CN117169503A - Application of SERPINA10 as molecular marker in preparation of products for diagnosing prostate cancer - Google Patents

Abstract

The application provides application of SERPINA10 as a molecular marker in preparing a product for diagnosing prostate cancer, and discovers that the SERPINA10 is used as an effective blood molecular marker for assisting in diagnosing prostate cancer, the plasma expression level of the SERPINA10 is closely related to the occurrence and the development of the prostate cancer, and a detection kit of the SERPINA10 can detect the expression level of the SERPINA10 in the plasma of a patient, so that the prostate cancer can be diagnosed rapidly, the sensitivity reaches over 78 percent, and the specificity reaches over 66 percent.

Description

Application of SERPINA10 as molecular marker in preparation of products for diagnosing prostate cancer

Technical Field

The application relates to the technical field of biomedical detection, in particular to application of SERPINA10 serving as a molecular marker in preparation of a product for diagnosing prostate cancer.

Background

Prostate cancer (PCa), the most common malignancy of the male reproductive system, increases in onset with age, and is the third leading cause of cancer death in men. Prostate cancer is characterized by abnormal division of prostate cells, resulting in abnormal growth of the prostate. Most men will not die from prostate cancer, but will either suffer from slow growing tumors or survive due to steadily improving and effective treatment. Prostate cancer death is primarily due to metastasis that occurs when cancer cells spread to other parts of the body, including pelvic and retroperitoneal lymph nodes, spinal cord, bladder, rectum, bone and brain.

Early detection of prostate cancer is important for effective cure, but the perfect test of early detection is not yet reliable. The detection of tumor markers such as Prostate Specific Antigen (PSA) in blood is used to find suspicious cases, but the test is criticized because it may miss some cancer cases. Several methods and techniques are available for diagnosing prostate cancer. It includes transrectal ultrasound (TRUS) to acquire images from areas selected for biopsy, but TRUS may not reliably detect all areas affected by prostate cancer. Color doppler ultrasound is an improved technique for detecting prostate cancer by measuring blood flow in the gland. Combinations of these techniques may be used to improve detection, including MRI in combination with TRUS-guided biopsies.

The recognition and benefit of the possibility of early discovery and treatment of prostate cancer increases the success rate of managing or controlling the disease, and therefore, the development of a specific biomarker for prostate cancer would be beneficial to the early discovery, early diagnosis and early treatment of this population, providing theoretical and technical support for improving its prostate function.

Disclosure of Invention

The application aims to provide an application of SERPINA10 as a molecular marker in preparing a product for diagnosing prostate cancer, and a detection kit of the SERPINA10 can be used for detecting the expression level of the SERPINA10 in the plasma of a patient, so that the expression level of the SERPINA10 in the plasma of the patient can be rapidly and conveniently detected. The single detection of the expression level of the plasma SERPINA10 distinguishes prostate cancer patients and normal control patients, the sensitivity reaches over 78 percent, and the specificity reaches over 66 percent.

In order to solve the technical problems, the application adopts the following technical scheme:

in a first aspect of the application there is provided the use of SERPINA10 as a molecular marker in the manufacture of a product for diagnosing prostate cancer.

Further, the product for diagnosing the prostate cancer comprises a detection kit of SERPINA10, and the detection kit of SERPINA10 comprises an ELISA detection kit or a real-time fluorescence quantitative PCR kit of SERPINA 10.

Further, the ELISA detection kit of SERPINA10 comprises:

(A) ELISA plate coated with SERPINA 10;

(B) Standard negative serum;

(C) Standard positive serum;

(D) Horseradish peroxidase-labeled enzyme-labeled secondary antibody: anti-IgG;

(E) Sample diluent, coating buffer, blocking solution, ELISA plate washing solution, antibody diluent, color developing solution and stop solution.

In a second aspect of the application there is provided the use of a detection kit of SERPINA10 in the manufacture of a product for diagnosing prostate cancer.

