CN104630338B - Application of RRM2B gene or protein thereof in liver cancer metastasis - Google Patents

Abstract

The invention discloses an application of RRM2B gene or protein thereof in liver cancer metastasis. Specifically, the invention provides an application of ribonucleotide reductase subunit M2B (riboside-diphosphate reductase subunit M2B, RRM2B) gene or protein detection reagent thereof in preparing a kit for diagnosing liver cancer cell metastasis. In addition, the invention also provides the effect of RRM2B on inhibiting liver cancer metastasis. Provides a new way for diagnosing and treating liver cancer metastasis.

Description

Application of RRM2B gene or protein thereof in liver cancer metastasis

Technical Field

The invention relates to the field of tumor diagnosis and treatment. In particular, the invention relates to application of RRM2B gene or protein thereof in diagnosis and treatment of liver cancer metastasis.

Background

Primary liver cancer is a malignant tumor seriously harming human health, and the east asia including china is a high incidence area of liver cancer, and has become the second lethal tumor in china due to high malignancy and poor prognosis. Wherein hepatocellular carcinoma (HCC) accounts for more than 90% of primary liver cancer. Research shows that the occurrence of liver cancer is closely related to viral hepatitis, food polluted by ingested aflatoxin and the like, and the occurrence and development of the liver cancer are closely related to abnormal expression of oncogene and inactivation of cancer suppressor gene. The molecular mechanism of HCC development is quite complex and is not fully understood at present. Therefore, the method further explores and researches the relationship between new gene function change and the occurrence and development of the liver cancer and the malignant characteristics of the liver cancer, and has important significance for disclosing the precise molecular mechanism of the occurrence and development, designing a reasonable treatment scheme, judging prognosis and further improving the treatment level of the liver cancer.

Nucleotides are building block molecules of DNA and RNA, and disorders in nucleotide biosynthesis widely affect physiological functions of normal cells, ultimately leading to malignant transformation of normal cells, for which the body must supply more nucleotides due to the capacity of tumor cells to proliferate indefinitely. Nucleotide metabolism therefore plays a very important role in the development and progression of tumors. Studies have shown that there are a number of key enzyme-regulated nucleotide syntheses and modifications, one of which is Ribonucleotide Reductase (RR).

In humans, RR consists of three subunits, a large subunit (RRM1) and two small subunits (RRM2 and RRM 2B). The role played by each subunit varies, as RRM1 is widely recognized as an oncogene suppressor, and RRM2 and RRM2B are thought to affect DNA repair through the modulation of p 53. However, the study of the specific functions and mechanisms of action of these two subunits is controversial, especially RRM2B, and the mechanism of action of this gene and the specific role played in cancer are not yet clear.

Therefore, there is an urgent need in the art to study tumor-associated genes, especially genes associated with liver cancer with a high incidence in China, elucidate the relationship between these genes and the occurrence and development of liver cancer, and provide a new concept for the diagnosis and treatment of liver cancer.

Disclosure of Invention

The invention provides a marker RRM2B for diagnosing liver cancer metastasis and application of RRM2B in treating liver cancer metastasis.

In a first aspect of the invention, the invention provides an application of ribonucleotide reductase subunit M2B (ribonucleotide reductase subunit M2B, RRM2B) gene or protein detection test agent thereof in preparing a kit for diagnosing liver cancer cell metastasis.

In another preferred embodiment, the metastasis includes intrahepatic and extrahepatic metastasis.

In another preferred embodiment, the extrahepatic metastasis includes pulmonary metastasis, bone metastasis, or brain metastasis.

In another preferred embodiment, the RRM2B gene or protein thereof is from a mammal, preferably, a rodent (mouse or rat) or primate (e.g. human); more preferably, it is of human origin.

