α-MSH as marker for screening liver cancer or other cancers

Abstract

Provided are a risk marker for screening cancers and the use thereof. Specifically, provided is the use of a gene, mRNA, cDNA or protein of a marker for the risk of cancer development or a detection reagent thereof in the preparation of a diagnostic agent or kit for determining the development risk, wherein the cancer risk marker comprises α-MSH. Experiments show that the cancer risk marker can be used as a marker for cancer development, and has high sensitivity and specificity.

Description

Alpha-MSH as a marker for screening of hepatocarcinoma or other cancers TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to alpha-MSH as a marker for screening of hepatocarcinoma or other cancers. BACKGROUND

[0002] Due to the insidious onset of hepatocarcinoma, it is difficult to be found early, easy to metastasize, and often resistant to drugs. Early diagnosis and early treatment are of great significance for inhibiting and delaying the development of hepatocarcinoma. At present, the detection of hepatocarcinoma in clinical practice mainly relies on AFP, and occasionally some third-class hospitals carry out alpha-fetoprotein heterogeneity (AFP-L3) and abnormal prothrombin (PIVKA). However, the sensitivity and specificity are not very high.

[0003] Therefore, there is an urgent need in the art to develop a method for early diagnosis of cancer with high sensitivity and specificity, in order to solve the problem of lack of early warning markers for hepatocarcinoma in the prior art, and the problem of insufficient specificity and sensitivity, which leads to decreased diagnostic ability and affects early detection and early treatment of hepatocarcinoma. SUMMARY

[0004] The purpose of the present application is to provide a method for early diagnosis of cancer with high accuracy and efficiency, and the application thereof.

[0005] In a first aspect of the present application, the use of a cancer risk marker gene, mRNA, cDNA, protein, or detection reagent thereof is provided for the preparation of a detection reagent or kit for diagnosing or assisting in the diagnosis of cancer.

[0006] In the present application, the cancer risk marker includes alpha-MSH.

[0007] In another preferred embodiment, the cancer is selected from the group consisting of hepatocarcinoma, lung cancer, colorectal cancer, thyroid cancer, gastric cancer, or a combination thereof.

[0008] In another preferred embodiment, the cancer includes hepatocarcinoma.

[0009] In another preferred embodiment, the cancer is hepatocarcinoma.

[0010] In another preferred embodiment, the gene, mRNA, cDNA, or protein of the cancer risk marker is derived from a human.

[0011] In another preferred embodiment, the detection reagent or kit is used to detect the expression level of the risk marker in the sample to be tested.

[0012] In another preferred embodiment, the expression level of the risk marker is the expression level in a biological sample.

[0013] In another preferred embodiment, the biological sample is selected from the group consisting of blood, plasma, serum, or a combination thereof.

[0014] In a second aspect of the present application, a kit is provided, which comprises a detection reagent for detecting a gene, mRNA, cDNA, or protein, or a combination thereof, of a cancer risk marker,

[0015] In another preferred embodiment, the cancer risk marker comprises a-MSH.

[0016] In another preferred embodiment, the kit is used for diagnosing or aiding diagnosis of cancer.

[0017] In another preferred embodiment, the cancer is selected from the group consisting of liver cancer, lung cancer, colorectal cancer, thyroid cancer, gastric cancer.

[0018] In another preferred embodiment, the cancer comprises liver cancer, lung cancer, colorectal cancer, thyroid cancer, gastric cancer.

[0019] In another preferred embodiment, the detection reagent comprises a specific antibody or a specific binding molecule against the cancer risk marker.

[0020] In another preferred embodiment, the subject to be detected is a human.

[0021] In another preferred embodiment, the subject to be detected is selected from the group consisting of a non-cancer patient or a cancer patient.

[0022] In another preferred embodiment, the detection is ex vivo detection of a sample.

[0023] In another preferred embodiment, the ex vivo sample is selected from the group consisting of a blood sample, a plasma sample, and a serum sample.

[0024] In another preferred embodiment, the detection reagent is coupled with or carries a detectable label.

[0025] In another preferred embodiment, the detectable label is selected from the group consisting of a chromophore, a chemiluminescent group, a fluorophore, an isotope, and an enzyme.

[0026] In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.

[0027] In another preferred embodiment, the diagnostic reagent comprises an antibody or a protein chip.

