Urine biomarker for ovarian cancer detection and screening method and application thereof

Abstract

The invention discloses a urine biomarker for ovarian cancer detection and a screening method and application thereof. Wherein the urine biomarker is selected from at least one of the following (1)-(5): (1) heterogeneous nuclear ribonucleoprotein A3; (2) a polyimmunoglobulin receptor; (3) small integrated membrane protein 24; (4) a V type collagen alpha 2 chain; and (5) a microtubule-associated protein 2. The urine polypeptide group biomarker has high sensitivity and specificity when being used for ovarian cancer detection, the detection process is simple and noninvasive, the early diagnosis rate of ovarian cancer can be increased, and the lifetime of an ovarian cancer patient can be prolonged.

Description

Technical Field

[0001] The present invention relates to the technical field of ovarian cancer diagnosis, and in particular to urine biomarkers for ovarian cancer detection and screening methods and applications thereof. Background Art

[0002] Ovarian cancer (OC) is the most deadly gynecological malignancy. Traditional diagnostic methods for ovarian cancer (such as radioimmunoassay, mass spectrometry, radioimmunoassay, immunoassay, polymerase chain reaction, and enzyme-linked immunosorbent assay) have problems such as long time and complex operation. At present, the commonly used diagnostic indicators for ovarian cancer are serum carbohydrate antigen 125 (CA125) and serum human epididymis protein 4 (HE4), but due to their poor specificity, they often lead to delayed diagnosis. At present, it is still difficult to achieve early diagnosis of ovarian cancer. The preferred treatment for ovarian cancer is surgery, followed by chemotherapy, biological therapy, hormone therapy, radiotherapy, etc., but it is still easy to relapse after systemic treatment, and the prognosis is extremely poor. Studies have reported that the 5-year survival rate of ovarian cancer in stages I, II, III, and IV is 90%, 70%, 40%, and 20%, respectively. If the early screening rate can be improved, the survival of patients can be significantly improved. Therefore, improving the early diagnosis rate of ovarian cancer and providing individualized treatment as early as possible is one of the important measures to improve the prognosis of ovarian cancer.

[0003] There are few reports on urinary biomarkers for ovarian cancer. In recent years, a large number of studies have confirmed that HE4 can be used as a tumor marker for ovarian cancer and endometrial cancer. The urine HE4 levels in each group are positively correlated with the serum HE4 levels. The sensitivity of urine HE4 to ovarian cancer is comparable to that of serum HE4, but the specificity is significantly higher than that of serum HE4. CA125 and HE4 are the most commonly used biomarkers for ovarian cancer, but due to their low sensitivity and specificity for early diagnosis, they are usually used for the diagnosis and monitoring of patients in the late stage.

[0004] Therefore, the existing technology still needs to be improved and developed. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide urine biomarkers for ovarian cancer detection and screening methods and applications thereof, aiming to solve the problems of insufficient existing urine biomarkers for ovarian cancer and low early diagnosis rate of ovarian cancer.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect, a urine biomarker for ovarian cancer detection is provided, wherein the urine biomarker is selected from at least one of the following (1) to (5):

[0008] (1) Heterogeneous nuclear ribonucleoprotein A3 (hnRNP A3);

[0009] (2) Polymeric immunoglobulin receptor (pIgR);

[0010] (3) Small integral membrane protein 24 (SMIM24);

[0011] (4) Collagen alpha-2(V) chain (COL5A2);

[0012] (5) Microfibrillar-associated protein 2 (MAP2).

[0013] In a second aspect, a method for screening urine biomarkers as described in the first aspect is provided, comprising the steps of:

[0014] The urine of ovarian tumor patients and normal healthy people was pre-treated for mass spectrometry to obtain samples to be tested;

[0015] Mass spectrometry is used to test the polypeptide components in the sample to be tested, and the peptide segments are assigned through database retrieval and analysis;

[0016] The differences in the degraded protein components between ovarian tumor patients and normal healthy people were compared, and urine biomarkers were screened.

[0017] The preferred technical solution, the mass spectrometry pre-treatment comprises the steps of:

[0018] Transfer the urine to a 3KD ultrafiltration centrifuge tube and centrifuge at 11,000 to 13,000 g for 8 to 12 minutes to obtain an ultrafiltration solution;

[0019] Desalting the ultrafiltered solution through a desalting column;

[0020] The polypeptide is eluted from the desalting column using an elution buffer to obtain an elution solution;

[0021] The eluted solution is centrifuged, concentrated and dried to obtain a sample to be tested.

