CN116482367A - Colorectal cancer detection method combining mSEPT9 detection and biomarker - Google Patents
Colorectal cancer detection method combining mSEPT9 detection and biomarker Download PDFInfo
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Abstract
A colorectal cancer detection method combining mSEPT9 detection and biomarker. The detection method is combined with mSEPT9 detection, differential proteins of patients with colorectal cancer, which are negative or positive for Septin9 gene methylation, are screened in a protein mass spectrometry analysis mode, colorectal cancer screening molecular markers are searched from the differential proteins, colorectal cancer combined detection is carried out on the colorectal cancer screening molecular markers and mSEPT9 detection, and colorectal cancer patients are detected according to the combined detection. According to the detection method, the protein mass spectrometry is adopted to screen differential proteins of patients with the negative or positive colorectal cancer of Septin9 gene methylation, biomarkers including CLNS1A, EPDR1 and RPL15 are searched, and combined detection is carried out with mSEPT9 detection, so that the specificity and sensitivity of detection of the patients with colorectal cancer can be remarkably improved.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a colorectal cancer detection method combining mSEPT9 detection with a biomarker.
Background
The occurrence and development of colorectal cancer (CRC) mostly follows the sequence of 'adenoma-carcinoma', and the time for the progress from precancerous lesions to cancers generally takes 5-10 years, so that early discovery, early diagnosis and early treatment can effectively reduce the incidence rate and the death rate of CRC, and bring obvious benefits to patients. In addition, epidemiological investigation shows that prognosis of colorectal cancer is closely related to diagnosis stage, the 5-year relative survival rate of stage I colorectal cancer exceeds 90%, while the 5-year relative survival rate of stage IV colorectal cancer with distant metastasis is below 15%; the population of the united states receiving enteroscopy screening has increased since around 1980, more patients were initially affected by the screening and colorectal cancer incidence was found to increase, but after that the incidence and mortality of colorectal cancer in the united states has decreased significantly, so early screening for CRC is important for people's health and welfare. However, due to factors such as economy, time and religious belief, the compliance of many patients to enteroscopy screening is poor, and the rise of tumor markers such as CEA and the like has a certain prompting effect on CRC, but the low specificity of the tumor markers can bring serious mental and economic burden to the patients and even damage the health of the patients, so that the CRC is often found to be late in many patients, especially in the crowds with poor compliance of enteroscopy screening, so that the search of a diagnosis test capable of ensuring high specificity and improving sensitivity earlier is significant for early diagnosis and life prolongation of colon cancer patients.
Septin9 is closely related to the occurrence of CRC, is highly methylated in cancer tissues, and is an important molecular feature in the development process of CRC. The Septin9 methylation (mSEPT 9) detection CRC has high sensitivity and specificity. One RESEPT study conducted in four hospitals in North China to opportunistic screening of Chinese mSEPT9 tests (symptomatic high risk group) indicated that the sensitivity of mSEPT9 test was 76.6%, the specificity was 95.9%, and the sensitivity of stage I and stage II CRC patients was 64.9% and 72.7%, demonstrating that mSEPT9 is suitable for screening of CRC general risk and high risk group with poor compliance of periodic enteroscopy screening. In addition, a great deal of researches show that Septin9 is related to tissue classification, differentiation degree, clinical stage and treatment prognosis of colorectal cancer, can be used for guiding CRC stage, can be used as a marker of tumor progress and treatment effect, can screen precancerous lesions and the like, and has wide market prospect. But the msrp 9 is affected by various factors such as race, age, circadian rhythm and other diseases such as diabetes and arthritis, and as the patient ages, the sensitivity and specificity of the msrp 9 are reduced, which has an effect on the clinical application of CRC with gradually rising prevalence with age, and limits the further popularization and application thereof. Thus, methods should be sought to improve the diagnostic test of mSEPT9 to increase its sensitivity and specificity.
In theory, the sensitivity and specificity of diagnostic tests are contradictory.
Disclosure of Invention
The invention aims to solve the technical problems and provide a colorectal cancer detection method combining mSEPT9 detection and biomarkers, wherein the detection method screens differential proteins of patients with colorectal cancer, including CLNS1A, EPDR1 and RPL15, of which the methylation of Septin9 genes is negative or positive, through a protein mass spectrometry analysis mode, searches for biomarkers including CLNS1A, EPDR and RPL15, and performs combined detection with mSEPT9 detection, so that the specificity and sensitivity of colorectal cancer patients can be remarkably improved.
The Septin9 gene is an oncogene and is located at 17q25.3, contains 17 exons, and Septin9 protein has multiple subtypes, such as Sept9-v1, v2, v3 and v4, and is related to cell functions of chromosome separation, DNA repair, migration, apoptosis and the like.
Methylated SEPT9 (mSEPT 9) detection has been approved by the FDA, european Union and CFDA as a detection tool for colorectal cancer (CRC) screening or early disease discovery. At present, screening of CRC high risk groups in a hospital mostly adopts a Fecal Immunochemistry Test (FIT) and a mSEPT9 test. Some preliminary studies suggest that msepat 9 detection may also detect hepatocellular carcinoma (HCC), suggesting that msepat 9 has the potential to detect other digestive system tumors.
