CN103487581A - Application of serum DDK1 in preparation of diagnosis reagent for alpha fetal protein negative hepatocellular carcinoma - Google Patents

Abstract

The invention relates to the use of serum DKK1 in preparing a diagnostic reagent for alpha-fetoprotein negative hepatocellular carcinoma. The present inventor analyzed the expression of DKK1 and AFP by analyzing a large number of hepatocellular carcinoma samples, and found for the first time that serum DKK1 has higher sensitivity and specificity in diagnosing AFP-negative hepatocellular carcinoma.

Description

Use of serum DKK1 in the preparation of diagnostic reagents for alpha-fetoprotein negative hepatocellular carcinoma

technical field

The invention belongs to the field of biomedicine; more specifically, the invention relates to the use of serum DKK1 in preparing a diagnostic reagent for alpha-fetoprotein-negative hepatocellular carcinoma. the

Background technique

Primary liver tumors include hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma, mixed hepatocellular carcinoma, cystadenoma of the bile duct, hepatoblastoma, hemangioma, carcinoid, lymphoma, epithelioid vascular endothelium Tumor, squamous cell carcinoma, teratoma, and leiomyosarcoma, fibrosarcoma, malignant fibrous histiocytoma, rhabdomyosarcoma and other liver sarcomas. At present, the etiology of hepatocellular carcinoma has not been fully clarified. Its occurrence is a process involving multi-factors, multi-stages, and multi-gene accumulation of mutations. The main factors are viral infection, aflatoxin intake, alcoholism, water pollution, and liver disease. Helicobacter and liver cirrhosis. Hepatitis B virus (HBV) infection is the main pathogenic factor of hepatocellular carcinoma, especially in Asia and Africa; Major contributors to the rising incidence of HCC in the country. Liver cirrhosis is the most dangerous factor for the occurrence of hepatocellular carcinoma, and it is also the main cause of death in patients with cirrhosis. Since most patients with hepatocellular carcinoma are in the middle and advanced stages when they are diagnosed, the best opportunity for treatment is lost. At present, only 30-40% of patients with hepatocellular carcinoma can receive effective surgical treatment, and the five-year survival rate is only 3-5%. Therefore, timely and effective diagnosis of HCC is one of the keys to improve the survival rate of patients. the

Currently, the most commonly used methods for screening and diagnosing HCC are imaging and serum alpha-fetoprotein (AFP) detection. Imaging includes ultrasound, CT, MRI, etc., but imaging detection is expensive, difficult to be widely used, and easily affected by operator experience, and it is difficult to distinguish liver cancer from non-malignant hyperplasia. AFP is currently the most widely used marker of hepatocellular carcinoma in the world. When its cut-off value is 20ng/mL, the sensitivity of AFP in diagnosing hepatocellular carcinoma is only 55-60%, resulting in a false negative rate of about 40%. This part of AFP-negative HCC patients is difficult to diagnose clinically, and it is difficult to evaluate its treatment effect and disease process. Therefore, the discovery of new and reliable diagnostic markers for hepatocellular carcinoma that can make up for the deficiency of AFP can greatly improve and improve the clinical comprehensive treatment effect, and help prolong the survival period and improve the quality of life of patients with hepatocellular carcinoma. An ideal tumor marker needs to have high specificity, be able to distinguish hepatocellular carcinoma from hepatitis, cirrhosis, liver regeneration nodules, etc., and also need to have high sensitivity, be able to diagnose early hepatocellular carcinoma, and It has the characteristics of easy detection, repeatability and non-invasiveness. the

The completion of the Human Genome Project, the development of high-throughput gene expression detection methods and the improvement of large-scale data analysis capabilities have enabled people to study the changes in gene expression during the development of hepatocellular carcinoma more deeply and better understand hepatocellular carcinoma. To screen and obtain clinically applicable biomarkers and drug targets. In the early stage, the inventors analyzed the differences in gene expression profiles between cancer tissues of patients with liver cancer and corresponding paracancerous liver tissues by cDNA expression profile chip (Affymetrix GeneChip Human Genome U133 Plus 2.0 Array) technology. It was further found that DKK1 is not expressed in various tissues of normal adults, and is only expressed in placental tissue, which has tumor specificity. the

DKK1 is an inhibitor of the Wnt signaling pathway. It was first cloned in 1998. It was found that it can inhibit the replication of the Wnt-induced axis and participate in the formation of the head in the early development of Xenopus laevis. The Wnt pathway is a signaling pathway that plays an important role in regulating embryonic development and participates in processes such as cell proliferation, differentiation, apoptosis, cell polarity, and motility. The mutation or abnormal expression of signaling molecules in the pathway is related to various diseases and cancers. The Wnt pathway is regulated by several secreted proteins, including DKK, WIF (Wnt-inhibitor factor) and SFRP (secret frizzled related protein). The DKK family contains four members (DKK1-4) that are relatively conserved in evolution. DKK1 encodes a secreted glycoprotein containing two cysteine-rich conserved regions (Cys1, Cys2), which can bind to low-density lipoprotein receptor-related protein 5/6 (low-density lipoprotein receptor-related protein 5/ 6, LRP5/6) binding, and with the participation of membrane protein Kremen1/2, by causing LRP5/6 endocytosis, inhibiting the formation of Wnt-Frizzled-LRP5/6 complex, and inhibiting the canonical Wnt signaling pathway. the

Subsequently, the inventors used the ELISA method to detect high concentrations of DKK1 protein in the culture supernatants of 10 different types of human tumor cells for the first time, suggesting that a variety of human tumor cells secrete and highly express DKK1, and DKK1 can be used in clinical serum of human malignant tumors diagnosis. So in 2005, the inventor applied for a national invention patent (CN200510110298.2) and an international PCT patent (PCT/CN2006/000382). The Chinese patent and the international patent of DKK1 for cancer serum diagnosis are the first domestic and international applications filed by the inventor. Although there have been previous studies on DKK1, the clinical significance of serum DKK1, especially the diagnostic ability of AFP-negative (serum AFP≤20ng/ml) hepatocellular carcinoma patients is not clear in this field. the

