CN112522402A - Diagnostic uses of genetic markers - Google Patents

Diagnostic uses of genetic markers Download PDF

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CN112522402A
CN112522402A CN202011440736.2A CN202011440736A CN112522402A CN 112522402 A CN112522402 A CN 112522402A CN 202011440736 A CN202011440736 A CN 202011440736A CN 112522402 A CN112522402 A CN 112522402A
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liver cancer
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杨承刚
宋宏涛
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Qingdao Yangshen Biomedical Co Ltd
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Abstract

The invention discloses diagnostic use of gene markers. The invention also discloses a reagent for detecting the gene marker, a detection product and a diagnostic system containing the gene marker.

Description

Diagnostic use of gene markers
Technical Field
The present invention relates to the field of disease diagnosis, more specifically, the present invention relates to the diagnostic use of gene markers.
Background
Primary liver cancer (primary cancer of liver, hereinafter referred to as liver cancer) is one of the common malignant tumors in China. According to statistics of 90 years in the 20 th century, the annual death rate of liver cancer in China is 20.37/10 ten thousand, the liver cancer occupies the 2 nd position in the death sequence of malignant tumors, and is only second to lung cancer in cities; second only to stomach cancer in rural areas. Due to the clinical application of serum alpha-fetoprotein (AFP) and the progress of various imaging technologies, especially the AFP and ultrasonic imaging are used for monitoring high risk group of liver cancer, so that the liver cancer can be diagnosed in the subclinical stage without symptoms and signs, and the prognosis of liver cancer is obviously improved compared with the past by the maturity of surgical operation technology and the development of various non-operative treatment methods such as local treatment and the like.
The clinical signs of primary liver cancer are extremely atypical, and the symptoms are usually much less obvious, especially in the early stage of the disease process. Usually, about 70% of small liver cancers below 5cm are asymptomatic, and about 70% of asymptomatic subclinical liver cancers are small liver cancers. Once symptoms appear, indicating that the tumor is already large, the progression of the disease is generally much more rapid, usually exhibiting cachexia within weeks, often failing to die within months to 1 year. The clinical picture is mainly the pathological changes of two aspects: the manifestations of cirrhosis, such as ascites, collateral circulation, hematemesis and edema of limbs; ② the symptoms caused by the tumor itself, such as weight loss, weakness of the whole body, pain of the liver area, enlargement of the liver, and the like.
It is also found in any part of the world that chronic liver diseases caused by any cause may play an important role in the development and progression of liver cancer. Epidemiological and experimental studies show that there is a specific relationship between viral hepatitis and primary liver cancer, and there are 3 types of viral hepatitis related to liver cancer, namely type B, type C and type D. Among them, hepatitis B is most closely related to liver cancer, in recent years, the number of HBsAg negative liver cancers increases and is related to hepatitis C, while the former Soviet Union is mostly D-type. Hepatitis B Virus (HBV) infection background exists in about 90% of liver cancer patients in China. Other risk factors include alcoholic cirrhosis, hepatic adenoma, long-term intake of aflatoxin, other types of chronic active hepatitis, Wilson's disease, tyrosinemia, and glycogen storage disease.
The currently used methods for liver cancer diagnosis include the following:
1. laboratory examination
(1) Detection of liver cancer markers in recent years serum markers for liver cancer detection are mainly: alpha-fetoprotein (AFP) and its heteroplasms; ② various serum enzymes such as gamma glutamyl transpeptidase isozyme II (GGT-II), alkaline phosphatase isozyme I (ALP-I), aldolase isozyme A (ALD-A), fucosidase (AFU), antitrypsin I (AAT), 5 '-nucleotide phosphodiesterase isozyme V (5' -NPD-V), pyruvate kinase isozyme (M2-PyK), glutathione S-transferase (GST), etc.; ③ abnormal prothrombin; and fourthly, ferritin and acidic ferritin. Of which AFP is of greatest diagnostic value. The combined application of the serum markers has certain diagnostic value for the diagnosis of AFP negative liver cancer.
2. Other auxiliary examinations
(1) Carrying out ultrasonic inspection; (2) computerized Tomography (CT); (3) magnetic Resonance Imaging (MRI); (4) hepatic artery angiography; (5) radionuclide imaging; (6) liver puncture biopsy.
