A bladder cancer urine exosome marker mRNA and uses thereof

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a bladder cancer urine exosome marker mRNA and a use thereof. The marker RNA comprises one or more of the following markers: MDK, KRT17, UBE2C and KLHDC7B. The sensitivity of the four urine exosome mRNAs to Bca detection can reach 88.9%, the specificity can reach 90.0%, and the AUC value can reach 0.957.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a bladder cancer urine exosome marker mRNA and its uses. Background Technology

[0002] Combining urinary cytology and transurethral cystoscopy is currently the commonly used clinical method for diagnosing bronchodilator-associated cancer (BCa). However, urinary cytology has low sensitivity for early diagnosing BCa, and cystoscopy is invasive and not suitable for frequent testing. Furthermore, due to the genomic instability and high mutation rate of BCa at the molecular level, tissue biopsy of a single tumor site may underestimate the heterogeneity of the entire tumor. Sequencing multiple regions of the tumor can identify mutated genes associated with tumor progression; these genes can serve as potential molecular markers for predicting disease progression and prognosis at the time of initial diagnosis.

[0003] Urine-based biomarker collection is an ideal liquid biopsy method for diagnosing BCA and may be more accurate than blood samples in reflecting tumor mutation profiles, especially for early-stage BCA. Urine biomarkers exist in various forms, such as proteins, metabolites, cell-free urinary DNA (UcfDNA), and different types of cell-free RNA. Multiple proteomics studies have identified urinary proteins with diagnostic and prognostic value. Nuclear matrix protein 22 (NMP22) and bladder tumor antigen (BTA) have been approved by the US Food and Drug Administration (FDA) as biomarkers for BCA diagnosis. The NMP22 BladderChek test for point-of-care monitoring is a qualitative method with a sensitivity of 68% and a specificity of 79%. Similarly, BTAstat and BTATRAK have been approved by the FDA for diagnosing BCA, with sensitivities of 66% and 70%, and specificities of 65% and 75%, respectively. In addition, metabolomics has been increasingly used in cancer research in recent years. Currently, urinary metabolic markers that can be used to distinguish between BCa and healthy controls include 5-hydroxyvalerate, cholesterol, 3-phosphoglycerate, glycolic acid, and carnitine C9:1. However, due to the lack of standardized sample collection procedures and unified analysis platforms, as well as the fact that environmental factors and food intake can strongly affect the composition of metabolites, these factors have led to a huge diversity in metabolomic profiles obtained from different laboratories.

[0004] In summary, existing methods for diagnosing BCA have the following limitations: Cystoscopy is an invasive procedure that may cause a series of complications, such as painful urination (50%), urinary frequency (37%), gross hematuria (19%), and infection (3%). Frequent and prolonged examinations place a heavy physical, psychological, and economic burden on patients. For advanced malignant tumors, urine cytology is an effective and non-invasive auxiliary examination; however, its sensitivity for early diagnosis of BCA is low (20%-53%), and a negative cytology result does not rule out the presence of a tumor. Furthermore, existing urine-based biomarker collection methods have moderate performance, high cost, and are prone to false positives under conditions of benign diseases such as inflammation and hematuria.

[0005] In conclusion, it is of great significance to discover a highly sensitive and specific urinary exosome marker for bladder cancer and a method for detecting that marker. Summary of the Invention

[0006] To address the above issues, this invention has developed a urinary exosome marker mRNA for bladder cancer, comprising one or more of the following markers: MDK, KRT17, UBE2C, and KLHDC7B. The expression levels of these four mRNAs in urinary exosomes are correlated with tumor stage, grade, and prognosis. Combining urinary exosome mRNAs can improve the ability to differentiate between bladder cancer (BCa) and other urinary system diseases, and can serve as a potential non-invasive tumor marker for the diagnosis and differentiation of BCa.

[0007] To achieve the above objectives, the present invention can adopt the following technical solutions:

[0008] In one aspect, the present invention provides a urinary exosome marker mRNA for bladder cancer, which includes one or more of the following markers: MDK, KRT17, UBE2C and KLHDC7B.

[0009] In another aspect, the present invention provides a PCR primer pair for detecting the above-mentioned urinary exosome marker mRNA of bladder cancer.

