CN108732351A - EPS8L2 is as tubercle and the biomarker of early warning liver cancer in instruction liver - Google Patents

Abstract

The present invention relates to EPS8L2 as a biomarker for indicating intrahepatic nodules and early warning of liver cancer. Specifically, the present invention provides the application of the following reagents in the preparation of kits for diagnosing intrahepatic nodules of subjects or whether a subject is in the critical stage of malignant transformation from hepatitis to liver cancer: (1) Reagents that specifically bind to EPS8L2 (2) an optional reagent that specifically binds to LTA4H; and (3) an optional reagent that specifically binds to PLA2G6. Based on the time series dynamics and network correlation analysis, this paper finds the critical critical stage of the transformation of inflammation and cancer, which provides a new method for clinical application in the diagnosis of early liver cancer in patients with hepatitis, whether it is in the critical stage of malignant transformation from hepatitis to liver cancer, and the prevention of the transformation of inflammation and cancer. direction.

Description

EPS8L2 is as tubercle and the biomarker of early warning liver cancer in instruction liver

Technical field

The present invention relates to EPS8L2 as tubercle and the biomarker of early warning liver cancer in instruction liver.

Background technology

Liver cancer threatens one of maximum malignant tumour as human life, receives global concern.It is counted according to global cancer Report, liver cancer has become the highest cancer of Chinese incidence, and wreaking havoc for hepatitis virus makes China become hepatitis and onset of liver cancer Severely afflicated area.Therefore, how effectively to prevent hepatitis and deteriorate one of the important issue for becoming studies in China person to liver cancer.

Recently, have studies have shown that chronic inflammation can induce the generation of the kinds cancer including liver cancer really.Although Researchers have been presented for much about inflammation promote cancer mechanism hypothesis, but mechanism from chronic hepatitis to liver cancer vicious transformation still It is completely not clear.Therefore, temporarily there are no more accurate and reliable biomarkers to realize hepatocarcinoma early diagnosis.Currently, main There are two types of the diagnostic modes wanted, and one is carry out clinical diagnosis by stages to hepatopathy based on medical image detection and doctors experience；Separately One is based on tissue biopsy, pathological grading is carried out to liver neoplasm with a small number of more accurate molecular markers.However, these are diagnosed Method is all defective, is on the one hand that the state of an illness is extremely serious, and tumour has grown sufficiently large, can just be differentiated by medical image instrument； On the other hand, tissue biopsy causes wound to patient, is not easy to extensive use.These diagnosis defects have seriously affected state of an illness understanding And then influence therapeutic quality and effect.

Therefore, this field still need in a hurry can to chronic inflammation to liver cancer vicious transformation (endogenous (and proto-oncogene swash It is living) and the chronic inflammation that causes of exogenous (virus infection) joint effect to liver cancer vicious transformation) molecule of progress early warning Marker and method.

Invention content

First aspect present invention provides EPS8L2 and optional LTA4H and optional PLA2G6 and exists as molecular marker Diagnose whether tubercle or diagnosis object in the liver of object are in application of the hepatitis into liver cancer vicious transformation critical stage.

In one or more embodiments, the present invention provides EPS8L2 and LTA4H as molecular marker in diagnosis pair Whether tubercle or diagnosis object are in application of the hepatitis into liver cancer vicious transformation critical stage in the liver of elephant.

In one or more embodiments, the present invention provides EPS8L2 and PLA2G6 as molecular marker in diagnosis pair Whether tubercle or diagnosis object are in application of the hepatitis into liver cancer vicious transformation critical stage in the liver of elephant.

In one or more embodiments, the present invention provides PLA2G6, LTA4H and EPS8L2 three as molecular marker Whether object tubercle or diagnosis object in the liver of diagnosis object are in application of the hepatitis into liver cancer vicious transformation critical stage.

Second aspect of the present invention provides and the reagent of EPS8L2 specific bindings, optional with LTA4H specific bindings Reagent and the optional reagent specifically bound with PLA2G6 tubercle or diagnosis object in the liver prepared for diagnosing object Whether application of the hepatitis into the kit of liver cancer vicious transformation critical stage is in.

In one or more embodiments, present invention offer is special with the reagent of EPS8L2 specific bindings and with LTA4H The reagent that the opposite sex combines prepare in liver for diagnosing object tubercle or diagnosis object whether in hepatitis to pernicious turn of liver cancer Change the application in the kit of critical stage.

In one or more embodiments, present invention offer is special with the reagent of EPS8L2 specific bindings and with PLA2G6 The reagent that the opposite sex combines prepare in liver for diagnosing object tubercle or diagnosis object whether in hepatitis to pernicious turn of liver cancer Change the application in the kit of critical stage.

In one or more embodiments, the present invention provides the reagent specifically bound with EPS8L2, special with LTA4H Property the reagent that combines and preparing tubercle or diagnosis pair in liver for diagnosing object with the reagent of PLA2G6 specific bindings As if it is no in application of the hepatitis into the kit of liver cancer vicious transformation critical stage.

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

Third aspect present invention provides a kind of detection kit, contains the reagent using EPS8L2 as detection object.

In one or more embodiments, the reagent using EPS8L2 as detection object includes the special of EPS8L2 Property antibody.

Also contain in one or more embodiments, in the detection kit using LTA4H and/or PLA2G6 as inspection Survey the reagent of object.

In one or more embodiments, the reagent using LTA4H and/or PLA2G6 as detection object includes The specific antibody of LTA4H and/or PLA2G6.

In one or more embodiments, the detection kit also contains detects EPS8L2 using ImmunohistochemistryMethods Methods Required reagent.

In one or more embodiments, the detection kit also contains detects LTA4H using ImmunohistochemistryMethods Methods And/or the reagent needed for PLA2G6.

Whether a kind of diagnosis object of fourth aspect present invention offer is with the presence or absence of tubercle in liver, or disliked to liver cancer in hepatitis Property conversion critical stage method, the method includes detecting the step of the expression quantity of the EPS8L2 in the object liver organization Suddenly；Wherein, the expression quantity prompt the patient there are tubercle in liver higher than the EPS8L2 expression quantity in normal population liver organization or It is in critical stage of the hepatitis to liver cancer vicious transformation.

In one or more embodiments, the method further include detect the object liver organization in LTA4H and/or The step of expression quantity of PLA2G6.

In one or more embodiments, tubercle is high-level atypical hyperplasia tubercle in the liver.

In one or more embodiments, the object is hepatitis.

Fifth aspect present invention provides EPS8L2 and optional LTA4H and optional PLA2G6 and exists as molecular marker Prepare diagnosis object liver in tubercle or diagnosis object whether in reagent from hepatitis to liver cancer vicious transformation critical stage and/ Or the application in kit.