In order to solve the problems in the prior art, the technical scheme of the invention is as follows:
a colorectal cancer detection method combining mSEPT9 detection and a biomarker, wherein the detection method is performed in combination with mSEPT9 detection, screening differential proteins of patients with negative or positive colorectal cancer of Septin9 gene methylation by means of protein mass spectrometry, searching colorectal cancer screening molecular markers from the differential proteins, performing colorectal cancer combined detection on the colorectal cancer screening molecular markers and mSEPT9 detection, and detecting colorectal cancer patients according to the combined detection;
the colorectal cancer screening molecular markers comprise one or more biomarkers including CLNS1A, EPDR1 and RPL 15;
the combined detection comprises a parallel test and a series of tests, wherein the parallel test is to make a plurality of tests at the same time, and only one test is positive, namely the test is positive, the parallel test can increase the sensitivity, the series of tests are a plurality of tests which are sequentially successive, all tests positive are needed to make positive judgment, and the series of tests can increase the specificity;
the detection method comprises the application method of a reagent for detecting the expression level of CLNS1A and/or EPDR1 and/or RPL15 in a sample in preparing a product for diagnosing colorectal cancer or evaluating the curative effect of colorectal cancer treatment, wherein the reagent comprises a reagent for detecting the mRNA expression level of CLNS1A and/or EPDR1 and/or RPL15 in the sample, a reagent for detecting the protein expression level of CLNS1A and/or EPDR1 and/or RPL15 in the sample, and a reagent for detecting the positive expression degree of CLNS1A and/or EPDR1 and/or RPL15 in the sample;
The evaluation of colorectal cancer treatment efficacy refers to the evaluation of the efficacy of a subject on a certain treatment method or treatment drug, and in a specific embodiment of the invention, the diagnosis of cancer and paracancerous normal tissues, which are positive or negative in detection of mSEPT9 by CLNS1A and/or EPDR1 and/or RPL15, is mainly referred to, and further evaluation shows that the efficacy of the subject on a certain treatment method or treatment drug is better in clinical application prospect.
Further, the reagent for detecting the mRNA expression level of CLNS1A and/or EPDR1 and/or RPL15 in the sample comprises a primer for specifically amplifying CLNS1A and/or EPDR1 and/or RPL15, a probe for specifically recognizing CLNS1A and/or EPDR1 and/or RPL15, the reagent for detecting the protein expression level of CLNS1A and/or EPDR1 and/or RPL15 in the sample comprises an antibody for specifically binding to a protein encoded by CLNS1A and/or EPDR1 and/or RPL15, an affinity protein for a protein encoded by CLNS1A and/or EPDR1 and/or RPL15, and the reagent for detecting the positive expression level of CLNS1A and/or EPDR1 and/or RPL15 in the sample comprises a reagent for detecting the positive expression level of CLNS1A and/or EPDR1 and/or RPL15 by an immunohistochemical assay;
the primer refers to a nucleic acid fragment comprising 5-100 nucleotides, preferably the primer comprises 15-30 nucleotides capable of initiating an enzymatic reaction, including an enzymatic amplification reaction;
By probe is meant a molecule capable of binding to a specific sequence or subsequence or other portion of another molecule, a polynucleotide probe capable of binding to another polynucleotide or target polynucleotide, also referred to as a target polynucleotide, by complementary base pairing, and a probe capable of binding to a target polynucleotide lacking complete sequence complementarity to the probe, depending on the stringency of the hybridization conditions, including but not limited to solution phase, solid phase, mixed phase or in situ hybridization assays.
Exemplary probes in the present invention include gene-specific DNA oligonucleotide probes, such as microarray probes immobilized on a microarray substrate, quantitative nuclease protection test probes, probes attached to molecular barcodes, and probes immobilized on beads.
The stringency of hybridization reactions can be readily determined by one of ordinary skill in the art and is generally empirically calculated based on probe length, wash temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of the variable DNA to re-anneal when the complementary strand is present in an environment below its melting temperature. The higher the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, it is inferred that higher relative temperatures will tend to make the reaction conditions more stringent, while lower temperatures will be less stringent.
Such agents that specifically bind to CLNS1A and/or EPDR1 and/or RPL15 encoded proteins include, but are not limited to: antibodies, affinity proteins, also including peptides, aptamers, and/or compounds that specifically bind to CLNS1A and/or EPDR1 and/or RPL15 encoded proteins, are well known in the art and refer to specific immunoglobulins directed against an antigenic site. The antibody of the present invention refers to an antibody specifically binding to the protein encoded by CLNS1A and/or EPDR1 and/or RPL15 of the present invention, and may be manufactured according to a conventional method in the art. Forms of antibodies include polyclonal or monoclonal antibodies, antibody fragments (such as Fab, fab ', F (ab') 2, and Fv fragments), single chain Fv (scFv) antibodies, multispecific antibodies (such as bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen binding site of an antibody, and any other modified immunoglobulin molecule comprising an antigen binding site so long as the antibody exhibits the desired biological binding activity.
The peptides have the ability to bind highly to target substances (CLNS 1A and/or EPDR1 and/or RPL15 encoded proteins of the present invention) and do not denature during heat or chemical treatment. Moreover, due to its small size, it can be used as a fusion protein by attaching it to other proteins. In particular, it can be used as a diagnostic kit and a drug delivery substance because it can be specifically attached to a high molecular protein chain.
The aptamer refers to a polynucleotide composed of a specific type of single-stranded nucleic acid (DNA, RNA or modified nucleic acid) which itself has a stable tertiary structure and has a property of being able to bind with high affinity and specificity to a target molecule (CLNS 1A and/or EPDR1 and/or RPL15 encoded protein according to the present invention). As described above, an aptamer can specifically bind to an antigenic substance like an antibody, but is more stable than a protein, has a simpler structure, and is composed of a polynucleotide that is easy to synthesize, and thus can be used instead of an antibody.
The reagent for detecting the positive expression level of CLNS1A and/or EPDR1 and/or RPL15 by the immunohistochemical assay includes, but is not limited to, any reagent required for detecting the positive expression intensity of CLNS1A and/or EPDR1 and/or RPL15 by the immunohistochemical assay, for example, fixative, buffer, color development solution, adhesive, sealing agent, enzyme digestion solution, sucrose solution. Wherein the fixative includes, but is not limited to: formaldehyde, glutaraldehyde, paraformaldehyde, ethanol, hneFIX, acetone; such buffers include, but are not limited to: PBS buffer solution, citrate buffer solution, EDTA buffer solution and TBS buffer solution; the color-developing liquid includes but is not limited to: DAB color development liquid, 4-chloro-1-20 Naphthol (4-Cl-1-Naphthol) color development liquid, 3-amino-9-ethylcarbazole (AEC) color development liquid, TMB color development liquid and NBT color development liquid; the adhesive includes, but is not limited to: gelatin, resin gum, polylysine, commercial adhesive; the sealant includes, but is not limited to: skimmed milk powder, BSA, serum and Fab fragment single chain secondary antibodies; the enzymatic digests include, but are not limited to: trypsin digest and pepsin digest.
