CN113215255B

CN113215255B - Biomarker for early diagnosis or prognosis of liver cancer and application thereof

Info

Publication number: CN113215255B
Application number: CN202110453708.2A
Authority: CN
Inventors: ***; 樊晋宇; 赵亚琪; 程雪; 池金鹏; 张诗薇
Original assignee: Shandong Tianchuan Precision Medical Technology Co ltd
Current assignee: Shandong Tianchuan Precision Medical Technology Co ltd
Priority date: 2021-04-26
Filing date: 2021-04-26
Publication date: 2022-04-05
Anticipated expiration: 2041-04-26
Also published as: CN113215255A

Abstract

The invention discloses a biomarker for early diagnosis or prognosis of liver cancer and application thereof. The biomarkers of the invention include at least one of SNPD1, SNOZ6, and ZXDC. The invention discovers that SNPD1, SNOZ6 or ZXDC can be used as a novel molecular marker for early diagnosis or prognosis of liver cancer and a gene target for drug therapy, and detection and further verification of clinical samples are carried out through qRT-RCR and immunohistochemistry. The invention provides a potential molecular marker for early diagnosis and prognosis of liver cancer, and has important practical application value.

Description

Biomarker for early diagnosis or prognosis of liver cancer and application thereof

Technical Field

The invention relates to a marker for early diagnosis and prognosis of liver cancer, in particular to a biomarker for early diagnosis or prognosis of liver cancer and application thereof.

Background

According to the latest national cancer report issued by the national cancer center, the incidence position of liver cancer (liver cancer) is listed as the fourth position of malignant tumor, the mortality rate is the second position, liver cancer is common malignant tumor, the initial symptoms are not obvious, and the liver cancer is classified into secondary or metastatic liver cancer. Secondary or metastatic liver cancer refers to liver metastasis of malignant tumor of multiple organ origins generally found in stomach, biliary tract, pancreas, colon and rectum, ovary, uterus, lung, and breast. And the onset age of the liver cancer patients tends to be younger, and most of the patients are diagnosed in middle and advanced stages for the first time. At present, active and effective methods and diagnostic reagents for early diagnosis or prognosis of liver cancer are still sought.

For example, CN110117652A discloses a method comprising sequencing a nucleic acid sample from a subject, aligning the sequencing data with sequences in a subject reference genome and an HBV reference genome database to identify whether the nucleic acid sample has a liver cancer-associated proto-oncogene variation, a liver cancer-associated tumor suppressor variation or an HBV integration site, wherein if the nucleic acid sample has one or more of the proto-oncogene variation, the tumor suppressor variation and the HBV integration site, the subject is suggested or at risk of having liver cancer.

For another example, CN109557312A discloses a method and a kit for early diagnosis of liver cancer, the method comprises: obtaining a biological sample from a subject; detecting the content of a plurality of biomarkers selected from two or more of AFP, AFP-L3, GP73, Fuc-GP73, DCP, CEA, CA19-9, TK1, DKK1, gamma-GT, ALP, AFU, ALT, and Fuc-Kininogen 1 in the biological sample; calculating a probability that the subject has liver cancer based on the detection result; comparing the probability with a set threshold, wherein when the probability is greater than or equal to the set threshold, the subject is considered to have liver cancer; and when the probability is smaller than a set threshold value, the subject is not considered to have liver cancer.

The marker plays an increasingly important role in the early diagnosis or prognosis of liver cancer and has potential clinical application value.

The information in this background is only for the purpose of illustrating the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art that is known to a person skilled in the art.

Disclosure of Invention

In view of the current situation in the prior art, the invention discovers that SNPD1, SNOZ6 or ZXDC can be used as a new molecular marker for early diagnosis or prognosis of liver cancer and a gene target for drug therapy after intensive research, and detection and further verification of clinical samples are carried out through qRT-RCR and immunohistochemistry. The present invention has been accomplished, at least in part, based on this. Specifically, the present invention includes the following.