[0010] In another aspect, the present invention provides a detection reagent for detecting bladder cancer, comprising the above-described PCR primer pair.

[0011] In another aspect, the present invention provides a detection kit for detecting bladder cancer, comprising the above-described PCR primer pair or the above-described detection kit for detecting bladder cancer.

[0012] In another aspect, the present invention provides the application of the above-mentioned bladder cancer urinary exosome marker mRNA, or the above-mentioned PCR primer pair, or the above-mentioned detection reagent in the preparation of a detection product for detecting bladder cancer.

[0013] In another aspect, the present invention provides a method for detecting urinary exosome marker mRNA in bladder cancer, characterized in that it includes: RNA sample extraction, cDNA preparation, and amplification using the above-mentioned PCR primer pair or the above-mentioned detection reagent, followed by detection.

[0014] The beneficial effects of this invention include at least the following: the sensitivity of the four urinary exosome candidate mRNAs combined in this invention for Bca detection can reach 88.9%, the specificity can reach 90.0%, and the AUC value can reach 0.957. Attached Figure Description

[0015] Figure 1 NTA results for the identification of exosomes extracted using the Qiagen kit;

[0016] Figure 2 Transmission electron microscopy for identification of exosomes extracted using the Qiagen kit;

[0017] Figure 3 Western blot results for identification of exosomes extracted using the Qiagen kit;

[0018] Figure 4 The expression of MDK in urinary exosomes of 40 BCa patients and 20 health controls (HCs) was detected by qRT-PCR.

[0019] Figure 5 The expression of KRT17 in urinary exosomes of 40 patients with BCa and 20 patients with HCs was detected by qRT-PCR.

[0020] Figure 6 The expression of UBE2C in urinary exosomes of 40 patients with BCa and 20 patients with HCs was detected by qRT-PCR.

[0021] Figure 7 The expression of KLHDC7B in urinary exosomes of 40 patients with BCa and 20 patients with HCs was detected by qRT-PCR.

[0022] Figure 8 ROC curves for the diagnostic efficacy of combining four urinary exosome mRNAs for early BCa. Detailed Implementation

[0023] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0024] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0025] This invention provides a combined detection kit for urinary exosome markers mRNA in bladder cancer, which includes one or more of the following markers: MDK, KRT17, UBE2C, and KLHDC7B.

[0026] It should be noted that the expression levels of the above four mRNAs in urinary exosomes are related to tumor stage, grade, and prognosis, and can serve as potential non-invasive tumor markers for the diagnosis and differentiation of BCa.

[0027] Another embodiment of the present invention provides a PCR primer pair for detecting the above-mentioned urinary exosome marker mRNA of bladder cancer.

[0028] In some specific embodiments, the reagents used to detect the above-mentioned bladder cancer urinary exosome marker mRNA can be PCR primer pairs. Preferably, the following primer pairs are used: PCR primer pairs for detecting MDK mRNA include: upstream primer: SEQ ID NO.1, downstream primer: SEQ ID NO.2; PCR primer pairs for detecting KRT17 mRNA include: upstream primer: SEQ ID NO.3, downstream primer: SEQ ID NO.4; PCR primer pairs for detecting UBE2C mRNA include: upstream primer: SEQ ID NO.5, downstream primer: SEQ ID NO.6; PCR primer pairs for detecting KLHDC7B mRNA include: upstream primer: SEQ ID NO.7, downstream primer: SEQ ID NO.8.

[0029] In another embodiment of the present invention, a detection reagent for detecting bladder cancer is provided, comprising the above-described PCR primer pair for detecting the above-described bladder cancer urinary exosome marker mRNA. It should be noted that the above-described reagent can be supplemented with auxiliary detection reagents to form a detection reagent for bladder cancer, including conventional reagents such as buffer solutions.

[0030] In some specific embodiments, the detection reagents described above may also include fluorescent probes or chemifluorescent materials.

[0031] It should be noted that, in order to further improve the efficiency and sensitivity of detection, chemifluorescent materials or fluorescent probes can be added to the PCR primer pairs. These materials or probes can label the synthesized double strands during the PCR amplification process, and the changes in fluorescence signals can be monitored using external devices, such as a PCR fluorescence detector, thereby allowing for real-time detection of the PCR amplification process.