[0022] According to a preferred technical solution, the elution buffer is prepared with water and contains 0.1 v / v% formic acid and 60 v / v% acetonitrile.

[0023] In a third aspect, a method for preparing an ovarian cancer diagnostic product is provided.

[0024] In a preferred technical solution, the diagnostic product is a reagent or a kit.

[0025] In a fourth aspect, a reagent for detecting ovarian cancer is provided, wherein the reagent comprises a product for detecting the urine biomarker as described in the first aspect.

[0026] In a fifth aspect, a kit for detecting ovarian cancer is provided, wherein the kit comprises a product for detecting the urine biomarker as described in the first aspect.

[0027] Beneficial effects: The present invention provides a urine biomarker for ovarian cancer detection. The urine biomarker has high sensitivity and specificity when used for ovarian cancer detection, and the detection process is simple and non-invasive, which is beneficial to improving the early diagnosis rate of ovarian cancer and improving the survival of ovarian cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the ROC curve of Heterogeneous nuclear ribonucleoprotein A3 for ovarian cancer detection.

[0029] Figure 2 This is the ROC curve of Collagen alpha-2(V)chain for ovarian cancer detection.

[0030] Figure 3 This is the ROC curve of Polymeric immunoglobulin receptor for ovarian cancer detection.

[0031] Figure 4 This is the ROC curve of Small integral membrane protein 24 for ovarian cancer detection.

[0032] Figure 5 This is the ROC curve of Microfibrillar-associated protein 2 for ovarian cancer detection.

[0033] Figure 6 It is a graph showing the results of the efficacy test of the urine biomarker of the present invention. DETAILED DESCRIPTION

[0034] The present invention provides urine biomarkers for ovarian cancer detection and screening methods and applications thereof. To make the purpose, technical solutions and effects of the present invention clearer and more specific, the present invention is further described in detail below.

[0035] The present invention provides a urine biomarker for ovarian cancer detection, wherein the urine biomarker is selected from at least one of the following (1) to (5):

[0036] (1) Heterogeneous nuclear ribonucleoprotein A3 (hnRNP A3);

[0037] (2) Polymeric immunoglobulin receptor (pIgR);

[0038] (3) Small integral membrane protein 24 (SMIM24);

[0039] (4) Collagen alpha-2(V) chain (COL5A2);

[0040] (5) Microfibrillar-associated protein 2 (MAP2).

[0041] The present invention provides a method for screening urine biomarkers as described above, comprising the steps of:

[0042] The urine of ovarian tumor patients and normal healthy people was pre-treated for mass spectrometry to obtain samples to be tested;

[0043] Mass spectrometry is used to test the polypeptide components in the sample to be tested, and the peptide segments are assigned through database retrieval and analysis;

[0044] The differences in the degraded protein components between ovarian tumor patients and normal healthy people were compared, and urine biomarkers were screened.

[0045] In one embodiment, the mass spectrometry pre-treatment comprises the steps of:

[0046] Transfer the urine to a 3KD ultrafiltration centrifuge tube and centrifuge at 11,000 to 13,000 g for 8 to 12 minutes to obtain an ultrafiltration solution;

[0047] Desalting the ultrafiltered solution through a desalting column;

[0048] The polypeptide is eluted from the desalting column using an elution buffer to obtain an elution solution;

[0049] The eluted solution is centrifuged, concentrated and dried to obtain a sample to be tested.

[0050] In one embodiment, the elution buffer is prepared in water and contains 0.1 v / v% formic acid and 60 v / v% acetonitrile.

[0051] The embodiment of the present invention provides the use of the above-mentioned urine biomarker in the preparation of an ovarian cancer diagnosis product.

[0052] In one embodiment, the diagnostic product is a reagent or a kit.

[0053] An embodiment of the present invention provides a reagent for detecting ovarian cancer, wherein the reagent comprises a product for detecting the urine biomarker as described above.

[0054] An embodiment of the present invention provides a kit for detecting ovarian cancer, wherein the kit comprises a product for detecting the urine biomarker as described above.

[0055] The present invention will be further described below by means of specific examples.

[0056] Example 1

[0057] 1. Collection of experimental materials:

[0058] The samples in this example were collected from Shanghai Fudan University Cancer Hospital, and 42 urine samples were collected from normal healthy people and patients with ovarian malignant tumors, and frozen at -80°C. The criteria for patients with ovarian malignant tumors were imaging and pathological sections. The inclusion criteria for the normal control group were age-matched healthy people.

