CN1292078C - Hepatic carcinoma tissue specific expression genes and use thereof - Google Patents

Abstract

The present invention provides a method for diagnosing individual susceptibility to tumors (such as liver cancer), which includes the steps of: detecting the expression level of liver cancer-related genes in the individual, and comparing it with the expression level of liver cancer-related genes in the normal population. The difference indicates that the possibility of the individual suffering from tumor is higher than that of the normal population, wherein the liver cancer-related gene is selected from Beclin1, Lis1, Pir51, RbAp48, or a combination thereof, and the difference is an up-regulation of expression. The invention also provides a corresponding detection kit.

Description

Specific expression genes of liver cancer tissue and its application

technical field

The invention relates to human liver cancer-related genes and applications. More specifically, the present invention relates to methods and kits for detecting tumor susceptibility by using up-regulated liver cancer-related genes in liver cancer.

Background technique

The mortality rate of malignant tumors ranks second only to heart and cerebrovascular diseases in my country. Among malignant tumors, the mortality rate of liver cancer ranks third after lung cancer and gastric cancer. Currently, there is no effective diagnosis and treatment for liver cancer. It is generally believed that carcinogenesis is a multi-factor and multi-step complex event, which is the result of the activation of various oncogenes and the inactivation of tumor suppressor genes.

About 110,000 people die of liver cancer every year in my country, including 80,000 men and 30,000 women, accounting for 45% of the world's liver cancer deaths. The trend of high incidence of liver cancer in our country is very serious, because there are 120 million HBV carriers in our country. The study of molecular biology of liver cancer is a new field that has developed rapidly in recent years, and it provides new materials for the occurrence and development of liver cancer and its relationship with hepatitis B virus. At present, the discovery of oncogenes and tumor suppressor genes of liver cancer, and the research on the infection of liver cancer and HBV have made rapid progress.

Normal human proto-oncogenes and tumor suppressor genes restrict each other and control the normal proliferation and differentiation of cells, tissue regeneration and individual development. A large part of the occurrence of cancer is due to the activation of proto-oncogenes into oncogenes and the inactivation of anti-cancer genes due to the action of viruses or chemical carcinogens, resulting in uncontrolled cell growth. The occurrence of cancer involves a variety of factors and is formed in multiple stages, so there may be activation or inhibition of multiple cancer-related genes in different periods. The activation or inhibition of cancer-related genes is manifested in abnormal gene expression compared with normal tissues or cells, increased or decreased expression, and even mutations in gene structure.

Because cancer is one of the main diseases that endanger human health. In order to effectively treat and prevent tumors (such as liver cancer), people have paid more and more attention to the early diagnosis and gene therapy of tumors. However, the number of liver cancer-related genes discovered so far is still very limited.

Therefore, there is an urgent need in this field to develop and research new cancer-related and/or human proteins with tumor suppressor functions, as well as corresponding detection kits.

Contents of the invention

The object of the present invention is to provide a new tumor-associated protein-human liver cancer-associated protein and fragments, analogs and derivatives thereof.

Another object of the present invention is to provide polynucleotides encoding these polypeptides.

Another object of the present invention is to provide a method and a kit for detecting tumor susceptibility using liver cancer-related genes.

In the first aspect of the present invention, there is provided a method for in vitro detection of whether the expression level of liver cancer-related genes in the cells to be tested is up-regulated, which includes the steps of:

(a) detecting the expression level of liver cancer-related genes in the cells to be tested, wherein the liver cancer-related genes are selected from: Beclin1, Lis1, Pir51, RbAp48, or a combination thereof;

(b) Comparing the expression level of the liver cancer-related gene in the test cell with the control, so as to determine whether the expression level of the liver cancer-related gene is up-regulated.

In another preferred example, the criterion for judging up-regulation in step (b) is that the expression level of liver cancer-related genes is 1.5-100 times higher than the expression level of liver cancer-related genes in normal population.

In another preferred example, the criterion for judging up-regulation in step (b) is that the expression level of liver cancer-related genes is 3-50 times higher than the expression level of liver cancer-related genes in normal population.

In another preferred example, the criterion for judging up-regulation in step (b) is that the expression level of liver cancer-related genes is 4-25 times higher than the expression level of liver cancer-related genes in normal population.

In another preferred example, in step (a), 2-4 liver cancer-related proteins among Beclin1, Lis1, Pir51 and RbAp48 are detected.

In another preferred embodiment, the cells to be tested in step (a) are hepatocytes.

In another preferred example, the detection in step (a) is performed by nucleic acid chip detection method, semi-quantitative PCR method, quantitative PCR method, protein chip detection method, or antigen-antibody detection method.

In the second aspect of the present invention, a kit for detecting tumor susceptibility is provided, which includes primers and instructions for specifically amplifying liver cancer-related gene transcripts, and the liver cancer-related genes are selected from the group consisting of Beclin1, Lis1 , Pir51, RbAp48, or a combination thereof.

In another preferred example, the primers are selected from the following group:

(i) Beclin1 specific primer, said primer has the sequence shown in SEQ ID NO: 1 and 2;

(ii) Lis1 specific primer, said primer has the sequence shown in SEQ ID NO: 3 and 4;

(iii) Pir51-specific primers, said primers have the sequences shown in SEQ ID NO: 5 and 6;

(iv) RbAp48 specific primer, described primer has the sequence shown in SEQ ID NO:7 and 8.