Further, the expression level refers to an absolute or relative amount of CLNS1A and/or EPDR1 and/or RPL15 of the present invention, and the expression level of CLNS1A and/or EPDR1 and/or RPL15 of the present invention may be determined by various techniques well known to those skilled in the art, and in particular, the absolute or relative amount of CLNS1A and/or EPDR1 and/or RPL15 of the present invention may be detected by using immunohistochemical detection methods well known to those skilled in the art.
By sample is meant a composition obtained or derived from a subject of interest comprising cellular entities and/or other molecular entities to be characterized and/or identified, e.g. based on physical, biochemical, chemical and/or physiological characteristics. The sample may be obtained from blood and other fluid samples of biological origin and tissue samples of the subject, such as biopsy tissue samples or tissue cultures or cells derived therefrom. The source of the tissue sample may be solid tissue, such as tissue from fresh, frozen and/or preserved organs or tissue samples, biopsy tissue or aspirates; blood or any blood component; body fluid; cells from any time of gestation or development of an individual; or plasma. The term sample includes biological samples that have been treated in any way after they have been obtained, such as by treatment with reagents, stabilization, or enrichment for certain components (such as proteins or polynucleotides), or embedding in a semi-solid or solid matrix for sectioning purposes.
The sample includes, but is not limited to, one or more of the following: tissue, blood, serum, plasma, blood-derived cells, lymph, synovial fluid, cerebral spinal fluid, pleural fluid, peritoneal fluid, bladder irrigation fluid, secretions, oral irrigation fluid, swabs, touch preparations, fine needle punctures, cell extracts, in particular embodiments of the present invention, the sample is preferably a tissue sample of subject 20 origin, more preferably a colorectal sample of subject origin.
The invention also provides a product for diagnosing colorectal cancer or assessing the efficacy of colorectal cancer treatment, the product comprising reagents for detecting CLNS1A and/or EPDR1 and/or RPL15 expression levels in a sample, the product comprising a detection kit, a biochip;
the detection kit comprises primers and probes which specifically bind to CLNS1A and/or EPDR1 and/or RPL 15;
the biochip comprises a solid support, and probes attached to the solid support that specifically recognize CLNS1A and/or EPDR1 and/or RPL 15.
Further, the detection kit further comprises one or more substances selected from the group consisting of: a container, instructions for use, positive control, negative control, buffer, adjuvant, or solvent;
The detection kit comprises: RT-PCR detection kit, ELISA detection kit, protein chip detection kit, rapid detection kit, DNA chip detection kit, immunohistochemical detection kit, or MRM (multiple reaction monitoring) detection kit;
the detection kit may further comprise elements necessary for the reverse transcription polymerase chain reaction;
the RT-PCR detection kit comprises a pair of primers specific for a gene encoding a marker protein, the primers being nucleotides having a nucleic acid sequence specific for the gene and may be about 7 to 50bp in length, more particularly about 10-39bp.
Further, the detection kit may further comprise primers specific for a nucleic acid sequence of a control gene;
the RT-PCR detection kit may also comprise a test tube or suitable vessel, reaction buffers (different pH values and magnesium concentrations), deoxynucleotides (dNTPs), enzymes (e.g., taq polymerase and reverse transcriptase), deoxyribonuclease inhibitors, ribonuclease inhibitors, DEPC-water, and sterile water.
The detection kit may comprise the elements necessary for manipulating a DNA chip, which may comprise a substrate to which a gene or cDNA or an oligonucleotide equivalent to a fragment thereof is bound, and reagents, agents and enzymes for constructing a fluorescently labeled probe, which may comprise a control gene or cDNA or an oligonucleotide equivalent to a fragment thereof.
In some embodiments, the presently disclosed detection kits may comprise the necessary elements for performing an ELISA. The ELISA detection kit may comprise antibodies specific for the proteins (the proteins encoded by CLNS1A and/or EPDR1 and/or RPL15 according to the invention). The antibodies have high selectivity and affinity for the marker protein, are non-cross-reactive with other proteins, and may be monoclonal, polyclonal or recombinant. Furthermore, the ELISA detection kit may comprise antibodies specific to a control protein. In addition, the ELISA detection kit may further comprise reagents capable of detecting the bound antibody, e.g., a labeled secondary antibody, a chromophore, an enzyme (e.g., conjugated to an antibody), and substrates thereof or substances capable of binding to the antibody.
The biochip, also called array, refers to a solid support comprising attached nucleic acid or peptide probes. The array typically comprises a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known locations. These arrays, also known as microarrays, can typically be produced using either mechanical synthesis methods or light-guided synthesis methods that combine a combination of photolithographic methods and solid-phase synthesis methods. The array may comprise a planar surface or may be a bead, gel, polymer surface, fiber such as optical fiber, glass or any other suitable nucleic acid or peptide on a substrate. The array may be packaged in a manner that allows for diagnosis or other manipulation of the fully functional device. Microarrays are ordered arrays of hybridization array elements, such as polynucleotide probes (e.g., oligonucleotides) or binding agents (e.g., antibodies), on a substrate. The substrate may be a solid substrate, for example, a glass or silica slide, beads, a fiber optic binder, or a semi-solid substrate, for example, a nitrocellulose membrane. The nucleotide sequence may be DNA, RNA or any arrangement thereof. In the invention, the biochip comprises a gene chip and a protein chip; the gene chip comprises a solid phase carrier; and an oligonucleotide probe orderly immobilized on the solid support, the oligonucleotide probe specifically corresponding to part or all of the sequences shown in CLNS1A and/or EPDR1 and/or RPL 15. The protein chip comprises a solid phase carrier, and specific antibodies or ligands of CLNS1A and/or EPDR1 and/or RPL15 encoded proteins immobilized on the solid phase carrier.