In a first aspect of the present invention, there is provided a method for early diagnosis or prognosis of liver cancer, comprising at least the step of detecting the expression level of at least one gene among SNPD1, SNOZ6, and ZXDC biomarkers.

In certain embodiments, according to the methods for early diagnosis or prognosis of liver cancer described herein, SNPD1, SNOZ6, and ZXDC gene expression levels include RNA expression levels or protein expression levels.

In certain embodiments, the method for the early diagnosis or prognosis of liver cancer according to the present invention comprises at least the following steps:

(1) a step of measuring the expression level of at least one of the SNPD1, SNOZ6, and ZXDC genes in a sample from a subject using a reagent to obtain a measurement value;

(2) a step of comparing the measured value with a control value; and

(3) if the measured value is higher than the control value, the subject is predicted to have liver cancer or to have high risk of liver cancer or to have poor prognosis, and if the measured value is lower than the control value, the subject is predicted to have no liver cancer or to have low risk of liver cancer or to have good prognosis.

In certain embodiments, the sample is a peripheral blood sample according to the methods for early diagnosis or prognosis of liver cancer described herein.

In certain embodiments, the method of the present invention for the early diagnosis or prognosis of liver cancer, wherein the liver cancer is a non-invasive liver cancer.

In certain embodiments, the reagent according to the method for early diagnosis or prognosis of liver cancer of the present invention comprises a primer and/or a probe for detecting an mRNA expression level of at least one of SNPD1, SNOZ6, and ZXDC genes, or an anti-SNPD 1, SNOZ6, and ZXDC antibody.

In certain embodiments, according to the method for early diagnosis or prognosis of liver cancer described in the present invention, the primer has a sequence as shown in SEQ ID No. 1-6, preferably further comprises a sequence as shown in SEQ ID No. 7-10.

In a second aspect of the present invention, there is provided a kit for early diagnosis or prognosis of liver cancer, comprising a primer and/or a probe for detecting the expression level of at least one of SNPD1, SNOZ6 and ZXDC genes, or an anti-SNPD 1, SNOZ6 and ZXDC antibody, wherein the primer has a sequence shown in SEQ ID No. 1-6, preferably further comprises a sequence shown in SEQ ID No. 7-10.

In a third aspect of the invention, there is provided a method for identifying a compound useful for liver cancer, comprising at least the steps of:

a. a step of measuring a gene expression level of at least one of SNPD1, SNOZ6, and ZXDC genes in a sample using a reagent to obtain a first measurement value;

b. a step of obtaining a second measurement value by the step of obtaining a measurement value from the expression level of at least one of the SNPD1, SNOZ6, and ZXDC genes in the sample after administration of the test compound using the reagent;

c. a step of comparing the first measured value with the second measured value, and

(ii) (i) identifying the test compound as useful for liver cancer if the second measurement is lower than the first measurement, i.e., the test compound decreases the expression level of at least one of the SNPD1, SNOZ6, and ZXDC genes; (ii) identifying the compound as not being useful for liver cancer if the second measurement is equal to or higher than the first measurement, i.e., the test compound has no effect or an elevated effect on the expression level of at least one of the SNPD1, SNOZ6, and ZXDC genes.

In certain embodiments, the compounds according to the methods of the present invention for identifying compounds useful for liver cancer further comprise other drugs with which the compounds are compatible and pharmaceutically acceptable carriers and/or excipients.

The invention discovers that SNPD1, SNOZ6 and ZXDC genes are closely related to liver cancer through intensive research. Clinical samples and verification are further utilized to show that the expression levels of SNPD1, SNOZ6 and ZXDC genes can be used for early diagnosis or prognosis of liver cancer. The invention provides a potential biomarker for early diagnosis and prognosis of clinical liver cancer, and has important practical application value.

Drawings

FIG. 1 shows the results of DNA electrophoresis of SNPD1, SNOZ6 and ZXDC genes.

FIG. 2 shows the results of expression difference of SNPD1, SNOZ6 and ZXDC in liver cancer tissue and para-cancer tissue detected by qRT-PCR.