In another preferred example, the kit also contains primers for amplifying other liver cancer-related gene transcripts, such as specific primers for amplifying aldolase b (SEQ ID NO: 9 and 10).

In another preferred example, the kit for detecting tumor susceptibility includes antibodies and instructions that specifically bind to the expression products of liver cancer-related genes, and the liver cancer-related genes are selected from the group consisting of Beclin1, Lis1, Pir51, RbAp48, or its combination.

In another preferred example, the tumor susceptibility kit includes a chip for detecting liver cancer-related genes and instructions, and the chip is selected from the following group:

(a) a DNA chip, the chip has a substrate and spotting DNA spotted on the spotting area on the substrate, the spotting DNA includes probes that specifically bind to liver cancer-related gene transcripts, and the liver cancer Related genes are selected from the group consisting of Beclin1, Lis1, Pir51, RbAp48, or a combination thereof;

(b) a protein chip, the chip has a substrate and a spotting protein that is spotted on the spotting area on the substrate, and the spotting protein includes an antibody that specifically binds to the expression product of a liver cancer-related gene, and the liver cancer The relevant gene is selected from the group consisting of Beclin1, Lis1, Pir51, RbAp48, or a combination thereof.

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

Description of drawings

Figure 1 shows the difference in gene expression between human liver cancer tissue and adjacent normal tissue analyzed by microarray hybridization technology.

Figure 2 shows the detection of the expression of four genes Beclin1, RbAp48, Aldolase b and Pir51 in liver cancer and adjacent normal tissues. T is liver cancer tissue, N is adjacent normal tissue.

Figure 3 shows the expression detection of Beclin1, RbAp48, Aldolase b, and Pir51 four genes in liver cancer cells.

Figure 4 shows the expression detection of Lis1 gene in normal liver (N), liver cancer (K) and adjacent normal liver tissue (L). The amount of total RNA on the membrane was 5 μg/point, 10 μg/point, and 20 μg/point, respectively. The hybridization result of β-actin (β-actin) gene was used as a control.

Detailed ways

After in-depth and extensive research, the inventor has newly discovered and isolated a group of liver cancer-related genes and their encoded proteins. This group of genes (Beclin1, Lis1, Pir51, RbAp48) are specifically highly expressed in liver cancer, so It can be used as a marker for the occurrence and development (especially early diagnosis) of liver cancer and a target for gene therapy.

In addition, the inventors have also discovered and isolated the liver cancer-related gene Aldolase b (aldolase b) that is specifically low-expressed (or even non-expressed) in liver cancer, so the gene can also be used as a marker for the occurrence and development of liver cancer (especially for early diagnosis). ) markers and targets for gene therapy.

Specifically, the present invention relates to the use of cDNA microarray technology to screen differentially expressed genes between normal liver and liver cancer, and the expression profiles of 8,400 genes have been obtained. Comparing the gene expression profiles of normal liver tissue and liver cancer tissue, 256 genes were up-regulated and 267 genes were down-regulated, and the up-regulation or down-regulation of these genes was more than 4 times. The present invention discloses that the 5 identified genes are differentially expressed in more than 40% of the samples. The differential expression percentages of these five five genes: 40% for Lis1, 50% for Beclin1, 50% for RbAp48, 60% for Pir51, and 70% for Aldolase b.

The present invention also adopts Northern blot method and RT-PCR method to detect the expression of the above gene in normal liver tissue and liver cancer tissue.

In the Northern blot method, the total RNA from the liver cancer tissue and adjacent normal liver tissue from the same patient was first separated and separated by 1% agarose electrophoresis containing formaldehyde, then transferred to a nylon membrane and fixed, and then gene Specific fragments are prepared for probe hybridization, and the expression strength of the gene is determined according to the hybridization signal.

The results showed that the expression levels of Beclin1 and RbAp48 were significantly increased in more than 50% of the selected tumor samples, but could not be detected in normal liver tissues; the expression level of Aldolase b (aldolase b) was significantly increased in 70% of the selected tumor samples reduce.

In the RT-PCR detection method, the isolated and prepared total RNA is firstly reverse-transcribed to synthesize cDNA, and then the gene-specific primers provided in our Table 1 are used for semi-quantitative PCR amplification, and the amplified products are subjected to agarose gel electrophoresis. Compare the intensity of electrophoresis bands to determine the level of gene expression products.

The results showed that the pir51 gene was upregulated in 60% of the selected tumor samples. The Lis1 gene was upregulated in 70% of selected tumor samples.

The inventor also used RT-PCR and Northern blotting to detect the expression of five genes in six liver cancer cell lines L02, Bel-7402, Bel-7404, SMMC-7721, HepG2, and Huh7.

It was found that Lis1 was expressed in 6 kinds of cells; Beclin1 was expressed in L02, Bel-7402, HepG2, SMMC-7721, but not in Bel-7404, Huh7; Pir51 was expressed in L02, Bel-7402, Bel-7404, Expressed in SMMC-7721, HepG2; RbAp48 expressed in L02, Bel-7404, Huh7, HepG2, Bel-7402 cells; Aldolase b was only weakly expressed in HepG2 cells, but not expressed in other liver cancer cells. These results are consistent with those detected in tissue samples.

