CN108732350A - PLA2G6 is as tubercle and the biomarker of early warning liver cancer in instruction liver - Google Patents

Abstract

The present invention relates to PLA2G6 as tubercle and the biomarker of early warning liver cancer in instruction liver.Specifically, the present invention provides whether following reagents tubercle or diagnosis object in the liver prepared for diagnosing object are in application of the hepatitis into the kit of liver cancer vicious transformation critical stage：(1) reagent specifically bound with PLA2G6；(2) the optional reagent with LTA4H specific bindings；(3) the optional reagent with EPS8L2 specific bindings.The crucial critical stage for having found the conversion of scorching cancer based on time-series dynamics and network association analysis herein, for clinical application in the diagnosis of hepatitis early liver cancer, whether to the critical stage of liver cancer vicious transformation and prevent scorching cancer and deteriorate to change to provide new direction in hepatitis.

Description

PLA2G6 as a biomarker indicating intrahepatic nodules and early warning of liver cancer

technical field

The present invention relates to PLA2G6 as a biomarker for indicating intrahepatic nodules and early warning of liver cancer.

Background technique

Liver cancer, as one of the most life-threatening malignant tumors, has attracted worldwide attention. According to the global cancer statistics report, liver cancer has become the cancer with the highest incidence rate in China, and the rampant hepatitis virus has made China the hardest hit area for hepatitis and liver cancer. Therefore, how to effectively prevent the deterioration of hepatitis to liver cancer has become one of the important topics for domestic researchers.

Recently, studies have shown that chronic inflammation can indeed induce the occurrence of various cancers, including liver cancer. Although researchers have put forward many hypotheses about the mechanism of inflammation promoting cancer, the mechanism of malignant transformation from chronic hepatitis to liver cancer is still not completely clear. Therefore, there is no more accurate and reliable biomarker for early diagnosis of liver cancer. At present, there are two main diagnostic methods, one is clinical diagnosis and staging of liver diseases based on medical imaging detection and doctor experience; the other is based on tissue biopsy, using a small number of more precise molecular markers for pathological grading of liver tumors. However, these diagnostic methods have drawbacks. On the one hand, the disease is extremely serious, and the tumor has grown large enough to be distinguished by medical imaging equipment; These diagnostic deficiencies seriously affect the understanding of the disease and thus affect the quality and effectiveness of treatment.

Therefore, there is still an urgent need in the field for early warning molecular markers for the malignant transformation of chronic inflammation to liver cancer [endogenous (proto-oncogene activation) and exogenous (viral infection) joint influence of chronic inflammation to malignant transformation of liver cancer] things and methods.

Contents of the invention

The first aspect of the present invention provides the application of PLA2G6, optional LTA4H and optional EPS8L2 as molecular markers in diagnosing intrahepatic nodules of a subject or diagnosing whether the subject is at a critical stage of malignant transformation from hepatitis to liver cancer.

In one or more embodiments, the present invention provides the use of PLA2G6 and LTA4H as molecular markers in diagnosing intrahepatic nodules of a subject or diagnosing whether a subject is at a critical stage of malignant transformation from hepatitis to liver cancer.

In one or more embodiments, the present invention provides the use of PLA2G6 and EPS8L2 as molecular markers in diagnosing intrahepatic nodules of a subject or diagnosing whether a subject is at a critical stage of malignant transformation from hepatitis to liver cancer.

In one or more embodiments, the present invention provides the use of PLA2G6, LTA4H and EPS8L2 as molecular markers in diagnosing intrahepatic nodules of a subject or diagnosing whether the subject is in the critical stage of malignant transformation from hepatitis to liver cancer.

The second aspect of the present invention provides a reagent that specifically binds to PLA2G6, an optional reagent that specifically binds to LTA4H, and an optional reagent that specifically binds to EPS8L2 in the preparation of intrahepatic nodules for diagnosing a subject or whether the subject is diagnosed The application of the kit in the critical stage of malignant transformation from hepatitis to liver cancer.

In one or more embodiments, the present invention provides a reagent that specifically binds to PLA2G6 and a reagent that specifically binds to LTA4H is used for diagnosing intrahepatic nodules of a subject or diagnosing whether the subject is at a critical stage of malignant transformation from hepatitis to liver cancer application in the kit.

In one or more embodiments, the present invention provides a reagent that specifically binds to PLA2G6 and a reagent that specifically binds to EPS8L2 is used for diagnosing intrahepatic nodules of a subject or diagnosing whether the subject is at a critical stage of malignant transformation from hepatitis to liver cancer application in the kit.

In one or more embodiments, the present invention provides a reagent that specifically binds to PLA2G6, a reagent that specifically binds to LTA4H, and a reagent that specifically binds to EPS8L2 is useful in preparing intrahepatic nodules for diagnosing a subject or diagnosing whether a subject is The application of the kit in the critical stage of malignant transformation from hepatitis to liver cancer.

In one or more embodiments, the reagent is an antibody or antigen-binding fragment thereof.

The third aspect of the present invention provides a detection kit, which contains a reagent with PLA2G6 as the detection object.

In one or more embodiments, the reagent for detecting PLA2G6 includes a specific antibody to PLA2G6.

In one or more embodiments, the detection kit further contains reagents for detecting LTA4H and/or EPS8L2.

In one or more embodiments, the reagents targeting LTA4H and/or EPS8L2 include specific antibodies to LTA4H and/or EPS8L2.

In one or more embodiments, the detection kit also contains reagents required for the detection of PLA2G6 by immunohistochemistry.

In one or more embodiments, the detection kit further contains reagents required for detecting LTA4H and/or EPS8L2 by immunohistochemical methods.

The fourth aspect of the present invention provides a method for diagnosing whether a subject has intrahepatic nodules, or whether he is in the critical stage of malignant transformation from hepatitis to liver cancer, the method includes the step of detecting the expression level of PLA2G6 in the liver tissue of the subject; wherein , the expression level is lower than the PLA2G6 expression level in the liver tissue of normal people, suggesting that the patient has intrahepatic nodules or is in the critical stage of malignant transformation from hepatitis to liver cancer.

In one or more embodiments, the method further includes the step of detecting the expression level of LTA4H and/or EPS8L2 in the subject's liver tissue.

In one or more embodiments, the intrahepatic nodule is a high grade dysplastic nodule.

In one or more embodiments, the subject is a hepatitis patient.

The fifth aspect of the present invention provides reagents and/or kits for PLA2G6, optional LTA4H and optional EPS8L2 as molecular markers for preparing intrahepatic nodules of diagnostic subjects or for diagnosing whether the subject is in the critical stage of malignant transformation from hepatitis to liver cancer in the application.

In one or more embodiments, the present invention provides PLA2G6 and LTA4H as molecular markers in reagents and/or kits for preparing intrahepatic nodules of diagnostic subjects or diagnosing whether the subject is in the critical stage of malignant transformation from hepatitis to liver cancer application.