[0028] In another preferred embodiment, the protein chip comprises a substrate and a specific antibody spotted on the substrate, wherein the specific antibody comprises a specific antibody against the cancer risk marker.

[0029] In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.

[0030] In another preferred embodiment, the kit comprises a protein of the cancer risk marker as a control or quality control.

[0031] In another preferred embodiment, the kit further comprises a label or instructions indicating that the kit is used for (a) judging the risk of cancer occurrence, and / or (b) evaluating the therapeutic effect of cancer.

[0032] In another preferred embodiment, the detection of the cancer risk marker is carried out by a method selected from the group consisting of antigen-antibody reaction.

[0033] In another preferred embodiment, the detection of the cancer risk marker is quantitative detection.

[0034] In another preferred embodiment, the kit comprises:

[0035] (f1) a first container containing streptavidin magnetic particles;

[0036] (f2) a second container containing biotin-labeled α-MSH antibody; and

[0037] (f3) a third container containing terpyridine ruthenium or acrid ester-labeled α-MSH antibody.

[0038] In a third aspect, the present application provides a detection method, comprising the steps of:

[0039] (a) providing a detection sample selected from the group consisting of blood samples;

[0040] (b) detecting the expression amount of the cancer risk marker gene in the detection sample, denoted as C1; and

[0041] (c) comparing the concentration C1 of the cancer risk marker with a control reference value C0;

[0042] In another preferred embodiment, the cancer comprises liver cancer.

[0043] In another preferred embodiment, if the detection result of the cancer risk of the detection object satisfies the following conditions, it is suggested that the cancer occurrence risk of the object is high:

[0044] The expression level of the marker is lower than the reference value or standard value C0, and the cancer occurrence risk of the detection object is high.

[0045] The expression level of the marker is lower than the reference value or standard value C0, and the cancer occurrence risk of the detection object is high.

[0046] In another preferred embodiment, the sample to be detected is plasma, and if the result of the detection of the cancer risk of the subject satisfies the following condition, it is suggested that the subject has a high risk of cancer:

[0047] The marker concentration C1 is ≤ 950 pg / ml, preferably ≤ 925 pg / ml, more preferably ≤ 850 pg / ml.

[0048] In another preferred embodiment, the method is an in vitro method.

[0049] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0050] It should be understood that, within the scope of the present application, each of the technical features of the present application described above and each of the technical features specifically described hereinafter (e.g., in the examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 shows the change of the concentration of α-MSH in the plasma of liver cancer patients.

[0052] Figure 2 shows the change of the concentration of β-MSH and γ-MSH in the plasma of liver cancer patients.

[0053] Figure 3 shows the standard curve of the kit of the present application.

[0054] Figure 4 shows the results of the validation experiment of the kit of the present application. DETAILED DESCRIPTION

[0055] The present inventors have made extensive and in-depth research, and through a large number of experiments and screening, for the first time, they have accidentally discovered a plasma marker for cancer screening. Specifically, experiments have shown that the level of α-MSH in plasma is highly correlated with the risk of cancer such as liver cancer. Taking liver cancer as an example, α-MSH is significantly down-regulated in the plasma of liver cancer patients. Therefore, α-MSH gene, mRNA, cDNA, protein, or its detection reagent can be used for diagnosing or assisting in diagnosing cancer. On this basis, the present application is completed.

[0056] TERMS

[0057] As used herein, the terms "transcriptional marker" or "transcriptional marker" are used interchangeably and both refer to one of the biomarkers that mark a specific disease state by detecting the level of gene transcription expression in tissues or cells. Biomarkers refer to biological indicators that can objectively measure physiological or pathological states, including changes at physiological, biochemical, immune, cellular, and molecular levels. Blood transcriptional markers refer to detecting the transcriptional expression level of a specific gene in a blood sample, thereby marking a specific disease state.

[0058] Transcriptional markers, for example, blood sample-based transcriptional markers, are based on detecting the transcriptional expression level of a single gene in blood, or detecting the transcriptional expression level of multiple genes and using a specific algorithm to calculate a marker score, which often has a significant difference between patients and healthy people, thus can be used to identify disease status. At present, transcriptional markers have been widely used in the diagnosis and screening of various diseases, such as various cancers, infectious diseases, etc. As used herein, the terms "RNA sequencing" or "RNA-seq" are used interchangeably and refer to a technology that can sequence all RNAs in cells or tissues at high throughput, by randomly breaking cDNA obtained by reverse transcription of mRNA, constructing a sequencing library, and then sequencing, the cDNA fragments are high-throughput sequenced, so as to detect the relative content of mRNA transcribed by all genes at the same time.