In another preferred embodiment, the coding sequence of the RRM2B gene is as set forth in SEQ ID No.:42 (Genbank ID: NM-015713.4):

(ATGGGCGACCCGGAAAGGCCGGAAGCGGCCGGGCTGGATCAGGATGAGAGATCATCTTCAGACACCAACGAAAGTGAAATAAAGTCAAATGAA GAGCCACTCCTAAGAAAGAGTTCTCGCCGGTTTGTCATCTTTCCAATCCAGTACCCTGATATTTGGAAAATGTATAAACAGGCACAGGCTTCCTTCTGGA CAGCAGAAGAGGTCGACTTATCAAAGGATCTCCCTCACTGGAACAAGCTTAAAGCAGATGAGAAGTACTTCATCTCTCACATCTTAGCCTTTTTTGCAGCCAGTGATGGAATTGTAAATGAAAATTTGGTGGAGCGCTTTAGTCAGGAGGTGCAGGTTCCAGAGGCTCGCTGTTTCTATGGCTTTCAAATTCTCATCGAG AATGTTCACTCAGAGATGTACAGTTTGCTGATAGACACTTACATCAGAGATCCCAAGAAAAGGGAATTTTTATTTAATGCAATTGAAACCATGCCCTATG TTAAGAAAAAAGCAGATTGGGCCTTGCGATGGATAGCAGATAGAAAATCTACTTTTGGGGAAAGAGTGGTGGCCTTTGCTGCTGTAGAAGGAGTTTTCTT CTCAGGATCTTTTGCTGCTATATTCTGGCTAAAGAAGAGAGGTCTTATGCCAGGACTCACTTTTTCCAATGAACTCATCAGCAGAGATGAAGGACTTCAC TGTGACTTTGCTTGCCTGATGTTCCAATACTTAGTAAATAAGCCTTCAGAAGAAAGGGTCAGGGAGATCATTGTTGATGCTGTCAAAATTGAGCAGGAGT TTTTAACAGAAGCCTTGCCAGTTGGCCTCATTGGAATGAATTGCATTTTGATGAAACAGTACATTGAGTTTGTAGCTGACAGATTACTTGTGGAACTTGG ATTCTCAAAGGTTTTTCAGGCAGAAAATCCTTTTGATTTTATGGAAAACATTTCTTTAGAAGGAAAAACAAATTTCTTTGAGAAACGAGTTTCAGAGTAT CAGCGTTTTGCAGTTATGGCAGAAACCACAGATAACGTCTTCACCTTGGATGCAGATTTTTAA)；

the sequence of the encoded protein is shown in SEQ ID No.: 43 (GenbankID: NP-056528.2)

(MGDRAAGDDRSSSDTNSIKSNRKSSRRVIIYDIWKMYKAASWTAVDSKDHWNKKADKYISHIAAASDGIVNNVRSVVARCYGIINVHSMYSIDTYI RDKKRNAITMYVKKKADWARWIADRKSTGRVVAAAVGVSGSAAIWKKRGMGTSNISRDGHCDACMYVNKSRVRIIVDAVKITAVGIGMNCIMKYIVADRV GSKVANDMNISGKTNKRVSYRAVMATTDNVTDAD)。

In another preferred example, the RRM2B gene or its protein detection reagent includes an antibody specific to RRM2B, a specific amplification primer, a probe or a chip.

In another preferred example, the detection reagent for detecting RRM2B protein or mRNA includes:

(a) an antibody specific against RRM2B protein; and/or

(b) A specific primer that specifically amplifies mRNA or cDNA of RRM 2B.

In another preferred embodiment, the specific antibody shown is Anti-p53R2 antibody (ab8105, available from Abcam).

In another preferred example, the specific primer sequence for specifically amplifying the mRNA or cDNA of RRM2B is as follows:

an upstream primer: AGGAGGTGCAGGTTCCAGAG (SEQ ID NO. 3)

A downstream primer: CTGCTATCCATCGCAAGGC (SEQ ID NO. 24)

In another preferred embodiment, the detection comprises enzyme-linked immunosorbent assay (ELISA) detection or time-resolved immunofluorescence assay (TRFIA) detection.