In one or more embodiments, the present invention provides PLA2G6 and EPS8L2 as molecular markers in the reagents and/or kits for preparing intrahepatic nodules of diagnostic subjects or diagnosing whether the subject is in the critical stage of malignant transformation from hepatitis to liver cancer application.

In one or more embodiments, the present invention provides PLA2G6, LTA4H and EPS8L2 as reagents and/or molecular markers for preparing intrahepatic nodules of diagnostic subjects or diagnosing whether the subject is in the critical stage of malignant transformation from hepatitis to liver cancer application in the kit.

Description of drawings

Figure 1: Flowchart for detection and quantification of liver proteomes in WHV/c-myc transgenic mice and wt-C57BL/6 mice as a reference.

Figure 2: Detection of early warning signals of malignant transformation of hepatitis to liver cancer based on dynamic network biomarkers.

Figure 3: Dynamic changes in network structure and expression variation of dynamic network biomarkers and their associated co-expressed molecules.

Figure 4: Images of HE-stained liver tissue sections.

Figure 5: Dynamic changes of the DNB-associated network (DNB-associated network) during the transformation of inflammation and cancer. A and B: The points or edges that overlap with differentially expressed proteins (DEP) or differentially co-expressed protein pairs (DCE) in the network were counted, and the hypergeometric test was used to evaluate the significance of the coincidence. C and D: A series of network diagrams showing the differentially expressed proteins (DEP) and differential coexpression involved in the DNB-related functional network of transgenic mice during the malignant transformation of inflammatory carcinoma (ie, between 3 and 7 months old) Dynamic changes in protein pairs (DCE).

Figure 6: Changes in the expression levels of PLA2G6, LTA4H and EPS8L2 at various time points (stages) during the process of malignant transformation from hepatitis to liver cancer.

Figure 7: Diagnosis of LTA4H applied to clinical patients.

Figure 8: Diagnosis of PLA2G6 applied to clinical patients.

Figure 9: The diagnosis of EPS8L2 applied to clinical patients.

Detailed ways

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form a preferred technical solution.

In order to imitate as much as possible the process of malignant transformation of inflammation and cancer caused by both endogenous and exogenous influences, this paper used c-myc transgenic mice that developed hepatitis to liver cancer under the infection of woodchuck hepatitis virus (WHV). Animal models of disease. Previous studies (Etiemble J, Degott C, Renard CA, etc., Liver-specific expression and high oncogenic efficiency of a c-myc transgene activated by woodchuck hepatitis virus insertion, Oncogene 1994, 9:727-737) showed that during the development of liver cancer In the study, the liver of WHV/c-myc mice has successively experienced pathological processes such as liver fibrosis, intrahepatic nodules, benign tumors and malignant tumors, which is very similar to the progression of liver cancer from the deterioration of hepatitis B patients. Therefore, based on previous studies (Etiemble J, Degott C, Renard CA, etc., Liver-specific expression and high oncogenic efficiency of a c-myc transgene activated by woodchuck hepatitis virus insertion, Oncogene 1994, 9:727-737), the present invention is based on different Five time points (2, 3, 5, 7, and 11 months after birth) were selected for the pathological stage of the mouse, and WHV/c-myc mice and wt-C57BL/6 mice were collected respectively. Liver samples were analyzed by mass spectrometry using Label-free quantitative technology and Tandem Mass Tag (TMT) quantitative technology to obtain time-series high-throughput proteomic quantitative data. At the same time, the method of finding dynamic network biomarkers (DNB) was applied (Chen L, Liu R, Liu ZP, Li M, Aihara K, Detecting early-warning signals for sudden deterioration of complex diseases by dynamical network biomarkers, Scientific reports 2012, 2: 342), identified the critical critical stage of the malignant transformation of hepatitis to liver cancer, and explored the early warning signals hidden in dynamic network biomarkers and their potential transformation mechanisms of inflammation and cancer.

Specifically, this paper analyzed the time-series proteome data of different symptoms of the WHV/c-myc transgenic mouse model in the process of malignant transformation from hepatitis to liver cancer, and found that five-month-old transgenic mice are in the critical stage of inflammatory cancer transformation , which is consistent with the results of histopathological morphology judgment; and a dynamic network marker (DNB) with early warning of this critical stage was also obtained. In order to further reveal the role of DNB in the process of inflammation and cancer transformation and the potential pathogenic mechanism, this paper constructed a DNB-associated network (DNB-associated network), and analyzed the dynamic changes of its network structure, and found that the DNB-associated network showed a flipping phenomenon. Therefore, it is inferred that PLA2G6, LTA4H and/or EPS8L2 as DNB members may induce subsequent abnormal inflammatory response and liver dysfunction, and finally promote inflammation to tumor progression. The results of histochemical experiments in this paper further confirmed the above inference.

At the same time, it has been reported that the liver nodule stage is the critical stage of malignant transformation from human hepatitis to liver cancer. In particular, high-grade dysplastic nodules (HGDN) are currently recognized as precancerous lesions of liver cancer, which are defined as "hepatic dysplasia but not up to the criteria for malignant tumors, with a diameter of 1 mm." above dysplastic nodules". Currently, HGDN is mainly detected by histopathology. The main pathological features of HGDN are: (1) increased cell density, (2) thickened liver plate, (3) increased nuclear-cytoplasmic ratio, (4) pseudoglandular cast formation, (5) mild nuclear atypia, (6) Increased cytoplasmic basophilic staining, (7) no stroma and portal vein infiltration.

In this paper, based on the time-series dynamics and network correlation analysis, the critical critical stage of the transformation of inflammation to cancer is found, which can be used clinically in the diagnosis of early liver cancer in patients with hepatitis (such as whether there are intrahepatic nodules, especially HGDN), whether it is in the malignant transformation of hepatitis to liver cancer. (that is, the critical stage of inflammatory cancer transformation) and the prevention of inflammatory cancer transformation provides a new direction.

Therefore, any one, any two (such as PLA2G6 and LTA4H, PLA2G6 and EPS8L2, or LTA4H and EPS8L2) or all three of PLA2G6, LTA4H and EPS8L2 are used as the detection object in this paper, and the protein expression in the liver of the subject is detected. Compared with the protein expression in the liver of normal people, it can be judged whether the subject has intrahepatic nodules, especially HGDN, and whether the subject is in the critical stage of malignant transformation from hepatitis to liver cancer.