In one or more embodiments, the present invention provides EPS8L2 and LTA4H and is examined in preparation as molecular marker Whether tubercle or diagnosis object are in reagent and/or reagent of the hepatitis to liver cancer vicious transformation critical stage in the liver of disconnected object Application in box.

In one or more embodiments, the present invention provides EPS8L2 and PLA2G6 and is examined in preparation as molecular marker Whether tubercle or diagnosis object are in reagent and/or reagent of the hepatitis to liver cancer vicious transformation critical stage in the liver of disconnected object Application in box.

In one or more embodiments, the present invention provides PLA2G6, LTA4H and EPS8L2 three as molecular marker Whether object tubercle or diagnosis object in the liver for preparing diagnosis object are in examination of the hepatitis to liver cancer vicious transformation critical stage Application in agent and/or kit.

Description of the drawings

Fig. 1：The detection of the hepatic protein group of WHV/c-myc transgenic mices and wt-C57BL/6 mouse as reference and Quantitative flow chart.

Fig. 2：Based on dynamic network biological marker analyte detection hepatitis to the early warning signal of liver cancer vicious transformation.

Fig. 3：Dynamic network biomarker and its relevant coexpression molecule are in terms of network structure and expression variance Dynamic change.

Fig. 4：The hepatic tissue section image of HE dyeing.

Fig. 5：The relevant functional network of dynamic network biomarker (DNB-associated network) turns in scorching cancer Dynamic change during change.A and B：It has counted respectively in network and has co-expressed albumen pair with differentially expressed protein (DEP) or difference (DCE) point or side overlapped is counted, and examines its coincidence conspicuousness of assessment using hypergeometry.C and D：A series of network Figure respectively shows the DNB correlation functions network institute of the transgenic mice (i.e. between 3 to 7 monthly ages) during scorching cancer vicious transformation Dynamic change of differentially expressed protein (DEP) and difference the coexpression albumen being related to (DCE).

Fig. 6：The table of PLA2G6, LTA4H and EPS8L2 each time point (stage) during hepatitis is to liver cancer vicious transformation Up to amount situation of change.

Fig. 7：LTA4H is applied to the diagnosis situation of clinical patient.

Fig. 8：PLA2G6 is applied to the diagnosis situation of clinical patient.

Fig. 9：EPS8L2 is applied to the diagnosis situation of clinical patient.

Specific implementation mode

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, to constitute preferred technical solution.

In order to imitate the process for the scorching cancer vicious transformation that endogenous and exogenous joint effect cause as far as possible, use herein Lower generation liver is infected in groundhog hepatitis virus (Woodchuck hepatitis virus, WHV) to c-myc transgenic mices Animal model of the inflammation to liver cancer lesion.Previous research (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) table Bright, during liver cancer genesis and development, the livers of WHV/c-myc mouse successively experienced liver fibrosis, tubercle in liver, benign swollen For tumor to pathologic processes such as malignant tumours, the process of this sb.'s illness took a turn for the worse with hepatitis B patient liver cancer is very similar.Therefore, it is based on previous Research (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 has chosen five times in different pathologic stages Point (postnatal 2nd, 3,5,7,11 month of mouse) acquires WHV/c-myc mouse and the wt-C57BL/6 as reference respectively The liver samples of mouse utilize non-marked (Label-free) quantitative technique and series connection label (Tandem Mass Tag, TMT) Quantitative technique is analyzed by mass spectrometry protein group, obtains the high throughput protein group quantitative data of sequential.Meanwhile using searching Method (Chen L, Liu R, Liu ZP, Li M, the Aihara K, Detecting of dynamic network biomarker (DNB) early-warning signals for sudden deterioration of complex diseases by Dynamical network biomarkers, Scientific reports 2012,2:342) hepatitis, is identified to liver cancer The crucial critical stage of vicious transformation, and excavate pre-warning signal and its potential scorching cancer that dynamic network biomarker implies It shifts to new management mechanisms.

Specifically, herein by WHV/c-myc transgene mouse models during hepatitis is to liver cancer vicious transformation The sequential proteome data of different symptom is analyzed, it is found that the transgenic mice at five monthly ages is in the crucial critical of scorching cancer conversion In the stage, this is consistent with Pathomorphologic judgement result；And also obtain the dynamic network mark with the early warning critical stage Will object (DNB).In order to further disclose the effect and potential pathogenic mechanism that DNB is played in scorching cancer conversion process, this paper structures DNB network of relation (DNB-associated network) has been built, and its network structure change has been analyzed, has been found There is flop phenomenon in DNB network of relation, thus infers that PLA2G6, LTA4H and/or EPS8L2 as DNB member may induce Subsequent inflammation abnormal reaction and hepatic insufficiency are so that finally inspired inflammation to tumor progression.The groupization experiment knot of this paper Fruit further demonstrates above-mentioned deduction.

Meanwhile it having been reported that and showing that liver nodal stage is critical stage of people's hepatitis to liver cancer malignant transformation.Especially, high Rank atypical hyperplasia tubercle (HGDN) be at present recognized by liver cancer precancerous lesion, be defined as that " hepatic tissue has abnormal shape Hyperplasia but not up to malignant tumour standard, with high malignancy potential, the atypical hyperplasia tubercle of diameter 1mm or more ".Mesh Before, HGDN is mainly detected by histopathology.The main pathological characteristic of HGDN has：(1) cell density increases, and (2) liver plate thickens, (3) karyoplasmic ratio increases, and (4) pseudoglandular formed, nuclear atypia that (5) are slight, and the dyeing of (6) endochylema basophilla increases, (7) without interstitial and Portal vein infiltration etc..

The crucial critical stage for having found scorching cancer conversion based on time-series dynamics and network association analysis herein, for clinic Applied to hepatitis early liver cancer diagnosis (such as whether there are tubercle in liver, especially HGDN), hepatitis whether is in liver cancer The critical stage and the scorching cancer of prevention of vicious transformation (i.e. scorching cancer conversion) deteriorate transformation and provide new direction.

Therefore, herein in PLA2G6, LTA4H and EPS8L2 any one, any two (such as PLA2G6 with LTA4H, PLA2G6 and EPS8L2 or LTA4H and EPS8L2) or all three for detection object, by detecting it in object liver Expressing quantity in dirty is tied compared with the expressing quantity in normal population liver to judge that object whether there is in liver Section, especially HGDN and whether be in critical stage of the hepatitis to liver cancer vicious transformation.