[0032] It should also be noted that chemifluorescent materials and fluorescent probes each have their advantages and disadvantages. Chemifluorescent materials are convenient to use, do not require complex probe design, and are relatively inexpensive. However, they also have drawbacks such as lack of template specificity, high requirements for primer specificity, inability to perform multiplex quantification, and relatively low sensitivity. Fluorescent probes, on the other hand, have advantages such as high specificity, good repeatability, high sensitivity, and the ability to perform multiplex quantification. However, they are only suitable for a specific target, are relatively expensive, and it is difficult to find probes with low background. When performing detection, the choice between chemifluorescent materials and fluorescent probes can be made based on the specific circumstances.

[0033] In some specific embodiments, the above-mentioned chemical fluorescent material can be a fluorescent dye known in the art, such as SYBR Green; the fluorescent probe can be designed by oneself according to methods known in the art, or it can be designed by a design company.

[0034] Another embodiment of the present invention provides a detection kit for detecting bladder cancer, which includes the above-described PCR primer pair or the above-described detection reagent for detecting bladder cancer.

[0035] It should be noted that the test kit is composed of conventional forms known in the art. For example, the test kit will include instructions, reagent bottles containing various test reagents, small compartments for placing the test reagent bottles, and a dropper for transferring the test reagents.

[0036] In another aspect, the present invention provides the application of the above-mentioned bladder cancer urinary exosome marker mRNA, or the above-mentioned PCR primer pair, or the above-mentioned detection reagent in the preparation of a detection product for detecting bladder cancer.

[0037] In some specific embodiments, the above-mentioned detection products include: detection reagents, test strips, detection kits, or gene chips.

[0038] Specifically, in addition to using the aforementioned PCR amplification primer pairs for conventional PCR amplification detection, followed by quantitative and qualitative analysis of the final PCR amplification products using gel electrophoresis, fluorescent PCR amplification detection can also be performed using the aforementioned detection reagents. This involves introducing a labeling reagent during PCR amplification to label the formed DNA double strands during annealing and extension. In this way, the PCR amplification process can be monitored in real time using the fluorescence signal emitted by the labeling reagent, allowing for qualitative and quantitative analysis of the DNA template to be detected. It should be noted that any method known in the art can be used for the qualitative and quantitative analysis of the sample to be tested.

[0039] In another aspect, the present invention provides a method for detecting urinary exosome marker RNA in bladder cancer, characterized in that it includes: RNA sample extraction, cDNA preparation, and amplification using the above-mentioned PCR primer pair or the above-mentioned detection reagent, followed by detection.

[0040] In some specific embodiments, the above amplification program includes: Stage 1: pre-denaturation, cycle: 1, 95°C, 30s; Stage 2: PCR reaction, cycle: 40, 95°C, 5s, 60°C, 34s.

[0041] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0042] I. Collection and Pretreatment of Urine Specimens

[0043] Collect 10ml-50ml of clean-catch midstream morning urine from the enrolled population. All urine samples are first centrifuged at 2000g for 10min to remove cells and sediment. Then, the supernatant is collected and centrifuged at 10000rpm for 15min to completely remove cell debris. The supernatant is collected, aliquoted into 15ml centrifuge tubes, and stored at -80℃ until exosome extraction. The entire process from sample collection to storage must be completed within 2 hours.

[0044] II. Extraction of RNA from urinary exosomes

[0045] Extraction was performed using the Qiagen miRNeasy Mini Kit (217084), according to the instructions for use:

[0046] Add 700 μl of QIAzol lysis reagent to the sample and vortex for 1 minute; place the tube containing the homogenate at room temperature (15-25℃) for 5 minutes to promote the dissociation of the nucleoprotein complex; add 1 / 5 volume of chloroform to the tube containing the homogenate, tighten the cap, and shake vigorously for 15 seconds to mix thoroughly; place the tube containing the homogenate at room temperature for 2-3 minutes; centrifuge at 12000g for 15 minutes at 4℃; after centrifugation, the sample is separated into three phases: a colorless aqueous phase containing RNA at the top, a white interfacial phase, and a red organic phase at the bottom;

