band, put it into the primary antibody (diluted with 5% BSA blocking solution), and incubate it in a 4°C refrigerator overnight. ⑥ Wash the PVDF membrane with 1X TBST solution; three times, 20 minutes each time. ⑦ Prepare the corresponding secondary antibody solution (diluted with 5% BSA blocking solution) and incubate at room temperature for 1-2 hours. ⑧ Wash the PVDF membrane three times with 1X TBST solution, 20 minutes each time. ⑨ Prepare the exposure solution and expose it in the machine. The primary antibodies used in the present invention: AP2S1 (abcom, ab128950), Tublin (Proteintech, 11224-1-AP).

[0029] Figure 5Figure 2 is the analysis result of AP2S1 protein level in cancer tissue and adjacent tissue in clinical samples. It can be seen that the AP2S1 protein level in clinical samples is significantly higher than that in adjacent tissue, and the AP2S1 protein level in muscle-invasive bladder cancer tissue is significantly higher than that in non-muscle-invasive bladder cancer tissue.

[0030] The above is only the preferred embodiment of the present application, which does not limit the protection scope of the present application. For those skilled in the art, the present application may have various changes and modifications. Within the spirit and principle of the present application, the changes, modifications, replacements, integrations and parameter changes of these embodiments by conventional substitutions or capable of achieving the same functions without departing from the principles and spirit of the present application shall fall within the protection scope of the present application.

Claims

1. Use of a reagent for detecting the expression level of the AP2S1 marker in the preparation of a product for bladder cancer diagnosis or / and prognosis prediction, characterized in that: The nucleotide sequence of AP2S1 is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that: The expression level of AP2S1 in bladder cancer tissue is higher than that in adjacent adjacent tissues.

3. The use according to claim 1, characterized in that: The reagents include reagents for detecting the expression level of the AP2S1 gene or its expression product by RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization or chip.

4. The use according to claim 3, characterized in that: The reagents for detecting the expression level of AP2S1 gene or its expression product by RT-PCR or real-time quantitative PCR include a forward primer and a reverse primer for specifically amplifying AP2S1 gene. The sequence of the forward primer is shown in SEQ ID NO.2, and the sequence of the reverse primer is shown in SEQ ID NO.

3.

5. The use according to claim 3, characterized in that: The expression product of AP2S1 gene is AP2S1 mRNA or AP2S1 protein.

6. The use according to claim 1, characterized in that: The product includes a detection chip or a test kit.

7. Application of AP2S1 as a target in the preparation of drugs for the treatment of bladder cancer.

8. The use according to claim 7, characterized in that: The drug for treating bladder cancer includes an inhibitor of the AP2S1 gene and / or its expression product; the inhibitor is an agent that inhibits the expression of the AP2S1 gene, or an agent that inhibits the stability of the AP2S1 gene expression product, or an agent that reduces the activity of the AP2S1 gene expression product, or an agent that inhibits the function of the AP2S1 gene expression product; the agent that inhibits the expression of the AP2S1 gene is an agent that inhibits the transcription of the AP2S1 gene, or an agent that inhibits the translation of the AP2S1 gene, or an agent that inhibits the content of the AP2S1 protein.

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