Herein, PLA2G6 refers to phospholipase A2 (VI groups), it is that a kind of 85KD is located at the intracytoplasmic phosphorus for not depending on calcium Lipase.It should be understood that include the various PLA2G6 that phospholipase A2 (VI groups) is defined as by those skilled in the art herein, such as PLA2G6 from different plant species (such as people or mouse or other animals).For example, the encoding gene of mouse PLA2G6 is located at mouse No. 15 On chromosome, specific chromosome segment is 15E1, and the gene number on ENSEMBL is ENSMUSG00000042632, Gene number on ENTREZ is No. 53357.And the encoding gene of people PLA2G6 is located on No. 22 chromosome, it is specific to dye Body section is 22q13.1, and the gene number on ENSEMBL is ENSG00000184381, and the gene number on ENTREZ is 8398 Number.Present invention particularly includes the detections of people's PLA2G6 albumen.It should be understood that the PLA2G6 albumen of different people may have one The difference of a or several amino acid residues, but this species diversity does not interfere the PLA2G6 albumen as biomarker, for this Purposes described in text and side.In certain embodiments, PLA2G6 albumen as described herein refers to identical in same species gene group The PLA2G6 albumen of gene code on position.

Herein, LTA4H refers to leukotriene A 4 hydrolase.Herein leukotriene is defined as by those skilled in the art including various The LTA4H of A4 hydrolases, such as the LTA4H from different plant species (such as people or mouse or other animals).For example, the volume of mouse LTA4H Code gene is located on No. 10 chromosome of mouse, and specific chromosome segment is 10C3, and the gene number on ENSEMBL is Gene number on ENSMUSG00000015889, ENTREZ is No. 16993.And the encoding gene of people LTA4H is located at No. 12 On chromosome, specific chromosome segment is 12q22, and the gene number on ENSEMBL is ENSG00000111144, ENTREZ On gene number be No. 4048.Present invention particularly includes the detections of people's LTA4H albumen.It should be understood that the LTA4H of different people Albumen may have the difference of one or several amino acid residues, but this species diversity does not interfere the LTA4H albumen as life Object marker is used for purposes as described herein and side.In certain embodiments, LTA4H albumen as described herein refers to same object The LTA4H albumen of gene code in kind genome in same position.

Herein, EPS8L2 refers to 8 sample albumen 2 of epidermal growth factor receptor kinase substrate.Include various by this field herein Technical staff is defined as the EPS8L2 of 8 sample albumen 2 of epidermal growth factor receptor kinase substrate, such as from different plant species (such as people Or mouse or other animals) EPS8L2.For example, the encoding gene of mouse EPS8L2 is located on No. 7 chromosome of mouse, it is specific to dye Body section is 7F5, and the gene number on ENSEMBL is ENSMUSG00000025504, and the gene number on ENTREZ is 98845 Number.And the encoding gene of people EPS8L2 is located on o.11 chromosome, specific chromosome segment is 11q15.5, on ENSEMBL Gene number be ENSG00000177106, gene on ENTREZ number is No. 64787.Present invention particularly includes people The detection of EPS8L2 albumen.It should be understood that the EPS8L2 albumen of different people may have one or several amino acid residues Difference, but this species diversity does not interfere the EPS8L2 albumen as biomarker, is used for purposes as described herein and side.? In certain embodiments, EPS8L2 albumen as described herein refers to the gene code in same position in same species gene group EPS8L2 albumen.

Herein, " object " refer to it is various need to judge its whether occur in liver tubercle and/or whether in hepatitis to liver cancer The animal of the critical stage of vicious transformation, including but not limited to mammal and rodent, such as people and mouse.Especially, In certain embodiments, " object " as described herein refers to people, more preferably once infected the people of hepatitis virus, hepatitis or Doubtful hepatitis.

PLA2G6, LTA4H in method well known in the art (such as ImmunohistochemistryMethods Methods) detection object liver organization can be used With in EPS8L2 any one, any two or whole three expression quantity.It is any can detect PLA2G6, LTA4H or The reagent of the expression quantity of EPS8L2 is utilized in the purposes of the present invention, method or kit, including it is various can with PLA2G6, The reagent of LTA4H or EPS8L2 specific bindings, such as antibody or its antigen-binding fragment.

ImmunohistochemistryMethods Methods are the methods of this field routine, usually utilize the specific binding of antigen and antibody, passing through Learning reaction makes the chromogenic reagent of labelled antibody, so that it is determined that the method for the presence and/or content of histocyte endoantigen.It can profit Implement this method or purposes with the respective specific antibody of PLA2G6, LTA4H and/or EPS8L2.This kind of specific antibody can To be known commercial antibody, for example, the antibody from Abcam.Alternatively, also can be according to known technology (such as hybridoma technology) Voluntarily prepare their own specific antibody.Antibody can be monoclonal antibody, can also be polyclonal antibody；It is preferred that Dan Ke Grand antibody.Illustrative ImmunohistochemistryMethods Methods are not limited to the side described in the part as described in " material and method " part herein Method.

Therefore, provided herein is a kind of detection kit, containing with PLA2G6, LTA4H and EPS8L2 in the detection kit In any one, any two or all three be detection object reagent.For example, the detection kit can contain with PLA2G6 be detect object reagent, or containing with LTA4H be detect object reagent, or containing with EPS8L2 be detect object Reagent, or contain arbitrary two classes reagent or whole three classes reagent.In certain embodiments, the detection kit of this paper contains With PLA2G6 be detect object reagent and with LTA4H be detect object reagent or PLA2G6 be detect object reagent with With EPS8L2 be detect object reagent, or with LTA4H be detect object reagent and with EPS8L2 be detect object reagent. As it was noted above, this kind of reagent can be the reagent of any expression quantity that can detect PLA2G6, LTA4H and EPS8L2, preferably Their own specific antibody.Further include antibody remain and the active antigen of the combination of PLA2G6, LTA4H or EPS8L2 Binding fragment.

Also contain the other reagents implemented needed for detection of the present invention in the detection kit of this paper, such as implements to exempt from Other reagents needed for epidemic disease group method.For example, when the object of processing is the histotomy of paraffin embedding, the detection of this paper tries Can also it contain in agent box：Reagent needed for slice dewaxing and hydration, such as dimethylbenzene, absolute ethyl alcohol, hydrogen peroxide methanol solution； Reagent needed for antigen retrieval, such as acid reparation liquid；BSA；Buffer solution, such as PBS buffer solution；Secondary antibody；It develops the color when using DAB When, DAB developing solutions；Haematoxylin；Ethanol solution hydrochloride；Carbolic acid；Resinene etc..The content of various reagents and dense in kit Degree can be determined conventionally.

The detection kit of this paper can be used for detecting the expression quantity of PLA2G6, LTA4H and/or EPS8L2 in sample, via The expression quantity, those skilled in the art can determine whether the sample to for object with the presence or absence of tubercle in liver, and/or whether be in Critical stage of the hepatitis to liver cancer vicious transformation.In general, can by testing result and normal population liver organization PLA2G6, The expression quantity of LTA4H and/or EPS8L2 is compared one by one.If it was found that in sample the expression quantity of PLA2G6 be less than, and/or Expression of the expression quantity of LTA4H and/or EPS8L2 higher than PLA2G6, LTA4H and/or EPS8L2 in normal population liver organization Amount, then can making the object, there are tubercles in liver, and/or the judgement in hepatitis to the critical stage of liver cancer vicious transformation.