Therefore, Beclin1, Lis1, Pir51, and RbAp48 are specifically highly expressed liver cancer-related genes in liver cancer, and aldolase b is a specifically low-expressed (or even non-expressed) liver cancer-related gene in liver cancer.

definition

As used herein, the term "liver cancer-related genes of the present invention" refers to Beclin1, Lis1, Pir51, RbAp48 whose expression is upregulated in liver cancer. Sometimes the term also includes the Aldolaseb (aldolase b) gene whose expression is downregulated in liver cancer.

As used herein, the term "hepatoma-associated protein of the present invention" refers to Beclin1, Lis1, Pir51, RbAp48 proteins whose expression is upregulated in liver cancer. Sometimes the term also includes Aldolase b (aldolase b) protein whose expression is downregulated in liver cancer.

As used herein, "nucleic acid" refers to a polymer of deoxyribonucleotides or ribonucleotides in single- or double-stranded form. Unless otherwise stated, the term includes analogs of natural nucleotides that are capable of functioning in a similar manner to natural nucleotides.

"Subsequence" refers to a nucleic acid sequence that contains a portion of a nucleic acid of a longer sequence of nucleic acids.

"Probe" or "nucleic acid probe" refers to a collection of one or more nucleic acid fragments whose hybridization to a target can be detected. The probe is labeled as described below so that its binding to the target can be detected. Probes are prepared from one or more specific portions of the genome, such as one or more clonally isolated complete chromosomes or chromosome fragments or polymerase chain reaction (PCR) products. Probes can be processed in some way, for example by blocking or removing repetitive nucleic acids or enriching for unique nucleic acids. The term "probe" thus refers not only to a detectable nucleic acid, but also to a detectable nucleic acid applied to a target.

"Hybridization" refers to the bringing together of two single-stranded nucleic acids through complementary base pairing.

"Substantially binds to" or "specifically binds to" or "selectively binds to" or "specifically hybridizes to" refers to a complementary hybridization reaction between an oligonucleotide and a target sequence, and may include minor mismatches, which Matching can be achieved by reducing the stringency of the hybridization medium to detect the desired target polynucleotide sequence. The term also refers to a molecule that binds, complexes or hybridizes to a specific nucleotide sequence under stringent conditions when the sequence is present in a complexed mixture (eg, total cellular NDA or RNA). The term "stringent conditions" refers to conditions under which a probe hybridizes to a targeted subsequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) of the putative sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which, at equilibrium, about 50% of probes complementary to the target sequence hybridize to the target sequence. Generally, for short probes, stringent conditions are those in which the salt concentration is at least about 0.02 Na ion concentration (or other salts), pH 7.0-8.3, and temperature is at least about 60°C. Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide.

Those skilled in the art will appreciate that the exact sequences of the specific probes described herein may be modified (modified) to some extent to produce probes that are "substantially identical" to the disclosed probes but retain substantially the ability to bind to the target sequence. probe. These modifications are covered by each probe herein. The term "substantially identical" to polynucleotides means that a sequence has at least 90%, more preferably at least 95%, sequence identity compared to a reference sequence.

Two nucleotide sequences are considered "identical" if, when aligned for maximum correspondence, the sequence of nucleotides in the two sequences is the same. As used herein, the term "complementary" means that the complementary sequence is sequential to all or a portion of the reference sequence.

Sequence comparison between two (or more) polynucleotides generally involves comparing the sequences of the two sequences within a "comparison window" to identify and compare local regions of sequence identity. As used herein, a "comparison window" refers to at least about 20 contiguous positions, usually about 50-200, and more usually about 100-150 contiguous positions, in which a sequence is compared with a sequence having the same Number of contiguous loci for comparison with the reference sequence.

"Percent sequence identity" is determined by comparing two optimally aligned sequences over a comparison window. Wherein, compared with a reference sequence (without insertion or deletion), in order to obtain an optimal alignment of the two sequences, the polynucleotide sequence in the comparison window may contain insertions or deletions (ie, gaps). Percentages are calculated by determining the number of identical nucleic acid bases or amino acid residues in the two sequences to give the number of matching positions. The percent sequence identity is then obtained by dividing the number of matching positions by the total number of positions in the comparison window and multiplying the result by 100%.

Another way to indicate that nucleotide sequences are substantially identical is to determine whether two molecules hybridize to the same sequence under stringent conditions. Stringent conditions are sequence-dependent and will be different in different circumstances.

Mark

Methods of labeling nucleic acid probes are well known to those skilled in the art. Preferred probes are those suitable for in situ hybridization. Nucleic acid probes can be detectably labeled prior to the hybridization reaction. Alternatively, a detectable label that binds to the hybridization product is used. Such detectable labels are well known in the art and include any substance having a detectable physical or chemical property.