[0059] As used herein, the terms "receiver operating characteristic curve" or "ROC" are used interchangeably and refer to the process of ranking samples according to the prediction results of the markers, and predicting each sample as a positive example in this order, and calculating the values of two important quantities (here, sensitivity and specificity) each time, and plotting them as horizontal and vertical coordinates, respectively, to obtain the ROC curve. The area under the ROC curve (AUC) reflects the ranking quality of the sample prediction, taking into account sensitivity and specificity, and can comprehensively reflect the ability of the marker to distinguish active tuberculosis patients from healthy people.

[0060] As used herein, the terms "sample" or "specimen" refer to material that is specifically associated with a subject from which certain information about the subject can be determined, calculated, or inferred. The sample can be composed in whole or in part of biological material from the subject.

[0061] As used herein, the term "expression" includes the production of mRNA from a gene or portion of a gene, and includes the production of a protein encoded by the RNA or gene or portion of a gene, and also includes the appearance of a detected substance associated with expression. For example, cDNA, binding of a binding partner (such as an antibody) to a gene or other oligonucleotide, protein or protein fragment, and color development of a color development moiety of a binding partner are all included within the scope of the term "expression". Thus, an increase in the intensity of a band on an immunoblot such as a Western blot is also within the scope of the term "expression" as used herein in reference to a biological molecule.

[0062] As used herein, the term "reference value" or "control reference value" refers to a value that is statistically associated with a particular result when compared to the results of an analysis. In preferred embodiments, reference values are determined from studies comparing mRNA expression and / or protein expression of the cancer risk marker and performing statistical analysis. Some such studies are shown in the Examples section herein. However, studies from the literature and user experience with the methods disclosed herein can also be used to generate or adjust reference values. Reference values can also be determined by taking into account conditions and results specifically relevant to the patient's ethnic group, medical history, genetics, age, and other factors.

[0063] α-MSH

[0064] Melanocyte stimulating hormone is a neuroendocrine peptide, mainly including α-MSH, β-MSH, γ-MSH, etc. The Chinese name of α-MSH is α-melanocyte stimulating hormone, and the chemical structural formula is: 77 H 109 N 21 O 19 S, and the natural sequence is SYSMEHFRWGKPV (SEQ ID NO: 1).

[0065] α-MSH is a neuroendocrine polypeptide secreted by the hypothalamic-pituitary neuroendocrine axis in the brain, which has very important physiological regulation function. Liver tissue and liver cancer cells do not express α-MSH.

[0066] Cancer risk marker

[0067] In the present application, the terms "cancer risk marker of the present application", "α-MSH of the present application", "α-melanocyte stimulating hormone of the present application", "liver cancer risk marker of the present application" can be used interchangeably, and all refer to the cancer risk marker of the present application.

[0068] It is understood that substitution of nucleotides in a codon is acceptable when the same amino acid is encoded. It is also understood that substitution of nucleotides to produce a conservative amino acid substitution is also acceptable.

[0069] In the case where the information of the cancer risk marker is obtained, a nucleic acid sequence encoding the same can be constructed therefrom, and a specific probe can be designed based on the nucleotide sequence. The nucleotide full-length sequence or a fragment thereof can be obtained by a PCR amplification method, a recombination method, or an artificial synthesis method. For the PCR amplification method, primers can be designed based on the nucleotide sequence of the cancer risk marker disclosed in the present application, particularly the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art can be used as a template to amplify the relevant sequence. When the sequence is long, it is often necessary to perform two or more times of PCR amplification, and then the fragments amplified each time are ligated in the correct order.

[0070] Once the relevant sequence is obtained, the recombination method can be used to obtain the relevant sequence in large quantities. This is usually performed by cloning the same into a vector, and then introducing the vector into a cell, and then isolating the relevant sequence from the proliferated host cell by a conventional method.

[0071] In addition, the relevant sequence can be synthesized by an artificial synthesis method, particularly when the length of the fragment is short. Usually, a long fragment of the sequence can be obtained by synthesizing a plurality of small fragments, and then ligating the same.