Herein, PLA2G6 refers to phospholipase A2 (Group VI), which is a calcium-independent phospholipase of 85KD located in the cytoplasm. It should be understood that various PLA2G6 defined as phospholipase A2 (group VI) by those skilled in the art are included herein, such as PLA2G6 from different species such as human or mouse or other animals. For example, the gene encoding mouse PLA2G6 is located on mouse chromosome 15, the specific chromosome segment is 15E1, the gene number on ENSEMBL is ENSMUSG00000042632, and the gene number on ENTREZ is 53357. The human PLA2G6 coding gene is located on chromosome 22, the specific chromosome segment is 22q13.1, the gene number on ENSEMBL is ENSG00000184381, and the gene number on ENTREZ is 8398. In particular, the invention includes the detection of human PLA2G6 protein. It should be understood that there may be one or several differences in amino acid residues between different human PLA2G6 proteins, but this difference does not prevent the PLA2G6 protein from being used as a biomarker for the purposes and methods described herein. In certain embodiments, the PLA2G6 protein described herein refers to the PLA2G6 protein encoded by a gene at the same position in the genome of the same species.

Herein, LTA4H refers to leukotriene A4 hydrolase. Included herein are various LTA4Hs defined by those skilled in the art as leukotriene A4 hydrolases, for example LTA4Hs from different species such as human or murine or other animals. For example, the gene encoding mouse LTA4H is located on mouse chromosome 10, the specific chromosome segment is 10C3, the gene number on ENSEMBL is ENSMUSG00000015889, and the gene number on ENTREZ is 16993. The gene encoding human LTA4H is located on chromosome 12, the specific chromosome segment is 12q22, the gene number on ENSEMBL is ENSG00000111144, and the gene number on ENTREZ is 4048. In particular, the invention includes the detection of human LTA4H protein. It should be understood that there may be one or several differences in amino acid residues between different human LTA4H proteins, but this difference does not prevent the LTA4H protein from being used as a biomarker for the uses and methods described herein. In certain embodiments, the LTA4H protein described herein refers to the LTA4H protein encoded by a gene at the same position in the genome of the same species.

Herein, EPS8L2 refers to epidermal growth factor receptor kinase substrate 8-like protein 2. Included herein are various EPS8L2s defined by those skilled in the art as EGFR-SK8-like protein 2, such as EPS8L2 from different species such as human or murine or other animals. For example, the gene encoding mouse EPS8L2 is located on mouse chromosome 7, the specific chromosome segment is 7F5, the gene number on ENSEMBL is ENSMUSG00000025504, and the gene number on ENTREZ is 98845. The gene encoding human EPS8L2 is located on chromosome 11, the specific chromosome segment is 11q15.5, the gene number on ENSEMBL is ENSG00000177106, and the gene number on ENTREZ is 64787. In particular, the invention includes the detection of human EPS8L2 protein. It should be understood that there may be one or several differences in amino acid residues between different human EPS8L2 proteins, but this difference does not prevent the EPS8L2 protein from being used as a biomarker for the uses and methods described herein. In certain embodiments, the EPS8L2 protein described herein refers to the EPS8L2 protein encoded by a gene at the same position in the genome of the same species.

Herein, "subjects" refer to various animals that need to be judged whether they have intrahepatic nodules and/or whether they are in the critical stage of malignant transformation from hepatitis to liver cancer, including but not limited to mammals and rodents, such as humans and mice. In particular, in certain embodiments, the "subject" described herein refers to a human, more preferably a human who has ever been infected with a hepatitis virus, a hepatitis patient or a suspected hepatitis patient.

The expression levels of any one, any two or all three of PLA2G6, LTA4H and EPS8L2 in the subject's liver tissue can be detected by methods known in the art (such as immunohistochemical method). Any reagent that can detect the expression level of PLA2G6, LTA4H or EPS8L2 can be used in the uses, methods or kits of the present invention, including various reagents that can specifically bind to PLA2G6, LTA4H or EPS8L2, such as antibodies or antigen-binding fragments thereof .

Immunohistochemical method is a routine method in the field, usually using the specific binding of antigen and antibody, through chemical reaction to make the chromogen of labeled antibody develop color, so as to determine the presence and/or content of antigen in tissue cells. The method or use can be carried out using antibodies specific for each of PLA2G6, LTA4H and/or EPS8L2. Such specific antibodies may be known commercially available antibodies, for example, antibodies from Abcam. Alternatively, their respective specific antibodies can also be produced by themselves according to known techniques (such as hybridoma technique). Antibodies may be monoclonal or polyclonal; monoclonal antibodies are preferred. Exemplary immunohistochemical methods are described in the "Materials and Methods" section herein, but are not limited to the methods described in this section.

Therefore, this paper provides a detection kit, which contains any one, any two or all three of PLA2G6, LTA4H and EPS8L2 as detection objects. For example, the detection kit may contain reagents for detection of PLA2G6, or reagents for detection of LTA4H, or reagents for detection of EPS8L2, or any two types of reagents or all three types of reagents. In certain embodiments, the detection kit herein contains a reagent for detection of PLA2G6 and a reagent for detection of LTA4H, or a reagent for detection of PLA2G6 and a reagent for detection of EPS8L2, or a reagent for detection of LTA4H Reagents for detection and reagents for detection of EPS8L2. As mentioned above, such reagents can be any reagents capable of detecting the expression levels of PLA2G6, LTA4H and EPS8L2, preferably their respective specific antibodies. Also included are antigen-binding fragments of antibodies that retain binding activity to PLA2G6, LTA4H, or EPS8L2.

The detection kit herein may also contain other reagents required for performing the detection of the present invention, such as other reagents required for performing immunohistochemical methods. For example, when the processed object is a paraffin-embedded tissue section, the detection kit herein may also contain: reagents required for section dewaxing and hydration, such as xylene, absolute ethanol, hydrogen peroxide methanol solution, etc.; Reagents required for antigen retrieval, such as acidic repair solution; BSA; buffer, such as PBS buffer; secondary antibody; when using DAB color development, DAB color development solution; hematoxylin; hydrochloric acid ethanol solution; carbolic acid; neutral resin . The content and concentration of various reagents in the kit can be determined according to conventional methods.

The detection kit herein can be used to detect the expression level of PLA2G6, LTA4H and/or EPS8L2 in the sample, and through the expression level, those skilled in the art can judge whether the sample has intrahepatic nodules for the target object, and/or whether it is in Critical stage of malignant transformation of hepatitis to liver cancer. Usually, the test results can be compared with the expression levels of PLA2G6, LTA4H and/or EPS8L2 in the liver tissue of normal people. If it is found that the expression level of PLA2G6 in the sample is lower, and/or the expression level of LTA4H and/or EPS8L2 is higher than the expression level of PLA2G6, LTA4H and/or EPS8L2 in the liver tissue of normal people, it can be concluded that the subject has intrahepatic Nodules, and/or judged to be in the critical stage of malignant transformation from hepatitis to liver cancer.

The expression levels of PLA2G6, LTA4H and EPS8L2 in the liver tissue of normal people can be investigated and counted using methods well known in the art, including the immunotransformation method described herein.