As used herein, a "label" is any substance detectable by spectroscopic, photoelectric, biochemical, immunochemical, or chemical methods. Labels that can be used in the present invention include: radioactive labels (such as ³² P, ¹²⁵ I, ¹⁴ C, ³ H, ³⁵ S), fluorescent dyes (such as fluorescein, rhodamine), electron-dense reagents (such as) gold, enzymes (such as enzymes commonly used in ELISA), colorimetric labels (such as colloidal gold), magnetic labels (such as Dynabeads ^TM ), etc. Examples of labels that are not directly detectable but can be detected through the use of direct detection labels include biotin, digoxin, and haptens and proteins for which labeled antisera or mAbs are available.

The particular marker used is not critical to the invention, however fluorescent markers (eg fluorescein-12-dUTP etc.) are preferred.

As used herein, a directly labeled probe is a probe to which a detectable label is attached. Because the direct label is already attached to the probe, no subsequent steps are required to associate the probe with a detectable label. In contrast, indirectly labeled probes are probes that carry a molecule that subsequently (usually after hybridization of the probe to a target nucleic acid) binds a detectable label.

In addition, the probe should detect as low a copy number as possible to maximize the sensitivity of the assay while maintaining a signal above background. Finally, it is essential to select probes that provide high localized signal, thus providing high spatial resolution in physically localizing staining results to chromosomes,

Labels can be coupled to probes using a variety of methods known to those skilled in the art. In preferred embodiments, nucleic acid probes are labeled by nick translation or random primer extension.

Those skilled in the art will understand that the probes of the present invention do not need to be absolutely specific to liver cancer-related genes. Instead, the probes are used to create "staining contrast". "Contrast" is determined by the ratio of the concentration of the probe in the target region of the genome to the concentration of the probe in other regions of the genome.

As described above, the detection of amplification of liver cancer-related genes is a marker of the presence and prognosis of various cancers, especially liver cancer.

In a preferred embodiment, the amplification of liver cancer-related genes is detected by hybridizing the probe of the present invention to a target nucleic acid (such as a chromosome sample). Suitable hybridization methods are known to those skilled in the art, including (but not limited to): various Northern blotting methods, in situ hybridization and quantitative amplification methods (such as quantitative PCR and semi-quantitative PCR).

In a preferred example, the amplification of liver cancer-related genes is identified by in situ hybridization. Generally, in situ hybridization includes the following main steps: (1) fixing the tissue or biological structure to be analyzed; (2) pre-hybridizing the biological structure to increase the accessibility (accessibility) of the target DNA and reduce non-specific binding; ( 3) hybridizing the nucleic acid mixture to nucleic acid in a biological structure or tissue; (4) performing a post-hybridization wash to remove unhybridized nucleic acid in the hybridization reaction; and (5) detecting hybridized nucleic acid fragments. Depending on the specific application, the reagents used in each step and the conditions of use can vary.

Examples of samples for testing include small biopsies from tumors, especially hepatocytes and liver tissue.

In Northern blotting, reverse transcribed cDNA is hybridized with probes specific for the target region. Hybridization signal intensities from probes directed to the target region are compared to control signal intensities to provide an estimate of the relative copy number of the target nucleic acid.

Detection method

Methods for detecting the expression level of a gene in a test cell are known in the art. Representative examples include: Northern hybridization, quantitative and semi-quantitative PCT (including RT-PCR), DNA chip detection method, protein chip detection method, antigen-antibody detection method (such as ELISA method). In addition, in situ hybridization can also be used. All of these methods described above can be used in the present invention. Related instruments and reagents can be purchased from companies such as Sigma.

Nucleic acid, protein and antibody of liver cancer-related genes

Based on the correlation between liver cancer-related genes and liver cancer and the sequence information of liver cancer-related genes disclosed in the present invention, use conventional techniques in the art to generate liver cancer-related proteins or fragments, and use corresponding liver cancer-related proteins or fragments to generate anti-liver cancer-related genes antibodies.

Human liver cancer-related genes (Beclin1, Lis1, Pir51, RbAp48 and Aldolase b) of the present invention are all known proteins before the present invention, and their basic information is as follows: Genbank accession number Beclin1 NM-003766 Lis1 NM-000430 Pir51 NM-006479 RbAp48 NM-005610 Aldolase b NM-000035

Beclin1 is a Bcl-2 binding protein, which is missing in breast cancer and ovarian cancer, and may be a tumor suppressor gene in breast cancer. In addition, the overexpression of Beclin1 in nerve cells can inhibit the replication of Sindbis virus and reduce the apoptosis of the central nervous system.

The Lis1 gene has been reported to be associated with the occurrence of human lissencephaly I. Lis1 protein is highly conserved during evolution and contains 7 WD repeats, which can mediate protein-protein interactions. Lis1 is a subunit of platelet-activating factor acetylhydrolase. In addition, Lis1 also binds to tubulin and directly affects the movement of microtubules. Overexpression of Lis1 can affect cell division by affecting the combination of spindle and chromosome.

Pir51 is the binding protein of human Rad51 recombinase. The Rad51 protein of eukaryotic cells plays an important role in homologous recombination and is also necessary for cell proliferation. Pir51 binds to Rad51 to regulate the function of Rad51.

