CN115825414A

CN115825414A - Blood or urine metabolic marker and application thereof in endometrial cancer early screening

Info

Publication number: CN115825414A
Application number: CN202310030787.5A
Authority: CN
Inventors: 曹冬焱; 陈君宇; 孙伟; 刘晓燕; 孙海丹; 俞梅; 周慧梅
Original assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Current assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Priority date: 2023-01-09
Filing date: 2023-01-09
Publication date: 2023-03-21
Anticipated expiration: 2043-01-09
Also published as: CN115825414B

Abstract

The invention belongs to the technical field of biology, and particularly relates to a blood or urine metabolic marker and application thereof in early screening of endometrial cancer, wherein the metabolic marker is selected from ADP-mannose, docosadienoic acid, hippuric acid or a combination thereof, and the metabolic marker can be used for early diagnosing patients with endometrial cancer, improving the survival rate of the patients, providing a new breakthrough for clinical diagnosis of the endometrial cancer, and having a better clinical application prospect.

Description

Blood or urine metabolic marker and application thereof in endometrial cancer early screening

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a blood or urine metabolic marker and application thereof in early screening of endometrial cancer.

Background

So far, differential metabolites including 3-hydroxybutyric acid, linoleic acid, stearic acid, myristic acid, threonine and valine in serum, phosphorylcholine, C14:2, 14-dienyl l-carnitine, C6 and caproyl-carnitine and the like in plasma, and porphobilinogen, acetylcysteine, n-acetylserine, imidazolidinoic acid and isobutyryl glycine and the like in urine have been found as predictors of Endometrial Cancer (EC). In addition, researches show that the urine of the EC patient contains higher estrogen and metabolite content thereof than normal women, which indicates that the high expression of the estrogen in the body of the patient can also be used as an EC screening index. However, a group of specific metabolites is not known. The reason for this is probably that the analysis of metabonomics is influenced by the fact that EC patients mostly combine with various metabolic complications. And most research samples have small quantity, and the diagnostic efficiency of the screened marker is more than 90 percent, so the effect is not good enough. In addition, different analytical platforms may also cause differences. Currently, the methods for metabonomic detection commonly used include mass spectrometry and Nuclear Magnetic Resonance (NMR) techniques. The NMR technology has the advantages that the sample pretreatment step is simple, nondestructive and unbiased analysis can be performed on the sample, but the sensitivity is poor, and the resolution is not high. Compared with NMR, the mass spectrometry has higher sensitivity, resolution and specificity, and a standard product library can be used for identification, but the specificity is slightly poor, the identification capability on a large number of spectrum peaks is not high, and the quantification of metabolites is easily influenced by the ionization degree. Therefore, mass Spectrometry is often combined with Chromatography that is accurate in quantitation, powerful in separation, but less powerful in qualitative analysis, including Gas Chromatography-Mass Spectrometry (GC-MS) and Liquid Chromatography-Mass Spectrometry (LC-MS). Wherein GC-MS is mainly suitable for analyzing samples with strong volatility, and LC-MS is mainly used for analyzing samples with weak volatility and poor thermal stability. In view of this, the invention adopts Ultra high throughput liquid chromatography-mass spectrometry (UPLC-MS) technology to perform combined analysis of blood and urine metabonomics on a sample to be researched, to search for differential metabolites of EC patients and healthy controls, and to establish a diagnostic model, so as to provide a new biomarker for early discovery and diagnosis of EC.

Disclosure of Invention

Based on the background, the invention provides a blood or urine metabolic marker and application thereof in early screening of endometrial cancer in order to solve the problems that no recognized marker exists, most research samples are small in quantity and diagnosis efficiency is poor.

In order to realize the purpose, the invention is realized by the following technical scheme:

the invention provides application of blood or urine metabolites as diagnostic markers in preparing products for early diagnosis of endometrial cancer, wherein the blood or urine metabolites are selected from ADP-mannose, docosadienoic acid, hippuric acid or a combination thereof.

Further, the product comprises a kit and a chip.

Further, the product can be used for diagnosing endometrial cancer by detecting the content levels of ADP-mannose, docosadienoic acid and hippuric acid.

Further, the detection method is a mass spectrometry identification method.

