CN106701902B - Application of FOXR2 gene and expression product in diagnosis and treatment of liver cancer - Google Patents

Abstract

The invention discloses the application of a FOXR2 gene and its expression product, which are used for preparing products for liver cancer diagnosis and treatment. The FOXR2 gene and its expression product of the present invention can be used as a specific marker gene for diagnosing liver cancer; the FOXR2 gene and its expression product of the present invention can also be used as a target gene for preparing a drug for treating cancer, especially liver cancer, providing a new cancer treatment approach .

Description

Application of FOXR2 gene and expression product in diagnosis and treatment of liver cancer

Technical Field

The invention belongs to the technical field of gene therapy and diagnosis, and particularly relates to an FOXR2 gene and application of an expression product thereof in diagnosis and treatment of liver cancer.

Background

Hepatocellular carcinoma (HCC) is one of the common malignant tumors in our country, and is the fifth most common tumor in the world on the global scale, and the third leading cause of tumor-related death. In China, the mortality rate of liver cancer is second to lung cancer and esophagus cancer and third. Because of the lack of sensitive and specific early diagnosis methods, the clinical findings are mostly late stage, the overall curative effect is poor, the death rate is high, and about 11 thousands of people die of liver cancer in China each year, accounting for 45% of the death rate of liver cancer all over the world. The initial symptoms of liver cancer are not obvious, and the advanced symptoms mainly include symptoms of liver pain, hypodynamia, emaciation, jaundice, ascites and the like. Due to the hidden onset and rapid development, most patients reach local advanced stage or have distant metastasis when diagnosed, and the natural life cycle is short. Early diagnosis of liver cancer is critical for effective treatment and long-term survival.

It is generally believed that carcinoembryonic and glycoprotein antigens, enzymes and isoenzymes, cytokines, genes can all be used as markers for primary liver cancer. Currently, the qualitative diagnosis of liver cancer in our country is based on the detection of serum AFP (alpha-fetoprotein). AFP is generally normal at values below 20. mu.g/L. Those with AFP > 500. mu.g/L for 1 month, or AFP > 200. mu.g/L for 2 months without evidence of liver disease activity, who can be excluded from gestational and gonadal embryonal carcinoma, should have a high suspicion of liver cancer, can be diagnosed by medical imaging diagnosis. In the history of early diagnosis and development of liver cancer, AFP opens the way of immune diagnosis of liver cancer, makes early diagnosis of liver cancer possible, and raises the heat tide of tumor marker research. However, liver cancer early diagnosis is developed by using AFP for screening diagnosis, so far, breakthrough progress cannot be made, namely, the cure rate or 5-year survival rate cannot be improved, and even the phenomenon that AFP is normal and tumors reach the early stage of liver cancer appears in the examination. Although the sensitivity and specificity of AFP are not satisfactory, AFP is still the liver cancer tumor marker which is currently most widely used worldwide, because the specificity of other tumor markers is not comparable to that of AFP at present. Until now, early diagnosis of liver cancer and molecular typing diagnosis for guiding personalized treatment are still great challenges, and finding new tumor markers with higher sensitivity and specificity is the key for improving the early diagnosis level of liver cancer.

The treatment effect of liver cancer has been greatly improved in the last two decades, and the improvement of the curative effect is mainly benefited by the improvement of the diagnosis level, particularly the development of the imaging technology, so that some cases can be discovered at an early stage and can be radically cured. However, for most of middle and advanced liver cancers, how to improve the curative effect is a difficult problem for various countries. Therefore, except for conventional treatments such as surgery, radiotherapy and intervention, the treatment means of liver cancer is relatively many, including local treatment means such as radio frequency ablation, intratumoral absolute alcohol injection and focused ultrasound thermotherapy, and a comprehensive treatment mode with surgical treatment as the main mode, local treatment as the auxiliary mode and multiple disciplines is formed at present. Among them, the chemotherapy drugs for liver cancer are basically used for treating other tumors, and have poor pertinence and poor overall curative effect, so that new action targets are urgently needed to be found, and new specific drugs for liver cancer are researched and developed to improve the specificity and effectiveness of chemotherapy.

Disclosure of Invention

The invention discloses a human FOXR2 gene and application of an expression product thereof in preparation of products for diagnosing and treating liver cancer.

The invention provides an application of FOXR2 gene or FOXR2 protein in preparing a reagent or a kit for detecting liver cancer;

in another preferred embodiment, the kit comprises: a reagent for quantitatively detecting FOXR2 protein or mRNA and a corresponding label or a specification.

In another preferred embodiment, the reagent comprises a FOXR2 specific primer, a specific antibody, a probe and/or a chip.

In another preferred embodiment, the reagent includes a detection chip including a nucleic acid chip and a protein chip.

In another preferred embodiment, the nucleic acid chip comprises a substrate and specific oligonucleotide probes of liver cancer related genes spotted on the substrate, wherein the specific oligonucleotide probes of the liver cancer related genes comprise probes specifically binding with FOXR2 gene or mRNA.

In another preferred embodiment, the protein chip comprises a substrate and specific antibodies of liver cancer related proteins spotted on the substrate, wherein the specific antibodies of the liver cancer related proteins comprise specific antibodies against FOXR2 protein.

In another preferred embodiment, the FOXR2 protein comprises a fusion protein and a non-fusion protein.

In a second aspect of the present invention, there is provided a diagnostic kit for detecting liver cancer, the kit comprising a container, wherein the container contains a detection reagent for detecting FOXR2 protein or mRNA; and a label or instructions indicating that the kit is for detecting liver cancer.

In another preferred embodiment, the label or instructions may indicate the following:

when the ratio of the expression quantity of FOXR2 relative to the mRNA of the reference protein in the detected object to the expression quantity of FOXR2 relative to the mRNA of the reference protein in the tissues beside the cancer is more than or equal to 1.5, the probability that the detected object suffers from liver cancer is higher than that of the ordinary people.

In another preferred embodiment, the reference protein comprises β -actin.