[0059] 2. Urine peptide separation method

[0060] (1) Transfer the urine sample solution (2-3 mL) to a 3KD ultrafiltration centrifuge tube and centrifuge at 12,000 g for 10 min;

[0061] (2) desalting the ultrafiltered solution using a C18 desalting column;

[0062] (3) Elute the sample with Elution buffer (0.1% FA, 60% ACN) and transfer the elution solution to a new EP tube;

[0063] (4) The eluted sample is concentrated and dried by centrifugation for mass spectrometry analysis.

[0064] 3. LC-MS / MS detection and database retrieval analysis

[0065] (1) LC-MS / MS detection

[0066] After desalting, the sample was centrifuged and dried, then redissolved in Nano-LC mobile phase A (0.1% formic acid / water) and loaded into a bottle for online LC-MS analysis. The dissolved sample was loaded onto a nanoViper C18 pre-column (3 μm, ), and then 20 μL of volume was used for washing and desalting. The liquid phase was UltiMate 3000RSLC nano nanoliter liquid phase system (ThermoFisher, USA). The sample was desalted and retained on the pre-column and then separated by the analytical column. The analytical column specification was C18 reverse phase chromatography column (Acclaim PepMap RSLC, 75 μm × 25 cm C18-2 μm ), the gradient used in the experiment was that the mobile phase B (80% acetonitrile, 0.1% formic acid) increased from 5% to 38% in 30 min. The mass spectrometer used the ThermoFisher Q Exactive plus system (ThermoFisher, USA) combined with the nanospray Nano Flex ion source (ThermoFisher, USA), the spray voltage was 1.9 kV, and the ion transfer tube heating temperature was 320 ° C. The mass spectrometer scanning mode was in the information-dependent acquisition working mode (DDA, Data Dependent Analysis), the primary mass spectrometer scanning resolution was 70000, the scanning range was 350-1500 m / z, and the maximum injection time was 100 ms. In each DDA cycle, a maximum of 20 secondary spectra with charges from 2+ to 5+ were collected, the maximum injection time of the secondary mass spectrometer ions was 50 ms, the collision cell energy (high energy collision-induced dissociation, HCD) was set to 28 eV, applicable to all precursor ions, and the dynamic exclusion was set to 25 seconds.

[0067] (2) Database retrieval analysis

[0068] The original raw data files collected by mass spectrometry were processed and searched using PEAKS Studio 8.5 (Bioinformatics Solutions Inc. Waterloo, Canada). The database was the Homo sapiens protein database downloaded from Uniprot, and the search parameters were set as follows: the mass tolerance of the primary mass spectrometry was 10 ppm, the secondary mass spectrometry was 0.03 Da, and the enzyme was set to none; variable modifications: protein N-terminal acetylation (Protein N-term), asparagine / glutamine deamination (Deamidation (NQ)), methionine oxidation (Oxidation (M)), glutamate pyroglutamination (Pyro-glu), glutamine pyroglutamyl cyclization (Pyro-Gln).

[0069] 4. Results

[0070] When conducting biomarker research, it is usually necessary to determine a critical value to determine whether a sample contains a specific protein. In this example, the number of peptides contained in the protein is set as the critical value. The sensitivity and specificity of each protein are calculated using this critical value. Sensitivity refers to the ability of a marker to correctly identify a disease (ovarian cancer in this example), while specificity refers to the ability of a marker to correctly identify a non-disease state.

[0071] This example compares the degradation proteins and their peptides in 32 ovarian cancer patients and 32 non-ovarian cancer control groups. In order to find the best biomarker combination, this example calculates the sum of the sensitivity and specificity of each protein, and then sorts them in descending order according to the value of this sum. Such a sorting can help identify proteins that are most likely to appear in ovarian cancer but not in all non-disease samples. Through this method, this example screened out several specific degradation proteins, and the results are as follows:

[0072] (1) Heterogeneous nuclear ribonucleoprotein A3, Collagen alpha-2(V)chain, Polymeric immunoglobulin receptor, Small integral membrane protein 24 and Microfibrillar-associated protein 2 were obtained by descending combination. The sensitivity and specificity of these five degradation proteins were higher than those of other proteins, and their AUC values ​​were all greater than 0.8. Therefore, they were considered as potential biomarkers. The ROC curves are shown in Figure 2. Figures 1 to 5 shown.