Contents of the invention

The object of the present invention is to provide the application of serum DKK1 in the preparation of a diagnostic reagent for alpha-fetoprotein-negative hepatocellular carcinoma. the

In the first aspect of the present invention, a use of the DKK1 protein or its coding gene in the preparation of a diagnostic reagent for alpha-fetoprotein-negative hepatocellular carcinoma is provided. the

In another aspect of the present invention, the use of DKK1 in the preparation of a diagnostic kit for alpha-fetoprotein-negative hepatocellular carcinoma is provided. the

In a preferred example, the diagnostic reagent is selected from (but not limited to):

Primers for specifically amplifying the gene encoding the DKK1 protein; or

A probe that specifically recognizes the gene encoding the DKK1 protein or its transcript; or

Antibodies specific to the DKK1 protein. the

In another preferred example, the primers for specifically amplifying the gene encoding the DKK1 protein are primer pairs, the nucleotide sequences of which are shown in SEQ ID NO:1 and SEQ ID NO:2. the

In another aspect of the present invention, there is provided a test kit for determining alpha-fetoprotein-negative hepatocellular carcinoma, which contains:

(1) A diagnostic reagent for detecting the expression or expression level of alpha-fetoprotein or its coding gene (preferably, alpha-fetoprotein or its coding gene in serum); and

(2) A diagnostic reagent for detecting the expression or expression level of DKK1 protein or its coding gene (preferably, DKK1 protein or its coding gene in serum). the

In a preferred example, in the kit, the diagnostic reagent of (1) is first used to detect the expression or expression level of alpha-fetoprotein or its coding gene in the test sample (preferably, serum); (2) The diagnostic reagent detects the expression or expression level of the DKK1 protein or its coding gene in the test sample (preferably, serum). When the alpha-fetoprotein test is negative, if the test is positive for DKK1, the The provider of the sample to be tested is at high risk of hepatocellular carcinoma. the

In another preferred example, the diagnostic reagents for detecting the expression or expression level of alpha-fetoprotein or its coding gene are selected from (but not limited to):

Primers that specifically amplify the gene encoding alpha-fetoprotein; or

A probe that specifically recognizes the gene encoding alpha-fetoprotein or its transcript; or

Antibodies specific to alpha-fetoprotein. the

In another preferred example, the primers for specifically amplifying the gene encoding alpha-fetoprotein are primer pairs, the nucleotide sequences of which are shown in SEQ ID NO:3 and SEQ ID NO:4. the

In another preferred example, the diagnostic reagent for detecting the expression or expression level of the DKK1 protein or its coding gene is selected from (but not limited to):

Primers for specifically amplifying the gene encoding the DKK1 protein; or

A probe that specifically recognizes the gene encoding the DKK1 protein or its transcript; or

Antibodies specific to the DKK1 protein. the

In another preference, the kit also includes:

Nucleic acid extraction reagents (such as nucleic acid extraction solution); and/or

Polymerase chain reaction reagents (such as dNTPs, Taq enzymes); and/or

Western blotting reagents; and/or

Enzyme chain immunoreaction reagents (such as chromogenic solution or hybridization solution). the

In another aspect of the present invention, a method for determining alpha-fetoprotein negative hepatocellular carcinoma is provided, the method comprising:

(a) detecting the expression or expression level of alpha-fetoprotein or its coding gene in the sample to be tested; and

(b) Detect the expression or expression of DKK1 protein or its coding gene in the sample to be tested. When the alpha-fetoprotein test is negative, if the test is positive for DKK1, the provider of the sample to be tested is at high risk of hepatocellular carcinoma . the

In a preferred example, the assay is performed using the kit. the

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. the

Description of drawings

Figure 1. Comparative analysis of the expression of DKK1 and AFP in liver tissue and corresponding serum. the

DKK1 (Figure A) and AFP (Fig. The mRNA expression of panel D) and the mRNA expression of DKK1 (panel B) and AFP (panel E) detected by qRT-PCR. The protein levels of DKK1 (Figure C) and AFP (Figure F) in the corresponding serum of these 28 liver tissues were detected by ELISA. the

Figure 2. Flow design of the research on whether DKK1 can be used as a diagnostic marker. the

Normal people (healthy controls, HC); chronic hepatitis B (chronic hepatitis B, CHB); liver cirrhosis (liver cirrhosis, LC); hepatocellular carcinoma (hepatocellular carcinoma, HCC); assay, ELISA); receiver operating characteristics (receiver operating characteristics, ROC); DKK1: dickkopf-1; AFP: alpha-fetoprotein. the

Figure 3. Concentrations of serum DKK1 protein and serum AFP protein in each group of test set and validation set. the

Figures A and C are the levels of serum DKK1 protein in the normal control group (healthy controls, HC), chronic hepatitis B group (chronic hepatitis B, CHB), liver cirrhosis group (liver cirrhosis, LC) and hepatocellular carcinoma group (HCC) concentration. Figures B and D are the concentration of serum AFP protein in these 4 groups. When the serum AFP concentration is higher than 1210ng/ml, its concentration is calculated as 1210ng/ml. *, P<0.001. the

Figure 4. Determination of serum DKK1 cutoff values for the diagnosis of hepatocellular carcinoma. the

Taking hepatocellular carcinoma (HCC) as the patient group, healthy controls (HC), chronic hepatitis B (CHB) and liver cirrhosis (LC) patients as the control group, a ROC curve was made to determine The cutoff value, sensitivity, specificity and area under the curve (AUC) of serum DKK1 in the diagnosis of hepatocellular carcinoma. the

Figure 5. The diagnostic ability of serum DKK1 for AFP-negative (≤20ng/ml) hepatocellular carcinoma. the

Panels A and C are serum DKK1 concentrations in AFP-negative hepatocellular carcinoma (AFP ⁻ -HCC) and AFP-positive hepatocellular carcinoma (AFP ⁺ -HCC). Figures B and D are AFP-negative HCC as the patient group, healthy controls (HC), chronic hepatitis B (CHB) and liver cirrhosis (LC) ) patients as the control group, ROC curve was made to evaluate the diagnostic value of serum DKK1 for AFP-negative hepatocellular carcinoma.