Furthermore, the mass spectrometry adopts a primary full-scanning mode for screening and combines secondary targeting analysis. Specifically, the method adopts an ultra-high liquid chromatography-high resolution mass spectrometry (UPLC-MS) to detect the blood or urine metabolites in a full scanning mode, wherein the full scanning mode is to simultaneously acquire primary information of all small molecules in a mass range of 50m/z to 1200m/z, then screen out the differential metabolites through multivariate statistical analysis (principal component analysis PCA and orthogonal partial least squares discriminant analysis OPLS-DA), further perform targeted secondary fragmentation on the differential metabolites, and finally determine the structures of the differential metabolites by combining a database secondary spectrogram.

The invention also provides an identification reagent for early diagnosis of endometrial cancer, wherein the identification reagent is a reagent for detecting blood or urine metabolites.

Further, the blood or urine metabolite is selected from ADP-mannose, docosadienoic acid, hippuric acid, or a combination thereof.

The invention also provides a kit for early diagnosis of endometrial cancer, which comprises an identification reagent for detecting ADP-mannose, docosadienoic acid, hippuric acid or a combination thereof.

The invention also provides a chip for early diagnosis of endometrial cancer, which comprises an identification reagent for detecting ADP-mannose, docosadienoic acid, hippuric acid or a combination thereof.

Compared with the prior art, the invention has the following beneficial effects:

the EC is researched through blood or urine combined metabonomics, the screened combined metabolic marker can distinguish the EC from a control, the AUC value can reach 0.983, the sensitivity and the specificity are better, the verification is better performed in a verification group, and the EC can be used as a potential tumor marker. In the population with long-term abnormal menstruation, abnormal uterine bleeding, occupation in uterine cavity, postmenopausal intimal thickening and the like, the blood or urine metabolic marker provided by the invention can be used for predicting canceration risk, screening out real high-risk population, and actively carrying out invasive examination to obtain definite diagnosis of intimal pathology.

Drawings

FIG. 1 is a classification chart of serum metabolism profiles of endometrial cancer group and normal control group (Panel A: PCA classification chart, panel B: OPLS-DA classification chart);

FIG. 2 is a classification chart of urine metabolism profiles of endometrial cancer group and normal control group (A: PCA classification chart, B: OPLS-DA classification chart);

FIG. 3 is a ROC curve for joint prediction of three metabolites in example 1;

FIG. 4 is a ROC curve for joint prediction of three metabolites in example 2.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Example 1 screening for differential metabolites

1. Instruments and reagents

1) The instrument comprises: waters ACQUITY H-Class liquid chromatograph (Waters corporation), triple TOF 5600 mass spectrometer (Thermofeisher Scientific corporation).

2) The main reagents are as follows: acetonitrile (Thermofisher Scientific); c18 reverse phase chromatography column (3.0 mm X100 mm, C18, 1.7 μm, waters corporation).

2. Sample collection

1) Blood and urine from 99 patients with endometrial cancer and 41 age and gender matched normal controls were selected from Beijing cooperative Hospital.

2) Blood collection: extracting fasting venous blood, centrifuging at 3000 g for 5min, and collecting supernatant.

3) Collecting urine: collecting fasting morning urine, centrifuging at 5000g for 30min, and removing precipitate.

3. Sample metabolite extraction

1) Blood metabolite extraction

Taking 50 mu L of serum sample, adding 150 mu L of mass spectrum water and 400 mu L of acetonitrile, vortexing, and standing for 1 hour at-20 ℃; centrifuging at 14000g for 10min, centrifuging and concentrating the supernatant, redissolving 50 μ L of 2% acetonitrile water, centrifuging at 14000g for 10min, and sampling 10 μ L after passing through a 10kD filter membrane.

2) Urine metabolite extraction

Collecting 200 μ L urine supernatant, adding 200 μ L acetonitrile, vortexing, standing at 4 deg.C for 30min, centrifuging 14000g for 10min, collecting supernatant, centrifuging, concentrating, redissolving with 200 μ L2% acetonitrile water, centrifuging 14000g for 10min, filtering with 10kD filter membrane, and sampling 10 μ L.

4. Liquid phase analysis

Liquid phase analysis was performed using a Waters acquisition H-Class model ultra performance liquid chromatography system, under the following conditions: and (3) chromatographic column: waters HSS T3C 18 (3.0 mm. Times.100 mm, 1.8 um), column temperature 40 ℃; the mobile phase A is 0.2 per mill formic acid water, the mobile phase B is 0.2 per mill formic acid acetonitrile solution, the elution mode is gradient elution, the elution time is 15 minutes, the flow rate is 0.5 mL/min, and the specific sample injection volume is 10uL. The specific information is shown in table 1 below.