RbAp48 is an Rb-binding protein. RbAp48 is also a subunit of chromatin assembly factors and a member of the histone deacetylase complex. RbAp48 also binds to CREB. The function of RbAp48 is very complex, so far, it is not very clear.

Aldolase b is a key enzyme in the metabolism of glucose and fructose, and a gene specifically expressed in the liver.

The nucleotide full-length sequence or its fragments of the human liver cancer-related gene of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the published relevant nucleotide sequences, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art can be used as Template, amplified to obtain related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

At present, the DNA sequence encoding the protein of the present invention (or its fragments, or its derivatives) can be completely chemically synthesized. This DNA sequence can then be introduced into various DNA molecules (such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

Fragments of the protein of the invention, in addition to being produced by recombinant methods, can also be produced by direct peptide synthesis using solid-phase techniques (Stewart et al., (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco; Merrifield J. ( 1963) J. Am Chem. Soc 85:2149-2154). Protein synthesis in vitro can be performed manually or automatically. For example, peptides can be synthesized automatically using an Applied Biosystems Model 431A Peptide Synthesizer (Foster City, CA). Fragments of a protein of the invention can be chemically synthesized separately and then chemically linked to produce a full-length molecule.

On the other hand, the present invention also includes polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies, specific to human liver cancer-related gene DNA or polypeptides encoded by its fragments. Here, "specificity" means that the antibody can bind to gene products or fragments of human liver cancer-related genes. Preferably, it refers to those antibodies that can bind to gene products or fragments of human liver cancer-related genes but do not recognize and bind to other irrelevant antigen molecules. Antibodies in the present invention include those molecules that can bind to and inhibit human liver cancer-related proteins, as well as those that do not affect the function of human liver cancer-related proteins. The present invention also includes those antibodies that bind to the gene products of human liver cancer-associated genes in modified or unmodified forms.

The present invention includes not only complete monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) ₂ fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules; Or chimeric antibodies, such as antibodies that have the binding specificity of a murine antibody but retain portions of the antibody from humans.

Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, purified human liver cancer-related gene gene products or antigenic fragments thereof can be administered to animals to induce polyclonal antibody production. Similarly, cells expressing human liver cancer-related genes or antigenic fragments thereof can be used to immunize animals to produce antibodies. Antibodies of the invention may also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology. The antibody of the present invention includes the antibody capable of blocking the function of the human liver cancer related gene and the antibody not affecting the function of the human liver cancer related gene. Various types of antibodies of the present invention can be obtained by conventional immunization techniques using fragments or functional regions of human liver cancer-related gene product. These fragments or functional regions can be prepared using recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to unmodified forms of human liver cancer-related gene gene products can be produced by immunizing animals with gene products produced in prokaryotic cells (such as E. Coli); antibodies that bind to post-translationally modified forms (such as glycosylation or phosphorylation (translated protein or polypeptide), which can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell (eg, yeast or insect cell).

Based on the specific anti-liver cancer-related protein antibody, the present invention also provides a method for detecting tumor or tumor susceptibility, which includes the steps of:

Under conditions suitable for the formation of antibody complexes, contact the antibodies containing specific anti-hepatoma-related proteins (Beclin1, Lis1, Pir51, RbAp48) with the DNA samples of the individual to be tested; detect whether the number of antibody complexes formed is higher than normal In the control, wherein the amount of antibody complexes formed is higher than that in the normal control, it means that the individual has a tumor or the susceptibility to the tumor is higher than that of the normal population.

In addition, this method can also be combined with the detection of other liver cancer-related genes (such as Aldolase b) whose expression is down-regulated in liver cancer. b) The antibody is contacted with the DNA sample of the individual to be tested; and detecting whether the amount of antibody complexes formed is lower than that of a normal control, wherein the amount of antibody complexes formed is lower than that of a normal control, which means that the individual has a tumor or a susceptibility to a tumor higher than the normal population.

Reagent test kit

The invention also provides a diagnostic kit for diagnosing liver cancer-related gene abnormalities. In a preferred example, a kit includes one or more probes for genes related to liver cancer. The kit may also contain blocking probes and instructional materials describing how to use the kit to detect genes associated with liver cancer. Kits may also contain one or more of the following: various labels or labeling reagents to aid in the detection of probes; reagents for hybridization (including buffers, etc.); and positive and negative hybridization controls, among others.

Another preferred test kit for detecting tumor susceptibility includes primers and instructions for specifically amplifying liver cancer-related gene transcripts, and the liver cancer-related gene is selected from the group consisting of Beclin1, Lis1, Pir51, RbAp48, Aldolase b, or combination.

Another preferred test kit for detecting tumor susceptibility includes antibodies and instructions that specifically bind to the expression products of liver cancer-related genes, and the liver cancer-related genes are selected from the group consisting of Beclin1, Lis1, Pir51, RbAp48, Aldolase b or its combination.