Detailed ways

The present inventor analyzed the expression of DKK1 and AFP by analyzing a large number of hepatocellular carcinoma samples, and found for the first time that serum DKK1 has higher sensitivity and specificity in diagnosing AFP-negative hepatocellular carcinoma. Therefore, DKK1 can be very effectively used in the diagnosis of AFP-negative hepatocellular carcinoma. the

In the present invention, the inventor first analyzed the expression of DKK1 and AFP in parallel at the tissue level, then designed a large-scale multi-center cohort study, and tested the test set cohort (including 213 normal people and 98 chronic hepatitis B patients). , 96 patients with liver cirrhosis and 424 patients with hepatocellular carcinoma (179 of which were AFP-negative)) serum DKK1 protein concentration, the first discovery at home and abroad that serum DKK1 has high sensitivity and specificity in diagnosing AFP-negative hepatocellular carcinoma , and in the validation set (including 99 normal subjects, 73 patients with chronic hepatitis B, 72 patients with liver cirrhosis and 209 patients with hepatocellular carcinoma (69 of which were AFP-negative)), the effect of serum DKK1 on AFP-negative liver Diagnostic value of cell carcinoma. the

As used herein, "AFP-negative hepatocellular carcinoma" refers to an indication, which is a hepatocellular carcinoma, but AFP is substantially absent or serum AFP is lower than 20 ng/ml. AFP is currently the most widely used marker of hepatocellular carcinoma. According to the existing technology, when the cut-off value of AFP is 20ng/mL, the sensitivity of AFP in diagnosing hepatocellular carcinoma is only 55-60%, resulting in a false negative rate of about 40%. . This part of AFP-negative HCC patients is difficult to diagnose clinically, and it is difficult to evaluate its treatment effect and disease process. the

At present, liver cancer is mainly divided into the following categories: (1) hepatocellular carcinoma, a malignant tumor originating from liver cells; (2) cholangiocarcinoma, a malignant tumor originating from intrahepatic and extrahepatic bile duct epithelial cells, accounting for about (3) mixed primary liver cancer, hepatocellular carcinoma and cholangiocarcinoma in the same liver cancer mass; (4) hepatic hemangioma, a relatively common benign liver tumor, It accounts for 5-20% of benign liver tumors; (5) Hepatic adenoma: in benign liver tumors, its incidence is second only to hepatic hemangioma, and its pathogenesis may be related to sexual endocrine disorders, mainly occurring in women; (6) Focal nodular hyperplasia of the liver, benign liver lesions, the etiology is unclear; (7) liver carcinoid, malignant tumor primary liver carcinoid is rare, secondary liver carcinoid mainly occurs in the digestive tract and other organs (8) Hepatoblastoma: a malignant embryonal tumor with multiple differentiation patterns, which is the most common liver tumor in children; (9) Metastatic liver cancer: primary A malignant tumor that has metastasized to grow in the liver in other parts of the body. the

In addition, there are chronic liver diseases (such as hepatitis and cirrhosis) that also manifest as liver discomfort, which is easy to be diagnosed as liver cancer. According to the characteristics of liver cancer and liver diseases above, it can be known that liver cancer is complicated clinically. How to differentiate and identify it and improve the accuracy of clinical diagnosis is an urgent problem to be solved. It is very necessary to be able to accurately diagnose hepatocellular carcinoma (especially the conventional method is difficult to accurately diagnose) markers for diagnosis of AFP-negative hepatocellular carcinoma, early-stage hepatocellular carcinoma, or small hepatocellular carcinoma). Although AFP has been used as a classic liver cancer marker, it cannot completely solve the problem of accurate subdivision and diagnosis in clinical practice, and the misdiagnosis rate remains high. As for the DKK1 marker, although the inventors have previously associated it with cancer, whether it can subdivide the types of liver cancer is unknown in previous studies. the

In the present invention, the amino acid sequence of the term "DKK1 protein" is substantially the same as the protein sequence provided by GenBank accession number AAQ89364, and also includes homologous proteins of DKK1 protein. The nucleotide sequence of the "gene encoding DKK1 protein" is substantially the same as the nucleotide sequence provided by GenBank Accession No. NM_012242.2 or its degenerate variants, including homologous genes of the DKK1 gene. The amino acid sequence of the term "AFP protein" is substantially identical to the protein sequence provided by GenBank Accession No. AAH27881.1. The nucleotide sequence of the "gene encoding AFP protein" is substantially identical to the nucleotide sequence provided by GenBank accession number NM_001134.1 or a degenerate variant thereof. the

Based on the new findings of the present inventors, the DKK1 protein or its coding gene can be used as a marker (marker) for determining hepatocellular carcinoma, especially AFP-negative hepatocellular carcinoma. By analyzing the expression of the DKK1 protein or its coding gene in the test sample (sample), the disease status of the subject can be known, and the basis for the diagnosis or prognosis of the disease can be provided. The sample to be tested or the sample to be tested is the patient's body fluid, preferably serum. the

Various techniques can be used to detect the expression of DKK1, and these techniques are all included in the present invention. Existing technologies available for detection of nucleic acid such as (but not limited to): gene chip technology, probe hybridization technology, polymerase chain reaction (PCR), Northern Blot and other methods. The protein can be detected by means of a mass spectrometer, or by methods such as Western Blot or ELISA. the