FIG. 3 shows the results of expression difference of SNPD1, SNOZ6 and ZXDC in liver cancer peripheral blood mononuclear cells and normal control group peripheral blood mononuclear cells detected by qRT-PCR.

FIG. 4 shows SNOZ6 immunohistochemistry results (where A is liver cancer tissue and A' is a 50 μm para-cancer tissue scale).

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.

[ method for early diagnosis or prognosis of liver cancer ]

The term "biomarker" is any gene whose expression level in a tissue or cell is altered compared to the expression level of a normal or healthy cell or tissue.

The term "kit" also comprises a label for labeling a DNA sample, and a substrate corresponding to the label. In addition, the kit may further include various reagents required for DNA extraction, PCR, hybridization, color development, and the like, including but not limited to: an extraction solution, an amplification solution, a hybridization solution, an enzyme, a control solution, a color development solution, a washing solution, and the like. In addition, the kit also comprises an instruction manual and/or chip image analysis software.

The term "prognosis" is used to predict the likelihood of progression (including recurrence, metastatic spread and drug resistance) of liver cancer of the present invention. This prediction is clinically useful to determine treatment by selecting the most appropriate therapy for any particular patient. Such a prediction may become a valuable tool in determining whether a patient is likely to respond positively to a treatment regimen, for example, or whether a patient will survive for a long period of time after surgery is completed. The term "better prognosis" may denote an increased likelihood of a positive clinical outcome as a clinical outcome, and the term "worse prognosis" may denote a decreased likelihood of a positive clinical outcome as a clinical outcome.

The term "differentially expressed gene" or "gene differential expression" refers to a gene that results in higher or lower levels of activation in a patient with liver cancer as compared to the expression of a normal or control target gene. In addition, "differentially expressed genes" or "differential gene expression" includes genes that result in higher or lower levels of activation in different stages of the same disease. Such differences may be evidenced, for example, by changes in mRNA levels, surface appearance, secretion, or other distribution of the polypeptide.

The present invention may utilize any method known in the art for determining gene expression. It will be appreciated by those skilled in the art that the means by which gene expression is determined is not an important aspect of the present invention. The expression level of the biomarker can be detected at the transcriptional level.

The term "probe" refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

The probe has a base sequence complementary to a specific base sequence of a target gene. Here, the term "complementary" may or may not be completely complementary as long as it is a hybrid. These polynucleotides usually have a homology of 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 100% with respect to the specific nucleotide sequence. These probes may be DNA or RNA, or may be polynucleotides in which part or all of the nucleotides are substituted with artificial nucleic acids such as PNA, LNA, ENA, GNA, TNA, etc.

In the present invention, the oligonucleotide probe for a gene may be DNA, RNA, DNA-RNA chimera, PNA or other derivatives. The length of the probe is not limited, and any length may be used as long as specific hybridization and specific binding to the target nucleotide sequence are achieved. The length of the probe may be as short as 25, 20, 15, 13 or 10 bases in length. Also, the length of the probe can be as long as 60, 80, 100, 150, 300 base pairs or more, even for the entire gene. Since different probe lengths have different effects on hybridization efficiency and signal specificity, the length of the probe is usually at least 14 base pairs, and at most, usually not more than 30 base pairs, and the length complementary to the nucleotide sequence of interest is optimally 15 to 25 base pairs.

The probe self-complementary sequence is preferably less than 4 base pairs so as not to affect hybridization efficiency.

In a first aspect of the present invention, there is provided a method for the early diagnosis or prognosis of liver cancer, comprising the step of detecting the expression level of at least one gene among SNPD1, SNOZ6, and ZXDC biomarkers. Preferably, the level of expression of at least one gene of the SNOZ6 and ZXDC biomarkers is detected. Also preferred is the step of detecting the level of gene expression in the SNOZ6 biomarker.