The expression quantity of PLA2G6, LTA4H and EPS8L2 in normal population liver organization can be used well known in the art Method, including Immune transform method as described herein carry out investigation statistics.

For example, in general, the expression quantity of PLA2G6 is the PLA2G6 expression quantity in normal population liver organization in sample 90% or less, 80% or less, 70% or less, 60% or less, 50% or less or 40% hereinafter, then it is believed that the object there are livers Interior tubercle (especially high-level atypical hyperplasia tubercle), and/or in hepatitis to the critical stage of liver cancer vicious transformation；With/ Or

In general, the expression quantity of LTA4H is at least the 1.3 of the expression quantity of the LTA4H in normal population liver organization in sample Again, at least 1.5 times, at least 1.7 times, at least 2 times, at least 2.5 times or at least 3 times, then it is assumed that there are tubercles in liver for the object (especially It is high-level atypical hyperplasia tubercle), and/or in hepatitis to the critical stage of liver cancer vicious transformation；And/or

In general, in sample EPS8L2 expression quantity be normal population liver organization in EPS8L2 expression quantity 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, then it is assumed that there are tubercles in liver for the object (especially high-level atypical hyperplasia tubercle), and/or in hepatitis to the critical stage of liver cancer vicious transformation.

In certain embodiments in addition PLA2G6, LTA4H in the liver organization of the doubtful illness of detectable object and/ Or the expression quantity of PLA2G6, LTA4H and/or EPS8L2 in the expression quantity of EPS8L2, with the object normal liver tissue carry out It compares one by one, if the expression quantity of PLA2G6 is less than, and/or the expression quantity of LTA4H and/or EPS8L2 in doubtful diseased liver tissue Higher than the expression quantity in its normal liver tissue, if for example, the expression quantity of PLA2G6 is that it is normal in doubtful diseased liver tissue 90% or less, 80% or less, 70% or less, 60% or less, 50% or less or 40% of PLA2G6 expression quantity in liver organization Below, and/or the expression quantity of LTA4H and/or EPS8L2 is at least 1.3 times of expression quantity in its normal liver tissue, at least 1.5 times, at least 1.7 times, at least 2 times, at least 2.5 times or at least 3 times, it is also contemplated that the object there are tubercles in liver (especially High-level atypical hyperplasia tubercle), and/or in hepatitis to the critical stage of liver cancer vicious transformation.

It should be understood that only needing a kind of expression quantity in detection PLA2G6, LTA4H and EPS8L2, you can obtain diagnosis Conclusion；But in some embodiments, it is preferred that detecting arbitrary two kinds in PLA2G6, LTA4H and EPS8L2, even all three Expression quantity.

The present invention will be hereafter illustrated in a manner of specific embodiment.It should be understood that these embodiments are only illustrative, and It is not intended to limit the invention.The various methods and material being previously mentioned in embodiment, unless otherwise stated, conventional for this field Method and material.

One, material and method

1, experimental animal and reagent

WHV/c-myc transgene mouse models and the raising of normal control C57BL-6 Strains of Mouse are in Chinese Academy of Sciences Shanghai Life science institute biochemistry and Institute of Cell Biology Experimental Animal Center, all experimental implementations follow strictly animal Ethics standard.It takes altogether after 5 different time points, including birth 2 months, 3 months, 5 months, 7 months, 11 months, each time It puts and respectively takes 5 WHV/c-myc transgenic mices and 5 normal C57 mouse, after sacrificed by decapitation, isolate liver organization.Use scissors Liver organization is shredded, first tissue fritter is carefully washed with the RPMI1640 buffer solutions of precooling, appropriate lysis buffer is added and splits Cell is solved, then carrying out mechanicalness to tissue fritter with homogenizer is crushed, further appropriate sonicated cells and soluble protein Matter, last 4 DEG C, 15000g centrifuges 60 minutes removal impurity such as DNA, RNA and insoluble cell fragment, and gained supernatant protein matter is molten Liquid is carefully transferred in new Eppendorf pipes, using Bradford methods (Bio-Rad) quantifying protein concentration, according to required After applied sample amount is dispensed, -80 DEG C save backup.

All buffer solutions use Milli-Q water (Millipore, Bedford, MA, USA) to prepare.Urea (Urea), bromine phenol Blue (Bromophenole blue), acrylamide (Acrylamide), methene double acrylamide (Bis), 3- [(3- courages amides third Base)-diethyl ammonium]-propane sulfonic acid (CHAPS), three (methylol) aminoethanes (Tris), dodecyl sodium sulfate (SDS), over cure The reagents such as sour ammonium (AP), tetramethylethylenediamine (TEMED) and Bradford standard measure liquid purchased from Bio-Rad companies (Hercules, CA,USA)；EDTA Dipotassium salt (EDTA), phenylmethylsulfonyl fluoride (PMSF) purchased from Amresco companies (Solon, OH, USA)；Protease inhibitors (Complete protease inhibitor cocktail tablets) is purchased from Roche companies (Indianapolis,IN,USA).Formic acid (Formic acid, FA) is purchased from Sigma companies (St.Louis, MO, USA)；HPLC Grade acetonitrile (ACN) and methanol (Methanol) are purchased from Fisher companies (Fair Lawn, NJ, USA)；Acetone (Acetone) and second Alcohol (Ethanol) is purchased from Shanghai Chemicals companies (Shanghai, China)；Sequencing grade trypsase (Trypsin, Sequencing grade) it is purchased from Promega companies (Madison, WI, USA).

2, the label-free of mouse liver proteins group (LFQ is quantitative)