As an option of the present invention, quantitative or semi-quantitative polymerase chain reaction (PCR) method is used to analyze the expression and expression level of DKK1 gene in the sample, so as to make a judgment. Preferably, detection is achieved by real-time quantitative Realtime-PCR. the

Based on the new discovery of the present invention, the present invention also provides a reagent for specifically recognizing the DKK1 protein or its coding gene. Any reagent that can recognize DKK1 protein or its coding gene is included in the present invention and used as a marker for detecting AFP-negative hepatocellular carcinoma. The reagents for specifically recognizing the DKK1 protein or its coding gene are, for example: primers for specifically amplifying the coding gene of the DKK1 protein; or probes for specifically recognizing the coding gene of the DKK1 protein or its transcript; or specific anti- Antibody to DKK1 protein. the

The present invention also provides the use of a reagent for specifically recognizing DKK1 protein or its coding gene for detecting AFP-negative hepatocellular carcinoma or high-risk population of AFP-negative hepatocellular carcinoma. the

As an embodiment of the present invention, the reagent is an anti-DKK1 antibody; more specifically, it is a monoclonal antibody or a polyclonal antibody. the

Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, purified antigen can be administered to animals such as rabbits, mice, rats, etc. to induce polyclonal antibody production. Various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant and the like. the

Antibodies of the invention may also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology. the

The antibody can be used in immunochemical techniques to detect the DKK1 level in the specimen, so as to diagnose AFP-negative hepatocellular carcinoma or judge the risk (susceptibility) of the disease. the

As another embodiment of the present invention, the reagent is a primer for specifically amplifying the DKK1 gene. After the nucleotide sequence of DKK1 is known, people can easily design primers based on it. As a more preferred mode of the present invention, the primers are a pair of primers having the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2. The primer pair can amplify to obtain an amplified product with a suitable length, and the detection specificity is good. the

Gene chip technology can also be used to detect DKK1. After knowing the nucleotide sequence of DKK1, people can easily design probes based on it. For example, if the solid phase carrier is a modified glass slide or a silicon wafer, and the 5' end of the probe contains amino-modified poly dT strings, the oligonucleotide probe can be formulated into a solution, and then the spotting instrument will spot it on The gene chip of the present invention can be obtained by modifying glass slides or silicon slices, arranging them into a predetermined sequence or array, and then fixing them by placing them overnight. If the oligonucleotide probe does not contain amino modification, its preparation method can also refer to the prior art. the

The level of DKK1 in body fluid can be detected by using the antibodies, probes or primers, so it can be used to detect AFP-negative hepatocellular carcinoma, can be used to predict the occurrence of early AFP-negative hepatocellular carcinoma, or can be used to prepare and detect AFP-negative hepatocellular carcinoma Preparations or kits for cancer, etc., used to prepare preparations or kits for predicting the occurrence of early AFP-negative hepatocellular carcinoma. the

The present invention also provides a kit for diagnosing AFP-negative hepatocellular carcinoma, which includes: a reagent for specifically recognizing DKK1 protein or its coding gene. More preferably, the kit also includes: a reagent that specifically recognizes the AFP protein or its coding gene; thereby the AFP-negative sample can be determined by first detecting the expression of AFP in the sample to be tested; further detecting the expression of DKK1 The situation; this detection method will make the detection of hepatocellular carcinoma more accurate, and can greatly reduce misdiagnosis and missed diagnosis. The reagents are, for example: the reagents that specifically recognize the DKK1 protein or its encoding gene are, for example: primers that specifically amplify the encoding gene of the DKK1 protein; or primers that specifically recognize the encoding gene of the DKK1 protein or its transcript Probe; or specific anti-DKK1 protein antibody (monoclonal antibody or polyclonal antibody). The reagent of described specific recognition AFP protein or its encoding gene is for example: the primer of the encoding gene of specific amplification AFP protein; Or the probe of the encoding gene of specific recognition AFP protein or its transcript; Or specific anti Antibodies (monoclonal or polyclonal) to AFP protein. the

The kit can also contain: nucleic acid extraction reagents (such as nucleic acid extraction solution); and/or polymerase chain reaction reagents (such as dNTP, Taq enzyme); and/or western blotting reagents; and/or enzyme Chain immunoreaction reagents (such as chromogenic solution or hybridization solution). the

As a preferred manner, the kit may also contain: reagents for immunochemical analysis, such as: secondary antibodies, staining agents, chromogenic reagents, and the like. In addition, the kit may also include instructions for use and the like. More specifically, the kit can be an enzyme-linked immunoassay (ELISA)-based kit for detecting AFP-negative hepatocellular carcinoma or predicting the occurrence of early AFP-negative hepatocellular carcinoma. ELISA techniques and detection reagents based on this technique will be apparent to those skilled in the art. the

As another preferred mode, the kit may also contain: (A) various reagents for PCR reactions, such as but not limited to: Taq enzyme, PCR buffer, dNTP, DNA polymerase, etc.; or (B) Various reagents required for extracting DNA or RNA (ie preparing PCR reaction template), such as but not limited to: phenol, chloroform, isoamyl alcohol, NaCl, etc.; or (C) kits for extracting DNA or RNA. the

The reagents for specifically recognizing DKK1 protein or its coding gene, or the reagents for specifically recognizing AFP1 protein or its coding gene can also be immobilized on test paper to prepare immunocolloidal gold test paper or similar detection materials. the

In addition, the kit may also contain instructions for use and/or standard operating procedures of the kit of the present invention. the

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods that do not indicate specific conditions in the following examples, usually according to the conditions described in J. Sambrook et al., Molecular Cloning Experiment Guide, Science Press, 2002, or according to the conditions suggested by the manufacturer . the