Such antibodies expressly include chimeric antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
The ligand may comprise a peptide, antibody or fragment thereof, or an aptamer or oligonucleotide capable of specifically binding to CLNS1A and/or EPDR1 and/or RPL 15. Antibodies against CLNS1A and/or EPDR1 and/or RPL15 encoded proteins used in the present invention are used in the broadest sense and specifically cover, for example, monoclonal antibodies, polyclonal antibodies, antibodies with multi-epitope specificity, multi-specific antibodies and antibody fragments. Such antibodies may be chimeric, humanized, human and synthetic. Provided that the fragment is capable of retaining the ability to bind to the CLNS1A and/or EPDR1 and/or RPL15 encoded protein.
Screening differential proteins of patients with Septin9 gene methylation negative or positive colorectal cancer by means of protein mass spectrometry, and searching colorectal cancer screening molecular markers from the differential proteins, wherein the colorectal cancer screening molecular markers comprise one or more biomarkers of CLNS1A, EPDR1 and RPL15, and the specific process is as follows:
Clinical sample collection
Colorectal cancer tissues obtained by surgical excision or biopsy are uniformly stored in a storage room after being embedded in paraffin according to a standard mode.
Proteomic analysis
Selecting 10 samples of colorectal cancer patients meeting inclusion and exclusion criteria for protein mass spectrometry analysis, carrying out research by using Label-free quantitative proteomics technology, and obtaining protein expression information of colorectal cancer and paracancerous normal tissues of each sample through the steps of protein extraction, peptide enzymolysis, chromatographic fractionation, liquid chromatography-tandem mass spectrometry (LC-MS/MS) data acquisition and database retrieval;
by mass spectrometry of 10 cases (5 cases of negative and positive mSEPT 9) colorectal cancer and paracancerous normal tissue proteins, respectively, the differentially expressed proteins of the negative and positive sets of mSEPT9 cancers and the paracancerous normal tissue and the differentially expressed proteins of the negative and positive sets of mSEPT9 cancers were obtained, and finally, by combined analysis, mammalian ependymal associated protein 1 (Mammalian ependymin-related protein 1, EPDR1) and 60S ribosomal protein L15 (60Sribosomal protein L15,RPL15) and a methylated subunit pICLn (Methylosome subunit pICLn, CLNS 1A) were proposed as tumor markers of colorectal cancer, and were verified by immunohistochemistry.
Third, immune group testing
Selecting 60 colorectal cancers and paracancerous normal tissues (30 cases of mSEPT9 negative and positive respectively) meeting inclusion and exclusion criteria, performing immunohistochemical verification on EPDR1, RPL15 and CLNS1A, and performing semi-quantitative analysis on the expression intensity of the colorectal cancers and paracancerous normal tissues, and analyzing the diagnostic value of the colorectal cancers and paracancerous normal tissues, wherein the specific steps are as follows:
(1) Immunohistochemical procedure
1. Slicing paraffin tissue according to 2.5 micrometers, spreading in a tablet spreading machine at 48 ℃, drying at 63 ℃ and baking for 1 hour;
2. dewaxing and hydrating tissues (dewaxing liquid I soaking for 15 minutes, dewaxing liquid II soaking for 15 minutes, absolute ethyl alcohol I soaking for 5 minutes, absolute ethyl alcohol II soaking for 5 minutes, 95% ethyl alcohol soaking for 2 minutes, 80% ethyl alcohol soaking for 1 minute, and distilled water washing for 3 times);
3.0.01M Phosphate Buffer (PBS) is soaked for 5 minutes for 3 times;
4. pressure cooker thermal antigen retrieval
1) Repair using citrate buffer (ph 6.0);
2) Preheating to boiling;
3) Placing the slices into corresponding pressure cooker, timing for 3min after EDTA is sprayed, and timing for 2.5min after citric acid is sprayed;
4) Cooling at room temperature for 20 minutes;
5) Soaking in 0.01M PBS for 5min, repeating for 3 times;
6) Soaking with 3% H2O2 for 10 min;
7) Soaking in 0.01M PBS again for 5min, repeating for 3 times;
8) After adding the primary antibody, placing the slices into a humidity box for incubation overnight at 37 ℃;
9) Soaking in 0.01M PBS for 5 min, repeating for 3 times;
10 Adding the secondary antibody, then placing the slices into a moisturizing box for incubation at 37 ℃ for 30 minutes;
11 Soaking in 0.01M PBS for 5 min, repeating for 3 times;
12 DAB color development (microscopy);
13 Hematoxylin counterstaining for 5 minutes, and then placing the mixture into a glass jar to be washed with water for 3 times;
14 Placing the color separation liquid into a glass jar for washing for 3 times after color separation;
15 Placing the blue-reversed liquid into a glass jar for water washing for 3 times after the blue-reversed liquid is reversed;
16 Microscopic examination, observing the staining condition of the cell nucleus;
17 Uplink dehydration, transparency (80% ethanol soaking for 1 min, 95% ethanol soaking for 2 min, absolute ethanol soaking for 5 min, transparent liquid soaking for 1 min, transparent liquid soaking for 5 min);
18 Neutral resin sealing piece and microscopic examination.
(2) Immunohistochemical outcome analysis
The immunohistochemical slides were placed under an optical microscope in this order, 3-5 high power fields were randomly selected and images were acquired, and then the average optical density values of RPL15, EPDR1 and CLNS1A were calculated using Image Pro Plus 6.0 software, respectively.
(3) ROC curve
According to the results of RPL15, EPDR1 and CLNS1A in mSEPT9 negative group and positive group cancer and paracancerous normal tissue, ROC curve is drawn, the diagnostic value of RPL15, EPDR1 and CLNS1A colorectal cancer is judged, and according to the results, the diagnostic value of RPL15, EPDR1 and CLNS1A in mSEPT9 detection negative group is good, and the sensitivity and specificity are higher.