In another preferred embodiment, the detection reagent comprises: specific primers, specific antibodies, probes and/or chips.

In another preferred embodiment, the kit is used for detecting a human liver tissue sample or a blood sample.

In another preferred embodiment, the liver tissue sample comprises liver cancer tissue, paracancerous tissue, or policy liver tissue.

In a third aspect of the invention, the invention provides an application of the FOXR2 protein, the FOXR2 gene or an inhibitor thereof in preparing a medicament for inhibiting the growth or proliferation of cancer cells or treating liver cancer.

In another preferred embodiment, the inhibitor comprises: an antibody of FOXR2, an antisense RNA of FOXR2 nucleic acid, siRNA, shRNA and an activity inhibitor of FOXR 2.

In another preferred embodiment, the inhibitor comprises si-1 or si-2 of the FOXR2 gene, preferably as shown in SEQ ID NO. 9-10 and/or SEQ ID NO. 11-12.

In a fourth aspect of the invention, there is provided an in vitro non-therapeutic method of inhibiting growth or proliferation of hepatoma cells, comprising the steps of: culturing the liver cancer cell in the presence of FOXR2 protein or its inhibitor, thereby inhibiting growth or proliferation of the liver cancer cell.

In another preferred embodiment, the method comprises adding the FOXR2 inhibitor to a culture system of the hepatoma cells, thereby inhibiting growth or proliferation of the hepatoma cells.

In a fifth aspect of the present invention, there is provided a method for screening a candidate compound for treating liver cancer, comprising the steps of:

(a) in the test group, adding a test compound into a culture system of cells, and observing the expression amount and/or activity of FOXR2 in the cells of the test group; in the control group, no test compound is added in the culture system of the same cells, and the expression amount and/or activity of FOXR2 in the cells of the control group are observed;

wherein, if the expression level and/or activity of FOXR2 of the cells in the test group is less than that of the control group, the test compound is a candidate compound for treating liver cancer and has the inhibition effect on the expression and/or activity of FOXR 2.

In another preferred example, the liver cancer cells include various commonly used liver cancer cell lines, such as HCC-LM3, WRL-68, YY-8103, Huh7, Hep3B, or L02.

In another preferred example, the method further comprises the steps of:

(b) further testing the candidate compound obtained in step (a) for its inhibitory effect on growth or proliferation of hepatoma cells.

In another preferred example, the step (b) includes the steps of: in the test group, adding a test compound into a culture system of cancer cells, and observing the number and/or growth condition of the cancer cells; in the control group, no test compound was added to the culture system of cancer cells, and the number and/or growth of cancer cells were observed; wherein, if the number or growth rate of the cancer cells in the test group is smaller than that in the control group, it is indicated that the test compound is a candidate compound for treating liver cancer having an inhibitory effect on the growth or proliferation of cancer cells.

In a sixth aspect of the present invention, there is also provided a method for inhibiting or treating liver cancer, comprising the steps of: use of a safe and effective amount of a FOXR2 inhibitor for administration to a subject (mammal) in need of such treatment.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIGS. 1A and B are schematic diagrams of the real-time quantitative PCR detection of the expression of FOXR2 gene in cancer tissues and paracarcinoma tissues of 41 patients with liver cancer in example 1, wherein "N" refers to the paracarcinoma tissues and "C" refers to the liver cancer tissues. The results showed that 28 patients (68.3%) had higher expression of FOXR2 gene in their cancer tissues than in the corresponding paracarcinoma tissues.

FIG. 1C shows that the immunohistochemical method detects the expression difference of FOXR2 gene between human liver cancer tissue sample and para-carcinoma tissue, wherein "N" refers to the para-carcinoma tissue, and "C" refers to the liver cancer tissue, and the expression level of FOXR2 gene in the patient's cancer tissue is higher than that in the corresponding para-carcinoma tissue.

FIG. 2A shows the cDNA sequence of the FOXR2 gene.

FIG. 2B shows the amino acid sequence of the protein encoded by the FOXR2 gene.

FIG. 3A shows the over-expression of FOXR2 in hepatoma cell lines Hep3B, Huh7, YY-8103 and L02, indicating that FOXR2 was successfully expressed in eukaryotic expression vectors.

FIG. 3B shows that the over-expressed FOXR2 protein enhances the growth ability of the hepatoma cell lines Hep3B, Huh7, YY-8103 and L02.

Figure 3C shows that over-expressed FOXR2 protein promotes proliferation of L02.

FIG. 4A shows that synthetic siRNA effectively interfered with the expression of FOXR2 gene.

FIG. 4B shows that silencing expression of FOXR2 by RNA interference inhibits growth of liver cancer cells WRL68 and HCC-LM 3.

FIG. 5 shows that silencing the expression of FOXR2 by RNA interference inhibits the clonogenic capacity of liver cancer cells WRL68 and HCC-LM3 in soft agar, thereby demonstrating that FOXR2 promotes malignant characterization of liver cancer cells.

FIG. 6A shows the ability of over-expressed FOXR2 gene to promote tumor formation of liver cancer cell YY-8103 subcutaneously in nude mice. The FOXR2 overexpression group was larger and heavier than the control group, both in volume and tumor weight, while the two groups were statistically significant.

FIG. 6B shows that silencing FOXR2 gene expression inhibits hepatoma cells WRL68 from forming tumors subcutaneously in nude mice. The FOXR2 silenced expression group was smaller and lighter than the control group both in volume and tumor weight, while both groups were statistically significant.

Detailed Description

The inventors of the present invention have conducted extensive and intensive studies, and unexpectedly found that FOXR2 is low expressed in cancer tissues and high expressed in cancer-adjacent tissues and normal tissues, so FOXR2 can be used as a marker for detecting liver cancer or as an auxiliary marker for detecting liver cancer. In addition, inhibitors of FOXR2 can inhibit the growth of cancer cells, especially liver cancer cells. The present invention has been completed based on this finding.