One or more technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

1. the application of the SERPINA10 serving as a molecular marker in preparing a product for diagnosing the prostate cancer provided by the application takes the SERPINA10 as an effective blood molecular marker for assisting in diagnosing the prostate cancer, and the plasma expression level of the SERPINA10 is closely related to the occurrence and development of the prostate cancer; the increased expression level of the marker SERPINA10 compared to healthy controls suggests that the patient has prostate cancer, and when the SERPINA10 content is 1.85 times higher than the normal SERPINA10 content, the patient is judged to be in the state of prostate cancer at the moment.

2. The application can prepare the kit for auxiliary diagnosis of the prostate cancer by using the detection kit of SERPINA10, thereby rapidly and conveniently screening or assisting in screening the prostate cancer.

3. The application uses the expression level of the plasma SERPINA10 to assist in diagnosing the prostate cancer patients, and independently detects the expression level of the plasma SERPINA10 to distinguish the prostate cancer patients from normal control patients, wherein the sensitivity is more than 78 percent, and the specificity is more than 66 percent.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic representation of the levels of SERPINA10 in patient serum; in fig. 1, group H is a healthy control group, and group C is a prostate cancer group;

FIG. 2 is a schematic representation of the ROC curve of SERPINA10 in patient serum. The area under the curve of the working characteristic curve of the test subject of SERPINA10 is more than or equal to 0.789;

FIG. 3 is a standard curve in which the ordinate is concentration (ng/ml) and the abscissa is absorbance OD.

Detailed Description

The advantages and various effects of the present application will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the application, not to limit the application.

Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In case of conflict, the present specification will control.

Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, etc., used in the present application are commercially available or may be obtained by existing methods.

The use of SERPINA10 of the application as a molecular marker for the preparation of a product for diagnosing prostate cancer will be described in detail below with reference to the examples and experimental data.

Example 1 obtaining of SERPINA10 as a diagnostic marker for prostate cancer

1. Experimental objects

Serum specimens and clinical information were collected from 29 prostate cancer patients and 25 healthy controls at the south-middle-arm university hospital at 2021, 12, to 2023, 6. The collected blood samples were centrifuged at 12000rpm in a centrifuge at 4℃for 15min, and the serum was separated and stored at-80 ℃. All patients incorporated were diagnosed based on their pathology report, and the judgment of tumor stage was referred to the united states joint committee for cancer (AJCC) seventh edition of handbook of stage cancer.

All experimental designs and procedures were approved by the ethics committee of the university of martial arts, south China hospital.

2. Experimental method

1. Assessing the patient's general condition (prostate volume, PSA level, MRI cues) and puncture outcome, determining whether the patient has prostate cancer; if yes, collecting serum of the patient;

2. and performing metabonomics analysis on the collected serum, screening out differential proteins, and performing diagnostic capability assessment on the differential proteins to obtain the diagnostic markers of the prostate cancer. The method specifically comprises the following steps:

(1) The collected serum is subjected to pretreatment, wherein the specific steps are that antibodies can be used for enrichment, purification and IP treatment in the pretreatment of serum samples, and then proteins to be detected are concentrated; 1D/2D-PAGE gel may also be used to isolate the proteins. After the concentrated serum protein is subjected to enzyme digestion, LC-MS/MS analysis is performed; the protein gel sample needs to cut off protein bands with the molecular weight consistent with that of the target protein, and then carries out in-gel digestion and mass spectrometry detection analysis on the protein.

(2) Then mass spectrum detection is carried out to obtain detection data;

(3) Screening the detection data for differential protein, wherein the screening principle is as follows: proteins with FC values greater than 2 or less than 0.5 and p values less than 0.05 were differential proteins in the prostate cancer group compared to the healthy group.

3. Experimental results

The biomarkers were evaluated for diagnostic ability (ELISA experiments with collected serum, results obtained for prostate cancer patients and healthy subjects, and differences between the two sets of data were analyzed using GraphPad Prism9.0, and plasma SERPINA10 expression levels for prostate cancer patients were found to be significantly higher than for normal controls (P < 0.0001)), resulting in the diagnostic marker SERPINA10 for prostate cancer (protein Z-dependent protease inhibitor, ZPI). The gene name is SERPINA10 gene, namely PZI; ZPI; gene ID 51156; the species homosapiens; DNA sequence number NM-016186.3 protein sequence number NP-057270.1.