In another preferred embodiment, the RRM2B protein or antibody specific thereto is coupled to or carries a detectable label.

In another preferred embodiment, the detectable label is selected from the group consisting of: chromophores, chemiluminescent groups, fluorophores, isotopes or enzymes.

In another preferred example, the antibody specific for RRM2B is a monoclonal antibody or a polyclonal antibody.

In another preferred embodiment, the detection is of liver cancer tissue, or a normal liver tissue sample.

In another preferred embodiment, the normal liver tissue comprises a tissue adjacent to cancer.

In a second aspect of the present invention, there is provided a diagnostic kit for detecting liver cancer cell metastasis, said kit comprising a container, said container containing a detection reagent for detecting RRM2B protein or mRNA; and a label or instructions indicating that the kit is for detecting liver cancer cell metastasis.

In another preferred embodiment, the detection of liver cancer cell metastasis refers to determining whether liver cancer cell metastasis occurs, and/or determining the probability (susceptibility) of liver cancer cell metastasis.

In another preferred embodiment, the metastasis includes intrahepatic and extrahepatic metastasis.

In another preferred embodiment, the extrahepatic metastasis includes pulmonary metastasis, bone metastasis, or brain metastasis.

In another preferred example, the judgment includes a preliminary judgment (prediction).

In another preferred example, the detection reagent for detecting RRM2B protein or mRNA includes:

(a) an antibody specific against RRM2B protein; and/or

(b) A specific primer that specifically amplifies mRNA or cDNA of RRM 2B.

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

when the ratio of the RRM2B expression quantity E1 in the liver cancer cells or tissues of the detection object to the RRM2B expression quantity E2 in the normal liver cells or tissues is less than or equal to 0.5, the probability of liver cancer cell transfer of the detection object is higher than that of the common people.

In another preferred example, the E2 is the RRM2B expression level of normal liver cells or tissues of normal people.

In another preferred embodiment, the normal liver cells or tissues comprise paracancerous liver cells or tissues.

In another preferred embodiment, the expression level is relative to the expression level of a control gene (e.g., β -actin).

In a third aspect of the invention, the application of the RRM2B gene, protein or promoter thereof is provided, and the RRM2B gene, protein or promoter thereof is used for preparing a pharmaceutical composition for inhibiting liver cancer or liver cancer cell metastasis.

In another preferred embodiment, the RRM2B gene or protein thereof is from a mammal, preferably, a rodent (mouse or rat) or primate (e.g. human); more preferably, it is of human origin.

In another preferred embodiment, the pharmaceutical composition comprises a therapeutically effective amount of the RRM2B gene, protein or promoter thereof, and a pharmaceutically acceptable carrier.

In another preferred embodiment, the medicament is administered by a mode of administration selected from the group consisting of: oral, intravenous, intramuscular, subcutaneous, sublingual, rectal, nasal, oral, topical or systemic transdermal administration.

In another preferred embodiment, the formulation of the drug is selected from the group consisting of: tablet, capsule, injection, granule, and spray.

In another preferred embodiment, the RRM2B inhibitor is administered to the mammal at a dose of 0.01-20mg/kg body weight (per time or per day).

In another preferred embodiment, the mammal includes a human, a mouse, a rat, and more preferably, a human.

In a fourth aspect of the present invention, there is provided a method for screening a candidate compound for inhibiting metastasis of hepatoma cells, comprising the steps of:

(a) in the test group, adding a test compound into a culture system of liver cancer cells, and observing the expression quantity and/or activity of RRM2B in the cells of the test group; in the control group, the test compound is not added to the culture system of the same cells, and the expression amount and/or activity of RRM2B in the cells of the control group are observed;

wherein, if the expression level and/or activity of RRM2B in the cells of the test group is higher than that of the control group, the test compound is a compound which can inhibit the metastasis of the liver cancer cells and has the promotion effect on the expression and/or activity of RRM 2B; and/or

(b) Testing the candidate compound obtained in step (a) for its inhibitory effect on metastasis of hepatoma cells.