FOXR2 proteins and polynucleotides

The forkhead box (Fox) protein family is a transcription factor with a wing-shaped helical structure in a DNA binding region, and at present, 19 subfamilies exist, which not only activate or inhibit the transcription activity of a target gene by binding DNA, but also can directly bind with heterochromatin to participate in the reconstruction, and participate in the transcription regulation in cooperation with other transcription factors. Fox protein plays a role in various biological processes such as embryonic development, cell cycle regulation, carbohydrate and lipid metabolism, biological aging, immune regulation and the like, and also plays an important role in the generation and development process of tumors. FOXR2 is an important member of the FOX protein family. So far, few reports on the function of the FOXR2 gene exist. The invention firstly expounds the expression condition of FOXR2 in liver cancer and proves that FOXR2 promotes the proliferation of liver cancer cells and is a potential oncogene through a research means of cell biology.

The invention aims to disclose an application of a human FOXR2 gene and an expression product thereof in preparation of a product for diagnosing and treating liver cancer. The FOXR2 gene and the expression product thereof can be used as a specific marker gene for diagnosing liver cancer, so that the diagnosis of the liver cancer is more accurate and rapid; the FOXR2 gene and its expression product can be used as molecular medicine for treating liver cancer, and provide new way for treating liver cancer. Therefore, the development of related proteins for liver cancer diagnosis is urgently needed in the field, and in order to effectively inhibit the growth of liver cancer cells, the development of drugs for inhibiting the growth of liver cancer cells is urgently needed in the field so as to improve the specificity and effectiveness of chemotherapy.

In the present invention, "the protein of the present invention", "the polypeptide of the present invention", "FOXR 2 protein" are used interchangeably and refer to abbreviated as FOXR 2). It is understood that the term also includes active fragments and derivatives of FOXR 2.

In the present invention, "gene of the present invention" and "polynucleotide of the present invention" refer to a nucleotide sequence encoding FOXR2 protein or active fragments and derivatives thereof, including sense and antisense nucleic acids. The FOXR2 gene is located in chromosome 6q15 of the cell, the whole length of cDNA is 786bp, and the gene codes a protein with 261 amino acids in the whole length.

In the present invention, the terms "FOXR 2 protein", "FOXR 2 polypeptide" or "liver cancer marker FOXR 2" are used interchangeably and all refer to a protein or polypeptide having the amino acid sequence of human protein FOXR 2.

FOXR2, also known as SRSF12(serine/arginine-rich cleaving factor 12, serine and arginine-rich cleavage factor 12), was named because it was originally found to be an inhibitor of SR and has a molecular weight of 35 kD. SR protein is a kind of shearing factor with RRM RNA binding structure domain rich in serine and arginine, and has the functions of regulating and controlling pre-mRNA transcript of eukaryotic cell to selectively shear intron and splice exon. In vivo studies found that FOXR2 was able to antagonize other SR proteins and activate the 5' -most selective cleavage OF pre-mRNA OF adenovirus E1A (Alison E, et al 2001.THE JOURNAL OF BIOLOGICAL CHEMISTRY).

The cDNA sequence of the FOXR2 gene is shown as SEQ ID NO. 1; genbank accession No. 135295, cDNA sequence CCDS47459.1 of FOXR2 and the amino acid sequence NP _542781.3 encoded thereby.

The amino acid sequence of the protein coded by the FOXR2 gene is shown in SEQ ID No. 2.

As used herein, "isolated" refers to a substance that is separated from its original environment (which, if it is a natural substance, is the natural environment). If the polynucleotide or polypeptide in the natural state in the living cell is not isolated or purified, but the same polynucleotide or polypeptide is isolated or purified if it is separated from other substances coexisting in the natural state.

As used herein, "isolated FOXR2 protein or polypeptide" means that the FOXR2 protein is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. One skilled in the art can purify FOXR2 protein using standard protein purification techniques. Substantially pure polypeptides are capable of producing a single major band on a non-reducing polyacrylamide gel. In the present invention, the FOXR2 protein includes fusion proteins and non-fusion proteins.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. The polypeptides of the invention may or may not also include an initial methionine residue.

The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

Polynucleotides encoding the mature polypeptide of FOXR2 include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide. The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to variants of the above polynucleotides which encode polypeptides having the same amino acid sequence as the present invention or fragments, analogs and derivatives of the polypeptides. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded polypeptide.

The invention also relates to nucleic acid fragments, including sense and antisense nucleic acid fragments, which hybridize to the sequences described above. As used herein, a "nucleic acid fragment" is at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides in length. The nucleic acid fragments can be used in nucleic acid amplification techniques (e.g., PCR) to determine and/or isolate a polynucleotide encoding the FOXR2 protein.

The human FOXR2 nucleotide full-length sequence or its fragment can be obtained by PCR amplification method, recombination method or artificial synthesis method. For the PCR amplification method, primers can be designed based on the disclosed nucleotide sequences, particularly open reading frame sequences, and the sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

A method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

The invention also relates to vectors comprising the polynucleotides of the invention, as well as genetically engineered host cells engineered with the vectors of the invention or the coding sequence for FOXR2 protein, and methods for producing the polypeptides of the invention by recombinant techniques.

The polynucleotide sequences of the present invention may be used to express or produce recombinant FOXR2 protein by conventional recombinant DNA techniques. Generally, the following steps are performed:

(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a human FOXR2 protein, or with a recombinant expression vector comprising the polynucleotide;

(2) a host cell cultured in a suitable medium;

(3) isolating and purifying the protein from the culture medium or the cells.

Methods well known to those skilled in the art can be used to construct an expression vector containing the human FOXR 2-encoding DNA sequence and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, bacterial cells of the genus streptomyces; fungal cells such as yeast; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, COS, or 293 cell.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

Antibodies

The invention also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, specific for human FOXR2 protein. As used herein, "specific" means that the antibody binds to the human FOXR2 gene product or fragment. Preferably, these antibodies bind to the human FOXR2 gene product or fragment, but do not recognize and bind to other unrelated antigenic molecules. The antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art.