ELISA detection kit for SERPINA10 of example 2 and use method

1. Coated ELISA plate

(1) Coating liquid: 0.05M pH 9.5 carbonate buffer

Na ₂ CO ₃ 1.50g

NaHCO ₃ 2.94g

Glutaraldehyde 0.10ml

The pH is adjusted to 9.5, and the volume is fixed to 1000ml.

(2) Coating washing liquid: 0.01M potassium phosphate buffer pH7.2

K ₂ HPO ₄ ·3H ₂ O 1.56g

KH ₂ PO ₄ 0.38g

The pH is adjusted to 7.2, and the volume is fixed to 1000ml.

(3) The coating method comprises the following steps: and (3) diluting the purchased antibody of the SERPINA10 protein with coating liquid until the protein content is 25 mug/ml, adding 100 mug of the antibody into each hole of a 96-hole ELISA plate, and placing the mixture at 2-8 ℃ for adsorption for 24 hours. The coating solution was removed by air, and the plate was washed 3 times with the washing solution.

(4) When the protein coating concentration is determined, firstly, the coating liquid is used for diluting the white until the protein content is 5 mu g/ml,10 mu g/ml,20 mu g/ml,25 mu g/ml,30 mu g/ml and 40 mu g/ml, the white is added into the micropores of the 96-well ELISA plate, 100 mu L of the white is added into each hole, and the mixture is adsorbed for 24 hours at the temperature of 2-8 ℃. From this an optimized coating concentration, i.e. 25. Mu.g/ml, was chosen.

2. Closing:

(1) Sealing liquid for coating:

the pH is adjusted to 7.2, and the volume is fixed to 1000ml.

(2) Closing operation: air-drying the coating solution, adding 150. Mu.L/well of the coating blocking solution, and standing at 2-8deg.C overnight. The blocking solution was removed and naturally dried for 24 hours.

3. Incubation of negative and positive serum (first antibody incubation)

(1) Serum dilution preparation: e.coli BL21 single colony is picked on the LB solid culture medium plate, transferred into a test tube containing LB liquid culture medium, and cultured for 12 hours at 37 ℃ to be used as seed bacterial liquid. The seed bacterial liquid is inoculated into 250ml LB liquid culture medium according to the proportion of 5 percent, and is cultivated for 7 hours at 37 ℃. The cells were collected in a centrifuge tube at 8000 rpm for 10 minutes. The cells were resuspended in PBS at a ratio of 1:10 (wt/vol) and sonicated. Centrifuging at 12000rpm for 10min, discarding the precipitate, and collecting supernatant. The supernatant was subjected to protein content measurement and diluted with PBS to a total protein content of 1.2mg/ml.

(2) Standard negative serum preparation: the separated serum was diluted 1:800 times for later use.

(3) Standard positive serum preparation: the sample is diluted 1:800 for later use.

(4) Washing liquid: (10 times concentration)

The pH is adjusted to 7.2, and the volume is fixed to 1000ml.

(5) The operation is as follows: the serum to be detected is diluted by PBS liquid as serum dilution liquid according to the proportion of 1:400, and is added into the coating plate hole for 100 mu L/hole. Standard positive serum or standard negative serum was directly aspirated into the wells of the coating plate, 100 μl/well. The ELISA plate was placed at 37℃for 30min.

The plate was washed 5 times with wash solution after the serum dilutions were emptied.

4. Incubation of enzyme-labeled secondary antibodies

Horseradish peroxidase-labeled enzyme-labeled secondary antibodies (IgG) were diluted to working concentration with serum dilution, 100. Mu.L/well was added to the wells of the ELISA plates, and the wells were incubated at 37℃for 30min.