[0047] Transfer the aqueous phase above to a new collection tube, add 1.5 volumes of anhydrous ethanol, mix by pipetting, do not centrifuge, and immediately proceed to the next step; transfer up to 700 μl of sample, including any precipitate that may form, into the RNeasy MinElute rotating column using a 2 ml collection tube; gently close the cap, centrifuge at ≥10,000 rpm for 15 seconds at room temperature, and discard the liquid in the collection tube; repeat the previous step using the remainder of the sample, discarding the liquid in the collection tube;

[0048] Add 700 μl of RWT buffer to the RNeasy MinElute spin column, gently close the cap, and centrifuge at ≥10,000 rpm for 15 seconds to wash the column. Discard the liquid in the collection tube. Add 500 μl of RPE buffer to the RNeasy MinElute spin column, centrifuge at ≥10,000 rpm for 15 seconds to wash the column. Discard the liquid in the collection tube. Transfer 500 μl of 80% ethanol to the RNeasy MinElute spin column, centrifuge at ≥10,000 rpm for 2 minutes to wash the spin column membrane, and discard the collection tube and the liquid in it.

[0049] Place the RNeasy MinElute rotating column into a new 2 mL collection tube. Open the column cap and centrifuge at full speed for 5 minutes to dry the membrane. Discard the collection tube and the liquid inside. Drying the rotating column membrane prevents residual ethanol from interfering with downstream reactions. Centrifuging with the cap open ensures that no ethanol is carried over during RNA elution.

[0050] Place the RNeasy MinElute spin column into a new 1.5 ml collection tube. Add 14 μl of RNase-free water directly to the center of the spin column membrane, gently close the cap, and centrifuge at full speed for 1 minute to elute RNA, yielding 12 μl of eluent.

[0051] RNA concentration and purity were determined using NanoDrop2000. RNA samples with A260nm / A280nm = 1.8-2.0 were used for subsequent experiments or stored at -80°C for later use.

[0052] III. Identification of Urinary Exosomes

[0053] In this embodiment of the invention, to determine the quality and purity of exosomes extracted from urine, nanoparticle tracking analysis (NTA) was performed to assess the size distribution of the exosomes. Figure 1 The NTA results showed that most of the particles extracted by ultracentrifugation had a diameter of ~128.3 nm, with 80% of the particles ranging from 85.6 nm to 221.4 nm. The particles extracted by the adsorption column kit had a diameter of ~133.3 nm, with 80% of the particles ranging from 91.6 nm to 198.1 nm. The extracted exosome samples were observed under a transmission electron microscope. The results showed that the exosomes varied in size, exhibiting a biconcave, saucer-like shape with a clearly visible double-membrane structure, and a diameter of approximately 30-150 nm. Figure 2 This is consistent with the results of NTA. Furthermore, Western blot analysis of exosome-specific protein markers revealed that CD9, CD81, and TSG101 were positive in exosome samples, while the negative marker Calnexin was negative. Figure 3 In summary, these results demonstrate that exosomes were successfully prepared and enriched, and can be used for downstream applications.

[0054] IV. qRT-PCR verification of candidate RNA expression levels

[0055] (1) Preparation of cDNA

[0056] 1) Incubate the RNA sample at 65°C for 5 minutes.

[0057] 2) Prepare a 100 μl RNA reverse transcription system on ice. The reverse transcription system is shown in Table 1.

[0058] Table 1 RNA reverse transcription system

[0059] reagents Added amount 5×PrimeScript RT Master Mix 20μl Total RNA 800ng <![CDATA[RNase Free dH2O]]> Add to 100μl

[0060] Mix thoroughly by pipetting up and down 40 times, centrifuge at 1000g for 30 seconds, and then place in a regular PCR instrument for reaction.

[0061] Reverse transcription conditions:

[0062] Table 2 Reverse transcription conditions

[0063] Step 1 step2 Step 3 37℃ 85℃ 4℃ 30min 5s ∞

[0064] 3) Aliquot the cDNA into two 200μl PCR tubes and store at -20℃ for later use, or use it directly for qRT-PCR detection.

[0065] (2) qRT-PCR

[0066] 1) Primer pairs for mRNA PCR were designed using Primer Express 3.0 software, and Pub-Med BLAST was used for result comparison; all primers were designed and synthesized by Shanghai Sangon Biotech Co., Ltd. Primer sequences are shown in Table 3.