The invention encompasses not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab)₂A fragment; an antibody heavy chain; an antibody light chain; a genetically engineered single chain Fv molecule; or a chimeric antibody.

The anti-human FOXR2 protein antibody can be used in immunohistochemical method to detect human FOXR2 protein in biopsy specimen.

Inhibitors and pharmaceutical compositions

By utilizing the protein of the invention, substances, particularly inhibitors and the like, which interact with the FOXR2 protein can be screened out by various conventional screening methods.

The inhibitor (including antibody, antisense nucleic acid and other inhibitors) of the FOXR2 protein can inhibit the expression and/or activity of the FOXR2 protein when being applied (dosed) on treatment, thereby inhibiting the growth or proliferation of liver cancer cells. Typically, these inhibitors will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, typically having a pH of from about 5 to about 8, preferably a pH of from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: enteral, intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.

Inhibitors useful in the present invention include: an antibody of FOXR2, an antisense RNA of FOXR2 nucleic acid, siRNA, shRNA and an activity inhibitor of FOXR 2. Among the typical FOXR2 inhibitors are siRNA and shRNA.

siRNAs useful in the present invention include a variety of siRNAs targeting FOXR2, which one skilled in the art can routinely screen based on, wherein one preferred siRNA comprises the sequence (si-1) as shown in SEQ ID NO 9 (sense strand) and SEQ ID NO 10 (antisense strand); or preferred siRNAs include the sequence shown as SEQ ID No. 11 (sense strand) and SEQ ID No. 12 (antisense strand) (si-2).

Typically, the technical scheme of using the FOXR2 gene as a target for preparing a liver cancer treatment drug comprises the following scheme:

1.the sequence specificity of the chemically synthesized double-stranded ribonucleic acid molecule is directed at the FOXR2 gene sequence, the double-stranded ribonucleic acid molecule is delivered into liver cancer cells by liposome encapsulation to interfere the expression of the FOXR2 gene, and the change of cell biological characteristics such as the forming capability of soft agar clone and cell proliferation is observed. The nucleic acid sequence (e.g., siRNA) specific for FOXR2 can be designed and synthesized using methods routine in the art.

2. Various vectors including DNA vectors and lentiviral vectors are utilized to interfere the expression of the FOXR2 gene, so that the effect of in vivo interference of the FOXR2 gene is achieved, and the treatment effect of the FOXR2 gene on subcutaneous tumor bodies of nude mice is detected, thereby realizing the purpose of inhibiting the proliferation of liver cancer.

3. The polypeptide and the monoclonal antibody which can specifically inhibit the FOXR2 gene transfer activity are obtained, so that the aim of inhibiting the FOXR2 activity is fulfilled, and the aim of inhibiting the liver cancer cell proliferation in vivo is fulfilled.

The invention also provides a pharmaceutical composition, which contains a safe and effective amount of the FOXR2 protein or the inhibitor thereof and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions, such as tablets and capsules, can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 1 microgram to 10 milligrams per kilogram of body weight per day.

Detection method and kit

The invention also relates to diagnostic assays for quantitative and in situ measurement of human FOXR2 protein levels or mRNA levels. These assays are well known in the art. Human FOXR2 protein levels detected in the assay can be used to diagnose liver cancer.

One method for detecting the presence or absence of FOXR2 protein in a sample is to use antibodies specific for FOXR2 protein, which comprises: contacting the sample with an antibody specific for FOXR2 protein; observing whether an antibody complex is formed, the formation of an antibody complex indicates the presence of FOXR2 protein in the sample.

The FOXR2 protein or its polynucleotide can be used for diagnosing and treating FOXR2 protein related diseases. A part or all of the polynucleotides of the present invention can be immobilized as probes on a microarray or DNA chip for analysis of differential expression of genes in tissues and gene diagnosis. anti-FOXR 2 antibodies can be immobilized on a protein chip for quantitative detection of FOXR2 protein in a sample.

The invention also provides a kit for detecting liver cancer, which contains a primer pair for specifically amplifying FOXR2 and/or an FOXR2 specific antibody, and preferred primers are shown as SEQ ID NO. 5 and SEQ ID NO. 6.

Screening method

The invention also provides a method for screening drugs based on FOXR 2. One method is to screen compounds which affect (promote) FOXR2 expression or activity, and then further test the screened compounds for inhibition of hepatoma carcinoma cells. One screening method can be based on the expression level of mRNA for FOXR 2.

Representative cancer cells include (but are not limited to): liver cancer cells.

The general method comprises the following steps:

(1) clinical tissue sample acquisition

Liver cancer and tissues adjacent to the cancer were obtained from surgically treated liver cancer patients, and informed consent was signed with the patients before obtaining the samples. Once the excised liver is separated, the primary tumor focus and the surrounding tissues beside cancer of 5cm are cut out quickly, put into liquid nitrogen for quick freezing, transferred to a refrigerator at minus 80 ℃ for storage, and stored in the liquid nitrogen during transportation. Both cancer and paracancerous tissue are ultimately diagnosed by a pathologist. Samples were classified as class I-III according to Edmondson classification criteria.