[0072] At present, the DNA sequence encoding the protein (or a fragment, derivative thereof) of the present application can be obtained by chemical synthesis. Then, the DNA sequence can be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.

[0073] The polynucleotide sequence of the present application can be used to express or produce a recombinant cancer risk marker by a conventional recombinant DNA technique.

[0074] Specific antibodies

[0075] In the present application, the terms "antibody of the present application" and "antibody specific to the cancer risk marker" are used interchangeably, and refer to an antibody that can specifically bind to and detect the cancer risk marker of the present application.

[0076] The antibody of the present application against the cancer risk marker includes a polyclonal antibody and a monoclonal antibody having specificity, particularly a monoclonal antibody.

[0077] The present application includes not only a complete monoclonal or polyclonal antibody, but also an antibody fragment having immunological activity, such as a Fab' or (Fab)2 fragment; an antibody heavy chain; an antibody light chain; a genetically engineered single chain Fv molecule (Ladner et al., U.S. Patent No. 4,946,778); or a chimeric antibody, such as an antibody having the binding specificity of a mouse antibody but retaining a portion of an antibody from a human.

[0078] Antibodies of the present application can be prepared by various techniques known to those skilled in the art. For example, purified gene products of human cancer risk markers or fragments thereof that are antigenic can be administered to an animal to induce the production of polyclonal antibodies. Similarly, cells expressing human cancer risk marker proteins or fragments thereof that are antigenic can be used to immunize an animal to produce antibodies. Antibodies of the present application can also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology.

[0079] Antibodies against human cancer risk marker proteins can be used in immunohistochemical techniques to detect human cancer risk marker proteins in a sample, particularly a tissue sample or a blood sample. Since cancer risk marker proteins are present in blood samples or tissue samples, their expression levels can be detected.

[0080] Detection methods

[0081] Based on the differential expression of cancer risk markers in tissue samples or blood samples, the present application also provides corresponding methods for determining cancer risk.

[0082] The present application relates to diagnostic test methods for quantitatively and qualitatively detecting the protein levels or mRNA levels of cancer risk markers. These tests are well known in the art. The protein levels or mRNA levels of human cancer risk markers detected in the tests can be used to determine (including assist in determining) whether there is a cancer risk.

[0083] A preferred method is to quantitatively detect mRNA or cDNA by PCR / qPCR / RT-PCR.

[0084] A preferred method is to quantitatively detect mRNA or cDNA by sequencing.

[0085] Polynucleotides of cancer risk markers can be used for the diagnosis of cancer risk. Part or all of the polynucleotides of the present application can be immobilized on a microarray or DNA chip as a probe for differential expression analysis and genetic diagnosis in analysis.

[0086] In addition, the present application can also be detected at the protein level. For example, antibodies against cancer risk markers can be immobilized on a protein chip for detecting cancer risk proteins in a sample.

[0087] Detection kits

[0088] Based on the correlation between cancer risk markers and cancer risk, cancer risk markers can be used as markers for determining cancer risk.

[0089] The present application also provides a kit for judging cancer risk, which comprises a detection reagent for detecting the gene, mRNA, cDNA, protein, or combination thereof of the cancer risk marker. Preferably, the kit comprises the antibody or immunoconjugate of the anti-cancer risk marker of the present application, or the active fragment thereof; or the primer or primer pair, probe or chip for specifically amplifying the mRNA or cDNA of the cancer risk marker.

[0090] In another preferred embodiment, the cancer comprises liver cancer.

[0091] In another preferred embodiment, the kit further comprises a label or instruction.

[0092] In another preferred embodiment, the kit comprises:

[0093] (f1) a first container containing streptavidin magnetic particles;

[0094] (f2) a second container containing biotin-labeled α-MSH antibody; and

[0095] (f3) a third container containing terpyridine ruthenium or acrid ester-labeled α-MSH antibody.

[0096] The main advantages of the present application include:

[0097] (1) The present application uses blood samples, which is more suitable for early screening diagnosis, and has the characteristics of faster, more convenient, and lower cost.

[0098] (2) Compared with the existing cancer screening methods, the marker of the present application has higher specificity, and the detection result is more accurate and stable.

[0099] (3) The kit of the present application is an α-MSH chemiluminescence kit, which has high accuracy, specificity and sensitivity.