According to the invention, the expression levels of SNPD1, SNOZ6 and ZXDC genes comprise two levels of RNA expression level or protein expression level. The invention can be used as the basis of the expression level of the SNPD1, SNOZ6 and ZXDC genes by detecting at least any one of the two layers.

The antibody of the present invention includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, nanobodies, humanized antibodies or fully human antibodies. The antibody of the invention may also be a single chain antibody. In the present invention, the modified antibody includes a chemical modified antibody and a conjugate of an antibody and another material. Examples of chemical modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, cross-linking cyclization, disulfide bonding, demethylation, covalent cross-linking, cysteinylation, pyroglutamylation, formylation, gamma-carboxylation, glycosylation, GPI anchoring, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolysis, phosphorylation of antibodies, and the like. Examples of the conjugate include, but are not limited to, conjugates with a nano polymer material, magnetic beads, and the like.

In certain embodiments, the reagents of the invention comprise an antibody and a detectable group, such as a fluorescent group, coupled thereto. In the present invention, in the case of a plurality of reagents, it is preferable that the detectable group of each reagent is different.

The method for early diagnosis or prognosis of liver cancer at least comprises the following steps:

(1) a step of measuring the expression levels of SNPD1, SNOZ6, and ZXDC genes in a sample from a subject using a reagent to obtain a measurement value;

(2) a step of comparing the measured value with a control value; and

In the present invention, a sample refers to any tissue, cell or derivative thereof derived from a subject, and examples thereof include, but are not limited to, diseased tissue (particularly liver cancer tissue), blood, plasma, serum, interstitial fluid, and the like. Preferably the sample is a peripheral blood sample.

In the present invention, the reference value may be a specific value point, or may be a value interval or range. The control value may be a value obtained by counting a large amount of clinical data on liver cancer, or may be a value of a tissue beside cancer or a normal tissue (preferably, a peripheral blood sample). The control value may be a value indicating, for example, the absolute amount of gene expression, or may be a value indicating, for example, the relative amount of gene expression.

In the present invention, the reagents for measuring the expression levels of SNPD1, SNOZ6, and ZXDC genes in a sample from a subject to obtain measured values may be known reagents. For example, reagents for real-time fluorescent PCR or immunoreactive reagents may be used. Reagents for real-time fluorescent PCR include primers and/or probes for SNPD1, SNOZ6, and ZXDC gene mRNA levels. The sequences of the primers and probes are not particularly limited as long as they are specific to the SNPD1, SNOZ6, and ZXDC genes, and those skilled in the art can freely design the primers and probes according to actual needs.

Examples

Identification of candidate genes

In order to search for biomarkers which can be used for predicting liver cancer, firstly, gene content related to liver cancer is searched from a GEO database, primary analysis is carried out on data by using GEO2R analysis, an Excel table is used for opening results, a P value is selected to be less than 0.05, logFC > -2 or < -2 >, and genes with obvious difference expression of liver cancer in gene chip data are screened out. And finally determining 3 obvious difference genes with high expression in liver cancer, namely SNPD1, SNOZ6 and ZXDC genes as genes for further research by combining the screening verification of GO, KEGG analysis, STRING analysis and the like.

II, identification of SNPD1, SNOZ6 and ZXDC

Primers for target genes SNPD1, SNOZ6, ZXDC and references beta-actin and GAPDH genes were designed using SnapGene software:

SNPD1-F：ACTCCTGGACTTGCCATCAA(SEQ ID NO.1)，

SNPD 1-R: GTACTCCACCTGGAACTAGT (SEQ ID NO.2), the size of the product being 120 bp;

snoZ6-F：TGGCCAAACCGTTCTTAAGG(SEQ ID NO.3)，

snoZ 6-R: AGTCCGGTTAACCGGTACAT (SEQ ID NO.4), the product size is 105 bp;

ZXDC-F：GGAATTCCAAGTCCGGAACT(SEQ ID NO.5)，

ZXDC-R: ATCTGAGGTTCAAGTCCGAT (SEQ ID NO.6), the product size is 110 bp;