I. Materials and methods

1. Liver tissue samples and corresponding serum collection

A total of 28 liver tissue samples were collected, including 4 normal human liver tissues from liver transplantation donors in the Liver Surgery Department of the Liver Cancer Institute of Fudan University; 8 liver tissue samples from patients with liver cirrhosis, from liver cirrhosis biopsy patients from the Department of Infectious Diseases, First Affiliated Hospital of Soochow University; 16 liver cancer tissues from patients with hepatocellular carcinoma (including 8 patients with serum AFP≤20ng/ml, and 8 patients with AFP>20ng/ml), were obtained from patients with hepatic resection in the Liver Surgery Department of the Liver Cancer Institute of Fudan University. At the same time, the corresponding serum of 28 cases was collected. the

2. RNA extraction

Extract the total cellular RNA with Trizol reagent, add Trizol and pipette until it is not viscous, add 200 μl chloroform per ml Trizol, shake vigorously for 15 seconds, then let stand at room temperature for 5 minutes; centrifuge at 10,000 rpm at 4°C for 15 minutes at high speed; Transfer the upper aqueous phase into a new RNase-Free centrifuge tube, add 500 μl isopropanol according to the volume of Trizol per milliliter, let stand at room temperature for 10 minutes, centrifuge at 10,000 rpm for 10 minutes at 4°C; Wash Trizol with at least 1ml of 75% alcohol), centrifuge at 8,000rpm at 4°C for 10min, discard the supernatant; dry the RNA pellet at room temperature for 5-10min (do not dry the RNA completely), and dissolve with RNase-Free H ₂ O at 55-60°C 10min, immediately put on ice. The spectrophotometer method was used to quantify and detect the purity, and a small amount of total RNA was taken for formaldehyde denaturation electrophoresis to check the integrity of the RNA.

3. Reverse transcription

Reverse transcription reaction system: 20 μl reaction volume, 1 μg total cellular RNA (Table 1). the

Table 1. Reverse transcription reaction system

Reaction conditions: 37°C for 45min (reverse transcription reaction), 85°C for 5sec (reverse transcriptase inactivation reaction), and store at 4°C. Add ddH ₂ O to dilute 40 times for later use.

4. Semi-quantitative PCR

The diluted cDNA template was used for semi-quantitative PCR amplification. The PCR reaction system is shown in Table 2. the

Table 2. Semi-quantitative PCR reaction system

Reaction conditions: 94°C for 5 min; 94°C for 30 sec, 57°C for 30 sec, 72°C for 30 sec, 32 cycles (DKK1 and AFP), 28 cycles (ACTB); 72°C for 5 min; 4°C storage. The PCR products were verified by 2% agarose gel electrophoresis. With ACTB (β-actin) as internal reference

The primer sequence is:

DKK1 (GenBank NM_012242.2):

Forward: 5'-GACCCAGGCTTGCAAAGTGAC-3'(SEQ ID NO:1),

Reverse: 5'-CGCTACCATCGCGACAAAGA-3'(SEQ ID NO:2);

AFP (GenBank NM_001134.1):

Forward: 5'-TGGGACCCGAACTTTCCAAG-3'(SEQ ID NO:3),

Reverse: 5'-CCTGCAGACAATCCAGCACAT-3'(SEQ ID NO:4);

ACTB (GenBank NM_001101.3):

Forward: 5'-TTGTTACAGGAAGTCCCTTGCC-3'(SEQ ID NO:5),

Reverse: 5'-ATGCTATCACCCTCCCCTGTGTG-3' (SEQ ID NO: 6). the

5. Real-time fluorescent quantitative PCR

The real-time fluorescent quantitative PCR reaction system is shown in Table 3. the

Table 3. Real-time fluorescent quantitative PCR reaction system

The PCR reaction was carried out according to the recommended conditions of the ABI 7300 instrument, using a two-step PCR amplification program: stage 1: 95°C for 30 sec; stage 2: 95°C for 5 sec, 60°C for 31 sec, 40 cycles; Dissociation Stage. Data analysis is automatically completed by ABI 7300 system software, and the Ct value (number of cycles required to reach the threshold value) is determined by making an amplification curve with the number of PCR cycles versus ΔRn. The relative expression of the target gene was corrected with ACTB as the internal reference and calculated according to the following formula: 2 ^-ΔΔCt (ΔCt=Ct(target gene)-Ct(β-actin)). The primer sequences are the same as before.

6. ELISA

(1) ELISA detection of serum DKK1 concentration

The DKK1 ELISA detection method was operated according to the instructions of R&D Company (Product No. DY1906), and the steps were as follows: 100 μl mouse anti-human monoclonal coating antibody (4 μg/ml) coated the ELISA plate, and left at room temperature overnight; the next day, use PBS containing 1% BSA The solution was blocked at 37°C for 1.5h; add serially diluted standards (the highest concentration was 4ng/ml, a total of 7 dilutions) and serum (100μl/well) diluted with PBS containing 10% calf serum, and incubated at 37°C for 1.5 h; then add biotin-labeled rabbit anti-human DKK1 polyclonal detection antibody (50ng/ml), and incubate at 37°C for 1.5h; then add streptavidin-coupled IgG-HRP, and incubate at 37°C for 20min; finally Add substrate hydrogen peroxide (H ₂ O ₂ ) and 3,3,5,5-tetramethyl benzidine (Tetramethyl benzidine, TMB), room temperature for 20min; 1M sulfuric acid to stop the reaction, and measure the absorption wavelength at 450nm with a microplate reader and 570nm reference wavelength. Wash three times with PBS solution containing 5‰ Tween-20 and pat dry between each step. The standard curve was fitted by a four-parameter regression method and the concentration of DKK1 was calculated.