[0100] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples, if not specified, are usually carried out according to the conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0101] Experimental materials

[0102] Bare magnetic microspheres (micron size): purchased from JSR Corporation; chloroauric acid purchased from Sigma.

[0103] Alpha-MSH capture antibody: mouse monoclonal antibody, purity >95%, concentration >5mg / mL.

[0104] Example 1: Low expression of alpha-MSH in liver cancer patients

[0105] 1.1 Method

[0106] Alpha-melanocyte-stimulating hormone ELISA kit (commercially available) Human Alpha Melanocyte-stimulating hormone Enzyme Immunoassay Kit (EIA-aMSH, RayBiotech) was used to detect the expression level of plasma alpha-MSH in clinical liver cancer patients and normal healthy controls.

[0107] According to the manufacturer's instructions, first label 7 microtubule standards with the following concentrations: 1000 pg / ml, 250 pg / ml, 62.5 pg / ml, 15.6 pg / ml, 3.9 pg / ml, 1 pg / ml and 0 pg / ml to calibrate and draw the standard curve, and the dilution ratio of the sample stock solution is 2 times. The following steps are followed: (1) Add 100 μl of anti-Alpha MSH to each well. Incubate at room temperature for 1.5 hours or at 4°C overnight. (2) Add 100 μl of diluted sample (or standard) to each well. Incubate at room temperature for 2.5 hours or at 4°C overnight. (3) Add 100 μl of prepared streptavidin solution. Incubate at room temperature for 45 minutes. (4) Add 100 μl of TMB one-step substrate reagent to each well. Incubate at room temperature for 30 minutes. (5) Add 50 μl of stop solution to each well. Immediately read the absorbance value at 450 nm.

[0108] 1.2 Results

[0109] As shown in Figure 1 and Table 1, the average plasma alpha-MSH in normal healthy controls was about 1050 pg / ml, and the average plasma alpha-MSH in liver cancer patients was about 850 pg / ml. The plasma alpha-MSH level in liver cancer patients was significantly lower than that in normal healthy controls, with statistical significance.

[0110] Table 1: Plasma alpha-MSH levels in healthy and liver cancer patients

[0111] The above results show that the plasma α-MSH level of liver cancer patients is significantly lower than that of the healthy group, and therefore, the plasma α-MSH can be used as a marker for diagnosing and / or screening liver cancer.

[0112] Preparation of the kit of Example 2

[0113] 2.1 Preparation of raw materials:

[0114] Preparation of an aqueous chloroauric acid solution: Dissolve chloroauric acid in deionized purified water to form a 2.5 x 10 M aqueous solution, store at 4°C, and use within 3 months. Before preparing the gold magnetic microparticles, immerse the glass instruments in a heavy chromic acid solution overnight, then wash with deionized water and dry. First, dilute 10 mL of bare magnetic microspheres solution with deionized purified water to 100 mL in a three-necked flask, slowly and uniformly add 2 mL of chloroauric acid solution through a feeding-4 tube to make the final concentration of chloroauric acid 5.0 x 10 M, and slowly and uniformly add 1 mL of reducing agent sodium borohydride solution through another feeding tube. Both solutions are added within 45 minutes, then immediately heat the reaction solution to boiling and maintain the boiling state for 1 hour. Stop stirring and cool to room temperature to obtain gold magnetic microparticles with a colloidal gold layer on the surface. Take 1 mg of gold magnetic microparticles and add to a 2 mL centrifuge tube, remove the supernatant through a magnetic separation frame, and then wash and separate by magnetism 3 times with 0.5 mL of magnetic bead washing solution (100 mM MES, 0.05% Tween 20, pH 6.0) to remove the supernatant.

[0115] Take 1 mg of gold magnetic microparticles and add to a 2 mL centrifuge tube, remove the supernatant through a magnetic separation frame, and then wash and separate by magnetism 3 times with 0.5 mL of magnetic bead washing solution (100 mM MES, 0.05% Tween 20, pH 6.0) to remove the supernatant; at the same time, take another 2 mL centrifuge tube and add a certain amount of α-MSH antibody solution, add 10 mM TCEP or 5 mM DTT dithiothreitol, mix well, and react at room temperature for 30 minutes to open the disulfide bonds between the heavy chains of the antibody. Remove the excess reducing agent with a desalting column, measure the protein concentration, dilute to volume, add 1 x coupling buffer to make the total volume 200 μL, and mix well.