For example, generally, if the expression level of PLA2G6 in the sample is less than 90%, less than 80%, less than 70%, less than 60%, less than 50% or less than 40% of the expression level of PLA2G6 in the liver tissue of the normal population, then the subject can be considered Presence of intrahepatic nodules (especially high-grade dysplastic nodules), and/or in the critical stage of malignant transformation of hepatitis to liver cancer; and/or

Usually, if the expression level of LTA4H in the sample is at least 1.3 times, at least 1.5 times, at least 1.7 times, at least 2 times, at least 2.5 times or at least 3 times the expression level of LTA4H in the liver tissue of the normal population, it is considered that the subject has liver disease. Internal nodules (especially high-grade dysplastic nodules), and/or in the critical stage of malignant transformation of hepatitis to liver cancer; and/or

Usually, if the expression level of EPS8L2 in the sample is at least 1.3 times, at least 1.5 times, at least 1.7 times, at least 2 times, at least 2.5 times or at least 3 times the expression level of EPS8L2 in the liver tissue of the normal population, it is considered that the subject has liver disease. Internal nodules (especially high-grade dysplastic nodules), and/or in the critical stage of malignant transformation from hepatitis to liver cancer.

In some embodiments, the expression level of PLA2G6, LTA4H and/or EPS8L2 in the suspected diseased liver tissue of the subject can even be detected, and compared with the expression level of PLA2G6, LTA4H and/or EPS8L2 in the normal liver tissue of the subject. For comparison, if the expression level of PLA2G6 in the suspected diseased liver tissue is lower, and/or the expression level of LTA4H and/or EPS8L2 is higher than that in the normal liver tissue, for example, if the expression level of PLA2G6 in the suspected diseased liver tissue The expression level is less than 90%, less than 80%, less than 70%, less than 60%, less than 50% or less than 40% of the expression level of PLA2G6 in normal liver tissue, and/or the expression level of LTA4H and/or EPS8L2 is its At least 1.3 times, at least 1.5 times, at least 1.7 times, at least 2 times, at least 2.5 times, or at least 3 times the expression in normal liver tissue, and the subject can also be considered to have intrahepatic nodules (especially high-grade dysplasia nodules), and/or at the critical stage of malignant transformation from hepatitis to liver cancer.

It should be understood that it is only necessary to detect the expression level of one of PLA2G6, LTA4H and EPS8L2 to draw a diagnostic conclusion; however, in some embodiments, it is preferred to detect any two of PLA2G6, LTA4H and EPS8L2, or even Expression levels of all three.

The present invention will be illustrated below in the form of specific examples. It should be understood that these examples are only illustrative, not intended to limit the present invention. Various methods and materials mentioned in the examples, unless otherwise stated, are conventional methods and materials in the art.

1. Materials and methods

1. Experimental animals and reagents

The WHV/c-myc transgenic mouse model and the normal control C57BL-6 strain mice were raised in the Experimental Animal Center of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and all experimental operations strictly followed animal ethics. A total of 5 different time points were taken, including 2 months, 3 months, 5 months, 7 months, and 11 months after birth. At each time point, 5 WHV/c-myc transgenic mice and 5 Normal C57 mice were sacrificed by decapitation, and the liver tissues were isolated. Cut the liver tissue into pieces with scissors, first carefully wash the small pieces of tissue with pre-cooled RPMI1640 buffer, add an appropriate amount of lysis buffer to lyse the cells, and then use a homogenizer to mechanically break up the small pieces of tissue. Dissolve the protein, and centrifuge at 15,000 g for 60 minutes at 4°C to remove impurities such as DNA, RNA, and insoluble cell debris. The resulting supernatant protein solution was carefully transferred into a new Eppendorf tube, and the protein concentration was quantified by the Bradford method (Bio-Rad). After aliquoting the required amount of sample, store at -80°C for later use.

All buffers were prepared with Milli-Q water (Millipore, Bedford, MA, USA). Urea, Bromophenol blue, Acrylamide, Bisacrylamide (Bis), 3-[(3-cholamidopropyl)-diethylammonium]-propanesulfonic acid ( CHAPS), tris(hydroxymethyl)aminoethane (Tris), sodium dodecylsulfonate (SDS), ammonium persulfate (AP), tetramethylethylenediamine (TEMED) and Bradford method quantitative liquid and other reagents Purchased from Bio-Rad (Hercules, CA, USA); ethylenediaminetetraacetic acid dipotassium salt (EDTA), phenylmethylsulfonyl fluoride (PMSF) were purchased from Amresco (Solon, OH, USA); protease inhibitors (Complete protease inhibitor cocktail tablets) were purchased from Roche Company (Indianapolis, IN, USA). Formic acid (FA) was purchased from Sigma Company (St.Louis, MO, USA); HPLC grade acetonitrile (ACN) and methanol (Methanol) were purchased from Fisher Company (Fair Lawn, NJ, USA); acetone (Acetone) and Ethanol (Ethanol) was purchased from Shanghai Chemicals Company (Shanghai, China); sequencing grade trypsin (Trypsin, sequencing grade) was purchased from Promega Company (Madison, WI, USA).

2. Label-free quantification of mouse liver proteome (LFQ quantification)

In label-free quantification (LFQ quantification) experiments, protein samples from each mouse were subjected to in-solution enzymatic digestion. First, add lysis buffer to the protein sample to a total volume of 100 μl, add 4 μl of 1M DTT, mix and react at 37°C for 2.5 hours, then add 10 μl of 1M IAA, mix and react in the dark for 45 minutes at room temperature. Then use 5 times the volume of -20°C pre-cooled protein precipitation solution for precipitation, and stand at -20°C overnight (16 hours). Then centrifuge the sample at 14,000g for 40 minutes to remove the supernatant, first wash the precipitate with -20°C pre-cooled acetone, centrifuge at 14,000g for 40 minutes to remove the supernatant, then wash the precipitate with -20°C pre-cooled 70% ethanol, and centrifuge at 14,000g for 40 minutes to remove the supernatant. Supernatant, samples were lyophilized in a vacuum lyophilizer. Add 200 μl of 100 mM ammonium bicarbonate, fully shake to redissolve the protein, then add 12 μg of trypsin, react for 4 hours at 37°C for enzymatic hydrolysis, then add 12 μg of trypsin, and shake in an oven at 37°C for overnight enzymolysis. After the enzymatic hydrolysis was completed, ultrafiltration was performed with a 10KD pore size ultrafiltration tube (Millipore), and the filtrate was collected, freeze-dried and stored at -80°C. The peptide mixture was separated online by pH continuous gradient elution system (pCOG) combined with strong cation exchange chromatography-reverse chromatography, and the raw data were collected by LFQ mass spectrometer (Thermo Finnigan) in positive ion mode. The peptide eluate was detected by the linear ion trap mass spectrometer directly through the ESI spray ion source, and the ion source parameters were set as: electrospray voltage, 3.0kV; capillary temperature, 170°C. The LTQ mass spectrometer collects data in the data-dependent mode under the control of AGC. The mass range of the full scan is m/z 400-1800. After each full scan, the 10 peaks with the highest intensity are taken for tandem mass spectrometry scanning, and the normalized collision energy Set to 35.0%, dynamic exclusion is set to repeat count of 2, repeat tolerance time is 30 seconds, and dynamic exclusion time is 90 seconds.