In another preferred example, step (b) includes the steps of: adding a test compound into a culture system of the liver cancer cells of the test group, and observing the number of moving distances and/or invasion conditions of the liver cancer cells; adding no test compound into the culture system of the control group liver cancer cells, and observing the number of moving distances and/or invasion conditions of the liver cancer cells; wherein, if the migration distance or the invasion number of the liver cancer cells in the test group is obviously smaller than that of the control group, the test compound is a compound which has an inhibitory effect on liver cancer cell metastasis.

In another preferred example, the step (a) further comprises one or more measurements of the expression levels of RRM2B and Egr1 in the control group and the test group.

In another preferred embodiment, the plurality of measurements comprises measuring the amount and/or activity of RRM2B and Egr1 and the tendency of RRM2B to change in expression and/or activity at the same time as the test compound is added, at 12 hours after the addition, and at 24 hours after the addition, respectively.

In another preferred example, according to the measurement result, a candidate compound in which the expression level and/or activity of Egr1 and RRM2B are simultaneously increased and the expression level and/or activity of RRM2B is not decreased is selected as the compound for inhibiting the metastasis of hepatoma cells.

In a fifth aspect of the invention, the application of the RRM2B inhibitor is provided, and the RRM2B inhibitor is used for preparing a composition or a reagent for promoting liver cancer cell metastasis.

In another preferred embodiment, the hepatoma cells comprise SMMC-7721, Huh7, HCC-LY10 cells.

In a sixth aspect of the invention, an expression vector of the RRM2B gene is provided, said expression vector having the following elements:

(i) a promoter element;

(ii) RRM2B coding sequence; and

(iii) a stop codon element;

wherein said promoter element does not bind to Egr 1.

In another preferred embodiment, the promoter element sequence is as set forth in SEQ ID No.: 44, shown in the figure:

(ggtcatggcaagatcccgtcaatctttcagcttcaaactacttaaacaaatctctcttccattttatttttgttag accaaactatttttttcaaaatatatttttttcaaaatatttgcctgactttagtaacgtgtccctctccccctcactcacc cgcattcacacacacacacacacactttcccccagacacttccccttttcggttgtttacgcacgaaaccagccagcccttt ccctagcgaagtgtttggcggtgtagctgtcccgtccccggcgcttcccacagcaacagcgcgttctcttcgcctcccgcag tctctcaggacaggccaggctagcgcaaagtaccgtgttcctggaacaccacgagggcgagctcgggaatctcgagccggcc tgcaggacacctgccgcaccaagtggctagagcccggggagggcgaggcgaggtgggacgaggcggggtggtgagcgggccg ggaaggcgaggccgcgcggactctgggatagctcctcaggagtgggcgcccggagtgggcgagcggaggaggcggggccgat gaggtgaggtggggccagctggagtaggctaagcagtccagagcagggggcgtccctcagtggaaagccgggcgactgggcg gtgcaacagagtaaggcggggccagcggaagcagggagatttccttaggccgcaggcggggaaatggggctggccggggtag gcggagccccgaggcaggggaggcgtggctggcagaggtaggcggtgccccaaggcaggcggggcttgccgagacagggata atcccttaggccacaggctgggaggcagggcaggccgaggcggggaggatcccttaggccgcagtaggggaggcgggtcggc cgaggcggggagaaacccttaggccgcaggcggggaggcggggctggccgaagttaggcggagccccgaggcgggggaggcg gggccgggccggcgcagggagagtcactcaatggacaggcgagaaagcaggaccggcgcggcggggcggggccggccgagtc cctagagctgggggcggggcggacccagcggaccagcggaccacctgggtgctgtcgtagttggaggtggcctgaggagctc agttccctcagcgcccgtagcttcggcggagtctg)。

in a seventh aspect of the present invention, there is provided a method for diagnosing liver cancer metastasis, comprising the steps of:

(i) preparing a test sample of the subject;

(ii) detecting the mRNA expression level E1 of RRM2B in the test sample relative to a control gene (such as beta-actin) and the RRM2B expression level E2 (namely a reference value) of normal liver cells or tissues, and when the ratio is less than or equal to 0.5, indicating that the probability of liver cancer metastasis of the test subject is higher than that of the common population.

In another preferred embodiment, the sample is a liver cancer cell or tissue.

In another preferred example, the reference value is the expression level of RRM2B in the non-liver cancer tissue sample.

In another preferred example, the detecting step (ii) comprises detecting the amount of RRM2B mRNA, or the amount of RRM2B cDNA; and/or detecting the amount of RRM2B protein.

In another preferred embodiment, the detecting step (ii) comprises detecting by RT-PCR or PCR methods.

In another preferred embodiment, the detecting step (ii) comprises detecting using an antibody against RRM2B protein.

In an eighth aspect of the present invention, there is provided a method for inhibiting metastasis of hepatoma cells, comprising the steps of: administering to a subject (mammal) in need of treatment a safe and effective amount of an RRM2B gene, protein or promoter thereof.

In another preferred embodiment, the metastasis includes intrahepatic metastasis and extrahepatic metastasis.

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

Figure 1 shows the expression of RRM2B in hepatocellular carcinoma and the effect of RRM2B on hepatocellular carcinoma cell invasion, migration and metastasis.

Figure 1A shows immunohistochemical detection of the expression levels of RRM2B and P53 in hepatocellular carcinoma tissues and the corresponding paracarcinoma, resulting in RRM2B being predominantly expressed in the cytosol and RRM2B being expressed in liver cancer less than the corresponding paracarcinoma tissues; p53 is mainly expressed in cell nucleus, and p53 is expressed in liver cancer higher than corresponding para-cancer tissues.

Fig. 1B shows that the expression of RRM2B protein in liver cancer tissues and tissues beside cancer was detected by immunoblotting, and as a result, RRM2B was observed to be less expressed in liver cancer than the corresponding tissues beside cancer.

FIG. 1C shows that the expression of RRM2B mRNA in liver cancer tissue and para-carcinoma tissue was detected by quantitative PCR, and that RRM2B in liver cancer was less expressed than that in corresponding para-carcinoma tissue in 30 pairs of liver cancer tissue and para-carcinoma tissue, and p < 0.05.

FIG. 1D shows the effect of Transwell assay on SMMC-7721 and HCC-LY10 cell migration and invasion following RRM2B gene silencing. The result shows that RRM2B gene can promote migration and invasion of liver cancer cells after being silenced, p is less than 0.05, and p is less than 0.01.

FIG. 1E shows the effect of Transwell assay on SMMC-7721 and Huh7 cell migration and invasion after overexpression of RRM2B gene. The result shows that the RRM2B gene can inhibit the migration and invasion of liver cancer cells after being over-expressed, p is less than 0.05, and p is less than 0.01.

FIG. 1F shows the effect of RRM2B gene silencing on tumor metastasis ability detected by nude mouse liver orthotopic transplantation tumor experiment, and the results show that RRM2B gene silencing can promote liver cancer cell orthotopic metastasis and distant lung metastasis of nude mouse liver, p is less than 0.05.

Fig. 2 shows the expression levels of RRM2B and p53 in liver cancer cell lines.

FIG. 3 shows mutations in liver cancer in the p53 gene.