(2) Tissue and cell RNA extraction

The RNA was extracted using TRIzol Reagent (Invitrogen) by the following procedure:

1) cleaning the containers such as mortar, pestle and homogenizer, and respectively using ddH₂O and DEPC H₂O rinsing, then baking in an oven at 180 ℃ for about 4 hours to remove RNase;

2) adding a proper amount of liquid nitrogen into a mortar for precooling, quickly taking out the tissue from the liquid nitrogen, cutting the tissue into 50-100mg, and grinding the tissue into powder in the mortar;

3) transferring the ground tissue powder to an RNase-free EP tube as completely as possible by using a curette, adding a proper volume (1ml) of TRIzol reagent in advance to the EP tube, and fully homogenizing;

4) standing at room temperature for 5 minutes, proportionally adding chloroform (200. mu.l/1 ml TRIzol) into a centrifuge tube, rapidly and violently shaking for 15 seconds, standing at room temperature for 2-3 minutes, and centrifuging at 4 ℃ under 12000 Xg for 15 minutes;

5) transferring the upper aqueous phase into a new RNA enzyme-free EP tube as far as possible, adding isopropanol with the same volume, reversing and uniformly mixing for 5 times, standing for 10 minutes at room temperature, centrifuging for 10 minutes at 4 ℃ under the condition of 12000 Xg, and then, detecting RNA precipitation;

6) pouring off the supernatant, adding 75% ethanol (1ml/1ml TRIzol), mixing, washing RNA, centrifuging at 4 deg.C for 5min at 7500 Xg;

7) discarding the supernatant, removing residual ethanol as much as possible, and naturally drying the precipitate for 5-10min (taking care not to completely dry); adding 30-50 μ l DEPC H₂O, blowing and sucking for several times, and dissolving RNA precipitate;

8) measuring the concentration and purity OD 260/280(1.8-2.0) of RNA by an enzyme-labeling instrument; gel electrophoresis was performed to observe whether degradation occurred or not, and the samples were stored at-80 ℃.

Extracting cell line RNA, collecting cells in logarithmic growth phase, sucking culture solution, adding TRIzol reagent (1ml TRIzol/10 cm) in corresponding amount according to the area of culture dish²) The cells were lysed and blown several times, and the lysed cells were collected in an RNase-free EP tube, and the remaining RNA was isolated and purified by the chloroform-isopropanol method according to the above steps 4) to 8).

(3) Reverse transcription of RNA

Reverse transcription was performed with M-MLV Reverse Transcriptase (Promega) as follows:

1) the following components were added to the nuclease-free EP tube:

the mixture was placed in a PCR apparatus at 70 ℃ for 5 minutes and then immediately cooled on ice for 5 min.

2) The following components are added into the system:

after mixing gently, the mixture was placed in a PCR instrument at 37 ℃ for 60 min.

The cDNA obtained by the reversion was stored at 4 ℃.

(4) Real-time quantitative PCR

Real-time quantitative PCR reaction use

Premix Ex Taq^TM(Perfect Real Time) kit (TaKaRa Biotechnology Co., Ltd. Dalian, China) using Thermal cycleDicce^TMReal Time System (TP800 Real-Time fluorescent quantitative PCR instrument, TaKaRa) was performed. The length of the amplification product of the quantitative PCR is preferably 80bp to 150bp (can be extended to 300 bp).

The reaction system is as follows:

reaction conditions are as follows:

dissolution curve analysis step:

95℃ 15sec

60℃ 30sec

95℃ 15sec

the dissociation time was 4 sec.

The fluorescence background signal and the threshold value adopt default values set by an instrument and are automatically generated after each PCR reaction is finished, the Ct value represents the number of cycles that the fluorescence signal in each reaction tube passes when the fluorescence signal reaches the set threshold value (10 times of the baseline fluorescence intensity), each template of the target gene FOXR2 is subjected to 3 multitubes, the obtained Ct value is averaged, the Ct average value of the FOXR2 gene subtracts the Ct average value of the internal reference gene (β -actin) of the corresponding template to obtain the Ct average value of the delta Ct. liver cancer groupThe delta Ct of corresponding paracancerous tissues is subtracted from the delta Ct to obtain a delta Ct value, and the fold relation of FOXR2 genes in the liver cancer group and the paracancerous group is 2^-ΔΔCtAnd (4) showing.

(5) Eukaryotic expression vector construction

1) Template: cDNA library of human liver immortalized cells L02.

2) Selection of eukaryotic expression vectors: PcdDNA^TM3.1/myc-His(-)A，5522nucleotides。

3) According to the sequence of FOXR2mRNA (NM-198451.3), the expression vector pcDNA is combined^TM3.1 designing a primer at the enzyme cutting site of myc-His (-) A, wherein the sequence of the primer is Forward:5-ccaccATGGACTTAAAACTAAAAGACTG (SEQ ID NO: 3); reverse:5-gttcGGTACCAAGATCAAAGAGAGAGGTCAAC-3(SEQ ID NO.: 4). Wherein the stop codon of FOXR2 in the reverse primer was removed, resulting in a C-myc and 6XHis tag at the C-terminus of FOXR 2. PrimeStar Using high fidelity DNA polymerase^TMHS DNA Polymerase (TaKaRa), using L02cDNA as template to amplify the full-length open reading frame of gene FOXR2, and 50 μ L total reaction system components as follows:

35 cycles of amplification were performed by a two-step PCR method (95 ℃, 10 sec; 60 ℃, 90 sec). The size of the PCR product is about 1.0kb, the size is identified by 1% agarose gel electrophoresis, and the PCR product which meets the size of the fragment is recovered by tapping (gel purification kit: MACHEREY-NAGEL).

4) PCR product and vector plasmid pcDNA were recovered by double digestion with EcoRV, KpnI (TaKaRa Biotechnology Inc. Dalian, China)^TM3.1/myc-His (-) A, the enzyme digestion reaction system is as follows:

carrying out enzyme digestion reaction at 37 ℃ for 1 hour; and (5) tapping and recovering the enzyme digestion product.

5) Connecting: mixing the PCR product and carrier at molar ratio of 4:1, linking with DNA ligase system containing 2.5 μ l of 4 × Solution I (TaKaRa) Code：D102A)，ddH₂Supplementing O to 10 μ l, and connecting at 16 deg.C for 2h or overnight;

6) and (2) transformation, namely mixing 10 mu l of the ligation product with 100 mu l of competent bacteria (TOP10 or DH5 α), standing on ice for 30min, thermally shocking at 42 ℃ for 90sec, immediately standing on ice for 5min, adding 800 mu l of LB culture solution without antibiotics, carrying out shaking culture at 37 ℃ and 200rpm for 30min to recover and amplify the thallus for one generation, centrifuging at 3000rpm for 2min, removing most of supernatant, reserving 50-100 mu l of bacterial solution, slightly blowing and beating the precipitate uniformly, then uniformly spreading the precipitate on an LB plate with ampicillin resistance (Amp +), and carrying out culture at 37 ℃ for 12-16 h.