The raw data collected by the mass spectrometer first used the BioWorks software (Thermo-Fisher) to extract the dta information, and then used the SEQUEST software to search the database. The database used the mouse IPI positive and negative library (v3.82) generated by the laboratory’s internal software. The parameters are set as follows: the mass deviation of the precursor ion is within 3 Da, the mass deviation of the product ion is within 1 Da, the protease is set to trypsin, the maximum allowable deletion enzyme cleavage site is 1, the fixed modification is set to carboxymethylation of cysteine, variable The modification was set to oxidation of methionine. The Sequest output results further use the BuildSummary software system independently developed by the laboratory for data integration and false positive control.

3. DNB algorithm

Based on the three conditions that the dynamic network markers required by the theoretical derivation of the mathematical model indicate that the system state is about to change, the present invention designs a corresponding implementation algorithm so as to be applied to the research of time-series high-throughput data for specific scientific problems. In order to detect a critical point (Criticalpoint) through a strong signal that comprehensively considers three conditions, the present invention defines the following integrated index (A composite index, CI) to design an algorithm:

CI=SD _d ×PCC _id /PCC _od

Among them, SD _d represents the mean value of the standard deviation of the concentration change of all molecules of the leading molecular group (Dominant group) or dynamic network biomarker (DNB) at the current time point compared with that from the corresponding reference (for example, the sample at the original time point or at the same time). The ratio of the mean value of the standard deviation of the concentration change of the negative control sample of the point. PCC _id is the ratio of the correlation (PCC) between two molecules inside the molecular group at the current time point to the mean value from the corresponding reference; and PCC _od is the correlation between each molecule inside the molecular group and its related molecules outside the molecular group ( PCC) to the mean from the corresponding reference at the current time point, respectively. It is worth noting that although the CI value is used here as an effective evaluation index for determining dynamic network markers and detecting critical critical points of state transitions, other evaluation indexes that can comprehensively meet the three conditions can also be constructed.

The specific algorithm process for determining dynamic network biomarkers based on the integrated index CI:

(1) Molecular fluctuation change screening (Deviation test): at the time point t, from several sample data at the same time point, select the observation value (gene expression product concentration, protein content, etc.) change fluctuation ratio corresponding to the reference observation value A group of molecular sets GtD with large fluctuations (high SD value);

(2) Intra-correlation test: At time point t, calculate the correlation (PCC) between two molecules in GtD, and use this to divide the internal molecules with high correlation at the current time point Subset GtI;

(3) Inter-correlation test between molecules and other related molecules (Inter-correlation test): At time point t, calculate the correlation (PCC) between each molecule in the molecule set GtI and other related molecules outside the molecular group, And use this to screen the external molecular set GtC that has a low correlation with the molecular set GtI;

(4) Determination of dynamic network biomarkers (DNB test): Based on the candidate molecular set GtI screened at each time point in the previous three steps and its corresponding external molecular set GtC, the corresponding CI values are calculated according to formula (3) , using significance analysis to judge that the molecular set GtI corresponding to the local highest CI value is the leading molecular group (Dominant group) or dynamical network biomarker (Dynamic network biomarker, DNB), and the corresponding time point is the state The critical critical stage of the upcoming transition (Critical point or Pre-transition state). It is worth noting that when there are many time points observed, there may be multiple state transition processes. At this time, multiple different markers and key critical stages may be obtained.

Referring to the theoretical derivation results of the mathematical model, when the system is close to the critical critical stage of state transition, both SD _d and PCC _id /PCC _od should reach the maximum value. However, it is worth noting that in most practical cases, we usually cannot obtain the data of the critical critical stage of the state transition very accurately, and can only observe the sample data of the time period near the critical point, so SD _d and PCC _id / PCC _od cannot reach the global maximum at the same time.

4. Immunohistochemical experiment

In this application, the immunohistochemical experiment adopts DAB chromogenic method. The main operation steps are as follows:

Cut paraffin-embedded tissue wax blocks into 4 μm sections;

Place the slices in a 60°C oven for 1 hour;

Section dewaxing and hydration (xylene 10 minutes → xylene 10 minutes → xylene 3 10 minutes → absolute ethanol 5 minutes → 95% ethanol 5 minutes → 85% ethanol 5 minutes → 75% ethanol 5 minutes → deionization water for 5 minutes);

Place the slices in a 3% methanol solution of hydrogen peroxide and let stand at room temperature for 20 minutes to remove peroxidase;

Wash with deionized water for 5 minutes, repeat three times;

Antigen restoration (place the slices in the acidic repair solution, boil on high heat until boiling, then turn to low heat, continue to cook for 2 minutes, stop the fire and wait for it to cool naturally);

Wash with deionized water for 5 minutes, repeat three times;

Add 1% BSA dropwise to the slices and place in a 37°C incubator to seal for 30 minutes;

Wash the blocking solution, add the corresponding primary antibody dropwise directly, place in a wet box, and freeze overnight at 4°C;

Rewarm at room temperature for 15 minutes;

Wash with PBS buffer for 5 minutes, repeat four times;

Drop the corresponding secondary antibody (Anti-Rabbit-HRP, Antibody Dignostica Inc.) onto the slices, place them in a wet box, and incubate in a 37°C incubator for 45-60 minutes;

Wash with PBS buffer for 5 minutes, repeat four times;

Use DAB color developing solution to develop color for 3-10 minutes, observe under the microscope, after the color development is successful, stop the color development with deionized water;

Elution with deionized water twice, 5 minutes each time;

Hematoxylin counterstained for 10 minutes, followed by HE staining;

Take out the slices and rinse them once or twice in tap water, then put them into hydrochloric acid ethanol solution and rinse them 2-3 times;

Rinse in running tap water for 20-30 minutes, take out the slices, and drain at room temperature;

Wash with deionized water for 5 minutes; wash in 95% ethanol, 100% ethanol 1, 100% ethanol 2, and xylene 1 for 10 minutes each;

In xylene and carbolic acid for 5 minutes each; finally add an appropriate amount of neutral resin dropwise, and seal the slide for observation.

Primary antibody dilution ratio: LTA4H (abcam ab133512): 1:400; EPS8L2 (abcam ab209339): 1:100; PLA2G6 (abcam ab103258): 1:100.

After the experiment was completed, the Aperio AT rurbo scanner (Leica Biosystems) and supporting software were used for image output and image analysis.