However, the defect of diagnosing liver cancer by using serum markers is insensitive, and the defect of diagnosing liver cancer by using other auxiliary tests cannot be diagnosed at the early stage of the disease, so that the condition is delayed. The key to improving the cure rate of liver cancer is to make an accurate diagnosis in early stage of onset, so the research of the application aims to search early stage molecular markers which can be used for liver cancer diagnosis on the gene level.
Disclosure of Invention
The invention aims to provide a marker for diagnosing liver cancer with high detection sensitivity and strong specificity.
The invention further aims to provide a diagnostic reagent/kit for detecting liver cancer with high sensitivity and strong specificity.
It is still another object of the present invention to provide a system for diagnosing liver cancer.
The invention provides a molecular marker combination for the first time, and the detected amount of the molecular marker combination has extremely high corresponding relation with liver cancer.
Specifically, the invention provides a marker for liver cancer diagnosis, which comprises PTK2B and SMAD 7.
In one embodiment of the invention, the markers include PTK2B, SMAD7, SOD 1.
In one embodiment of the invention, the markers include EGFR, PTK2B, SMAD7, SOD 1.
The invention also provides a detection reagent of the marker.
Further, the detection reagent includes a reagent for detecting the mRNA expression level of the marker and a reagent for detecting the protein expression level of the marker.
Furthermore, the detection reagent comprises a fluorescent quantitative PCR primer, a fluorescent quantitative PCR probe, an antibody functional fragment and a coupling antibody.
The invention also provides a detection product of the detection reagent.
Furthermore, the detection product comprises a kit, a chip, test paper and a high-throughput sequencing platform.
Furthermore, the kit comprises a qPCR kit, an immunoblotting detection kit, an immunochromatography detection kit, a flow cytometry analysis kit, an immunohistochemical detection kit, an ELISA kit and an electrochemiluminescence detection kit.
The invention also provides a diagnosis system for liver cancer, which comprises:
a detection means: the detection component is used for detecting the expression level of the marker;
a result judgment means: the result judging component is used for outputting the disease result of the liver cancer patient according to the result of the expression level of the marker detected by the detecting component.
Further, the expression level of the marker includes the mRNA expression level of the marker and the protein expression level of the marker.
As an alternative embodiment, the result judging means comprises an input module, an analysis module and an output module; the input module is used for inputting the expression quantity of the marker; the analysis module is used for analyzing the possibility of the disease risk result of the liver cancer patient according to the expression quantity of the marker; the output module is used for outputting the analysis result of the analysis module.
As an alternative embodiment, the detection means includes qPCR kit, immunoblot detection kit, immunochromatography detection kit, flow cytometry kit, immunohistochemical detection kit, ELISA kit, electrochemiluminescence detection kit, qPCR apparatus, immunoblot detection apparatus, flow cytometer, immunohistochemical detection apparatus, ELISA detection apparatus, electrochemiluminescence detection apparatus.
In certain embodiments, the sample to which the detection product or diagnostic system of the invention is directed is tissue, body fluid, or feces.
In one embodiment, the tissue is liver tissue.
In certain embodiments, the bodily fluid is blood, extracellular fluid, interstitial fluid, lymphatic fluid, cerebrospinal fluid, or aqueous humor.
As a preferred embodiment, the body fluid is blood.
As a more preferred embodiment, the body fluid is plasma.
In certain embodiments, the fecal material is sputum, saliva, urine, or feces.
The primers included in the product can be prepared by chemical synthesis, appropriately designed with reference to known information by using a method known to those skilled in the art, and prepared by chemical synthesis.
The antibody in the detection product or the diagnostic system of the present invention may be an antibody or a fragment thereof of any structure, size, immunoglobulin class, origin, etc., as long as it binds to the target protein. The antibodies or fragments thereof included in the assay products of the invention may be monoclonal or polyclonal. An antibody fragment refers to a portion of an antibody (partial fragment) or a peptide containing a portion of an antibody that retains the binding activity of the antibody to an antigen. Antibody fragments may include F (ab')2Fab', Fab, single chain fv (scfv), disulfide-bonded fv (dsfv) or polymers thereof, dimerized V regions (diabodies), or CDR-containing peptides.
Antibodies can be obtained by methods well known to those skilled in the art. For example, mammalian cell expression vectors that retain all or part of the target protein or incorporate polynucleotides encoding them are prepared as antigens. After immunizing an animal with an antigen, immune cells are obtained from the immunized animal and myeloma cells are fused to obtain hybridomas. The antibody is then collected from the hybridoma culture. Finally, a monoclonal antibody against the marker protein can be obtained by subjecting the obtained antibody to antigen-specific purification using the marker protein or a portion thereof used as an antigen. Polyclonal antibodies can be prepared as follows: an animal is immunized with the same antigen as above, a blood sample is collected from the immunized animal, serum is separated from the blood, and then antigen-specific purification is performed on the serum using the above antigen. The antibody fragment can be obtained by treating the obtained antibody with an enzyme or by using sequence information of the obtained antibody.