[0067] Table 3 Primer List

[0068]

[0069] The primers were diluted with RNase-free water to a working solution of 10 μM for the experiment.

[0070] 2) Prepare the qRT-PCR reaction system (20 μl) on ice. The qRT-PCR reaction system is shown in Table 4.

[0071] Table 4. qRT-PCR reaction system (20 μL)

[0072] reagents Added amount TB Green Premix Ex Taq II(Tli RNaseH Plus)(2×) 10μl PCR forward primer (10 μM) 0.8μl PCR reverse primer (10 μM) 0.8μl ROX Reference Dye or Dye II(50×) 0.4μl cDNA 2μl Sterilized water 6μl

[0073] Mix by pipetting up and down 40 times, centrifuge at 1000g for 30s, and then place in an ABI-7500 real-time PCR instrument for reaction.

[0074] 3) Standard procedure for two-step PCR amplification:

[0075] Stage 1: Pre-denaturation, Cycle: 1, 95℃, 30s;

[0076] Stage 2: PCR reaction, cycles: 40, 95℃, 5s, 60℃, 34s

[0077] Melting curve stage

[0078] 4) Data processing

[0079] Each sample was tested in triplicate. GAPDH was used as an internal control. The relative expression level of the target gene was calculated using the Ct method, as shown in the following formula.

[0080] ΔCt=Ct 目的基因 -Ct GAPDH

[0081] The relative fold increase of the target gene expression in each group of samples: fold = 2 -ΔCt .

[0082] The expression levels of MDK, KRT17, UBE2C, and KLHDC7B in urinary exosomes of 40 patients with BCa and 20 patients with HCs were detected using the above method. The results are as follows: Figure 4 , Figure 5 , Figure 6 and Figure 7 As shown in the figure. The results showed that, compared with HCs, the expression of MDK, KRT17, UBE2C and KLHDC7B in the urinary exosomes of BCa patients was significantly upregulated (all P<0.001).

[0083] To calculate the diagnostic performance of the exosomal mRNA combination, this embodiment of the invention used the formula to calculate the predictive probability of BCa diagnosis, as follows: Logit(P) = 9.716 × MDK + 0.386 × KRT17 + 0.597 × UBE2C + 4.241 × KLHDC7B. The highest AUC was 0.957 (95% CI, 0.910–1.000), and the sensitivity and specificity were improved to 88.9% and 90.0%, respectively. Figure 8 The differences between different ROC curves were compared using De. Long's algorithm, and the results showed that the diagnostic efficiency of the combination of four exosomal mRNAs was significantly higher than that of any single molecule (Table 5). Therefore, the combination of exosomal mRNAs can serve as a potential non-invasive biomarker for the diagnosis of BCa.

[0084] Table 5. Difference between the ROC curve of the combined diagnosis and the diagnosis of the four mRNAs independently (De. Long method)

[0085] mRNA AUC difference 95% CI P value MDK 0.096 0.763-0.959 0.0343 KRT17 0.151 0.690-0.921 0.0031 UBE2C 0.232 0.590-0.860 0.0003 KLHDC7B 0.068 0.800-0.977 0.0204

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The application of reagents for detecting urinary exosome marker mRNA in bladder cancer in the preparation of detection products for bladder cancer, characterized in that, The bladder cancer urinary exosome marker mRNA consists of MDK, KRT17, UBE2C, and KLHDC7B.

2. The application according to claim 1, characterized in that, The reagents include PCR primer pairs or kits.

3. The application according to claim 2, characterized in that, The PCR primer pairs for detecting MDK mRNA include: upstream primer: SEQ ID NO.1, downstream primer: SEQ ID NO.2; the PCR primer pairs for detecting KRT17 mRNA include: upstream primer: SEQ ID NO.3, downstream primer: SEQ ID NO.4; the PCR primer pairs for detecting UBE2C mRNA include: upstream primer: SEQ ID NO.5, downstream primer: SEQ ID NO.6; and the PCR primer pairs for detecting KLHDC7B mRNA include: upstream primer: SEQ ID NO.7, downstream primer: SEQ ID NO.

8.

4. The application according to claim 1, characterized in that, The detection products include detection reagents, test strips, or gene chips; the detection reagents also include fluorescent probes or chemifluorescent materials.

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