5. Acquisition of in vitro stable isotope labeling quantification (TMT quantification) data of mouse liver proteome

In the in vitro stable isotope labeling quantification (TMT quantification) experiment, 200 μg of mixed liver protein samples from mice at the same time point were taken at each time point, and the membrane-assisted enzymatic hydrolysis method was adopted. The enzymatic hydrolysis process was as follows: first add UAbuffer (8M urea, 0.1M Tris/HCl pH 8.5), centrifuge at 5000g for about 1h, repeat once; then add 50mM IAA (prepared with UA), place in the dark at room temperature for 30min, then centrifuge at 5000g for 1h; then use 0.5M TEAB solution Wash three times; finally add Trypsin according to the ratio of 1:50, and put it in a shaker or incubator at 37°C for 16-20 hours for enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the peptides were collected for TMT. Two groups of TMT reagents were used to label the enzymatic products of liver tissue of transgenic mice or control mice, and then the different labeled samples of the two groups of experiments were mixed and freeze-dried.

The Angilent 3100OFFGEL Fractionator system (Agilent Technologies) was used for fractionation, 50μA constant current, and the peptides were collected for about 48h. Each of the two samples was fractionated into 6 components, and a total of 12 samples were subjected to LFQ-Orbitrap Velos mass spectrometry detection (Thermo Finnigan, San Jose, CA), each sample was loaded 3 times. The reversed-phase column is a self-made needle reversed-phase column in the laboratory. The eluents of the reversed-phase elution system are 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The flow rate is 250nL/min, using 4 Linear gradient from %B to 30%B. The peptides eluted from the reversed-phase column enter the linear ion trap-electrostatic field orbitrap combined mass spectrometer LTQ Orbitrap-Velos mass spectrometer for analysis. The data-dependent data acquisition mode is followed by 10 first-order spectra. For the secondary spectrum, the dynamic exclusion is set to 90 seconds, the parent ion fragmentation mode is HCD mode, and the resolutions of the primary spectrum and secondary spectrum are set to 30000 and 7500, respectively.

The original files collected by mass spectrometry were analyzed with the default parameters of MaxQuant1.3.0.5 software, and the database used was UniProt mouse database (downloaded in June 2012). The search parameters are set as follows: Protease is set to Trypsin, TMT-labeled peptide screening is selected, fixed modification is set to carboxymethylation of cysteine, variable modification is set to oxidation of methionine, protein N-terminal acetylation, The false positive rate for both proteins and peptides was set to 0.01.

2. Experimental results

1. Dynamic changes of protein expression levels in WHV/c-myc transgenic mice during the development of liver cancer

In order to obtain high-throughput proteome data suitable for the research purpose of this work, as shown in the figure, the present invention designed five corresponding Sampling time points (the 2nd, 3rd, 5th, 7th and 11th months after the mice were born); at each time point, 5 WHV/c-myc transgenic mice and 5 WHV/c-myc transgenic mice under the same feeding conditions As a reference, the liver tissues of wt-C57BL/6 mice were collected separately; then, for each liver tissue sample, the protein was separated and purified and enzymatically digested into peptides, and the non-labeled (Label-free) peptides were passed through Multidimensional liquid chromatography fractionation followed by tandem mass spectrometry (LC-MS/MS) detection (Zhou H, Dai J, Sheng QH et al., A fully automated 2-D LC-MS method utilizing online continuous pH and RPgradients for global proteome analysis, Electrophoresis 2007 , 28:4311-4319), to obtain the final quantitative protein data. In this batch of quantitative data, by running 570 LC-MS/MS detection cycles, a total of high-precision (based on the false positive detection rate (False discovery rate, FDR) of 0.4% peptides as the standard) was identified. 42,689 different peptides of 8,713 proteins; but considering the accuracy and stability of subsequent calculations and analysis, the present invention only selected 50 samples at more than the total number (a total of 5 time points, 25 of which were WHV/c-myc 3372 proteins were identified in half of the samples from transgenic mice and 25 wt-C57BL/6 mice (ie, these proteins were quantified in more than 25 mouse liver samples).

2. Identification of critical stages of malignant transformation from hepatitis to liver cancer

In order to accurately determine the critical critical stage of malignant transformation of inflammatory cancer, the present invention uses the method of finding dynamic network biomarkers (Dynamic network biomarker, DNB) to analyze this set of time-series high-throughput proteome data. After analysis by using the DNB algorithm described herein, the present invention found that a strong signal of CI value appeared at the time point of 5 months of age, which indicated that WHV/c-myc transgenic mice were at the critical point of malignant transformation from hepatitis to liver cancer at this time stage (that is, it warns that malignant transformation of inflammatory carcinoma is about to occur). This result is consistent with the liver pathological symptoms of WHV/c-myc transgenic mice roughly divided in the period from 0 to 11 months of age. From the perspective of the three criteria required for the theoretical derivation of dynamic network biomarker identification (see Chen L et al., Scientific reports 2012), the 48 proteins that make up this group of lead molecules meet the increased inter-molecular correlation (using PCC _id ), molecules had decreased molecular relatedness to other non-lead molecular populations (measured by PCC _od ), and the largest fluctuations in molecular expression (measured by SD _d ) (see Figure 2).

At the same time, in order to more intuitively observe the dynamic changes in the network structure and expression fluctuations of the dynamic network biomarkers (DNB) at these five time points, the present invention calculates the corresponding Pearson correlation coefficient (PCC) based on the actually measured proteome data. ) to construct a protein co-expression network, rather than a network of known molecular interactions in mice based on data collection (see Figure 3). As shown in Figure 3, at the fifth month (i.e., the critical stage of the predicted malignant transformation of inflammatory carcinoma), compared with the reference mice, the proteins that make up the DNB (arranged on the inner circle represent the correlation between the dots) The correlation between the DNB protein in the inner ring and other co-expressed proteins in the outer ring was significantly reduced (the connection between the inner ring point and the outer ring point was blue), and at the same time The expression of DNB protein in the inner ring fluctuates violently (the dots in the inner ring are in red).

Thereafter, in order to judge the accuracy of the prediction of the key critical point of inflammatory cancer transformation from histopathological morphology, the present invention collects the liver tissues of WHV/c-myc transgenic mice and wt-C57BL/6 mice of the same age at each time point Samples, tissue sections were prepared and stained with hematoxylin and eosin (Hematoxylin and eosin, HE) (see Figure 4). As shown in Figure 4, the liver tissue morphology of the 2-month-old transgenic mice was similar to that of the reference normal mice, both showing the classic hepatic lobular structure (for example, hepatocytes arranged radially around the central vein in an orderly manner). liver cell plate formation); 3-5 month-old transgenic mice showed intermittent intervals of hepatic lobular boundary plates accompanied by local debris-like necrosis, which indicated that an inflammatory response had occurred; 7-month-old transgenic mice Bridge necrosis and large areas of necrosis accompanied by expansion of hepatic sinusoids appeared in the liver tissue of mice, which indicated the appearance of early liver cancer symptoms; when the transgenic mice were 11 months old, the structure of the hepatic lobular cell plate was destroyed, the central vein was narrowed, and the volume of hepatic cells increased. Large, enlarged nuclei and darkened staining, and vacuoles appear, these phenomena indicate that cancer has occurred in the liver tissue. These liver histopathological results indicated that the 5-month-old transgenic mice were in the critical critical period of inflammatory cancer transformation.