7) Cloning and identification: selecting bacterial colonies which grow after ampicillin resistance screening, carrying out amplification culture in a liquid culture medium added with ampicillin, extracting plasmids for enzyme digestion identification: taking 1-2 mu g of the small-size plasmid, carrying out double digestion by using EcoRV and KpnI, carrying out agarose gel electrophoresis to identify the size of the digestion fragment, and carrying out vector pcDNA^TMThe size of the 3.1/myc-His (-) A fragment is about 5.5kb, the size of the FOXR2 reading frame fragment is about 1000bp, and the clones with the same size are sequenced to confirm the correctness of the sequence of the inserted fragment.

(6) Determination of cell growth curves

1) Different kinds of HCC cells are grown according to 3-5 × 10³The total amount of cells was calculated per 100. mu.l/well, and after digesting the cells sufficiently, the cells were diluted to the desired concentration and seeded in a 96-well plate. Inoculating cells in each group with three wells every day for 5-7 days;

2) cell status and number were observed after the cells were substantially adherent. Color reaction was carried out with CCK-8 developer (Cell Counting Kit-8, DOJINDO, Japan) by adding 10. mu.l of CCK-8 to 100. mu.l of the culture medium at 37 ℃ with 5% CO₂And (3) placing the incubator for incubation for 1h, measuring the absorbance at 450nm by using an enzyme-labeling instrument, recording, and determining the actual initial density of the cells as a relative zero point of growth.

3) Changing the liquid half a day or every other day, which is determined by the experiment requirement;

4) observing the cell morphology under a microscope, measuring at fixed time intervals, and recording the cell growth condition;

5) typically 5 to 7 days. After the measurement is finished, data are collected and processed, and a graph is drawn by Excel.

(7) Cell clone formation assay

1) Transfection: using Lipofectamine^TM2000(Invitrogen), overexpressing or silencing expression of FOXR2 gene in the cells;

2) after culturing the transfected cells in a 6-well plate or a 35mm culture dish for 24h by using a normal culture solution, digesting and counting the cells, inoculating the cells to a 100mm culture dish according to a certain number (different cell strains are different in number), continuously culturing for 24h, and then adding G418 (600-;

3) culturing for 2-3 weeks, changing fresh culture solution every 3-5 days, and adding G418 for screening until macroscopic cell clone is formed;

4) sucking out the culture solution in the culture dish, washing twice with 1 × PBS, dyeing with Coomassie brilliant blue R-250 for 2h, washing with water, and decolorizing with Coomassie brilliant blue dye-decolorizing solution for 30-60 min;

5) colony formation staining results were photographed and cell clones on each dish were counted according to the same criteria (cell clone size).

(8) Western Blot (Western Blot)

1) Protein sample preparation: the cultured cells were aspirated from the culture supernatant, washed twice with pre-cooled 1XPBS, added with 2 XSDS lysate (100mM Tris-Cl, pH 6.8, 4% SDS, 20% glycerol), lysed thoroughly, heated in a boiling water bath for 10min, centrifuged at 12000 Xg for 10min, the supernatant was transferred to a new tube,

the BCA Protein Assay Kit quantifies the obtained Protein and stores the Protein at-80 ℃;

2) protein electrophoretic separation: adding a proper amount of loading buffer solution (loadingbuffer) containing 200mM DTT into the protein sample, heating in a boiling water bath for 10min, slightly centrifuging, and carrying out SDS-PAGE protein gel electrophoresis separation on the sample;

3) film transfer: the electrophoresis gel, nitrocellulose membrane, thick (thin) filter paper pad were immersed in membrane transfer buffer (24mM Tris, 192mM glycine, 20% methanol) for equilibration for 15-20 min. Filter paper backing plate according to thickness of positive electrode-1 layer-nitrocellulose membrane-electrophoretic gel-2 layers of thin filter paper backing plate-negative electrode, placing in sequence, and wet-turning apparatus (XCell SureLock)^TMInvitrogen) 30V film transfer for 30-40 min;

4) and (3) sealing: sealing with 5% skimmed milk powder/0.1% PBST as sealing liquid for 30min-2h at room temperature by horizontal shaking table;

5) a first antibody: diluting the primary antibody with blocking solution (concentration recommended by reference to antibody specification), incubating at room temperature for 2h or at 4 deg.C overnight, washing with 0.1% PBST for three times (5 min each time);

6) secondary antibody: diluting the fluorescent secondary antibody with blocking solution (1:1000), incubating at room temperature for 30min, and washing with 0.1% PBST for three times, each time for 5 min;

7) sweeping the membrane: the ODYSSEY infrared imaging system scans the nitrocellulose membrane and stores the image.

(9) Soft agar colony formation assay

1) Preparing 1% and 2% low-melting-point Agarose (TaKaRa), and sterilizing at high temperature and high pressure;

2) preparing 2 × DMEM culture solution (2.5 × DMEM containing 20% FBS);

3) mixing 2% Agarose and 2 × DMEM culture solution with the same volume at 37 deg.C, adding 0.5ml per well into 24-well plate, placing in 4 deg.C refrigerator, and solidifying for use;

4) fully digesting the cultured cells into single cells, counting and diluting to the same concentration (3000-;

5) mixing the cell suspension with 1% Agarose at 37 deg.C, adding into 24-well plate with 0.5ml of glue, and placing in refrigerator at 4 deg.C for 10 min;

6) adding 0.2ml culture medium to the coagulated soft agar, and standing at 37 deg.C with 5% CO₂Continuously culturing for 2-3 weeks in an incubator;

7) the growth of cell clones in each well was observed under a microscope, counted and analyzed.