Another preferred test kit for detecting tumor susceptibility includes a chip for detecting liver cancer-related genes and instructions, and the chip is selected from the following group:

(a) a DNA chip, the chip has a substrate and spotting DNA spotted on the spotting area on the substrate, the spotting DNA includes probes that specifically bind to liver cancer-related gene transcripts, and the liver cancer Related genes are selected from the group consisting of Beclin1, Lis1, Pir51, RbAp48, Aldolase b or combinations thereof;

(b) a protein chip, the chip has a substrate and a spotting protein that is spotted on the spotting area on the substrate, and the spotting protein includes an antibody that specifically binds to the expression product of a liver cancer-related gene, and the liver cancer Related genes are selected from the group consisting of Beclin1, Lis1, Pir51, RbAp48, Aldolase b or combinations thereof.

The main advantage of the present invention is that: the present invention discovers new liver cancer-related genes for the first time, which not only fills the blind area of detection, but also these liver cancer-related genes can be used as targets for liver cancer diagnosis, gene therapy and drug therapy.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions.

Example 1

To establish the gene differential expression profiles of HCC and adjacent normal liver tissues.

Liver cancer and adjacent normal liver tissues preserved in liquid nitrogen were taken, and total RNA was separated and extracted according to conventional methods. Trizol reagent (Life Teclonologies, Inc, N.Y., USA) was used for RNA extraction. For every 50-100mg tissue sample, add 1ml Trizol, homogenate, and stand at room temperature for 5 minutes; then, add 0.2ml chloroform, shake vigorously, mix well, and let stand at room temperature for 2-3 minutes; centrifuge at 12,000g, 4°C for 15 minutes; Pipette the supernatant into another Eppendorf tube, add an equal volume of isopropanol, mix well, and let stand at room temperature for 10 minutes. Total RNA was collected by centrifugation at 12,000 g at 4°C, washed once with 75% ethanol, drained, and dissolved in sterile water. Total RNA was used for mRNA preparation after denatured formaldehyde agarose electrophoresis to check that there was no price reduction. 200ug of total RNA was used in one reaction to isolate and prepare mRNA, and the Oligotex mRNA minkit (Qiagen, Hilden, Germany) kit was used for the isolation of mRNA.

The cDNA microarray adopts LifeGrid 1.0 microarray membrane purchased from Incyte Genomic Company, which contains about 8,400 cDNA PCR products and 27 controls, and is printed on a 12×22cm nylon membrane in a double-dot pattern. 33P-labeled cDNA probes were generated by reverse transcription of mRNA from liver cancer and adjacent normal liver tissue in the presence of a-33P dCTP. The percentage of label incorporation is not less than 40%. For the hybridization conditions of each cDNA probe to the microarray, please refer to the instructions of the LifeGrid 1.0 microarray. After hybridization overnight, the membrane was dried at room temperature, exposed and scanned by a phosphor imager, and the scanning results, namely the computer analysis and interpretation of the hybridization signal, were completed by Incyte.

According to the computer analysis results provided by Incyte, the expression profiles of 8,400 genes in liver cancer and adjacent normal liver tissues were obtained (Fig. 1). Comparing the gene expression profiles of normal liver tissue and liver cancer tissue, removing genes whose expression is missing in both normal liver tissue and liver cancer tissue, and removing genes whose difference between two points of the same cDNA is greater than 2.5 times and one point is less than 2 times the background. Finally, 6542 gene expressions were detected in normal liver tissue and liver cancer tissue. Through comparative analysis, it was found that 256 genes were up-regulated and 267 genes were down-regulated in liver cancer. These up-regulated or down-regulated genes all refer to expression differences greater than 4 times. From these differentially expressed genes, genes related to cell division, gene expression regulation, signal transduction, metabolism, etc. were selected for verification. Beclin1, Lis1, Pir51, RbAp48, and Aldolase b are five genes with high or low expression in liver cancer.

Example 2

Expression detection of four genes Beclin1, RbAp48, Aldolase b and Pir51 in liver cancer and adjacent normal tissues

Total RNA was prepared according to the method of Example 1, and cDNA was prepared with SuperscripII reverse transcriptase (Life Technologies Inc, NY, USA). The reverse transcription conditions were as specified in the kit, and 3ug of total RNA was added to 20ul of the reaction mixture. PCR amplification was then performed. According to the primers provided in Table 1, PCR amplification was carried out according to the conditions listed in Table 1 to obtain fragments of the corresponding genes. β-actin gene encoding actin was used as experimental control. Gene Primer sequence (5'-3') SEQ ID NO: PCR product size (bp) Annealing temperature ℃ number of cycles Beclin1 Forward: cttaccacagcccaggcgaaac Reverse: gccagagcatggagcagcaa 12 814 58 30 Lis1 Forward: aattacagccaagatggtg Reverse: aatcaacggcactcccacac 34 1200 62 30 Pir51 Forward: gtggaagatgatgttggtggtg Reverse: aaggcggagactctgattgg 56 527 58 32 RbAp48 Forward: gaactgcctttctttcaatc Reverse: atggctcagacacctacctc 78 826 58 30 Aldolase b Forward: gccactctcaacctcaatgc Reverse: tctccttcccaacctaccac 910 423 55 32 β-actin Forward: tgacggggtcacccaacactgtgcc Reverse: cttagaagcattgcggtggacgatg 1112 666 60 25

(a) Northern blot analysis

Isotope α- ³² P-dCTP labeled gene fragments were used as probes for Northern blot analysis: about 15ug of total RNA from liver cancer tissue or adjacent normal tissue was separated by formaldehyde-containing 1% agarose gel electrophoresis in each lane, and then Transfer to Hybond-N+ nylon membrane (Amersham Pharmacia Biotech). Bake the membrane at 80°C for 1 hour to immobilize the RNA on the membrane. Hybridization was performed at 68° C. for 3 hours in ExpressHyb hybridization solution (ClonTech Inc, California, USA). Wash the membrane once with 2×SSC containing 0.1% SDS at room temperature, then wash twice with 0.1×SSC containing 0.1% SDS at 68°C, 20 minutes each time, and finally press for 1 to 6 days at -80°C , autoradiography. For the specific operation of Northern blot, see the laboratory manual of 'Molecular Cloning' written by Sambrook et al.