[0073] (2) The peptide belonging to Heterogeneous nuclear ribonucleoprotein A3 is M(+42.01)EVKPPP (SEQ ID NO.1), and its related peptides and their derivative peptides (M(+42.01)EVKPPPGRPQ PDSGRRR, M(+42.01)EVKPPPGRPQPDSGRR, M(+42.01)EVKPPPGRPQPDSGR, etc.) were detected in 19 patients;

[0074] The peptide belonging to Collagen alpha-2(V) chain is GPEGPPGEPGPPGPP (SEQ ID NO. 2), which was detected in 10 patients and its related peptides, such as (GPEGPPGEPGPPG, GPEGPPGEPGPP, GPEGPPGEPGP, etc.);

[0075] The peptide belonging to Polymeric immunoglobulin receptor is EEKAVADTRDQADGSRASVDSGS SEEQGGSSRA (SEQ ID NO.3), and its related peptides (EEKAVADTRDQAD GSRASVDSGSSEEQGGSSRALVSTLVPLG, EEKAVADTRDQADGSRASVDSGSSEEQGGSS RALVSTLVP, EEKAVADTRDQADGSRASVDSGSSEEQGGSSRALVSTL, etc.) were detected in 10 patients;

[0076] The peptide belonging to Small integral membrane protein 24 was Q(-17.03)QATEHRLKP (SEQ ID NO. 4), and its related peptides (Q(-17.03)QATEHRLKPWLVG, Q(-17.03)QATE HRLKPWL, and Q(-17.03)QATEHRLKPW, etc.) were detected in 13 patients;

[0077] The peptide belonging to Microfibrillar-associated protein 2 is PLPPFPDHV (SEQ ID NO. 5), and its related peptides (PLPPFPDHV, LPPFPDH, and LPPFPDHV) were detected in 22 patients.

[0078] 5. Biomarker efficacy testing

[0079] (1) Detection method

[0080] Directly compare the qualitative differences in the types of degraded proteins in the urine of healthy people and ovarian cancer patients.

[0081] (2) Test results

[0082] In the urine of another 10 ovarian cancer patients and the control group, the detection values ​​of the above five degradation proteins showed significant differences. The results are as follows Figure 6 As shown. Figure 6 It can be seen that the five degradation proteins proposed in this example have a minimum frequency of 6 and a maximum of 10 in the 10 verified ovarian cancer samples. Although two proteins were detected in the control group, it cannot be ruled out that they have a potential risk of ovarian cancer. This result shows that the urine biomarker proposed in this example can accurately predict ovarian cancer.

[0083] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A urine biomarker for ovarian cancer detection, characterized in that: The urine biomarker is selected from at least one of the following (1) to (5): (1) Heterogeneous nuclear ribonucleoprotein A3; (2) polymeric immunoglobulin receptor; (3) small integral membrane protein 24; (4) Type V collagen α2 chain; (5) Microtubule-associated protein 2.

2. The method for screening urine biomarkers according to claim 1, characterized in that: Includes steps: The urine of ovarian tumor patients and normal healthy people was pre-treated for mass spectrometry to obtain samples to be tested; Mass spectrometry is used to test the polypeptide components in the sample to be tested, and the peptide segments are assigned through database retrieval and analysis; The differences in the degraded protein components between ovarian tumor patients and normal healthy people were compared, and urine biomarkers were screened.

3. The screening method according to claim 2, characterized in that The mass spectrometry pre-treatment comprises the steps of: Transfer the urine to a 3KD ultrafiltration centrifuge tube and centrifuge at 11,000 to 13,000 g for 8 to 12 minutes to obtain an ultrafiltration solution; Desalting the ultrafiltered solution through a desalting column; The polypeptide is eluted from the desalting column using an elution buffer to obtain an elution solution; The eluted solution is centrifuged, concentrated and dried to obtain a sample to be tested.

4. The screening method according to claim 3, characterized in that The elution buffer was prepared with water and contained 0.1 v / v% formic acid and 60 v / v% acetonitrile.

5. Use of the urine biomarker according to claim 1 in the preparation of an ovarian cancer diagnostic product.

6. The use according to claim 5, characterized in that: The diagnostic product is a reagent or a kit.

7. A reagent for detecting ovarian cancer, characterized in that: The reagent comprises a product for detecting the urine biomarker according to claim 1.

8. A kit for detecting ovarian cancer, characterized in that: The kit comprises products for detecting the urine biomarker according to claim 1.

Images