5. Identification by mass spectrometry

Eluted blood or urine metabolites were analyzed using Triple TOF 5600 mass spectrometer with data pattern DDA. The parameters are as follows: the primary full scan range is 50-1200m/z, the integration time is 0.25s, the secondary integration time is 0.1s, GS1 is 55, GS2 is 55, curtain Gas is 35, the temperature is set to 550, and Ionspray Voltage flowing is 4500.

The raw data obtained from the UPLC-LTQ orbitrap was processed using the commercial omics analysis software progenesis QI (Version 2.0, nonlinear Dynamics, UK) from Waters corporation. The software can automatically complete pretreatment procedures such as peak alignment, peak identification, peak correction and the like, and finally output a three-dimensional matrix, namely a spectrum peak index variable consisting of retention time and accurate mass-to-charge ratio, a sample name and peak intensity/area. The obtained data matrix is imported into multivariate statistical software SIMCA-P software 14.0 (Umetrics AB, umea, sweden) for PCA analysis, and the change trend among groups is visualized. Variables with non-reference test p-values less than 0.05 were considered as significantly different variables between groups and were screened as early potential markers of endometrial cancer. And (3) performing secondary fragmentation on the screened differential variables, and selecting 204060eV energy according to specific metabolites by adopting an HCD (High collision dissociation) fragmentation mode. Deconvoluting the secondary fragment by using progenesis QI software, searching HMDB (HUMAN METABOLOME DATABASE) DATABASE, and determining the structure of the differential metabolite.

6. Screening results

Blood metabolites: unsupervised PCA plots showed that the endometrial cancer group and the normal control group exhibited some discrimination (see fig. 1A); and a model is further constructed by adopting a supervision OPLS-DA, and the two groups are more distinct (see FIG. 1B). 39 differential metabolites were screened between the endometrial cancer group and the normal control group.

Metabolites of urine: the PCA plot showed that the endometrial cancer group was clearly distinguished from the normal control group (see fig. 2A), and OPLS-DA showed that the two groups were more clearly distinguished (see fig. 2B). 78 differential metabolites were screened between the endometrial cancer group and the normal control group.

The differential metabolite obtained by primary screening of blood or urine metabolites is subjected to targeted dipolar analysis to determine 3 metabolite structures, namely ADP-mannose, docosadienoic acid and hippuric acid. And the 3 metabolites are combined and used for the regional endometrial cancer group and the normal control group to achieve a better prediction effect by adopting a logistic regression calculation method, the AUC value of an ROC curve is 0.983 (see figure 3), and the sensitivity and the specificity are shown in a table 2.

Example 2 validation of differential metabolites obtained by screening

And (3) verifying the screened metabolites as markers for early diagnosis of endometrial cancer.

Alternatively 47 examples of blood or urine metabolites from patients with endometrial cancer and 18 benign controls were used as samples and blood or urine metabolites were extracted as in example 1. Performing full-scan mode detection on blood or urine metabolites by using UPLC-MS, and quantitatively analyzing three differential metabolites between an endometrial cancer group and a benign control group based on the primary spectrum peak area of a marker: ADP-mannose, docosadienoic acid and hippuric acid. The ROC curve was used to evaluate the predictive effect of three metabolites on endometrial cancer. The results show that the AUC value predicted by the combination of the three metabolites is 0.971 (see figure 4), the prediction effect is good, and the prediction effect is remarkably superior to that of a single metabolite.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. The application of blood or urine metabolite as a diagnostic marker in the preparation of products for early diagnosis of endometrial cancer is characterized in that: the blood or urine metabolite is selected from ADP-mannose, docosadienoic acid, hippuric acid or a combination thereof.

2. Use according to claim 1, characterized in that: the product comprises a kit and a chip.

3. Use according to claim 1, characterized in that: the product can be used for diagnosing endometrial cancer by detecting the content levels of ADP-mannose, docosadienoic acid and hippuric acid.

4. Use according to claim 3, characterized in that: the detection method is a mass spectrometry identification method.

5. Use according to claim 4, characterized in that: the mass spectrometry identification method adopts a primary full-scanning mode for screening and combines secondary targeting analysis.

6. An identification reagent for use in the early diagnosis of endometrial cancer, comprising: the identification reagent is a reagent for detecting blood or urine metabolites; the blood or urine metabolite is selected from ADP-mannose, docosadienoic acid, hippuric acid or a combination thereof.

7. A kit for early diagnosis of endometrial cancer, comprising: the kit comprises the identification reagent of claim 6.

8. A chip for early diagnosis of endometrial cancer, comprising: the chip comprises the identification reagent of claim 6.