[0116] Coating of α-MSH antibody: 200 μL of the above activated antibody solution was added to the gold magnetic microparticle tube after washing and removing the supernatant, mixed and incubated at 180 rpm for 30 min at room temperature. After the reaction, the tube was washed with 500 μL of 1 x PBST washing buffer for 3 times to remove the non-stable bound antibody, and the supernatant was removed by magnetic separation. Blocking: 1 mL of inactivated alkaline phosphatase was added as blocking agent to the above centrifuge tube, mixed and incubated at 180 rpm for 1 h in a constant temperature (37°C) incubator, and then placed at 2-8°C for 10 h. Finally, the tube was washed with 500 μL of 1 x PBST washing buffer for 3 times to remove the excess blocking agent and non-stable bound substances, and the supernatant was removed by magnetic separation. Preservation: 1 mL of preservation solution was added to the blocked immunomagnetic beads, mixed and placed at 2-8°C for preservation.

[0117] 2.2 Preparation of tripyridine ruthenium / acrid ester labeled antibody:

[0118] 0.5 mg of custom-made α-MSH antibody was added to 0.2 mL of antibody activation agent solution (100 mM Tris, 0.3% NaCl, 5 mM EDTA, 10 mg / mL 2lT crosslinking agent), and activated at room temperature for 10-20 min. The activated antibody was purified by molecular sieve chromatography. The activated α-MSH antibody was then coupled with alkaline phosphatase or chemiluminescent reagent. Tripyridine ruthenium or acrid ester and activated antibody were mixed according to the mass ratio, 0.01 mL of 1 M MgCl2 was added, and the reaction was carried out at 2-8°C for 10 h. After the reaction, the enzyme-labeled secondary antibody complex was obtained by purification with molecular sieve. The enzyme-labeled antibody R was obtained by adding glycerol according to a volume ratio of 1:1, and was stored at -20°C.

[0119] 2.3 Preparation of magnetic bead dilution solution reagent and standard:

[0120] The antibody coated magnetic beads were diluted with M dilution solution (100 mM PB, 0.1% Tween 20, 0.5% BSA, 0.1% PC-300, pH 7.5) according to a ratio of 1:20 to obtain the magnetic bead dilution solution reagent.

[0121] 2.4 Preparation of calibration: α-MSH antigen was prepared into concentrations of 0 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 250 ng / mL, 500 ng / mL, and 1000 ng / mL with calibration dilution solution (50 mM Tris, 0.9% NaCl, 0.2% Tween 20, 0.2% casein, 1% BSA, 0.1% PC-300, pH 7.5).

[0122] Example 3

[0123] 3.1 Method

[0124] Beta and Gamma Melanocyte-stimulating hormone Beta-MSH and Gamma-MSH ELISA Kit (commercially available) Human Alpha Melanocyte-stimulating hormone Enzyme Immunoassay Kit (EIA-beta MSH and EIA-gamma MSH, RayBiotech) was used to detect the plasma beta and gamma-MSH expression levels of clinical liver cancer patients and normal healthy controls.

[0125] According to the manufacturer's instructions, first label 7 microtubes of standard with the following concentrations: 1000 pg / ml, 250 pg / ml, 62.5 pg / ml, 15.6 pg / ml, 3.9 pg / ml, 1 pg / ml and 0 pg / ml to calibrate and draw the standard curve, and the dilution ratio of the sample stock solution was 2 times. The following steps were followed: (1) Add 100 μl of beta or gamma MSH antibody to each well. Incubate at room temperature for 1.5 hours or at 4°C overnight. (2) Add 100 μl of diluted sample (or standard) to each well. Incubate at room temperature for 2.5 hours or at 4°C overnight. (3) Add 100 μl of prepared streptavidin solution. Incubate at room temperature for 45 minutes. (4) Add 100 μl of TMB one-step substrate reagent to each well. Incubate at room temperature for 30 minutes. (5) Add 50 μl of stop solution to each well. Immediately read the absorbance value at 450 nm.

[0126] 3.2 Results

[0127] As shown in Figure 2, the average plasma beta and gamma-MSH median values in the normal healthy control group were 978 pg / ml and 947 pg / ml, respectively, and the average plasma beta and gamma-MSH median values in liver cancer patients were 957 pg / ml and 939 pg / ml, respectively. There was no significant difference in plasma beta and gamma-MSH levels in liver cancer patients compared with normal healthy controls, p>0.05, which was not statistically significant.