Further, the detection method further comprises the steps of combining colorectal cancer diagnosis reagents or kits to find colorectal cancer screening molecular markers, wherein the colorectal cancer screening molecular markers comprise one or more biomarkers of CLNS1A, EPDR and RPL15, respectively adding primary antibodies of CLNS1A, EPDR and RPL15 in an immunohistochemical process, placing the sections into a constant temperature box with a humidity box at 37 ℃ for incubation overnight, observing through an optical microscope, collecting images under high-power visual field, and performing semi-quantitative analysis on the expression results;
further, the detection method further comprises searching for colorectal cancer screening molecular markers including one or more biomarkers of CLNS1A, EPDR, RPL15 in combination with a colorectal cancer stage prediction reagent or kit.
The colorectal cancer detection method combining mSEPT9 detection and biomarker has the beneficial effects that:
1. the detection method is carried out based on the mSEPT9 detection technology, a novel colorectal cancer screening molecular marker is found by comparing the gene sequences of the colorectal cancer with the colorectal cancer without the mSEPT9, and the colorectal cancer screening molecular marker is combined with the mSEPT9, so that the colorectal cancer detection rate can be improved, the specificity and the sensitivity are both higher, colorectal cancer patients can be identified early, the diagnosis efficiency is high, and the colorectal cancer can be rapidly screened;
2. The detection method solves the problems of low sensitivity and specificity of colorectal cancer detection, poor patient compliance caused by various reasons and the like in the prior art, brings convenience for screening and preventing colorectal cancer, can be used as a potential target point of colorectal cancer, and plays a comprehensive role in diagnosis, treatment and prognosis judgment of colorectal cancer.
Drawings
FIG. 1 shows a photograph of immunohistochemical staining of CLNS1A in a colorectal cancer detection method combining mSEPT9 detection and biomarker according to the present invention;
FIG. 2 is a histogram of semi-quantitative analysis of CLNS1A expression in a colorectal cancer detection method combining mSEPT9 detection with biomarkers of the present invention;
FIG. 3 shows the results of a CLNS1A immunohistochemical semi-quantitative analysis in a colorectal cancer detection method combining mSEPT9 detection and biomarker according to the present invention and the ROC curve for diagnosing colorectal cancer;
FIG. 4 is a photograph of RPL15 immunohistochemical staining in a colorectal cancer detection method combining mSEPT9 detection and biomarker according to the present invention;
FIG. 5 is a histogram of semi-quantitative analysis of RPL15 expression in a colorectal cancer detection method combining mSEPT9 detection with biomarkers of the present invention;
FIG. 6 shows the result of immunohistochemical semi-quantitative analysis of RPL15 protein in a colorectal cancer detection method combining mSEPT9 detection and biomarker and the ROC curve for diagnosing colorectal cancer according to the present invention;
FIG. 7 is a photograph of RPL15 immunohistochemical staining in a colorectal cancer detection method combining mSEPT9 detection and biomarker according to the present invention;
FIG. 8 is a histogram of semi-quantitative analysis of RPL15 expression in a colorectal cancer detection method combining mSEPT9 detection with biomarkers of the present invention;
FIG. 9 shows the result of immunohistochemical semi-quantitative analysis of RPL15 protein in a colorectal cancer detection method combining mSEPT9 detection and biomarker and the ROC curve for diagnosing colorectal cancer according to the present invention;
FIG. 10 is a ROC curve of combined diagnosis of the results of immunohistochemical semi-quantitative analysis of CLNS1A, EPDR and RPL15 proteins in a colorectal cancer detection method combining mSEPT9 detection and biomarkers according to the invention.
Detailed Description
The invention is further illustrated below with reference to examples.
Selection of experimental samples:
samples of this study were all colorectal cancer paraffin embedded tissue surgically excised between 2019-2022. All samples were diagnosed by the physician of the pathology department as colorectal cancer, and the patient had no other malignant tumor, no antitumor treatment prior to surgery, and the mSEPT9 test was a pre-surgery test. The study was approved by the medical ethics committee of the hospital and 10 CRC patients screened for informed consent of the subject or subject family (guardian) were grouped according to msepat 9 results (5 cases each in msepat 9 positive and negative groups), with msepat 9 negative colorectal cancer labeled T1 and paracancerous normal tissue labeled N1; colorectal cancers positive for mSEPT9 are marked as T2 and paracancerous normal tissues are marked as N2.
Proteomics study-protein mass spectrometry analysis:
the research is carried out by using Label-free quantitative proteomics technology, and the mass spectrometry experimental analysis flow mainly comprises the steps of protein extraction, peptide fragment enzymolysis, chromatographic fractionation, liquid chromatography-tandem mass spectrometry (LC-MS/MS) data acquisition, database retrieval and the like, and the steps are as follows.
(1) Protein extraction and peptide fragment enzymolysis
Dewaxing: xylene was added to each EP tube and soaked for 30 minutes and then blotted with a pipette, this procedure was repeated three times, next each EP tube was sequentially added with gradient alcohol (90%, 80%, 70%), each gradient for 30 minutes, then blotted with a pipette, hydrated 3 times, 10 minutes each time, and negative 80 ℃ low temperature storage was awaited for mass spectrometry. 200ul of each group is taken to evaluate the total protein content of the paraffin specimen by an SDS-PAGE method, and after the protein content meets the following experiment, the following experiment can be carried out.
Cleavage and quantification of protein: after the samples were taken at-80℃the proteins were extracted by SDT (4% (w/v) SDS,100mM Tris/HCl pH7.6,0.1 MDTT) lysis. The supernatant was collected after centrifugation, and the protein concentration was measured by BCA method.
Enzymolysis of protein peptide segments: an appropriate amount of protein was taken from each sample and subjected to trypsin enzymatic hydrolysis using the Filter aided proteomepreparation (FASP) method.
(2) LC-MS/MS data acquisition:
each sample was separated using a nanoliter flow HPLC liquid phase system Easy nLC. Wherein the buffer solution A was 0.1% formic acid aqueous solution, and the buffer solution B was 0.1% formic acid acetonitrile aqueous solution (84% acetonitrile). The chromatographic column is equilibrated with 95% solution A, the sample is loaded onto the loading column by an autosampler, and separated by an analytical column at a flow rate of 300nL/min. The sample was chromatographed and then mass analyzed using a Q-exact series mass spectrometer.