Binding of the marker to the antibody or fragment thereof can be carried out by methods generally known in the art. For example, proteins or peptides may be fluorescently labeled as follows: the protein or peptide is washed with phosphate buffer, a dye prepared with DMSO, a buffer, or the like is added, and the solution is mixed and left at room temperature for 10 minutes. In addition, labeling may be carried out using commercially available labeling kits, such as biotin labeling kit, e.g., biotin labeling kit-NH 2, biotin labeling kit-SH (Dojindo laboratories); alkaline phosphatase labeling kits such as alkaline phosphatase labeling kit-NH 2, alkaline phosphatase labeling kit-sh (dojindo laboratories); peroxidase labeling kits such as peroxidase labeling kit-NH 2, peroxidase labeling kit-NH 2(Dojindo Laboratories); phycobiliprotein labeling kits such as phycobiliprotein labeling kit-NH 2, phycobiliprotein labeling kit-SH, B-phycoerythrin labeling kit-NH 2, B-phycoerythrin labeling kit-SH, R-phycoerythrin labeling kit-NH 2, R-phycoerythrin labeling kit SH (dojindo laboratories); fluorescent labeling kits such as fluorescein labeling kit-NH 2, HiLyte Fluor (TM)555 labeling kit-NH 2, HiLyte Fluor (TM)647 labeling kit-NH 2(Dojindo Laboratories); and DyLight 547 and DyLight647(Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody labeling kit, Qdot (TM) antibody labeling kit (Invitrogen Corporation) and EZ-marker protein labeling kit (Funakoshi Corporation). For proper labeling, a suitable instrument can be used to detect the labeled antibody or fragment thereof.
The invention also provides a method for diagnosing liver cancer, which comprises the following steps:
(1) obtaining a sample from a subject;
(2) detecting the expression level of the aforementioned marker in a sample from the subject;
(3) correlating the measured expression level of the standard with the presence or absence of disease in the subject.
(4) If the expression level of the marker is decreased as compared to a normal control, the subject is judged to have a predisposition to have liver cancer, or has already suffered from liver cancer, or the liver cancer patient is judged to have a relapse, or the liver cancer patient is judged to have a poor prognosis.
In the context of the present invention, "diagnosis of liver cancer" includes determining whether a subject has liver cancer, determining whether a subject is at risk of having liver cancer, determining whether a liver cancer patient has relapsed, and determining the prognosis of a liver cancer patient.
SOD1 was Gene ID:6647 in NCBI.
COCH is Gene ID:1060 in NCBI.
PRDM2 at NCBI Gene ID: 7799.
VTN is Gene ID 7448 in NCBI.
PTK2B Gene ID:2185 in NCBI
EGFR is Gene ID 1956 in NCBI.
SMAD7 is Gene ID:4092 in NCBI.
ATG7 Gene ID:10533 in NCBI.
SERPINA3 Gene ID:12 in NCBI.
PLP2 Gene ID:5355 in NCBI.
The invention has the advantages and beneficial effects that:
the marker combination has extremely high association degree with liver cancer, and the sensitivity and specificity can reach more than 0.75, namely the liver cancer diagnostic reagent or the kit has extremely high sensitivity and specificity; the kit is very rare in clinic, can be used for early detection of liver cancer, strives for time for patients, starts treatment as soon as possible, improves survival rate of the patients, reduces death rate, and has great significance for reducing medical burden in China.
Drawings
Fig. 1 shows a boxplot of PTK2B gene mRNA expression, wherein a: TCGA; b: GEO;
fig. 2 shows a boxplot of SMAD7 gene mRNA expression, where a: TCGA; b: GEO;
fig. 3 shows a boxplot of SOD1 gene mRNA expression, wherein, a: TCGA; b: GEO;
fig. 4 shows a boxplot of EGFR gene mRNA expression, wherein a: TCGA; b: GEO;
fig. 5 shows boxplot of VTN gene mRNA expression, wherein a: TCGA; b: GEO;
fig. 6 shows a boxplot of PRDM2 gene mRNA expression, where a: TCGA; b: GEO;
fig. 7 shows a boxplot of PLP2 gene mRNA expression, wherein a: TCGA; b: GEO;
fig. 8 shows a boxplot of SERPINA3 gene mRNA expression, where a: TCGA; b: GEO;
fig. 9 shows the ROC plot for the PTK2B + SMAD7 combination, where a: TCGA; b: GEO;
FIG. 10 shows ROC plots for the combination PTK2B + SMAD7+ SOD1, where A: TCGA; b: GEO;
FIG. 11 shows ROC plots for the combination of EGFR + PTK2B + SMAD7+ SOD1, where A: TCGA; b: GEO.