The detection results are shown in Figure 2. The detection of Beclin1, RbAp48 and Aldolase b was performed by the Northern blot method. The expression level of pir51 was too low, and a more sensitive PT-PCR method was used. The expression levels of Beclin1 and RbAp48 were significantly increased in tumor samples, but could not be detected in normal liver tissues; the expression level of Aldolase b was significantly decreased in 70% of the selected tumor samples.

(b) RT-PCR detection method

Firstly, the isolated and prepared total RNA was reverse-transcribed to synthesize cDNA, and then semi-quantitative PCR amplification was performed with the gene-specific primers provided in Table 1, and the amplified products were subjected to agarose gel electrophoresis to compare the strength of the electrophoresis bands. Determine the level of gene expression products.

The results showed that the pir51 gene was upregulated in 60% of the selected tumor samples.

Example 3

Expression detection of four genes Beclin1, RbAp48, Aldolase b, and Pir51 in liver cancer cells

The expression of Beclin1, RbAp48, pir51 and Aldolase b in 6 kinds of liver cell lines (L02, Bel-7404, SMMC-7721, Bel-7402, HuH7, HepG2) were detected by the same RT-PCR method as in Example 2.

The results found that Beclin1 was expressed in L02, Bel-7402, HepG2, SMMC-7721 cells, but not in Bel-7404 and Huh7 cells; RbAp48 was expressed in L02, Bel-7404, Huh7, HepG2, Bel-7402 cells expressed in different kinds of cells, but not in SMMC-7721 cells; Pir51 was expressed in L02, Bel-7402, Bel-7404, SMMC-7721, and HepG2 cells, but not in HuH7 cells; and Aldolase b was only expressed in It is weakly expressed in HepG2 cells, but not in other liver cancer cells. This result is consistent with the detection results in tissue samples, that is, Beclin1, RbAp48 and Pir51 are expressed in most liver cancer cells, while Aldolase b is not expressed or lowly expressed in most liver cancer cells (Figure 3).

Example 4

Detection of Lis1 Gene Expression in Liver Cancer and Paracancerous Tissues

We used Northern dot blotting to detect the expression of Lis1 gene in HCC and paracancerous tissues. Total RNA was prepared according to the method in Example 1, and total RNA from various sources was spotted on Hybond-N+ nylon membrane (Amersham Pharmacia Biotech) with 5 μg, 10 μg, and 20 μg of each point respectively. Bake the membrane at 80°C for 1 hour to immobilize the RNA on the membrane. Hybridization was carried out at 68°C for 3 hours in ExpressHyb hybridization solution (ClonTech Inc, California, USA). Wash the membrane once with 2×SSC containing 0.1% SDS at room temperature, then wash twice with 0.1×SSC containing 0.1% SDS at 68°C for 20 minutes each time, and finally press for 1 to 3 days at -80°C , autoradiography. For the specific operation of Northern Dot Blot, see "Molecular Cloning: A Laboratory Manual" by Sambrook et al.

The experimental results showed that the expression of Lis1 gene was up-regulated in liver cancer tissues, but not or lowly expressed in normal liver tissues and paracancerous tissues ( FIG. 4 ).

Example 5

Liver Cancer Susceptibility Detection Kit

A kit is prepared which contains:

Name Beclin1 Primer Concentration

Forward primer SEQ ID NO: 1 Dry powder 20D

Reverse primer SEQ ID NO: 2 Dry powder 20D

PCR reaction solution containing Taq enzyme dNTP magnesium ion PCR reaction buffer

Example 6

Liver Cancer Susceptibility Detection Kit

A kit is prepared which contains:

Name Lis1 Primer Concentration

Forward primer SEQ ID NO: 3 Dry powder 20D

Reverse primer SEQ ID NO: 4 Dry powder 20D

PCR reaction solution containing Taq enzyme dNTP magnesium ion PCR reaction buffer

Example 7

Liver Cancer Susceptibility Detection Kit

A kit is prepared which contains:

Name Pir51 Primer Concentration

Forward primer SEQ ID NO: 5 Dry powder 20D

Reverse primer SEQ ID NO: 6 Dry powder 20D

PCR reaction solution containing Taq enzyme dNTP magnesium ion PCR reaction buffer

Example 8

Liver Cancer Susceptibility Detection Kit

A kit is prepared which contains:

Name RbAp48 Primer Concentration

Forward primer SEQ ID NO: 7 Dry powder 20D

Reverse primer SEQ ID NO: 8 Dry powder 20D

PCR reaction solution contains Taq enzyme dNTP magnesium ion PCR reaction buffer

Example 9

Liver cancer susceptibility detection kit with multiple detection factors

A kit is prepared which contains:

Name Primer Concentration for Beclin1, Lis1, Pir51, RbAp48

Forward primer SEQ ID NO: 1, 3, 5, 7 Each Yu powder 20D

Reverse primer SEQ ID NO: 2, 4, 6, 8 Each dry powder 20D

PCR reaction solution containing Taq enzyme dNTP magnesium ion PCR reaction buffer

Example 10

Liver Cancer Susceptibility Detection Kit

A kit is prepared which contains:

Name Primer Concentration for Aldolase b

Forward primer SEQ ID NO: 9 Dry powder 20D

Reverse primer SEQ ID NO: 1O Dry powder 20D

PCR reaction solution containing Taq enzyme dNTP magnesium ion PCR reaction buffer

Take a small amount of hepatocytes from the male patient to be tested, use conventional methods (or use a specific kit) to extract mRNA from the hepatocytes, and reverse transcribe it into cDNA. The PCR primers in the liver cancer detection kit of Examples 5-9 were diluted to 1 μmol/μl, and the obtained cDNA was used as a template to carry out PCR reaction with the provided primers. The amplified products were subjected to agarose gel electrophoresis, and the intensity of the electrophoresis bands was compared to determine the level of gene expression products.

The detection results showed that the expression levels of Beclin1, Lis1, Pir51, and RbAp48 were up-regulated and the expression of aldolase b was down-regulated, indicating that the susceptibility of liver cancer was higher than that of normal people.

Example 11

Liver cancer susceptibility detection kit with multiple detection factors

A kit is prepared which contains:

Name Beclin1, Lis1, Pir51, RbAp48 Concentration

Primers for Aldolase b and

Forward primer SEQ ID NO: 1, 3, 5, 7, 9 Each dry powder 20D

Reverse primer SEQ ID NO: 2, 4, 6, 8, 10 Each dry powder 20D

PCR reaction solution containing Taq enzyme dNTP magnesium ion PCR reaction buffer

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

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Claims (11)

1. a method for in vitro detection of liver cancer-related gene expression levels in cells to be tested, characterized in that, the cells to be tested are hepatocytes, and the method comprises the steps of: (a) detecting the expression level of liver cancer-related genes in the cells to be tested, wherein the liver cancer-related genes are selected from: Beclin 1, Lis 1, Pir51, RbAp48, or a combination thereof; (b) Comparing the expression level of the liver cancer-related gene in the test cell with the control, so as to determine whether the expression level of the liver cancer-related gene is up-regulated. 2. The method according to claim 1, wherein the criterion for judging up-regulation in step (b) is that the expression level of liver cancer-related genes is 1.5-100 times higher than the expression level of liver cancer-related genes in normal population. 3. The method according to claim 1, wherein the criterion for judging up-regulation in step (b) is that the expression level of liver cancer-related genes is 3-50 times higher than the expression level of liver cancer-related genes in normal population. 4. the method for claim 1, is characterized in that, detects 2-4 kinds in Beclin 1, Lis 1, Pir51, RbAp48 in step (a). 5. The method according to claim 1, wherein the criterion for judging up-regulation in step (b) is that the expression level of liver cancer-related genes is 4-25 times higher than the expression level of liver cancer-related genes in normal population. 6. The method according to claim 1, wherein the detection in step (a) is carried out by nucleic acid chip detection method, semi-quantitative PCR method, quantitative PCR method, protein chip detection method, or antigen-antibody detection method . 7. the purposes of a kind of liver cancer related gene, described liver cancer related gene is selected from: Beclin 1, Lis 1, Pir51, RbAp48 or its combination, it is characterized in that, described liver cancer related gene is used for preparing and detecting liver cancer or liver cancer Susceptibility Kit. 8. The use according to claim 7, said kit comprising primers and instructions for specifically amplifying said liver cancer-related gene transcripts. 9. purposes as claimed in claim 8, is characterized in that, described primer is selected from the group: (i) Beclin 1 specific primer, said primer has the sequence shown in SEQ ID NO: 1 and 2; (ii) Lis 1 specific primer, said primer has the sequence shown in SEQ ID NO: 3 and 4; (iii) Pir51-specific primers, said primers have the sequences shown in SEQ ID NO: 5 and 6; (iv) RbAp48 specific primer, described primer has the sequence shown in SEQ ID NO:7 and 8. 10. The use according to claim 7, characterized in that the kit comprises an antibody specifically binding to the expression product of the liver cancer-related gene and instructions. 11. The use according to claim 7, wherein the kit includes a chip for detecting liver cancer-related genes and instructions, and the chip is selected from the group consisting of: (a) a DNA chip, the chip has a substrate and spotting DNA spotted on the spotting area on the substrate, the spotting DNA includes probes that specifically bind to the liver cancer-related gene transcripts; (b) a protein chip, the chip has a substrate and a spotting protein spotted on the spotting area on the substrate, and the spotting protein includes an antibody specifically binding to the expression product of the liver cancer-related gene.

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