β-actin-F：GAGCACAGAGCCTCGCCTTT(SEQ ID NO.7)；

β-actin-R：TCATCATCCATGGTGAGCTGG(SEQ ID NO.8)；

GAPDH-F：TGCAACCGGGAAGGAAATGA(SEQ ID NO.9)；

GAPDH-R：GCCCAATACGACCAAATCAGA(SEQ ID NO.10)。

the qRT-PCR reaction program was 95 ℃ for 3min, 30 cycles (95 ℃ for 30s, 56 ℃ for 30s, 72 ℃ for 30 s). The specificity and the band size of the PCR product are detected by 1.5% agarose gel, the result is shown in figure 1, M represents the band used by the nucleic acid and indicates a marker, the result shows that the product fragment is single and specific, and the band size of the PCR product is consistent with the expected size.

The PCR products of SNPD1, SNOZ6 and ZXDC are sent to a sequencing company for sequencing, and the correctness of the PCR products is determined.

Expression difference of SNPD1, SNOZ6 and ZXDC in liver cancer tissue and PBMC of HCC patient

3.1 sample Collection

Inclusion criteria for primary liver cancer patients:

1) the patient is older than 18 years and younger than or equal to 70 years;

2) no extrahepatic metastases;

3) no history of other malignant tumors;

4) after liver tumor resection, there is sufficient residual liver volume;

5) radical tumor resection;

6) clear diagnosis of pathology and primary hepatocellular carcinoma.

Exclusion criteria:

1) incomplete clinical and pathological data;

2) missed visits within 60 days after discharge;

3) the operation mode is non-radical liver cancer resection;

4) patients with TACE or portal vein embolization prior to surgery.

According to the above criteria, liver cancer tissues, tissues around cancer and peripheral blood of 17 cases of primary liver cancer patients were collected in infectious disease hospitals in the new county city, all samples were approved by patients and family members, and after signing an informed consent, collected from tissues taken from liver cancer resection, and benign tissues around cancer at a distance of 5cm or more from the tumor margin in the above liver cancer cases were taken as a control group. All pathologies were confirmed by clinical symptoms, signs, serum AFP values, imaging (liver B-ultrasound, CT or MRI examination) and pathological diagnosis.

In addition, in the peripheral blood sample collection of 17 healthy people and the clinical practice of Xinxiang medical college, 10ml of peripheral blood EDTA-anticoagulation tube was collected by each test subject, PBMCs were obtained by density gradient centrifugation (3 ml of lymphocyte separation medium was added to a centrifuge tube in advance, anticoagulation was slowly added to the centrifuge tube containing separation medium along the centrifuge tube wall, centrifuged at 2500rpm/min for 25min, the second layer of leukocyte layer was aspirated by a pipette), washed, centrifuged, dissolved in 1ml of Trizol reagent, and after repeated aspiration and dissolution, frozen in liquid nitrogen for use.

3.2 Total RNA extraction and reverse transcription:

3.2.1 extraction of sample RNA

Grinding 0.1g of tissue sample into powder in liquid nitrogen, adding 1mL of Trizol, and uniformly mixing to completely crack cells; after the collected PBMCs were sampled out, they were left to dissolve at room temperature for 3 min. Adding chloroform, shaking vigorously, standing at room temperature for 10-20min to crack fully, centrifuging at 4 deg.C and 12,000rpm for 15min, taking the upper aqueous phase, transferring to a new EP tube of 1.5mL, adding equal volume of 70% ethanol, transferring to Spin Column RM, extracting with RNA kit, and dissolving RNA in 30ul DEPC water. The purity of the RNA detected by the NanoDrop is 1.8-2.1 in absorbance 260/280, and the purity of the extracted RNA is high.

3.2.2 Synthesis of cDNA by reverse transcription

Reverse Transcription was carried out using the Reverse Transcription System kit from Promega, the total amount of Reverse transcribed RNA per sample was 1ug, the conditions of Reverse Transcription were (15 minutes at 37 ℃, 5s at 85 ℃ and 10 minutes at 4 ℃), and the cDNA was stored at-20 ℃ for use.