3. The flip phenomenon of dynamic network biomarker-associated network (DNB-associated network) in the process of inflammation and cancer transformation

Previous work of the present invention has shown that dynamic network biomarkers (DNB) serve as a group of lead molecules that have an early warning effect on state transitions, although they are different from traditional biomarkers (such as differentially expressed proteins DEPs or differentially co-expressed protein pairs) DCEs) are different, but they have a functional synergistic relationship with them—that is, DNBs are considered to be a group of molecules that play an inducing role (Inducer or regulator) in the period of state transition, while DEPs (or DCEs) are biological agents of state change. Function performer or embodiment.

In order to explore the synergistic relationship between DNB member proteins and DEPs and DCEs, according to the background network of known intermolecular interactions in mice (data collected in KEGG, BioGRID, TRED database), the present invention integrates DNB member proteins and Its first-order neighbor proteins (or functional linker proteins) construct DNB-associated network (DNB-associated network, which involves a total of 164 proteins and 183 edges). Then count the points or edges that coincide with differentially expressed proteins or differentially coexpressed protein pairs in the network, respectively, and use the hypergeometric test to evaluate the significance of the coincidence.

The statistical results show that there is a significant regulatory relationship between DNB and differentially expressed proteins (see Figure 5, A) and differentially co-expressed protein pairs (see Figure 5, B) during the transition from inflammation to cancer; at the same time, it also indirectly shows that This DNB-associated network plays an important role in the transition from inflammation to cancer.

Then, the present invention draws a corresponding network diagram to visually observe, for transgenic mice, in the process of malignant transformation of inflammation and cancer, the protein expression in the DNB-associated network (see Figure 5, C) and the dynamic changes of the regulatory (or co-expression) relationship between DNB and its first-order neighbor proteins (see Figure 5, D). It can be observed from Figure 5(C) that during the malignant transformation of inflammatory carcinoma, the expression level of DEPs functionally linked to DNB was significantly reversed—that is, for transgenic mice, from Between 3 (or 5) months and 5 (or 7) months, the expression level of a certain DEP changed significantly from low (high) to high (low). As for the differential co-expression relationship involved in DNB protein (that is, DNB protein-related DCEs), it can be seen from Figure 5(D) that during the malignant transformation of inflammatory cancer, its regulatory (or co-expression) relationship also occurs. There was a significant reversal—that is, for transgenic mice, from 3 (or 5) months of age to 5 (or 7) months of age, the correlation between DNB protein and a certain protein changed from positive (negative) Significant change to negative (positive). To sum up, for transgenic mice, during the malignant transformation of inflammation and cancer, the expression levels of 75 of the 164 first-order neighbor proteins of DNBs changed significantly and 183 of the functional connection edges of DNBs had significant changes. Eighty-six items showed the flipping phenomenon (Fig. 5, C and D), which not only strongly suggested that DNB member proteins (especially, PLA2G6, LTA4H and/or EPS8L2) might regulate or affect functionally related proteins to mediate the inflammatory tendency. Cancer malignant transformation, and may also provide accurate candidate molecular markers for the early diagnosis of inflammatory cancer malignant transformation in terms of clinical application.

4. Validation of the reliability of TMT-labeled quantitative protein data against non-labeled quantitative protein data

In order to verify the reliability of the analyzed data and its results, the present invention adopts a technical repetition method to batch test the real situation of the proteome change trend in the process of malignant transformation of inflammatory cancer, that is, the present invention adopts a more accurate TMT marker quantitative method Requantitative analysis was performed on the proteome of the same batch of mouse samples. Different from the non-labeled quantitative sample detection strategy used in previous calculations, this verification is no longer a quantitative analysis of the liver tissue samples of each mouse, but to eliminate the impact of individual differences in mice, each time After the samples of the same kind of mice were mixed in equal amounts to form a sample, the TMT marker quantitative technology was used for analysis. As shown in Figure 6, the proteins PLA2G6, LTA4H and EPS8L2 involved in the DNB-associated network, which may have played an important role in the process of inflammation and cancer transformation, have the same expression level change trend in the two sets of data Very high, with high reliability.

5. PLA2G6, LTA4H and EPS8L2 play an important biological function in the process of inflammation and cancer transformation

Although many hypotheses about the mechanism of inflammation-promoting cancer have been put forward, the mechanism of malignant transformation from chronic hepatitis to liver cancer is still not completely clear. In order to further reveal the potential mechanism of malignant transformation of inflammation and cancer, on the one hand, the present invention focuses on analyzing the abnormal dynamic changes of immune-related functions enriched in DNB-related networks. The Fc gamma R-mediated phagocytosis that DNB protein PLA2G6 participates in is abnormal in the early stage of malignant transformation of inflammatory cancer (that is, between 3 and 5 months old); moreover, the present invention finds that DNB-related functional networks (especially, PLA2G6 and Its related differentially expressed proteins DEPs and differentially co-expressed proteins have an interactive effect on the regulation of lipid metabolism and TRP channels involved in DCEs) on inflammatory mediators, which may lead to subsequent abnormal inflammatory responses and liver dysfunction that eventually promote inflammation to tumor progression. Simultaneously, PLA2G6, a member of the DNB protein identified by the present invention that has an early warning effect on state changes, is just regulated by the oncogenic driver of the transgenic mouse model - c-Myc transcriptional regulation (Figure 6), which shows that the development of The DNB found by the mathematical model is so close to the carcinogenic driving source that it can more timely and effectively warn the state of the imminent transition of inflammation to cancer. In addition, because the present invention considers DNB protein as a leading molecule to respond preferentially to the imminent change of the system state, therefore, in the biological pathway involved, starting from the biological metabolism affected by PLA2G6, through the level of important lipid metabolism molecules downstream of it, Linked signal transmission and then lead to orderly abnormalities of inflammatory response and liver detoxification function.

In the LOX pathway, the present invention also found that another DNB member LTA4H (leukotriene A4 hydrolase) is involved in arachidonic acid metabolism to synthesize leukotrienes (LT). LTA4H is a pro-inflammatory enzyme that generates leukotriene B4 (LTB4) (Snelgrove et al., 2010), which is an important inflammatory mediator that can stimulate the growth of human pancreatic cancer cells through the MAPK and PI-3 kinase pathways (Tong et al., 2005). Interestingly, in the transgenic mice, LTA4H had a completely opposite expression pattern and direct regulation pattern during the critical period from March to July, but its expression was downregulated by November, so the role of LTA4H was still unknown. Not sure. Recent studies have elucidated the dual role of LTA4H, which is to resist pro-inflammatory effects through production of LTB4 and anti-inflammatory effects through Pro-Gly-Pro (PGP) degradation (Snelgrove et al., 2010). The results here suggest an anti-inflammatory role for LTA4H, as prolyl endopeptidase (PREP), which specifically cleaves collagen in the lung to produce the neutrophil chemoattractant peptide PGP, in 11-month-old transgenic mice (Snelgrove et al., 2010) was upregulated. All these results suggest the importance of discovering the dual function of LTLA4H in preventing inflammatory transformation and implicating new therapeutic strategies.