(3) Protein identification and quantitative analysis:
and (3) carrying out database checking identification and quantitative analysis by adopting MaxQuant software, wherein the original data of mass spectrometry are RAW files. According to the quantitative result, the protein with obvious difference expression between each comparison group is screened out according to the principle of Fold Change (FC) and obvious difference analysis (P value), and the protein is subjected to clustering analysis, subcellular localization analysis, structural domain analysis, enrichment analysis such as GO, KEGG and the like.
Protein identification and quantification results
Before protein mass spectrometry analysis, firstly, quality control is carried out on a sample, and the next operation is carried out after the sample meets the detection requirement of the sample.
By mass spectrometry of 10 cases (5 cases of negative and positive mSEPT 9) of colorectal cancer and paracancerous normal tissue proteins, we obtained the differential expression proteins of the negative and positive group of mSEPT9 cancers and paracancerous normal tissue and the differential expression proteins of the negative and positive group of mSEPT9 cancers, respectively. By pooled analysis, mammalian cell membrane associated protein 1 (Mammalian ependymin-associated protein 1, epdr 1) and 60S ribosomal protein L15 (60S ribosomal protein L15,RPL15) and the methylated subunit plcln (Methylosome subunit plcln, CLNS 1A) were finally proposed as tumor markers for colorectal cancer and validated by immunohistochemistry.
To further demonstrate the significant differences in proteins of each comparison group, the proteins in each comparison group were plotted against both fold difference in expression and P value, proteins with significantly down-regulated expression were marked green (FC <0.60 and P < 0.05), proteins with significantly up-regulated expression were marked red (FC >1.5 and P < 0.05), and the remaining proteins were gray.
By protein mass spectrometry analysis and comparison we obtained significant differentially expressed proteins of T1/N1, T2/N2 and T1/T2, respectively. Wherein 406 significantly different proteins occur in both T1/N1 and T2/N2, 283 up-regulating proteins in the negative group, 123 down-regulating proteins in the positive group, 282 up-regulating proteins in the positive group, and 124 down-regulating proteins in the positive group. The 406 significantly different proteins were compared to the significantly differentially expressed protein of T1/T2 to select a total of 7 core different proteins, wherein mammalian cell membrane associated protein 1 (Mammalian ependymin-associated protein 1, epdr 1) and 60S ribosomal protein L15 (60Sribosomal protein L15,RPL15) were all up-regulated in the three sets of comparisons. In addition, we found that the methylated subunit pICLN (Methylosome subunit pICLN, CLNS 1A) only had quantitative results in the T1 group among proteins that appeared on only one side of the comparison group. Therefore, we considered RPL15, EPDR1 and CLNS1A as tumor markers for colorectal cancer and validated by immunohistochemistry.
Example 1:
1-3, a colorectal cancer detection method combining mSEPT9 detection and a biomarker, wherein the detection method is performed in combination with mSEPT9 detection, screening differential proteins of patients with negative or positive colorectal cancer of Septin9 gene methylation by means of protein mass spectrometry, searching colorectal cancer screening molecular markers from the differential proteins, performing colorectal cancer combined detection on the colorectal cancer screening molecular markers and mSEPT9 detection, and detecting colorectal cancer patients according to the combined detection;
the colorectal cancer screening molecular marker is a CLNS1A biomarker, fig. 3 is a CLNS1A immunohistochemical semi-quantitative analysis result and ROC curve of colorectal cancer diagnosis in this example, in fig. 1-10, sensitivity is Sensitivity, specificity is Specificity, group1 is a Septin9 gene methylation negative Group, group2 is a combination of a Septin9 gene methylation positive Group and a negative Group, group3 is a Septin9 gene methylation positive Group, AUC is 0.8730, about dengue index is 0.774 in the Septin9 gene methylation negative Group, sensitivity is 83.87%, and Specificity is 93.55%; in the Septin9 gene methylation negative group and positive group combined, the AUC is 0.5956, the about dengue index is 0.317, the sensitivity reaches 80.00%, and the specificity is 51.67%; in the Septin9 gene methylation positive group, the AUC is 0.6556, the about dengue index is 0.40, the sensitivity is 83.33%, and the specificity is 56.67%.
Example 2:
as shown in fig. 4 to 6, a colorectal cancer detection method combining mSEPT9 detection and a biomarker is performed in combination with mSEPT9 detection, screening differential proteins of patients with colorectal cancer having negative or positive methylation of the Septin9 gene by means of protein mass spectrometry, searching for a colorectal cancer screening molecular marker from the differential proteins, performing colorectal cancer combined detection on the colorectal cancer screening molecular marker and the mSEPT9 detection, and detecting colorectal cancer patients according to the combined detection;
the colorectal cancer screening molecular marker is an EPDR1 biomarker, fig. 6 is an EPDR1 immunohistochemical semi-quantitative analysis result and an ROC curve for diagnosing colorectal cancer in this example, in fig. 6, group1 is a Septin9 gene methylation negative Group, group2 is a combination of a Septin9 gene methylation positive Group and a negative Group, group3 is a Septin9 gene methylation positive Group, AUC is 0.9289, about log index is 0.8667 in the Septin9 gene methylation negative Group, and the sensitivity reaches 90.00% at this time, and the specificity is 96.67%; in the Septin9 gene methylation negative group in the positive group, the AUC is 0.7656, the about dengue index is 0.60, the sensitivity reaches 88.33%, and the specificity is 71.67%; in the Septin9 gene methylation positive group, the AUC was 0.5989, the about dengue index was 0.3333, and at this time, the sensitivity was 90.00%, and the specificity was 43.33%.