In label-free (LFQ is quantitative) experiment, enzymolysis in solution is carried out from the protein example of every mouse.It is first First, protein example is added into lysis buffer to 100 μ l of total volume, the 1M DTT of 4 μ l is added, react 2.5 for 37 DEG C after mixing Hour, the 1M IAA of 10 μ l are then added, room temperature is protected from light 45 minutes after mixing.Then with -20 DEG C of precoolings of 5 times of volumes Protein precipitation liquid is precipitated, and -20 DEG C stand overnight (16 hours).Then sample 14000g is centrifuged 40 minutes and removes supernatant, Precipitation first is cleaned with -20 DEG C of pre- cold acetones, 14000g, which is centrifuged, removes supernatant for 40 minutes, then 70% ethyl alcohol being pre-chilled with -20 DEG C cleans Precipitation, 14000g centrifugations go supernatant, vacuum freeze-drying machine that sample is lyophilized after forty minutes.The ammonium hydrogen carbonate of the 100mM of 200 μ l is added, Fully albumen is redissolved in oscillation, adds 12 μ g trypsase, and 37 DEG C of enzyme digestion reactions add trypsase 12 μ g again after 4 hours, and 37 DEG C baking oven oscillation enzymolysis is overnight.Super filter tube (Millipore) ultrafiltration that 10KD pore sizes are used after the completion of enzymolysis, collects filtration Liquid, -80 DEG C of preservations after freeze-drying.It is reversed using pH continuous gradients eluent system (pCOG) joint strong cation exchange chromatography- Chromatography detaches peptide fragment mixture online, and LFQ mass spectrographs (Thermo Finnigan) is used in combination to acquire original number using positive ion mode According to.Peptide fragment eluate is directly entered linear ion hydrazine Mass Spectrometer Method through ESI esi ion sources, and source parameters is set as：EFI Mist voltage, 3.0kV；Capillary temperature, 170 DEG C.LTQ mass spectrographs controlled with AGC under data-dependent patterns into line number According to acquisition, full scan mass range is m/z 400~1800, takes 10 highest peaks of intensity to carry out series connection matter after each full scan Spectrum scanning, normalization collision energy are set as 35.0%, and dynamic excludes to be set as number of repetition to be 2, and it is 30 to repeat patient time Second, the dynamic exclusion time is 90 seconds.

The collected initial data of mass spectrum extracts dta information first with BioWorks softwares (Thermo-Fisher), so Afterwards database search, the positive and negative libraries mouse IPI that database is generated using laboratory internal software are carried out using SEQUEST softwares (v3.82), it is as follows to search library parameter setting：Within parent ion mass deviation 3Da, within product ion mass deviation 1Da, protease is set It is set to trypsase, maximum allowable missing restriction enzyme site is 1, and fixed modification is set as the carboxy methylation of cysteine, can turn revisionism Decorations are set as the oxidation of methionine.Sequest outputs result further uses the BuildSummary of laboratory independent development Software architecture carries out Data Integration and false positive control.

3, DNB algorithms

Three items that dynamic network marker forecasting system state based on the requirement of mathematical model theory deduction will change Part, the present invention devise corresponding realization algorithm, so as to applied to the time series high throughput data for specific problem in science Research in.In order to which the strong signal that can consider three conditions by one detects crucial critical point (Critical Point), invention defines carry out algorithm for design with next integral index (A composite index, CI)：

CI=SD_d×PCC_id/PCC_od

Wherein, SD_dRepresent the institute of guide's molecular group (Dominant group) or dynamic network biomarker (DNB) Have molecule time point concentration instantly change the mean value of standard deviation with to should refer to (e.g., the sample of original time point or same The negative control sample at time point) concentration variation standard deviation the ratio between mean value.PCC_idIt is two intermolecular phases inside molecular group Closing property (PCC) is respectively at time point instantly and from the ratio between the mean value to should refer to；And PCC_odIt is each molecule inside molecular group Correlation (PCC) with its relevant molecule outside molecular group is respectively at time point instantly and from the ratio between the mean value to should refer to. Although it is worth noting that, changing crucial critical point as determining dynamic network marker and detecting state used here as CI values Effective evaluation index, but other evaluation indexes that can integrate the three big conditions that meet can also be built.

The specific algorithm flow of dynamic network biomarker is determined based on integral index CI：

(1) molecule fluctuating change screening (Deviation test)：At time point t, from several samples at same time point In data, picks out observation (gene expression product concentration, protein content etc.) variation fluctuation and compare the observation that should refer to One group of Molecule Set GtD of variation fluctuation big (SD values are high)；

(2) intermolecular relativity evaluation (Intra-correlation test)：At time point t, two points are calculated in GtD Correlation (PCC) between son, and the subset GtI between time point interior molecules instantly with high correlation is divided in this；

(3) relativity evaluation (Inter-correlation test) of molecule and other relevant molecules：In time point t Under, the correlation (PCC) of each molecule and other relevant molecules outside the molecular group in Molecule Set GtI is calculated, and sieve with this Choosing has the outer portion subset GtC of low correlation with Molecule Set GtI；

(4) determination (DNB test) of dynamic network biomarker：It is screened at every point of time based on three step of front Outer portion subset GtC corresponding candidate molecules collection GtI, calculates corresponding CI values according to formula (3) respectively, using notable Property analysis choose wherein part most high cl values corresponding to Molecule Set GtI be that guide's molecular group (Dominant group) Or dynamic network marker (Dynamical network biomarker, DNB), and corresponding time point is state The crucial critical stage (Critical point or Pre-transition state) that will change.It is worth noting that, working as When the time point of observation is more, it is understood that there may be multiple state transition processes, at this point, multiple and different marks may be obtained Object and crucial critical stage.

With reference to the mathematical model theory deduction as a result, when the crucial critical stage of system proximity state transformation, SD_dWith PCC_id/PCC_odMaximum value should all be reached.It should be noted, however, that in most practical cases, we generally can not The data for accurately obtaining very much the crucial critical stage of state transformation, only it is observed that the sample number of Near The Critical Point period According to, thus SD_dAnd PCC_id/PCC_odGlobal maximum cannot be reached simultaneously.

4, immunohistochemical experiment

Immunohistochemical experiment uses DAB development processes in the application.Main operational steps are as follows：

The paraffin embedded tissues of paraffin embedding are cut into 4 μm of slice；

Slice is placed in 60 DEG C of ovens 1 hour；

Slice dewaxing and hydration (one 10 minutes → dimethylbenzene of dimethylbenzene, 2 10 minutes → dimethylbenzene 3 10 minutes → anhydrous second 5 minutes → 95% ethyl alcohol of alcohol, 5 minutes → 85% ethyl alcohol, 5 minutes → 75% 5 minutes → deionized water of ethyl alcohol 5 minutes)；

Slice is placed in a concentration of 3% hydrogen peroxide methanol solution, is stored at room temperature 20 minutes, removes peroxidase；

Deionized water washing 5 minutes, in triplicate；

Slice (is positioned in acid reparation liquid, big fire is boiled to boiling, and small fire is adjusted to, and continues to boil 2 points by antigen retrieval Clock, truce wait for its natural cooling)；

Deionized water washing 5 minutes, in triplicate；

A concentration of 1% BSA is added dropwise on tangential section, is placed in 37 DEG C of incubators and closes 30 minutes；

Confining liquid is cleaned, corresponding primary antibody is directly added dropwise, is placed in wet box, 4 DEG C of refrigerator overnights；

Rewarming 15 minutes under room temperature environment；

It is washed 5 minutes, is repeated four times using PBS buffer solution；

It is added dropwise on corresponding secondary antibody (Anti-Rabbit-HRP, Antibody Dignostica Inc.) to slice, is just placed in In wet box, 37 DEG C of incubators are incubated 45-60 minutes；

It is washed 5 minutes, is repeated four times with PBS buffer solution；

It develops the color 3-10 minutes with DAB developing solutions, under the microscope, after developing the color successfully, deionized water color development stopping；

Deionized water elutes twice, 5 minutes every time；

Haematoxylin is redyed 10 minutes, and HE dyeing is led in subsequent operation；

It takes out slice and rinses one in tap water twice, put it into ethanol solution hydrochloride rinse 2-3 times later；

It is rinsed 20-30 minutes in the tap water of flowing, takes out slice, room temperature drains；

Deionized water is washed 5 minutes；It is respectively washed in 95% ethyl alcohol, 100% ethyl alcohol one, 100% ethyl alcohol two, dimethylbenzene one 10 minutes；

Each 5 minutes in dimethylbenzene two and carbolic acid；Appropriate resinene, mounting observation is finally added dropwise.