(10) Nude mouse tumorigenesis experiment

1) The mice are 5-6 week male BLAB/c nu nude mice, provided by Shanghai Si Laike laboratory animals Co., Ltd, and are bred in southern model animal culture center;

2) the treated cells are taken and inoculated under the skin of the mouse in the same quantity (different cell types are inoculated in different numbers), and in order to avoid errors caused by individual differences, the same cells can be inoculated in the same mouse in a left-right symmetrical mode through different treatments;

3) after a visible tumor appears, the size of the tumor is monitored every 3 days, the long diameter and the short diameter of the tumor are read by a vernier caliper, and the tumor volume is calculated according to the following formula: volume is long diameter x short diameter²；

4) After continuously monitoring for about 8-9 times, the data are collated and counted.

(11) Antibody acquisition and immunoassay

1) Antigen protein acquisition

Obtaining a cDNA sequence of human FOXR2 gene from a Genebank database, obtaining a coding frame through PCR amplification, inserting the coding frame into a prokaryotic organism or eukaryotic organism expression vector to express the FOXR2 protein, and purifying the protein according to a purification system of a gene engineering expression product.

2) Antibody preparation

Antibodies can be prepared by several methods:

a cell fusion method: animals (including rabbit, goat, etc.) are immunized with the prepared FOXR2 protein, spleen cells are obtained, and then fused with myeloma cells, and monoclonal antibodies are prepared according to conventional monoclonal antibody preparation technology.

b, cloning spleen IgG variable regions of immune animals by utilizing a phage surface display library and expressing the spleen IgG variable regions into a genetic engineering monoclonal antibody.

And c, immunizing animals by using the purified protein to prepare multi-antiserum.

3) Detection of

a, using the prepared antibody (polyclonal antibody or monoclonal antibody) to carry out pathological detection of liver cancer by a histochemical method, wherein a negative signal is liver cancer.

b, taking serum of the patient, detecting by an ELISA method, and determining the negative reaction as the suspicious patient of the liver cancer.

And c, using the FOXR2 antibody as one of probes of a protein chip for various tumor diagnoses.

Example 1: expression pattern of FOXR2 in clinical samples

In 41 clinical samples, 28 samples of liver cancer tissues have higher FOXR2 expression than corresponding tissues beside the cancer by real-time quantitative PCR detection, and the up-regulation rate reaches 68.3%, and statistical analysis shows that p is less than 0.01 (FIG. 1A) to illustrate the reliability of the result, meanwhile, the expression pattern of FOXR2 in a certain number of liver cancer tissues is detected by an immunohistochemical method, as shown in FIG. 1C, the FOXR2 gene expression in the cancer tissues of patients is higher than that in the corresponding tissues beside the cancer, and the primer sequences of real-time quantitative PCR for detecting FOXR2 expression used in the experiment are 5-TCAGTGTGCAGGAGATCTAC-3(SEQ ID NO: 5) and 5-AAGATCAAAGAGAGAGGTCAAC-3(SEQ ID NO: 6) and the primer sequences of β -actin as internal references are 5-cctggcacccagcacaatg-3(SEQ ID NO: 7) and 5-368 (SEQ ID NO: 26).

Example 2: over-expression FOXR2 gene for promoting proliferation of liver cancer cell

The cDNA sequence of FOXR2, CCDS 35308.1 (fig. 2A) (SEQ ID No.:1) and its encoded amino acid sequence NP _940853.1 (fig. 2B) (SEQ ID No.:2) were searched online by NCBI. To verify the function of FOXR2 in liver cancer development, we first constructed its eukaryotic expression vector, and cloned the cDNA of FOXR2 into pcDNA^TM3.1/myc-His (-) A, and the plasmid constructed by transfection enters liver cancer cells and is subjected to western blot detection, and the FOXR2 is successfully expressed (figure 3A). The subsequent functional experiment shows that the excessive expression of FOXR2 promotes the proliferation and clonogenic capacity of hepatoma cells Hep3B, Huh7, YY-8103 and L02 (FIG. 3B, C). The primer sequence for constructing the FOXR2 eukaryotic expression vector is Forward:5-ccaccATGGACTTAAAACTAAAAGACTG-3(SEQ ID No.: 3); reverse:5-gttcGGTACCAAGATCAAAGAGAGAGGTCAAC-3(SEQ ID NO.: 4).

Example 3: silencing expression of FOXR2 inhibits cell growth

To further validate the cell growth promoting function of FOXR2, changes in cell growth were examined by silencing their expression by RNA interference. siRNA for interfering with FOXR2 expression was synthesized by shanghai ge code pharmaceuticals, ltd, with the sequence: si-1 sense strand 5-GCUCCCUAGAUGAGAUACAdTdT-3(SEQ ID No.: 9); si-1 antisense strand 5-UGUAUCUCAUCUAGGGAGCdTdT-3(SEQ ID NO: 10). si-2 sense strand 5-GGUGUUAAGUUAGAGAdTdT-3 (SEQ ID No.: 11); si-2 antisense strand 5-UCUAACUUACUUAACACCdTdT-3 (SEQ ID NO: 12). The NC nonspecific nucleotide sequence used as an interference control was: sense strand 5-UUCCCGAACGUCACGUdT-3 (SEQ ID No.: 13); the antisense strand 5-ACGUGACACGUCGGAGAAdTdT-3 (SEQ ID No.: 14). Real-time quantitative PCR detection shows that the artificially synthesized interfering siRNA can effectively reduce the expression level of FOXR2 (figure 4A), and growth curve experiments also prove that the down-regulation of FOXR2 can inhibit the growth of liver cancer cells WRL68 and HCC-LM3 (figure 4B), thereby strongly supporting the conclusion that FOXR2 promotes the growth of tumor cells.

Example 4: FOXR2 promotes malignant characterization of hepatoma carcinoma cells

The soft agar clone forming ability is a strong in vitro index reflecting the malignancy degree of tumor cells. To examine the effect of FOXR2 on malignancy of hepatoma cells, WRL-68 and HCC-LM3 (purchased from cell banks of the Central institute of sciences) were selected as subjects for silencing expression. Through the analysis of the number of cell clones growing in soft agar (figure 5), the final determination that the silence expression FOXR2 can effectively inhibit the malignancy of WRL-68 and HCC-LM3 cells shows that FOXR2 promotes the malignancy of liver cancer cells.