5. Color development:

50. Mu.L of substrate solution A and 50. Mu.L of substrate solution B are added, mixed by shaking, and reacted at 37℃for 10 minutes.

(1) Substrate solution A

Urea hydrogen peroxide 0.5g

The pH is adjusted to 5.0, and the volume is fixed to 1000ml.

(2) Substrate solution B

The pH is adjusted to 5.0, and the volume is fixed to 1000ml.

6. And (3) terminating:

stop solution: 2mol/L H ₂ SO ₄ 。

After the completion of the color development, 100. Mu.L of a stop solution was added to each well.

7. Reading a plate:

the light absorption value of the ELISA plate was read at a wavelength of 450 nm.

Example 3 abnormal elevation of plasma SERPINA10 expression levels in prostate cancer patients

1. Detection of the expression level of plasma SERPINA10 by the method of example 2

Multiple samples of groups a-H were prepared and plasma SERPINA10 expression levels were measured using the method described in example 2 and OD values are shown in table 1.

TABLE 1

Quantitative determination by serial dilution of known amounts of standard followed by ELISA kit measurement (shown in Table 2), standard curve drawing, as shown in FIG. 3, y= 1513.5x-304.3, R ² ＝0.9966。

TABLE 2

The OD values shown in table 1 were substituted into the standard curve to obtain the concentrations of the sample to be tested and the control group (normal SERPINA 10).

When the SERPINA10 content is 1.85 times higher than the normal SERPINA10 content, judging that the prostate cancer is in the state at the moment;

when the SERPINA10 content is less than or equal to 1.85 times the normal SERPINA10 content, it cannot be judged.

2. Statistical analysis:

the diagnostic value of plasma SERPINA10 in prostate cancer was assessed using the subject work profile (Receiver-operator characteristic, ROC). All statistical analyses were performed using GraphPad prism9.0 and IBM SPSS Statistics 26.0.26.0. The difference of P <0.05 is statistically significant.

As shown in fig. 1, plasma SERPINA10 expression levels were significantly higher in prostate cancer patients than in normal controls (P < 0.0001).

As shown in fig. 2, ROC curve analysis results showed that plasma SERPINA10 levels were useful for distinguishing prostate cancer patients from normal controls (auc=0.789, p < 0.0001), sensitivity of 79.3% and specificity of 66.7%.

In conclusion, the application discovers that the expression of plasma SERPINA10 is closely related to the occurrence of prostate cancer for the first time through experiments. The research result shows that from healthy people to prostate cancer patients, the expression level of the plasma SERPINA10 is gradually increased, the expression level of the plasma SERPINA10 can be used for distinguishing the prostate cancer patients from healthy control people, the diagnosis value is higher, and the SERPINA10 is further proved to be an effective blood molecular marker for assisting in diagnosing the prostate cancer. Meanwhile, the ELISA detection kit is used for preparing the kit for auxiliary diagnosis of the prostate cancer, and the expression level of the SERPINA10, a blood molecular marker for auxiliary diagnosis of the prostate cancer, is detected, so that the diagnosis of the prostate cancer is more convenient and easy to implement.

Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present application have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the application.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (4)

1. Use of serpina10 as a molecular marker for the preparation of a product for diagnosing prostate cancer.
2. 2. The use according to claim 1, wherein the product for diagnosing prostate cancer comprises a detection kit for SERPINA10, and the detection kit for SERPINA10 comprises an ELISA detection kit for SERPINA10 or a real-time fluorescent quantitative PCR kit.
3. 3. The use according to claim 2, wherein the SERPINA10 ELISA detection kit comprises:

   (A) ELISA plate coated with SERPINA10 antibody;

   (B) Standard negative serum;

   (C) Standard positive serum;

   (D) Horseradish peroxidase-labeled enzyme-labeled secondary antibody: anti-IgG;

   (E) Sample diluent, coating buffer, blocking solution, ELISA plate washing solution, antibody diluent, color developing solution and stop solution.
4. Use of a serpina10 detection kit for the preparation of a product for diagnosing prostate cancer.