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

After the completion of experiment, carried out using Aperio AT rurbo scanners (Leica Biosystems) and software kit Image output and image analysis.

5, the stabilization in vitro isotope labelling of mouse liver proteins group quantifies the acquisition of (TMT is quantitative) data

In stabilization in vitro isotope labelling quantitative (TMT is quantitative) experiment, each time point is fetched derived from time point mouse Each 200 μ g of hepatic protein mixed in equal amounts sample, and take filter membrane assistance enzymolysis method, enzymolysis flow is：It is firstly added UA Buffer (8M urea, 0.1M Tris/HCl pH 8.5), 5000g centrifuge about 1h, are repeated once；Then 50mM IAA are added (being prepared with UA), dark at room temperature place 30min, and then 5000g centrifuges 1h；It is washed three times with 0.5M TEAB solution respectively again；Most Afterwards according to 1:Trypsin is added in 50 ratios, is placed in 37 DEG C of shaking tables or insulating box enzymolysis 16-20h.After enzymolysis is completed, peptide is collected Section is used for TMT, and two groups of TMT reagents mark transgenic mice or the hepatic tissue enzymolysis product of control mice, then mix respectively respectively It closes the not isolabeling sample of two groups of experiments and is freeze-dried.

Angilent 3100OFFGEL Fractionator systems (Agilent Technologies) are used to carry out again Classification, 50 μ A constant currents, 48h or so collect peptide fragment, and two samples are each classified as 6 components, and totally 12 samples carry out LFQ- Orbitrap Velos Mass Spectrometer Methods (Thermo Finnigan, San Jose, CA), each sample simultaneously repeat loading 3 times.Instead Phase column is that nozzle needle reversed-phase column is made in laboratory by oneself, and the eluent of reverse phase eluent system is the aqueous solution (A) and 0.1% of 0.1% formic acid The acetonitrile solution (B) of formic acid, flow velocity 250nL/min, using the linear gradient of 4%B~30%B.Under being eluted from reversed-phase column Peptide fragment enter linear ion hydrazine-electrostatic field orbit trap combination mass spectrum LTQ Orbitrap-Velos mass spectrographs and analyzed, The data acquisition scheme of data-dependent, 10 two level spectrograms are closely followed after every level-one spectrogram, and dynamic excludes to be set as 90 Second, parent ion fragmentation mode takes HCD patterns, level-one spectrogram and two level spectra resolution rate to be respectively set to 30000 and 7500.

The collected original document of mass spectrum is analyzed with the default parameters of MaxQuant1.3.0.5 softwares, the number of use It is UniProt mouse databases (in June, 2012 download) according to library.It is as follows to search library parameter setting：Protease is set as Trypsin selects TMT label peptide fragment screenings, fixed modification to be set as the carboxy methylation of cysteine, and variable modification is set as first Oxidation, the protein N terminal acetylation of methyllanthionine, the false positive rate of protein and peptide section are disposed as 0.01.

Two, experimental result

1, during liver cancer genesis and development WHV/c-myc transgenic mices protein expression level dynamic change

In order to obtain the high throughput protein group data for being suitable for this work sutdy purpose, as shown in Figure 1, the present invention is in WHV/ The different pathological stages that c-myc transgenic mices are undergone during liver cancer genesis and development devise five corresponding samplings Time point (mouse postnatal 2nd, 3,5,7 and 11 months)；Under at every point of time, to 5 under same rearing conditions WHV/c-myc transgenic mices and 5 be only used as reference wt-C57BL/6 mouse, have collected their liver organization respectively；With Afterwards, it to every a liver tissue sample, by protein separation and digests into peptide fragment, non-marked (Label-free) peptide fragment After the classification of multidimensional liquid chromatogram (Zhou H, Dai J, Sheng QH etc., A are detected using tandem mass spectrum (LC-MS/MS) fully automated 2-D LC-MS method utilizing online continuous pH and RP Gradients for global proteome analysis, Electrophoresis 2007,28:4311-4319), it obtains Obtain final quantitative protein data.In this batch of quantitative data, by having run 570 LC-MS/MS detection cycles, Zong Gongjian Surely arrive high accurancy and precision (with the false positive recall rate (False discovery rate, FDR) of 0.4% peptide fragment for standard) The different peptide fragments of 42689 of 8713 kinds of protein；But in view of the follow-up Stability and veracity calculated and analyze, the present invention is only It has chosen and (five time points, 50 samples is being amounted to, wherein 25 WHV/c-myc transgenic mices and 25 wt- more than sum C57BL/6 mouse) half sample in 3372 protein all identifying (that is, these protein are in more than 25 Mouse Livers It is quantified in dirty sample).

2, identification of the hepatitis to the crucial critical stage of liver cancer vicious transformation

In order to accurately determine the crucial critical stage of scorching cancer vicious transformation, present invention uses find dynamic network biology The method of marker (Dynamical network biomarker, DNB) analyzes this set sequential high throughput protein group data. After DNB Algorithm Analysis as described herein, present invention finds a CI value strong signals to appear in this time at 5 monthly ages Point, this show WHV/c-myc transgenic mices at this time be in critical stage from hepatitis to liver cancer vicious transformation (that is, its early warning Scorching cancer vicious transformation will occur).This result is thick on 0 to 11 this period at monthly age with WHV/c-myc transgenic mices The hepatic pathology symptom slightly divided is consistent.Meet simultaneously required by the theory deduction that dynamic network biomarker is identified From the point of view of three standards (referring to Chen L etc., Scientific reports 2012), 48 eggs of this guide's molecular group are formed Intermolecular correlation raising is met in vain (uses PCC_idTo weigh), the molecular relativities of molecule and other non-guide's molecular groups reduces (it is used for PCC_odWeigh) and developed by molecule fluctuation is maximum (uses SD_dTo weigh) (see Fig. 2).