(2) ELISA detection of serum AFP concentration

The method of AFP ELISA detection of serum is operated according to the instructions of the AFP kit of Shanghai Kehua Bioengineering Co., Ltd. The brief steps are as follows: add standard and serum samples (50μl/well) to the pre-coated plate, incubate at 37°C for 20min; add peroxide Enzyme-coupled detection antibody (50 μl/well), incubate at 37°C for 20 minutes; add substrates hydrogen peroxide (H ₂ O ₂ ) and 3,3,5,5-tetramethylbenzidine (TMB), room temperature for 10 minutes; 1M sulfuric acid was used to terminate the reaction, and the absorption wavelength of 450nm and the reference wavelength of 570nm were measured on a microplate reader. Wash three times with PBS solution containing 5‰ Tween-20 and pat dry between each step. A standard curve was fitted and serum AFP concentrations were calculated.

7. Collection of serum samples

(1) Serum samples

The test set contains 831 serum samples, including 213 healthy controls (HC) serum, 98 chronic hepatitis B (CHB) serum and 96 liver cirrhosis (LC) serum from Soochow University. Infectious Diseases Department of an affiliated hospital, 424 cases of hepatocellular carcinoma patients’ serum were obtained from the Liver Surgery Department of Zhongshan Hospital Affiliated to Fudan University; a total of 453 cases of serum samples were collected in the validation set, including 99 cases of normal human serum, 73 cases of chronic hepatitis patients’ serum and 72 cases of liver cirrhosis patients, Sera from 209 patients with hepatocellular carcinoma were obtained from the Dongfang Hepatobiliary Surgery Hospital Affiliated to the Second Military Medical University. Serum was collected at the time of clinical diagnosis, from October 2008 to June 2011. Serum was collected in coagulation-promoting tubes, centrifuged at 2,000-3,000rpm for 18-20min, the supernatant was drawn into centrifuge tubes, and stored at -80°C. The clinicopathological characteristics of patients with hepatocellular carcinoma, hepatitis and liver cirrhosis in the two cohorts are shown in Table 4-6. the

(2) Inclusion criteria

Normal human serum is the serum of voluntary blood donors, with normal liver function, no history of liver disease, and no other malignant diseases. Chronic hepatitis was diagnosed according to the guidelines for the diagnosis and treatment of chronic hepatitis B (Lok AS, McMahon BJ. Chronic hepatitis B: update 2009. Hepatology 2009; 50: 661-2). The diagnostic criteria of liver cirrhosis refer to the 12th edition of "Practical Internal Medicine" (edited by Chen Haozhu), including imaging diagnosis, clinical index detection, pathological diagnosis, etc. The diagnostic criteria for hepatocellular carcinoma are carried out according to the AASLD (American Association for the Study of Liver Diseases) guidelines (Bruix J, Sherman M. Management of hepatocellular carcinoma. Hepatology 2005;42:1208-36), including imaging diagnosis, clinical index detection, Pathological diagnosis, etc. the

(3) Determination of various clinical indicators

(a) AFP positive: The serum AFP content is detected by radioimmunoassay or enzyme immunoassay, and if it is greater than 20ng/ml, it is positive, and if it is less than 20ng/ml, it is negative. the

Liver function grading was based on the Child-Pugh grading method (Van Deusen MA, Abdalla EK, Vauthey JN, Roh MS. Staging classifications for hepatocellular carcinoma. Expert Rev Mol Diagn 2005;5:377-83). the

(b) Tumor staging is based on Barcelona Clinic Cancer (BCLC) grading standards (Llovet JM, Di Bisceglie AM, Bruix J, et al.Design and endpoints of clinical trials in hepatocellular carcinoma.J Natl Cancer Inst 2008;100:698-711) . BCLC stage 0+A is early stage, BCLC stage B+C+D is late stage. the

(c) The degree of tumor differentiation is diagnosed according to the tissue samples of each case and the results of follow-up visits, and according to the Edmondson criteria (Wittekind C.[Pitfalls in the classification of liver tumors]. Pathologe 2006;27:289-93), Edmondson Ⅰ-Ⅱ For well-differentiated, Ⅲ-Ⅳ grades for poorly differentiated. the

(d) Tumor thrombus will be revisited based on the histopathological sections of each case. Microscopic tumor thrombus is microscopic tumor thrombus. Preoperative CT/MRI suggests portal vein tumor thrombus and is confirmed during operation as macroscopic tumor thrombus. Both of them were cancer thrombus positive group, and the others were cancer thrombus negative. the

8. Statistical analysis

Data analysis was performed using SPSS 15.0 software (SPSS Inc., USA). Continuous variables are represented by median (Median) and mean ± standard deviation (Mean ± SD); comparison of measurement data between groups is by Mann-Whitney U test (non-parametric statistics) or T-Test; count data is by chi-square test or Fisher's exact test. The diagnostic ability was evaluated by drawing the receiver operating characteristic (ROC) curve and calculating the corresponding area under the curve (AUC). The optimal cutoff value was selected as the value corresponding to the maximum sum of sensitivity and specificity and the minimum error [(1-sensitivity) ² +(1-specificity) ² square root]. MedCale10.4.7.0 software was used to compare the difference of the area under the curve (AUC). P<0.05 (both sides) means there is a statistical difference. R software was used to analyze the test performance and sample size, and the test performance was considered to be effective if the test performance was greater than or equal to 0.8.

9. Clinical characteristics of patients

The clinical characteristics of HCC patients in the test set and validation set are shown in Table 4. the

Table 4

Abbreviations: AFP: alpha-fetoprotein, alpha-fetoprotein; HbsAg: hepatitis B surface antigen, hepatitis B virus surface antigen; HbeAg: hepatitis B e antigen, hepatitis B virus core antigen; ALT: alanine aminotransferase, alanine aminotransferase; GGT: gamma glutamyl Transpeptidase, γ-glutamyl transpeptidase; BCLC: Barcelona Clinic Liver Cancer, Barcelona Clinic Liver Cancer Criteria. the

The clinical characteristics of patients with chronic hepatitis B in the test set and validation set are shown in Table 5. the

table 5

Abbreviation: HBV: hepatitis B virus, hepatitis B virus; AFP: alpha-fetoprotein, alpha-fetoprotein; HbsAg: hepatitis B surface antigen, hepatitis B virus surface antigen; HbsAb: hepatitis B surface antibody, hepatitis B surface antibody; HbeAg: hepatitis B e antigen, hepatitis B virus e antigen; HBeAb, hepatitis B e antibody, hepatitis B virus e antibody; HbcAb: hepatitis B core antibody, hepatitis B virus core antibody. the

^a , Among the 98 patients with chronic hepatitis B in the test set, 57 patients without AFP value were not included in the calculation.