Also of interest is the present discovery that another DNB member, EPS8L2, is also involved in the regulation of TF effects of both c-Myc and PLA2G6 starting from the growth factor-stimulated MAPK pathway, which is predominantly found in actin cells Plays an important role in membrane fluctuations and remodeling of the cytoskeleton. Some c-Myc targets such as DKC1, MAPK3 are also involved.

6. Histochemical experiment results

From Figure 5, it can be observed that the DNB-related network is significantly reversed before and after the critical stage of inflammatory cancer transformation (WHV/c-myc mice at 5 months old), which suggests that PLA2G6, LTA4H and EPS8L2 are early warning of malignant transformation of inflammatory cancer The role may hold great promise in clinical application.

High-grade dysplastic nodules (HGDN) are currently recognized as precancerous lesions of liver cancer. Typical hyperplastic nodules".

Antibodies were purchased and the histochemical experiments described herein were done in human HGDN tissue. The results are shown in Figure 7-9 and Table 1-2 below. Among them, Table 1 shows the age, gender and health status of some patients, Figures 7 and 8 show the antibody histochemical staining effect of patient number S2-136902, and Figure 9 shows the antibody histochemical staining effect of patient number S10-120093 , Table 2 summarizes the immunohistochemical data of 11 patients.

These results showed that there were obvious expression differences in normal liver tissue, HGDN and cancer tissue. Among them, the expressions of LTA4H, PLA2G2 and EPS8L2 were up-regulated in HGDN tissues, which were higher than those in corresponding cancer (HCC) tissues and normal liver tissues. Among them, it is worth noting that although the expression of PLA2G6 appears to be locally elevated in the nodules shown in tissue sections, in the high-throughput data obtained from the global liver tissue (that is, non-specific nodular areas), the expression of PLA2G6 The amount decreased significantly in the critical stage or nodular stage (Fig. 6), which may be caused by the complex regulatory mechanism of the protein. Both LTA4H and EPS8L2 were consistently detected in global liver tissue (Fig. 6) and in specific nodular regions.

Table 1

Table 2

Note: "Normal" means the patient's normal liver tissue; "HGDN" means the liver tissue from the patient's HGDN; "tumor" means the tissue from the patient's liver tumor; "/" means there is no HCC on the sample block organize. "-" means negative. "+" means positive, and each additional "+" means that the expression level increases by at least 1 times.

Claims (10)

1. The application of the following reagents in the preparation of a kit for diagnosing intrahepatic nodules of a subject or diagnosing whether a subject is in the critical stage of malignant transformation from hepatitis to liver cancer: (1) A reagent that specifically binds to PLA2G6; (2) an optional reagent that specifically binds to LTA4H; and (3) An optional reagent that specifically binds to EPS8L2. 2. application as claimed in claim 1, is characterized in that, described application is the reagent that specifically binds with PLA2G6 and the reagent that specifically binds with LTA4H is used for preparing the intrahepatic nodule of diagnostic object or whether diagnostic object is in Application in the kit for the critical stage of malignant transformation of hepatitis to liver cancer; or The application is the application of a reagent specifically binding to PLA2G6 and a reagent specifically binding to EPS8L2 in preparing a kit for diagnosing intrahepatic nodules of a subject or diagnosing whether a subject is at a critical stage of malignant transformation from hepatitis to liver cancer; or The application is that the reagents that specifically bind to PLA2G6, the reagents that specifically bind to LTA4H, and the reagents that specifically bind to EPS8L2 are used in the preparation of intrahepatic nodules for diagnosing objects or whether the object is in the critical stage of malignant transformation from hepatitis to liver cancer application in the kit. 3. The application according to any one of claims 1-2, wherein the reagent is an antibody, including monoclonal antibody and polyclonal antibody, or an antigen-binding fragment of an antibody. 4. A detection kit, characterized in that, the detection kit contains: (1) Reagents using PLA2G6 as the detection object; (2) an optional reagent that uses LTA4H as the detection object; and (3) An optional reagent for detecting EPS8L2. 5. detection kit as claimed in claim 4, is characterized in that, described detection kit contains: (a) a reagent with PLA2G6 as the detection object and a reagent with LTA4H as the detection object; (b) a reagent with PLA2G6 as the detection object and a reagent with EPS8L2 as the detection object; or (c) Reagents for detection of PLA2G6, reagents for detection of LTA4H, and reagents for detection of EPS8L2. 6. The detection kit according to claim 4 or 5, wherein the reagent is an antibody, including monoclonal antibody and polyclonal antibody, or an antigen-binding fragment of an antibody. 7. The detection kit according to any one of claims 4-6, characterized in that, the kit also includes reagents required for implementing the immunohistochemical method. 8. A method for diagnosing whether an object has intrahepatic nodules or whether it is at a critical stage of malignant transformation from hepatitis to liver cancer, characterized in that the method includes the step of detecting the expression of PLA2G6 in the liver tissue of the object; wherein, The expression level is lower than the PLA2G6 expression level in the liver tissue of normal people, indicating that the patient has intrahepatic nodules or is in the critical stage of malignant transformation from hepatitis to liver cancer; Optionally, the method further includes the step of detecting the expression level of LTA4H and/or EPS8L2 in the liver tissue of the subject; Preferably, the intrahepatic nodules are high-grade dysplastic nodules; Preferably, the subject is a hepatitis patient. 9. The application of molecular markers selected from the following in the preparation of reagents and/or kits for the diagnosis of intrahepatic nodules of the subject or whether the subject is in the critical stage of malignant transformation from hepatitis to liver cancer: (1) PLA2G6; (2) optional LTA4H; and (3) Optional EPS8L2. 10. The application according to claim 9, wherein the application comprises: The application of PLA2G6 and LTA4H as molecular markers in the preparation of reagents and/or kits for intrahepatic nodules of diagnostic subjects or whether diagnostic subjects are in the critical stage of malignant transformation from hepatitis to liver cancer; or The application of PLA2G6 and EPS8L2 as molecular markers in the preparation of reagents and/or kits for intrahepatic nodules of diagnostic subjects or whether diagnostic subjects are in the critical stage of malignant transformation from hepatitis to liver cancer; or Application of PLA2G6, LTA4H and EPS8L2 as molecular markers in the preparation of reagents and/or kits for diagnosing intrahepatic nodules of objects or whether the objects are in the critical stage of malignant transformation from hepatitis to liver cancer.