Meanwhile in order to more intuitively observe dynamic network biomarker (DNB) in this five time point network structures and The dynamic change for expressing fluctuation, the present invention is based on actually measured proteome datas by calculating corresponding pearson correlation system Number (PCC) structure protein co-expresses network, and is not based on interactive network between mouse known molecular obtained by data collection (see Fig. 3).As shown in figure 3, five month (critical stage of the scorching cancer vicious transformation predicted), with compared with mouse Afterwards, it forms correlation between the albumen (be arranged in point indicate) in inner ring of DNB and increases (line takes on a red color between inner ring midpoint) and interior (line is in indigo plant between interior circling point and outer circling point for the correlation significant decrease of other of the DNB albumen of ring and outer shroud coexpression albumen Color), while the expression fluctuation of inner ring DNB albumen is acutely (point of inner ring takes on a red color).

Hereafter, in order to judge the accuracy of the above-mentioned crucial critical point prediction of scorching cancer conversion, this hair from histopathology form The bright hepatic tissue sample that WHV/c-myc transgenic mices and wt-C57BL/6 mouse of the same age are acquired to each time point, respectively It prepares histotomy and it is dyed with h and E (Hematoxylin and eosin, HE) (see Fig. 4).Such as Shown in Fig. 4, the liver organization form of the transgenic mice at 2 monthly ages is similar to the normal mouse of reference, has and shows classics Lobuli hepatis structure (e.g., liver cell is in emitting shape ordered arrangement centered on central vein, forms liver cell plate)；3 to 5 monthly ages There are lobuli hepatis limiting plate intermittent intervals and with localized debris shape necrosis phenomena in transgenic mice blood lipid, this shows Existing inflammatory reaction；Then there is the necrosis of bridge shape in the transgenic mice hepatic tissue at 7 monthly ages and large stretch of necrotic zone expands along with hepatic sinusoid , this shows that primary liver cancer symptom occurs；When 11 monthly age of transgenic mice, lobuli hepatis cell plate structure destroys, and central vein becomes Narrow, liver cell volume increases, and nucleus increases and dyeing is deepened, and vacuole occurs, these phenomenons show that canceration has occurred for hepatic tissue. These level of fibrosis are the result shows that the transgenic mice at 5 monthly ages is in the crucial critical period of scorching cancer conversion.

3, dynamic network biomarker correlation function network (DNB-associated network) is transformed in scorching cancer Flop phenomenon in journey

The previous work of the present invention is it has been shown that dynamic network biomarker (DNB) will change state as one group Guide's molecular group with forewarning function, although (e.g., differentially expressed protein DEPs or difference co-express with traditional biological marker Albumen is to DCEs) it is different, but there is function synergic relationships with them --- that is, DNB is considered as that will be converted in state Period plays the molecular group of inducing action (Inducer or regulator), and DEPs (or DCEs) is the biology that state changes Function follower or agent.

In order to explore existing conspiracy relation between DNB member proteins and DEPs and DCEs, according to molecule known to mouse Between interaction relationship backgroundnetworks (data collection is in KEGG, BioGRID, TRED databases), the present invention pass through integrate DNB Member protein and its single order neighbours albumen (or functional connection albumen) structure DNB network of relation (DNB-associated Network, the network are related to 164 albumen and 183 sides in total).Then respectively in network with differentially expressed protein or difference Different coexpression albumen counts the point of coincidence or side, and examines its coincidence conspicuousness of assessment using hypergeometry.

The statistical result shows that DNB is co-expressed with differentially expressed protein (see Fig. 5, A) and difference in scorching cancer transfer process There are significant regulation relationships between (see Fig. 5, B) for albumen；Meanwhile also showing that the DNB network of relation turns in scorching cancer indirectly Important function has been played during changing.

Then, the present invention draws corresponding network intuitively to watch, and for transgenic mice, is disliked in scorching cancer In property conversion process, expressing quantity (see Fig. 5, C) in DNB correlation functions network (DNB-associated network) and The dynamic change situation of regulation and control (or coexpression) relationship (see Fig. 5, D) between DNB and its single order neighbours' albumen.From Fig. 5 (C) observable It arrives, during experience scorching cancer vicious transformation, significant overturning occurs in DEPs its expression being connect with the functional property of DNB Phenomenon --- that is, for transgenic mice, from 3 (or 5) monthly ages to 5 (or 7) monthly ages, the table of some DEP The significant changes from low (height) to high (low) have occurred up to amount.And (i.e. for the difference coexpression relationship involved by DNB albumen The relevant DCEs of DNB albumen) for, it can be seen that from Fig. 5 (D), during scorching cancer vicious transformation, regulation and control (or coexpression) are closed Significant overturning also has occurred in system --- that is, for transgenic mice, from 3 (or 5) monthly ages to 5 (or 7) monthly ages, The correlation of DNB albumen and some albumen has occurred from positive (negative) significant changes to negative (just).In conclusion for transgenosis For mouse, during scorching cancer vicious transformation, there is the expression quantity of 75 albumen in single order neighbours' albumen of 164 DNB There are 86 flop phenomenon (Fig. 5, C and D) occur in the functional connection side of significant changes and 183 DNB, this is not only consumingly Imply that DNB member proteins (especially, PLA2G6, LTA4H and/or EPS8L2) may be functionally correlated by regulating and controlling or influencing Albumen with transmitting inflammation to cancer vicious transformation, and may also be examined early stage in terms of clinical application being scorching cancer vicious transformation It is disconnected to provide accurately candidate molecules marker.

4, TMT labels are quantitative to label-free protein data reliability demonstration

In order to verify the data of analysis and its reliability of result, present invention employs technology repetitive modes to examine in bulk The truth of protein group variation tendency during scorching cancer vicious transformation is tested, i.e., the present invention is using the higher TMT marks of precision Remember that quantitative approach carries out big ration analysis to the protein group with batch mouse sample.With calculate the label-free used before Sample detection strategy is different, this verification is no longer the hepatic tissue sample progress quantitative analysis to every mouse, but in order to disappear Except the influence that individual mice difference is brought, after carrying out mixed in equal amounts into a sample to the congenic mouse sample of each time point, TMT label quantitative techniques are reused to be analyzed.As shown in fig. 6, important function may have been played in scorching cancer conversion process Albumen PLA2G6, LTA4H and EPS8L2 involved by DNB network of relation (DNB-associated network), in two tricks Consistency according to the horizontal variation tendency of middle expression quantity is very high, has very high reliability.