Detailed Description
The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.
Example 1 screening of genes differentially expressed in liver cancer
1. Data source
Data are from the TCGA and GEO databases. The sample size in the TCGA database was parahepatic carcinoma: liver cancer 50:371 (test set), the GEO database used the GSE14520 dataset, with the sample size for the liver cancer side: liver cancer 220:225 (validation set).
2. Data pre-processing
After data download for TCGA, annotation was performed using the reference genome, version GRCh38, and finally normalization was performed using the Voom method. The standardized gene expression matrix is downloaded from the GEO data, annotation is carried out through a Platform file, a plurality of probes correspond to the same gene, and the average value is taken as the expression quantity of the gene.
3. Differential expression analysis
The data for TCGA and GEO of liver cancer were analyzed for differential expression using R-package "limma" (version 3.36.5) with a screening criterion of FDR < 0.05. There are 12845 differentially expressed genes in TCGA and 10204 differentially expressed genes in GEO. Among them, 5710 differentially expressed genes sharing the same expression trend were found.
4. Results
The expression levels of the differentially expressed genes PTK2B, SMAD7, SOD1, EGFR, VTN, PRDM2, PLP2 and SERPINA3 involved in the invention are shown in FIGS. 1-8;
the logFC of the differentially expressed gene COCH was 2.6755 (para-carcinoma vs, P ═ 0.00000000038), and 1.4548 in GEO (para-carcinoma vs, P ═ 3.78115491966993E-35).
The logFC of the differentially expressed gene ATG7 in TCGA was 0.2232 (para-vs, P0.000252335); the logFC in GEO is 0.3312 (para-carcinoma vs. P1.28280890970982E-18).
Example 2 diagnostic Performance validation
Receiver Operating Curves (ROCs) were plotted using the R package "pROC" (version 1.15.0) and AUC values, sensitivity and specificity were analyzed. The results are shown in tables 1-3 and FIGS. 9-11.
TABLE 1 Single Gene diagnostic Performance statistics
Marker substance TCGA AUC GEO AUC
SERPINA3 0.735 0.720
SOD1 0.771 0.745
COCH 0.778 0.779
PLP2 0.735 0.783
ATG7 0.700 0.743
PRDM2 0.715 0.743
VTN 0.765 0.743
SMAD7 0.757 0.766
PTK2B 0.769 0.764
EGFR 0.764 0.756
TABLE 2 diagnostic Performance statistics of Gene combinations in test set TCGA databases
Figure BDA0002822011550000081
Figure BDA0002822011550000091
TABLE 3 diagnostic Performance statistics of Gene combinations in the validation set GEO database
Marker substance AUC Sensitivity of the composition Specificity of
PTK2B+SMAD7 0.829 0.756 0.786
ATG7+PRDM2 0.631
PTK2B+SMAD7+SOD1 0.894 0.836 0.823
COCH+SERPINA3+SMAD7 0.523
EGFR+PTK2B+SMAD7+SOD1 0.909 0.827 0.905
PLP2+PRDM2+SMAD7+VTN 0.507
It can be seen from the results of this example that the diagnosis effect of any combination of several markers on liver cancer is not better than that of a single marker, but the specific combination of markers has better diagnosis effect.