Example 5: FOXR2 influences the tumorigenicity ability of liver cancer cells under nude mouse skin

In vitro experiments clearly show that FOXR2 promotes the growth of hepatoma cells, and then a tumor-bearing nude mouse model is used for researching the influence of the expression of FOXR2 on the subcutaneous tumor forming capability of hepatoma cells of nude mice. Will be 1 × 10⁶YY-8103 (sourced from Zhongshan Hospital in Shanghai city) cells over-expressing FOXR2 and control cells were injected into nude mice subcutaneously at the abdomen, the growth of tumor bodies was observed, and the length and width of the tumor bodies were recorded every 3 days with a vernier caliper. After 1 month, the nude mice were sacrificed and the tumor bodies were taken out and weighed. The results indicate that overexpression of FOXR2 effectively promoted the tumorigenic capacity of YY-8103 cells subcutaneously in nude mice (fig. 6A). Similarly, 1 × 10⁶A WRL68 cell and a control cell which silence expression of FOXR2 are symmetrically injected into the abdominal skin of a nude mouse, and the growth of a tumor body is observed and recorded. The final results show that there is a down-regulation of FOXR2The ability of WRL68 cells to form subcutaneous tumors was effectively inhibited (FIG. 6B).

Experiments prove that the expression of the FOXR2 gene in a liver cancer tissue is obviously higher than that of a tissue beside the cancer, and the growth of liver cancer cells can be obviously promoted by the over-expression of the exogenous FOXR2 gene, so that the FOXR2 gene and an expression product thereof can be used as a marker for diagnosing liver cancer and a drug target for treating the liver cancer, and the liver cancer can be diagnosed more accurately and quickly. In a word, the FOXR2 gene and the application thereof provide a new therapeutic target spot and an effective new medicine for preventing and treating liver cancer.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (18)

1. Use of a detection reagent for FOXR2 gene or a detection reagent for FOXR2 protein, characterized in that it is used to prepare a reagent or kit for detecting liver cancer. 2 . The use according to claim 1 , wherein the kit comprises: reagents for quantitatively detecting FOXR2 protein or mRNA and corresponding labels or instructions. 3 . 3. The use according to claim 1, wherein the reagents comprise FOXR2-specific primers, specific antibodies, probes and/or chips. 4. The use of claim 3, wherein the reagents comprise nucleic acid chips and protein chips. 5. purposes as claimed in claim 4 is characterized in that, described nucleic acid chip comprises substrate and the specific oligonucleotide probe of liver cancer-related gene spotted on the substrate, described liver cancer-related gene Specific oligonucleotide probes include probes that specifically bind to FOXR2 mRNA. 6. The use according to claim 4, wherein the protein chip comprises a substrate and a specific antibody for liver cancer-related protein spotted on the substrate, and the specific antibody for the liver cancer-related protein includes an anti-HCC antibody. Antibodies specific for FOXR2 protein. 7. The use according to claim 1, wherein the FOXR2 protein comprises fusion protein and non-fusion protein. 8. purposes as claimed in claim 2 is characterized in that, the following content is indicated in described label or instruction manual: When the ratio of the mRNA expression of FOXR2 relative to the reference protein of the test object to the mRNA expression of FOXR2 relative to the reference protein in the adjacent tissue is greater than or equal to 1.5, it indicates that the probability of the test object suffering from liver cancer is higher than that of the general population. 9. Use of a FOXR2 protein inhibitor or a FOXR2 gene inhibitor, characterized in that it is used to prepare a drug for inhibiting the growth or proliferation of liver cancer cells. 10. The use according to claim 9, wherein the inhibitor comprises: an activity inhibitor of FOXR2. 11. The use of claim 10, wherein the FOXR2 activity inhibitor comprises FOXR2 antibody, FOXR2 nucleic acid antisense RNA, siRNA, and shRNA. 12. The use according to claim 10, wherein the inhibitor is si-1 of the FOXR2 gene, the sequence of which is shown in SEQ ID NO.: 9-10. 13. The use according to claim 10, wherein the inhibitor is si-2 of the FOXR2 gene, the sequence of which is shown in SEQ ID NO.: 11-12. 14. An in vitro non-therapeutic method for inhibiting the growth or proliferation of liver cancer cells, comprising the step of: culturing liver cancer cells in the presence of an inhibitor of FOXR2 protein, thereby inhibiting the growth or proliferation of liver cancer cells. 15. The method of claim 14, wherein the method comprises adding a FOXR2 inhibitor to the culture system of liver cancer cells, thereby inhibiting the growth or proliferation of liver cancer cells. 16. A method for screening candidate compounds for the treatment of liver cancer, wherein the method comprises the steps of: (a) In the test group, the test compound was added to the cell culture system, and the expression and/or activity of FOXR2 in the cells of the test group were observed; in the control group, the test compound was not added to the same cell culture system compound, and observe the expression and/or activity of FOXR2 in the cells of the control group; Wherein, if the FOXR2 expression and/or activity of the cells in the test group is lower than that of the control group, it indicates that the test compound is a candidate compound for treating liver cancer with inhibitory effect on the expression and/or activity of FOXR2. 17. The method of claim 16, further comprising the step of: (b) The candidate compound obtained in step (a) is further tested for its inhibitory effect on the growth or proliferation of liver cancer cells. 18. The method of claim 17, wherein the step (b) comprises a step: in the test group, the test compound is added to the culture system of the cancer cells, and the number and/or growth of the cancer cells are observed In the control group, the test compound was not added to the culture system of cancer cells, and the number and/or growth of cancer cells were observed; wherein, if the number or growth rate of cancer cells in the test group was lower than that of the control group, it indicated that the The test compound is a candidate compound for the treatment of liver cancer that has an inhibitory effect on the growth or proliferation of cancer cells.

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