Example 3:
referring to FIGS. 7 to 9, a colorectal cancer detection method combining mSEPT9 detection and a biomarker is performed in combination with mSEPT9 detection, screening differential proteins of patients with negative or positive colorectal cancer for Septin9 gene methylation by means of protein mass spectrometry, searching colorectal cancer screening molecular markers from the differential proteins, performing colorectal cancer combined detection on the colorectal cancer screening molecular markers and mSEPT9 detection, and detecting colorectal cancer patients according to the combined detection;
the colorectal cancer screening molecular marker is an RPL15 biomarker, fig. 9 is an RPL15 immunohistochemical semi-quantitative analysis result and an ROC curve for diagnosing colorectal cancer in the example, in fig. 9, group1 is a Septin9 gene methylation negative Group, group2 is a combination of a Septin9 gene methylation positive Group and a negative Group, group3 is a Septin9 gene methylation positive Group, AUC is 0.910, about log index is 0.8334 in the Septin9 gene methylation negative Group, sensitivity reaches 86.67%, and specificity is 96.67%; in the Septin9 gene methylation negative group and positive group combined, the AUC is 0.850, the about dengue index is 0.7334, the sensitivity reaches 76.67%, and the specificity is 96.67%; in the Septin9 gene methylation positive group, the AUC was 0.7578, the about dengue index was 0.6334, and the sensitivity was 66.67% and the specificity was 96.67%.
Example 4:
a colorectal cancer detection method combining mSEPT9 detection and a biomarker, wherein the detection method is performed in combination with mSEPT9 detection, screening differential proteins of patients with negative or positive colorectal cancer of Septin9 gene methylation by means of protein mass spectrometry, searching colorectal cancer screening molecular markers from the differential proteins, performing colorectal cancer combined detection on the colorectal cancer screening molecular markers and mSEPT9 detection, and detecting colorectal cancer patients according to the combined detection;
the colorectal cancer screening molecular markers are three biomarkers of CLNS1A, EPDR and RPL15, FIG. 10 shows the result of immunohistochemical semi-quantitative analysis of the three biomarkers of CLNS1A, EPDR and RPL15 in the example and the ROC curve for diagnosing colorectal cancer, FIG. 10 shows that Group1 is a Septin9 gene methylation negative Group, group2 is a combination of the Septin9 gene methylation positive Group and the negative Group, group3 is a Septin9 gene methylation positive Group, and in the Septin9 gene methylation negative Group, the AUC is 0.9689, the about dengue index is 0.90, the sensitivity is 93.33%, and the specificity is 96.67%; in the Septin9 gene methylation negative group and positive group combined, the AUC is 0.9056, the about dengue index is 0.6834, the sensitivity reaches 81.67%, and the specificity is 86.67%; in the Septin9 gene methylation positive group, the AUC was 0.8278, the about dengue index was 0.6334, and the sensitivity was 66.67% and the specificity was 96.67%.
The foregoing detailed description of the invention has been presented for purposes of illustration and description, but is not intended to limit the scope of the invention, i.e., the invention is not limited to the details shown and described.
Claims (10)
1. A colorectal cancer detection method combining msepat 9 detection and biomarkers, characterized in that:
the detection method is carried out in combination with mSEPT9 detection, differential protein of a Septin9 gene methylation negative or positive colorectal cancer patient is screened by a protein mass spectrometry analysis mode, colorectal cancer screening molecular markers are searched from the differential protein, colorectal cancer combined detection is carried out on the colorectal cancer screening molecular markers and mSEPT9 detection, and colorectal cancer patients are detected according to the combined detection;
the colorectal cancer screening molecular markers comprise one or more biomarkers including CLNS1A, EPDR1 and RPL 15;
the combined detection comprises a parallel test and a series of tests, wherein the parallel test is to make a plurality of tests at the same time, and only one test is positive, namely the test is positive, the parallel test can increase the sensitivity, the series of tests are a plurality of tests which are sequentially successive, all tests positive are needed to make positive judgment, and the series of tests can increase the specificity;
The detection method comprises the application method of a reagent for detecting the expression level of CLNS1A and/or EPDR1 and/or RPL15 in a sample in preparing a product for diagnosing colorectal cancer or evaluating the curative effect of colorectal cancer treatment, wherein the reagent comprises a reagent for detecting the mRNA expression level of CLNS1A and/or EPDR1 and/or RPL15 in the sample, a reagent for detecting the protein expression level of CLNS1A and/or EPDR1 and/or RPL15 in the sample, and a reagent for detecting the positive expression degree of CLNS1A and/or EPDR1 and/or RPL15 in the sample;
the evaluation of colorectal cancer treatment efficacy refers to the evaluation of the efficacy of a subject on a certain treatment method or treatment drug, and the evaluation of colorectal cancer treatment efficacy refers to the diagnosis of cancer positive or negative to mSEPT9 detection by CLNS1A and/or EPDR1 and/or RPL15 and further evaluation of the efficacy of a subject on a certain treatment method or treatment drug.
2. A colorectal cancer detection method in combination with msepat 9 detection and biomarkers according to claim 1, wherein the reagent for detecting the mRNA expression level of CLNS1A and/or EPDR1 and/or RPL15 in the sample comprises a primer for specifically amplifying CLNS1A and/or EPDR1 and/or RPL15, a probe for specifically recognizing CLNS1A and/or EPDR1 and/or RPL15, and the reagent for detecting the protein expression level of CLNS1A and/or EPDR1 and/or RPL15 in the sample comprises an antibody that specifically binds to CLNS1A and/or EPDR1 and/or RPL15 encoded protein, an affinity protein that specifically binds to CLNS1A and/or EPDR1 and/or RPL15 encoded protein, and the reagent for detecting the positive expression level of CLNS1A and/or EPDR1 and/or RPL15 in the sample comprises a reagent for detecting the positive expression level of CLNS1A and/or EPDR1 and/or RPL15 by an immunohistochemical assay;
The primer refers to a nucleic acid fragment comprising 5-100 nucleotides, preferably the primer comprises 15-30 nucleotides capable of initiating an enzymatic reaction, including an enzymatic amplification reaction;
by probe is meant a molecule capable of binding to a specific sequence or subsequence or other portion of another molecule, a polynucleotide probe capable of binding to another polynucleotide or target polynucleotide, also referred to as a target polynucleotide, by complementary base pairing, and a probe capable of binding to a target polynucleotide lacking complete sequence complementarity to the probe, depending on the stringency of the hybridization conditions, including but not limited to solution phase, solid phase, mixed phase or in situ hybridization assays;
such agents that specifically bind to CLNS1A and/or EPDR1 and/or RPL15 encoded proteins include, but are not limited to: antibodies, affinity proteins, further including peptides, aptamers, and/or compounds that specifically bind to CLNS1A and/or EPDR1 and/or RPL15 encoded proteins;
the antibody refers to a specific immunoglobulin against an antigenic site, the peptide has a high binding ability to a target substance and does not undergo denaturation during heat treatment or chemical treatment, and the aptamer refers to a polynucleotide composed of a specific type of single-stranded nucleic acid which itself has a stable tertiary structure and has a property of being able to bind to a target molecule with high affinity and specificity.
