CN112695090A - Use of marker combinations - Google Patents

Abstract

The invention discloses an application of a marker combination. Marker combinations of the invention include PRDM2, SMAD7, PPARGC1A, FGFR1, CFD. The marker combination of the invention has high diagnostic sensitivity and specificity and is suitable for clinical application.

Description

Use of marker combinations

Technical Field

The present invention relates to the field of disease diagnosis, more specifically the present invention relates to the use of marker combinations.

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 PRDM2 and SMAD 7.

In one embodiment of the invention, the markers include PRDM2, SMAD7, PPARGC 1A.

In one embodiment of the invention, the markers include PRDM2, SMAD7, PPARGC1A, FGFR 1.

In one embodiment of the invention, the markers comprise PRDM2, SMAD7, PPARGC1A, FGFR1, CFD.

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.

COCH is Gene ID:1060 in NCBI.

PRDM2 at NCBI Gene ID: 7799.

VTN is Gene ID 7448 in NCBI.

SMAD7 is Gene ID:4092 in NCBI.

PPARGC1A is Gene ID:10891 in NCBI.

FGFR1 is Gene ID:2260 in NCBI.

CFD is Gene ID:1675 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 PRDM2 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 is a boxplot showing the mRNA expression of the PPARGC1A gene, wherein A: TCGA; b: GEO;

fig. 4 shows boxplots of FGFR1 gene mRNA expression, wherein a: TCGA; b: GEO;

fig. 5 shows a boxplot of CFD gene mRNA expression, wherein a: TCGA; b: GEO;

fig. 6 shows boxplot of VTN gene mRNA expression, wherein 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 ROC plots for the PRDM2+ SMAD7 combination, where a: TCGA; b: GEO;

FIG. 10 shows a ROC plot for the combination PPARGC1A + PRDM2+ SMAD7, where A: TCGA; b: GEO;

fig. 11 shows ROC plots for FGFR1+ PPARGC1A + PRDM2+ SMAD7 combinations, where a: TCGA; b: GEO;

fig. 12 shows ROC plots for the CFD + FGFR1+ PPARGC1A + PRDM2+ SMAD7 combination, wherein 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 PRDM2, SMAD7, PPARGC1A, FGFR1, CFD, VTN, PLP2 and SERPINA3 involved in the invention are shown in FIG. 1-FIG. 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. 9-12.

TABLE 1 Single Gene diagnostic Performance statistics

Marker substance	TCGA AUC	GEO AUC
			SERPINA3	0.735	0.720
ATG7	0.700	0.743
			COCH	0.778	0.779
PLP2	0.735	0.783
			PRDM2	0.715	0.743
VTN	0.765	0.743
			SMAD7	0.757	0.766
PPARGC1A	0.786	0.762
			FGFR1	0.776	0.712
CFD	0.777	0.748

TABLE 2 diagnostic Performance statistics of Gene combinations in test set TCGA databases

TABLE 3 diagnostic Performance statistics of Gene combinations in the validation set GEO database

Marker substance	AUC	Sensitivity of the composition	Specificity of
				PRDM2+SMAD7	0.805	0.631	0.850
ATG7+PRDM2	0.631
				PPARGC1A+PRDM2+SMAD7	0.865	0.733	0.873
COCH+SERPINA3+SMAD7	0.523
				FGFR1+PPARGC1A+PRDM2+SMAD7	0.882	0.782	0.864
PLP2+PRDM2+SMAD7+VTN	0.507
				CFD+FGFR1+PPARGC1A+PRDM2+SMAD7	0.902	0.782	0.914

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. A marker for liver cancer diagnosis, wherein the marker comprises PRDM2, SMAD 7;

preferably, the markers include PRDM2, SMAD7, PPARGC 1A;

preferably, the markers include PRDM2, SMAD7, PPARGC1A, FGFR 1;

preferably, the markers include PRDM2, SMAD7, PPARGC1A, FGFR1, CFD.

2. A detection reagent for the marker of claim 1;

preferably, the detection reagent comprises a reagent for detecting the mRNA expression level of the marker, a reagent for detecting the protein expression level of the marker;

preferably, the detection reagent comprises a fluorescent quantitative PCR primer, a fluorescent quantitative PCR probe, an antibody functional fragment and a coupling antibody.

3. A test product comprising the test reagent of claim 2; preferably, the detection product comprises a kit, a chip, test paper and a high-throughput sequencing platform;

preferably, the kit comprises a qPCR kit, an immunoblotting detection kit, an immunochromatography detection kit, a flow cytometry kit, an immunohistochemical detection kit, an ELISA kit and an electrochemiluminescence detection kit.

4. A system for diagnosing liver cancer, comprising:

a detection means: the detection means for detecting the expression level of the marker of claim 1;

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;

preferably, the expression level of the marker includes the mRNA expression level of the marker and the protein expression level of the marker.

5. The diagnostic system of claim 4, wherein the result determination component 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.

6. The diagnostic system of claim 4, wherein the detection means comprises a qPCR kit, an immunoblot detection kit, an immunochromatographic detection kit, a flow cytometric assay kit, an immunohistochemical detection kit, an ELISA kit, an electrochemiluminescent detection kit, a qPCR instrument, an immunoblot detection device, a flow cytometer, an immunohistochemical detection device, an ELISA detection device, an electrochemiluminescent detection device.

7. The use of the detection reagent according to claim 2 for the preparation of a product for diagnosing liver cancer.

8. Use according to claim 7, wherein the test product is a test product according to claim 3.

9. Use of the detection reagent according to claim 2 for preparing a diagnostic system for liver cancer.

10. Use according to claim 9, wherein the diagnostic system is a diagnostic system according to any one of claims 4-6.

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