3.2.3Real-time PCR assay

According to the instruction of the kit, performing qRT-PCR by using a SYBR Super Mix kit, wherein the reaction system is an ABI 7500Real-Time PCR system, and finally 2^-△△CtThe expression levels of SNPD1, SNOZ6 and ZXDC were calculated.

The expression statistics of the final SNPD1, SNOZ6, ZXDC in the HCC group and the normal control group are shown in fig. 2 and fig. 3. FIG. 2 shows the results of expression difference of SNPD1, SNOZ6 and ZXDC in liver cancer tissues and tissues adjacent to the cancer detected by qRT-PCR; FIG. 3 shows the results of qRT-PCR detection of the expression difference of SNPD1, SNOZ6 and ZXDC in HCC PBMCs and normal control PBMCs. The results show that the expression of the SNOZ6 and ZXDC genes is higher than that of paracarcinoma in most liver cancer samples, and simultaneously, the expression of the SNPD1, SNOZ6 and ZXDC genes, especially the SNOZ6 gene, is higher than that of the normal group in liver cancer PBMC, and has extremely obvious difference (P <0.01)

4. Detection and verification of SNOZ6 gene expression product by immunohistochemistry

(1) Antigen retrieval: dewaxing and hydrating by conventional method, soaking pathological section in 0.01M sodium citrate buffer solution, repairing antigen under high pressure for 15min, naturally cooling to room temperature, washing with PBS for 3 times, each for 3 min;

(2) serum blocking: dropping normal sheep serum, standing at 37 deg.C for 30min to block nonspecific sites;

(3) primary antibody incubation: dropwise adding primary antibody working solution, incubating at 4 deg.C overnight, standing at 37 deg.C for 1h, washing with PBS for 3 times, each time for 3 min;

(4) adding a biotinylated secondary antibody: adding secondary antibody dropwise, standing at 37 deg.C for 30min, washing with PBS for 3 times, each for 3 min;

(5) color development: dropping SABC, standing at 37 deg.C for 30min, washing with PBS for 3 times, 3min each time, dropping DAB color developing agent, observing under microscope, washing with flowing water when yellow appears, and stopping color development;

(6) dewatering and sealing: after the slices are dehydrated, the slices are sealed by neutral gum, placed in a fume hood and dried.

The results are shown in fig. 4, wherein the left side of fig. 4 shows the expression result of SNOZ6 in the liver cancer tissue, and the right side of fig. 4 shows the expression result of SNOZ6 in the para-carcinoma tissue. The result shows that SNOZ6 is expressed in cell nucleus, the positive expression of the liver cancer tissue observed under a microscope is obviously higher than that of the tissue beside the cancer, and most cells of the cancer tissue show positive reaction.

While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Many modifications and variations may be made to the exemplary embodiments of the present description without departing from the scope or spirit of the present invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.

Claims

1. Use of reagents for the preparation of a kit for the early diagnosis of liver cancer, characterized in that the diagnosis comprises detectionZXDCA step of detecting the gene expression level of a biomarker, the reagent comprising a probe for detectingZXDCPrimers and/or probes for the mRNA expression level of a gene, or anti-ZXDC antibodies, said primers being:

ZXDC F：GGAATTCCAAGTCCGGAACT；

ZXDC R：ATCTGAGGTTCAAGTCCGAT。

2. use according to claim 1, characterized in that it comprises the following steps:

(1) measuring in a sample from a subject using a reagentZXDCA step of obtaining a measurement value of a gene expression level of a gene;

(2) a step of comparing the measured value with a control value; and

3. Use according to claim 1, characterized in that saidZXDCThe gene expression level includes RNA expression level or protein expression level。

4. The use according to claim 2, wherein the sample is a peripheral blood sample or a liver cancer sample.

5. The use of claim 4, wherein the liver cancer is a non-invasive liver cancer.