3. A colorectal cancer detection method in combination with msepat 9 detection and biomarkers according to claim 2, wherein the reagents for detecting CLNS1A and/or EPDR1 and/or RPL15 positive expression levels by immunohistochemical assays include, but are not limited to, any reagents required to detect CLNS1A and/or EPDR1 and/or RPL15 positive expression levels by immunohistochemical assays, including fixatives, buffers, color-developing solutions, adhesion agents, sealing agents, enzymatic digests, sucrose solutions.
4. A colorectal cancer detection method combining msepat 9 detection and biomarkers according to claim 3, wherein the fixative comprises, but is not limited to: formaldehyde, glutaraldehyde, paraformaldehyde, ethanol, hneFIX, acetone;
such buffers include, but are not limited to: PBS buffer solution, citrate buffer solution, EDTA buffer solution and TBS buffer solution;
the color-developing liquid includes but is not limited to: DAB color development liquid, 4-chloro-1-20 naphthol color development liquid, 3-amino-9-ethylcarbazole color development liquid, TMB color development liquid and NBT color development liquid;
the adhesive includes, but is not limited to: gelatin, resin gum, polylysine, commercial adhesive; the sealant includes, but is not limited to: skimmed milk powder, BSA, serum and Fab fragment single chain secondary antibodies;
The enzymatic digests include, but are not limited to: trypsin digest and pepsin digest.
5. Colorectal cancer detection method in combination with msepat 9 detection and biomarkers according to claim 1, wherein the expression level refers to the absolute or relative amount of CLNS1A and/or EPDR1 and/or RPL15 in the invention, which can be detected by using an immunohistochemical detection method.
6. Colorectal cancer detection method in combination with msepat 9 detection and biomarkers according to claim 1, wherein the sample refers to a composition obtained or derived from a target subject comprising cellular entities and/or other molecular entities to be characterized and/or identified, e.g. based on physical, biochemical, chemical and/or physiological characteristics;
the sample includes, but is not limited to, one or more of the following: tissue, blood, serum, plasma, blood-derived cells, lymph, synovial fluid, cerebrospinal fluid, pleural fluid, peritoneal fluid, bladder irrigation fluid, secretions, oral irrigation fluid, swabs, touch preparations, fine needle punctures, cell extracts.
7. A product for diagnosing colorectal cancer or assessing the efficacy of colorectal cancer treatment, the product comprising reagents for detecting CLNS1A and/or EPDR1 and/or RPL15 expression levels in a sample, the product comprising a detection kit, a biochip;
the detection kit comprises primers and probes which specifically bind to CLNS1A and/or EPDR1 and/or RPL 15;
the biochip comprises a solid support, and probes attached to the solid support that specifically recognize CLNS1A and/or EPDR1 and/or RPL 15.
8. A product for diagnosing colorectal cancer or assessing the efficacy of a treatment of colorectal cancer according to claim 7, wherein the detection kit further comprises one or more substances selected from the group consisting of: a container, instructions for use, positive control, negative control, buffer, adjuvant, or solvent;
the detection kit comprises: RT-PCR detection kit, ELISA detection kit, protein chip detection kit, rapid detection kit, DNA chip detection kit, immunohistochemical detection kit, or MRM (multiple reaction monitoring) detection kit;
the detection kit may further comprise elements necessary for the reverse transcription polymerase chain reaction;
The RT-PCR detection kit comprises a pair of primers specific for a gene encoding a marker protein, the primers being nucleotides having a nucleic acid sequence specific for the gene and may be 7 to 50bp in length, more particularly 10-39bp.
9. A product for diagnosing colorectal cancer or assessing the efficacy of a treatment of colorectal cancer according to claim 8, wherein the detection kit further comprises primers specific for the nucleic acid sequence of the control gene;
the RT-PCR detection kit may further comprise a test tube or a suitable vessel, a reaction buffer, deoxynucleotide, enzyme, deoxyribonuclease inhibitor, ribonuclease inhibitor, DEPC-water, and sterile water;
the detection kit may comprise the elements necessary for manipulating a DNA chip, which may comprise a substrate to which a gene or cDNA or an oligonucleotide equivalent to a fragment thereof is bound, and reagents, agents and enzymes for constructing a fluorescently labeled probe, which may comprise a control gene or cDNA or an oligonucleotide equivalent to a fragment thereof.
10. A product for diagnosing colorectal cancer or assessing the efficacy of a treatment of colorectal cancer according to claim 7, wherein said biochip, also called array, refers to a solid support comprising linked nucleic acid or peptide probes;
The biochip comprises a gene chip and a protein chip, wherein the gene chip comprises a solid-phase carrier and an oligonucleotide probe orderly fixed on the solid-phase carrier, the oligonucleotide probe specifically corresponds to part or all of sequences shown by CLNS1A and/or EPDR1 and/or RPL15, and the protein chip comprises the solid-phase carrier and a specific antibody or ligand of proteins coded by CLNS1A and/or EPDR1 and/or RPL15 fixed on the solid-phase carrier;
the antibodies expressly include chimeric antibodies in which a portion of the heavy and/or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the heavy and/or light chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies;
the ligand may comprise a peptide, antibody or fragment thereof, or an aptamer or oligonucleotide capable of specifically binding to CLNS1A and/or EPDR1 and/or RPL 15.
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