^b , Among the 73 patients with chronic hepatitis B in the validation set, 18 patients without AFP value were not included in the calculation.

采用Fisher精确检验；其它差异分析采用Chi-square检验。 

Fisher's exact test was used; other differences were analyzed using Chi-square test.

The clinical characteristics of patients with liver cirrhosis in the test set and validation set are shown in Table 6. the

Table 6

Abbreviation: HBV: hepatitis B virus, hepatitis B virus; AFP: alpha-fetoprotein, alpha-fetoprotein; HbsAg: hepatitis B surface antigen, hepatitis B virus surface antigen; HbsAb: hepatitis B surface antibody, hepatitis B surface antibody; HbeAg: hepatitis B e antigen, hepatitis B virus e antigen; HBeAb, hepatitis B virus e antibody; HbcAb: hepatitis B core antibody, hepatitis B virus core antibody. the

*, Among the 96 patients with liver cirrhosis in the test set, 22 patients without relevant data were not included in the calculation. the

II. Embodiment

Example 1, Comparative analysis of the expression of DKK1 and AFP in non-cancerous liver tissue and hepatocellular carcinoma tissue and corresponding serum

The present inventors compared and analyzed DKK1 and AFP in 12 cases of non-cancerous liver tissues (4 cases were normal human liver tissues, 8 cases were liver cirrhosis tissues) and 16 cases of hepatocellular carcinoma liver tissues (8 cases of patients with serum AFP≤20ng /ml, and the expression in serum AFP>20ng/ml of the other 8 cases). The results of semi-quantitative reverse transcription polymerase chain reaction (semi-qPCR) and real-time quantitative polymerase chain reaction (qRT-PCR) experiments showed that compared with AFP, DKK1 was more effective in 4 cases of healthy controls (HC) and 8 cases. In cases of liver cirrhosis (liver corrosion, LC), the expression was almost undetectable, but it was highly expressed in most liver tissues of hepatocellular carcinoma (Fig. 1A-B, 1D-E). the

Enzyme-linked immunosorbent assay detects the protein levels of DKK1 and AFP in the serum of patients corresponding to hepatocellular carcinoma tissue, with 20ng/ml as the cutoff value of AFP and 2.153ng/ml as the cutoff value of DKK1 (the cutoff value used in this patent, See below for the specific determination method), the results showed that in 4 cases of normal people (HC), both were negative; in 8 cases of liver cirrhosis patients (LC), 1 case was positive; in 8 cases of AFP-negative liver Among patients with cellular carcinoma (serum AFP≤20ng/ml, code 1-8), 7 cases showed positive serum DKK1, and among 8 patients with AFP-positive hepatocellular carcinoma (serum AFP>20ng/ml, code 9-16), Six cases showed positive serum DKK1 (Fig. 1C, F). the

Therefore, DKK1 can detect AFP-negative HCC. the

Example 2. Serum DKK1 is a serum protein marker for the diagnosis of hepatocellular carcinoma

The present inventors evaluated the diagnostic ability of serum DKK1 protein for AFP-negative hepatocellular carcinoma according to the research design process in Fig. 2 . the

Firstly, the expression of serum DKK1 and AFP in each group of the test set was analyzed. As shown in Figure 3A, the serum DKK1 protein level in the hepatocellular carcinoma (HCC) group was significantly higher than that in the healthy controls (HC), chronic hepatitis B (CHB) control group and liver cirrhosis (liver cirrhosis, LC) control group (P<0.001), the median (mean ± standard deviation) was 3.08 (3.48 ± 2.33) ng/ml, while there was no significant difference among the three control groups. Serum AFP levels in the HCC group were also higher than those in the other three groups (P<0.001), but their levels in the chronic hepatitis B (CHB) and liver cirrhosis (LC) groups were significantly higher than those in the normal control group (HC) (P <0.001) (Figure 3B). See Table 4 for the concentrations of serum DKK1 and AFP in each group. the

Therefore, serum DKK1 may be used as a serum protein marker for the diagnosis of HCC. the

Table 4. Concentrations of serum DKK1 protein and serum AFP protein in each group in the test set and validation set

Abbreviations: DKK1: dickkopf-1; AFP: alpha-fetoprotein, alpha-fetoprotein; HC: healthy controls, normal controls; CHB: chronic hepatitis B, chronic hepatitis B; LC: liver cirrhosis, liver cirrhosis; HCC: hepatocellular carcinoma, hepatocellular carcinoma. the

Example 3. Determination of the cutoff value of serum DKK1 in the diagnosis of hepatocellular carcinoma

Subsequently, the inventor took hepatocellular carcinoma (HCC) as the patient group, and normal people (HC), chronic hepatitis B (CHB) and liver cirrhosis (LC) patients as the control group to make ROC curves to determine the diagnosis of serum DKK1 and AFP The cutoff value, sensitivity, specificity and area under the curve (AUC) of hepatocellular carcinoma. the

The results are shown in Figure 4. The optimal cutoff value of serum DKK1 was 2.153ng/ml, the sensitivity was 69.1%, the specificity was 90.6%, and the AUC was 0.848 (95%CI: 0.820-0.875). The optimal cutoff value of AFP is 15.35ng/ml, the sensitivity is 59.4%, and the specificity is 87.4%; when the AFP cutoff value is 20ng/ml, the sensitivity is 57.8%, the specificity is 88.0%, and there is no statistical difference between the two (P=0.104), so in the present invention, we choose 20ng/ml, a commonly used clinical value, as the cutoff value of AFP. the