[0128] Example 4

[0129] 4.1 Method

[0130] The kit prepared in Example 2 was used to detect alpha-MSH standards of known concentration and serially diluted, and a standard curve was generated.

[0131] 4.2 Results

[0132] The results are shown in Figure 3. The minimum concentration of α-MSH that can be detected using the chemiluminescence kit of the present application is 0.02 pg / mL. Compared with the ELISA kit of Raybiotech, the kit of the present application has higher sensitivity, wider linear range and better sample correlation.

[0133] Example 5

[0134] 5.1 Method

[0135] The applicant collected 130 samples of fasting plasma of liver cancer patients aged 40-60 years and healthy people of the same age at around 8:00 in the morning. The samples were verified using the kit prepared in Example 2, and the sensitivity and specificity of the method were analyzed by ROC curve.

[0136] 5.2 Results

[0137] The results are shown in Figure 4. The concentration of α-MSH in the plasma of liver cancer patients is significantly lower than that of healthy people. The ROC curve analysis of the concentration of α-MSH in the plasma of liver cancer patients detected by the chemiluminescence kit of the present application has an AUC value of more than 0.95, and the sensitivity and specificity are 90.8% and 89.6%, respectively.

[0138] Discussion

[0139] The applicant found that the concentration of α-MSH in the plasma of liver cancer patients is significantly lower than that in the plasma of normal healthy people, but the concentrations of β and γ-MSH in the plasma of liver cancer patients did not change significantly (p>0.05). It has been verified by experiments that α-MSH can be used as a marker for diagnosing and / or screening liver cancer. Moreover, through ROC (receiver operating characteristic) curve analysis, it is found that the AUC is equal to 0.958, the sensitivity is more than 90%, and the specificity is close to 90%. The present application provides a reliable technology and method for non-invasive screening and even early screening of liver cancer.

[0140] All the documents mentioned in the present application are incorporated herein by reference as if each document were individually incorporated by reference. In addition, it should be understood that various changes and modifications can be made to the present application by those skilled in the art upon reading the above description of the present application, and such equivalent forms are also within the scope of the appended claims.

Claims

1. Use of a gene, mRNA, cDNA, protein, or detection reagent thereof of a cancer risk marker, characterized in that, A detection reagent or a kit for preparing a detection reagent or a kit for diagnosing or aiding in diagnosing cancer; The cancer risk marker comprises α-MSH.

2. Use according to claim 1, characterized in that, The cancer is selected from the group consisting of liver cancer, lung cancer, colorectal cancer, thyroid cancer, gastric cancer, or a combination thereof.

3. Use according to claim 1, characterized in that, The cancer comprises liver cancer.

4. The use according to claim 1, characterized in that, The gene, mRNA, cDNA, or protein of the cancer risk marker is derived from a human.

5. The use according to claim 1, characterized in that, The detection reagent or the kit is used for detecting the expression level of the risk marker in a sample to be tested.

6. A kit characterized in that, The kit comprises a detection reagent for detecting a gene, mRNA, cDNA, or protein of a cancer risk marker, or a combination thereof, The cancer risk marker comprises α-MSH.

7. The kit of claim 6, wherein The kit is used for diagnosing or aiding in diagnosing cancer.

8. The kit of claim 6, wherein The detection reagent comprises a specific antibody or a specific binding molecule for the cancer risk marker.

9. A method of detection, characterized in that, The method comprises the steps of: (a) providing a detection sample selected from a blood sample; (b) detecting the expression amount of a cancer risk marker gene in the detection sample, denoted as C1; and (c) comparing the concentration C1 of the cancer risk marker with a control reference value C0; The cancer risk marker comprises α-MSH.

10. The method of claim 9, wherein, The cancer comprises liver cancer.

11. The method of claim 9, wherein, If the detection result of the cancer risk of the detection object satisfies the following condition, it is suggested that the object has a high risk of cancer: The expression level of the marker is lower than the reference value or the standard value C0, and the detection object has a high risk of cancer.

12. The method of claim 9, wherein, If the detection result of the cancer risk of the detection object satisfies the following condition, it is suggested that the object has a high risk of cancer: The marker concentration C1 is less than or equal to 950 pg / ml.

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