5, PLA2G6, LTA4H and EPS8L2 have played important biological function effect in scorching cancer conversion process

It is suggested although having and much promoting the hypothesis of cancer mechanism about inflammation, chronic hepatitis is to liver cancer vicious transformation Mechanism is completely clear not yet.Further to disclose the potential mechanism of scorching cancer vicious transformation, on the one hand, selective analysis of the present invention The Anomalous dynamics for the immune correlation function that DNB network of relation is enriched with change.The Fc gamma R that DNB albumen PLA2G6 is participated in There is vicious transformation early period (i.e. between 3 to 5 monthly ages) function in scorching cancer abnormal in the phagocytosis of mediation；Moreover, of the invention It was found that (especially, PLA2G6 and its relevant differentially expressed protein DEPs and difference co-express albumen pair to DNB correlation functions network DCEs lipid-metabolism and the channels TRP involved by) may result in subsequent inflammation reaction to the regulation and control reciprocal effect of inflammatory mediator Abnormal and hepatic insufficiency is so that finally inspired inflammation to tumor progression.Meanwhile the present invention identify will to state Variation has the DNB protein members of forewarning function --- PLA2G6 is exactly the carcinogenic driver by transgene mouse model --- c- Myc transcriptional controls (Fig. 6), the DNB that this explanation is found based on the mathematical model developed of the present invention from carcinogenic driving source very closely with As for more timely and effectively early warning inflammation cancer imminent state can be changed.Further, since it is considered herein that DNB albumen is as first It leads molecule preferentially system mode will be changed and make a response, therefore, influenced with PLA2G6 in involved biological approach Biological metabolism sets out, and causes inflammatory reaction and liver by the cascade signal of important lipid-metabolism molecule downstream transmits The orderly exception of dirty function of detoxification.

In LOX accesses, the present invention is it has also been found that another DNB member LTA4H (leukotriene A 4 hydrolase) participates in peanut four In olefin(e) acid metabolism synthesis leukotriene (LT).LTA4H is proinflammatory enzyme, generates leukotriene B4 (LTB4) (Snelgrove etc., 2010), The latter is an important inflammatory mediator, can via MAPK and PI-3 kinase pathway stimulate human pancreatic cancer cell growth (Tong etc., 2005).Interestingly, in transgenic mice, in this critical period in March to July, LTA4H, which has, to completely contradict Expression pattern and direct shaping modes, but lowered to its expression when November, therefore the effect of LTA4H is unclear.Recently Research illustrate the double action of LTA4H, i.e., resist pro-inflammatory effect by generating LTB4, and pass through Pro-Gly-Pro (PGP) it degrades and realizes anti-inflammatory effect (Snelgrove etc., 2010).The result of this paper has prompted the anti-inflammatory effect of LTA4H, because For in the transgenic mice at 11 monthly ages, collagen in specificity cutting lung, to generate the dried meat of neutrophilia Chemotactic Peptide PGP Peptide acyl endopeptidase (PREP) (Snelgrove etc., 2010) is raised.All these result prompts find the dual function of LTLA4H Importance in terms of prevention of inflammation converts and is related to new therapeutic strategy.

Equally interestingly, present invention discover that another DNB member EPS8L2 also assists in c-Myc and PLA2G6 two Person originates in the adjusting of the TF effects of the MAPK accesses of factors stimulated growth, and the access is mainly in actin cytoskeleton Film is fluctuated to play an important role with reconstruct.Some c-Myc targets such as DKC1, MAPK3 are also assisted in wherein.

6, groupization experimental result

By Fig. 5 can be observed DNB network of relation before scorching cancer conversion critical stage (5 monthly age of WHV/c-myc mouse) and Notable flop phenomenon later, it prompts PLA2G6, LTA4H and EPS8L2 to act on the early warning of scorching cancer vicious transformation may There is very big foreground in terms of clinical application.

High-level atypical hyperplasia tubercle (HGDN) is the precancerous lesion of liver cancer recognized at present, is defined as " liver Group is woven with dysplasia but not up to malignant tumour standard, with high malignancy potential, the increasing that is not true to type of diameter 1mm or more Raw tubercle ".

Purchase antibody simultaneously completes as described herein group of change experiment in people's HGDN tissues.As a result such as Fig. 7-9 and following table Shown in 1-2.Wherein, table 1 shows that age, gender and the health status of some patients, Fig. 7 and 8 show that number is respectively The antibody group coloring of S2-136902 patient, Fig. 9 display numbers are the antibody histochemical staining effect of S10-120093 patient Fruit, table 2 summarize the immunohistochemistry data of 11 patients.

These are the results show that there are obvious differential expressions in normal liver tissue, HGDN and cancerous tissue.Its In, tri- albumen of LTA4H, PLA2G2 and EPS8L2 up-regulated expression in HGDN tissues, higher than corresponding cancer (HCC) tissue and normally Hepatic tissue.Wherein, it is notable that although the expression quantity of PLA2G6 shows that tubercle position part occurs and increases in histotomy Phenomenon, but in the high-throughput data that global hepatic tissue (i.e. nonspecific knuckle areas) obtains, the expression quantity of PLA2G6 is facing Boundary's stage or tubercle phase are substantially reduced (Fig. 6), and this phenomenon may be caused by the protein regulation mechanism complexity.LTA4H and EPS8L2 is consistent in the detection of global hepatic tissue (Fig. 6) and specific knuckle areas.

Table 1

Table 2

Note：" normal " indicates the normal liver tissue of the patient；" HGDN " indicates the liver group of the HGDN from the patient It knits；" tumour " indicates the tissue of the liver neoplasm from the patient；"/" indicates to organize without HCC on the sample wax stone."-" indicates It is negative."+" indicates positive, wherein often increasing a "+" indicates that expression quantity at least increases 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) Reagents that specifically bind to EPS8L2; (2) an optional reagent that specifically binds to LTA4H; and (3) An optional reagent that specifically binds to PLA2G6. 2. The application according to claim 1, characterized in that, the application is that the reagent specifically binding to EPS8L2 and the reagent specifically binding to LTA4H are used in the preparation of intrahepatic nodules for diagnosing the subject or whether the diagnosing subject 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 the reagent specifically binding to EPS8L2 and the reagent specifically binding to PLA2G6 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 EPS8L2, the reagents that specifically bind to LTA4H, and the reagents that specifically bind to PLA2G6 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 with EPS8L2 as the detection object; (2) an optional reagent that uses LTA4H as the detection object; and (3) An optional reagent using PLA2G6 as the detection object. 5. detection kit as claimed in claim 4, is characterized in that, described detection kit contains: (a) reagents with EPS8L2 as the detection object and reagents with LTA4H as the detection object; (b) a reagent with EPS8L2 as the detection object and a reagent with PLA2G6 as the detection object; or (c) Reagents for detection of EPS8L2, reagents for detection of LTA4H, and reagents for detection of PLA2G6. 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 in the critical stage of malignant transformation from hepatitis to liver cancer, characterized in that the method includes the step of detecting the expression of EPS8L2 in the liver tissue of the object; wherein, The expression level is higher than the expression level of EPS8L2 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 PLA2G6 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) EPS8L2; (2) optional LTA4H; and (3) Optional PLA2G6. 10. The application according to claim 9, wherein the application comprises: The application of EPS8L2 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 EPS8L2 and PLA2G6 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.