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims (10)

1.一种肝癌诊断的标志物，其特征在于，所述标志物包括PTK2B、SMAD7；1. a marker for liver cancer diagnosis, characterized in that, the marker comprises PTK2B, SMAD7; 优选地，所述标志物包括PTK2B、SMAD7、SOD1；Preferably, the markers include PTK2B, SMAD7, SOD1; 优选地，所述标志物包括EGFR、PTK2B、SMAD7、SOD1。Preferably, the markers include EGFR, PTK2B, SMAD7, SOD1. 2.如权利要求1所述的标志物的检测试剂；2. the detection reagent of the marker as claimed in claim 1; 优选地，所述检测试剂包括检测所述标志物的mRNA表达量的试剂、检测所述标志物的蛋白表达量的试剂；Preferably, the detection reagent includes a reagent for detecting the mRNA expression of the marker, and a reagent for detecting the protein expression of the marker; 优选地，所述检测试剂包括荧光定量PCR引物、荧光定量PCR探针、抗体、抗体功能性片段、偶联抗体。Preferably, the detection reagents include fluorescent quantitative PCR primers, fluorescent quantitative PCR probes, antibodies, antibody functional fragments, and conjugated antibodies. 3.包含权利要求2所述的检测试剂的检测产品；优选地，所述检测产品包括试剂盒、芯片、试纸、高通量测序平台；3. The detection product comprising the detection reagent of claim 2; preferably, the detection product comprises a test kit, a chip, a test paper, and a high-throughput sequencing platform; 优选地，所述试剂盒包括qPCR试剂盒、免疫印迹检测试剂盒、免疫层析检测试剂盒、流式细胞分析试剂盒、免疫组化检测试剂盒、ELISA试剂盒和电化学发光检测试剂盒。Preferably, the kits include qPCR kits, immunoblotting detection kits, immunochromatographic detection kits, flow cytometry analysis kits, immunohistochemical detection kits, ELISA kits and electrochemiluminescence detection kits. 4.一种肝癌的诊断系统，其特征在于，所述诊断系统包含：4. A diagnostic system for liver cancer, wherein the diagnostic system comprises: 检测构件：所述检测构件用以检测权利要求1所述的标志物的表达量；Detection component: the detection component is used to detect the expression level of the marker according to claim 1; 结果判断构件：所述结果判断构件用于根据所述检测构件所检测的所述标志物的表达量的结果，输出肝癌患者疾病结果；Result judging component: the result judging component is used to output the disease result of the liver cancer patient according to the result of the expression level of the marker detected by the detecting component; 优选地，所述的标志物的表达量包括所述标志物的mRNA表达量、所述标志物的蛋白表达量。Preferably, the expression level of the marker includes the mRNA expression level of the marker and the protein expression level of the marker. 5.如权利要求4所述的诊断系统，其特征在于，所述结果判断构件含有输入模块、分析模块和输出模块；输入模块用于输入所述标志物的表达量；分析模块用于根据所述标志物的表达量，分析出肝癌患者疾病风险结果的可能性；输出模块用于输出分析模块的分析结果。5. The diagnostic system according to claim 4, wherein the result judging component comprises an input module, an analysis module and an output module; the input module is used to input the expression level of the marker; the analysis module is used to The expression level of the above-mentioned markers is used to analyze the possibility of disease risk results of liver cancer patients; the output module is used to output the analysis results of the analysis module. 6.如权利要求4所述的诊断系统，其特征在于，所述检测构件包括qPCR试剂盒、免疫印迹检测试剂盒、免疫层析检测试剂盒、流式细胞分析试剂盒、免疫组化检测试剂盒、ELISA试剂盒、电化学发光检测试剂盒、qPCR仪、免疫印迹检测装置、流式细胞仪、免疫组化检测装置、ELISA检测装置、电化学发光检测装置。6. The diagnostic system of claim 4, wherein the detection component comprises a qPCR kit, an immunoblot detection kit, an immunochromatographic detection kit, a flow cytometry analysis kit, and an immunohistochemical detection kit Box, ELISA kit, electrochemiluminescence detection kit, qPCR instrument, western blot detection device, flow cytometer, immunohistochemical detection device, ELISA detection device, electrochemiluminescence detection device. 7.如权利要求2所述的检测试剂在制备肝癌的诊断产品中的应用。7. The application of the detection reagent according to claim 2 in the preparation of a diagnostic product for liver cancer. 8.如权利要求7所述的应用，其特征在于，所述检测产品是权利要求3所述的检测产品。8 . The application according to claim 7 , wherein the test product is the test product according to claim 3 . 9.如权利要求2所述的检测试剂在制备肝癌的诊断系统中的应用。9. The application of the detection reagent according to claim 2 in the preparation of a diagnostic system for liver cancer. 10.如权利要求9所述的应用，其特征在于，所述诊断系统是权利要求4-6中任一项所述的诊断系统。10. The application of claim 9, wherein the diagnostic system is the diagnostic system of any one of claims 4-6.
CN202011440736.2A 2020-12-08 2020-12-08 Diagnostic uses of genetic markers Withdrawn CN112522402A (en)

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