Example 4, the diagnostic ability of serum DKK1 to AFP-negative (≤20ng/ml) hepatocellular carcinoma

Taking 2.153ng/ml as the DKK1 cutoff value and 20ng/ml as the AFP cutoff value, the positive rate of serum DKK1 in the AFP-negative HCC patients in the test set was 70.4% (126/179). In AFP-negative HCC patients and AFP-positive HCC patients, serum DKK1 levels were not statistically different between the two groups (P = 0.42) (Fig. 5A). The ROC curve results showed that the sensitivity (sensitivity) of DKK1 in diagnosing AFP-negative HCC was 70.4%, the specificity (specificity) was 90.0%, and the area under the curve (AUC) was 0.841 (95% CI: 0.801-0.882) (Fig. 5B and Table 5). the

Therefore, for patients with AFP-negative (≤20ng/ml) hepatocellular carcinoma, serum DKK1 has good sensitivity and specificity. the

Table 5. Diagnostic value of serum DKK1 for AFP-negative (serum AFP≤20ng/ml) hepatocellular carcinoma ^*

Abbreviations: DKK1: dickkopf-1; AFP: alpha-fetoprotein; AUC: area under the curve, area under the curve; 95%CI: 95%confidence interval, 95% confidence interval; PPV: positive predictive value, positive predictive value; NPV : negative predictive value, negative predictive value; +LR: positive likelihood ratio, positive likelihood ratio; -LR=negative likelihood ratio, negative likelihood ratio; HCC: hepatocellular carcinoma, hepatocellular carcinoma; CHB: chronic hepatitis B, chronic hepatitis B ; LC: liver cirrhosis, liver cirrhosis; vs.: versus. the

*, the cutoff value of serum DKK1 is 2.153ng/ml, and the cutoff value of serum AFP is 20ng/ml. the

Example 5, Verification of the diagnostic ability of serum DKK1 on AFP-negative hepatocellular carcinoma

In order to verify the diagnostic ability of serum DKK1 for AFP-negative HCC, the inventors detected the serum DKK1 concentration in another independent sample cohort (n=453) (Figure 2, validation set). As shown in Fig. 3C, same as the test group, in the validation group, serum DKK1 was significantly elevated in hepatocellular carcinoma compared with the other three control groups. Taking 2.153ng/ml determined in the test set as the cutoff value of serum DKK1, the positive rate of serum DKK1 in AFP-negative HCC in the validation set was 68.1% (47/69). There was no statistical difference in serum DKK1 concentration between the AFP-negative HCC patient group (AFP ^- -HCC) and the AFP-positive HCC patient group (AFP ⁺ -HCC) in the validation set (Fig. 5C). The sensitivity (sensitivity) for diagnosing AFP-negative HCC was 66.7%, the specificity (specificity) was 87.2%, and the area under the curve (AUC) was 0.869 (95% CI: 0.815-0.923) (Fig. 5D).

The results of the validation set were similar to those of the test set, demonstrating the diagnostic value of serum DKK1 for AFP-negative HCC. the

in conclusion

As a new tumor serum protein marker, serum DKK1 has diagnostic value for AFP-negative (AFP≤20ng/ml) HCC patients. the

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application. the

Claims (10)

1. Use of DKK1 protein or its coding gene in the preparation of diagnostic reagents for alpha-fetoprotein-negative hepatocellular carcinoma. 2. The use of DKK1 in the preparation of a diagnostic kit for alpha-fetoprotein-negative hepatocellular carcinoma. 3. purposes as claimed in claim 1 or 2, is characterized in that, described diagnostic reagent is selected from: Primers for specifically amplifying the gene encoding the DKK1 protein; or A probe that specifically recognizes the gene encoding the DKK1 protein or its transcript; or Antibodies specific to the DKK1 protein. 4. purposes as claimed in claim 3, is characterized in that, described primer is primer pair, and nucleotide sequence is as shown in SEQ ID NO:1 and SEQ ID NO:2. 5. A test kit for measuring alpha-fetoprotein negative hepatocellular carcinoma, characterized in that, the test kit contains: (1) A diagnostic reagent for detecting the expression or expression level of alpha-fetoprotein or its coding gene; and (2) A diagnostic reagent for detecting the expression situation or expression amount of DKK1 protein or its coding gene. 6. kit as claimed in claim 5, is characterized in that, the diagnostic reagent of described detection alpha-fetoprotein or its coding gene expression situation or expression amount is selected from: Primers that specifically amplify the gene encoding alpha-fetoprotein; or A probe that specifically recognizes the gene encoding alpha-fetoprotein or its transcript; or Antibodies specific to alpha-fetoprotein. 7. test kit as claimed in claim 5, is characterized in that, the diagnostic reagent of described detection DKK1 albumen or its coding gene expression situation or expression amount is selected from: Primers for specifically amplifying the gene encoding the DKK1 protein; or A probe that specifically recognizes the gene encoding the DKK1 protein or its transcript; or Antibodies specific to the DKK1 protein. 8. The test kit according to claim 5, further comprising: nucleic acid extraction reagents; and/or polymerase chain reaction reagents; and/or Western blotting reagents; and/or Enzyme chain immunoreaction reagents. 9. A method for measuring alpha-fetoprotein negative hepatocellular carcinoma, characterized in that the method comprises: (a) detecting the expression or expression level of alpha-fetoprotein or its coding gene in the sample to be tested; and (b) Detect the expression or expression of DKK1 protein or its coding gene in the sample to be tested. When the alpha-fetoprotein test is negative, if the test is positive for DKK1, the provider of the sample to be tested is at high risk of hepatocellular carcinoma . 10. The method of claim 9, wherein the assay is performed using the kit of claim 5.

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