68 cases of liver cancer tissues were analyzed by sequencing for mutation of exon 7 of p53 gene, and as a result, it was found that AGG → AGT point mutation occurs at codon 249 of exon 7 of p53 gene, which results in arginine mutation to serine. The mutation was found in 20 out of 68 liver cancer tissues, and the p53 gene was mutated by sequencing of liver cancer tissues positive for immunohistochemical staining p 53.

Figure 4 shows that RRM2B inhibits hepatoma cell EMT-like changes.

FIG. 4A shows the corresponding morphological changes in SMMC-7721 and HCC-LY10 cells after RRM2B gene silencing.

FIG. 4B shows the detection of the expression levels of E-cadherin, N-cadherin and Slug after the gene silencing of SMMC-7721 and RRM2B in HCC-LY10 cells.

FIG. 4C shows SMMC-7721 and Huh7 cells RRM2B gene overexpression, detection of E-cadherin, N-cadherin and Slug expression levels.

FIG. 4D shows the detection of the expression levels of E-cadherin, N-cadherin and RRM2B by immunohistochemistry in nude mouse tumorigenic tissues from SMMC-7721 cells silenced with RRM2B gene.

FIG. 4E shows SMMC-7721 and Huh7 cells with over-expressed or gene-silenced RRM2B treated with doxorubicin (2. mu.g/ml) for 24 hours and tested for viability in MTT assay.

Figure 5 shows that RRM2B inhibits expression of the liver cancer stem cell markers CD133 and CD 44.

Fig. 5A shows SMMC-7721 and Huh7 cells with stable RRM2B overexpression or gene silencing, and quantitative PCR to detect the expression levels of CD133, CD44, notch1, manog, and Oct 4.

Figure 5B shows SMMC-7721 and Huh7 cells overexpressing stable RRM2B, with immunoblots detecting expression levels of CD133, CD44, and RRM 2B.

Figure 5C shows RRM2B gene silenced SMMC-7721 cells, immunoblotted to detect expression levels of CD133, CD44, and RRM 2B.

Figure 6 shows that RRM2B affects phosphorylation of Akt1 by modulating expression levels of Egr-1 and PTEN.

FIG. 6A shows immunoblot detection of expression levels of stably overexpressed RRM2B in Huh7 and SMMC-7721 cells PTEN, phosphorylated Akt (p-Akt), total Akt, and RRM 2B.

FIG. 6B shows expression levels of PTEN, p-Akt, total Akt and RRM2B in SMMC-7721 and HCC-LY10 cells tested by immunoblotting for RRM2B gene silencing.

FIG. 6C shows quantitative PCR detection of PTEN mRNA levels following overexpression or gene silencing of RRM2B in hepatoma cells SMMC-7721, Huh7 and HCC-LY 10.

FIG. 6D shows the transfection of reporter genes comprising the PTEN promoter in hepatoma cells SMMC-7721 and Huh7, and the effect of RRM2B on PTEN promoter activity was examined.

FIG. 6E shows quantitative PCR detection of Egr-1mRNA levels following overexpression or gene silencing of RRM2B in hepatoma cells SMMC-7721, Huh7 and HCC-LY 10.

FIG. 6F shows the co-transfection of SMMC-7721 and Huh7 cells with pcDNA3.1-Egr-1 plasmid or empty vector pcDNA3.1 and PTEN-Luc plasmid to detect the effect of Egr-1 on PTEN promoter activity.

FIG. 7 shows the effect of interfering with Akt1 expression or the Akt1 phosphorylation inhibitors LY294002 and wortmannin on hepatoma cell migration and invasion.

FIG. 7A shows siRNA of SMMC-7721 cells with RRM2B gene silencing transfected with Akt1, and immunoblotting to detect expression levels of Akt1, E-cadherin, and N-cadherin.

FIG. 7B shows that LY294002 (10. mu.M) and wortmannin (1. mu.M) act on SMMC-7721 cells with RRM2B gene silencing for 12 hours, and the expression levels of Akt1, E-cadherin and N-cadherin were detected by immunoblotting.

Figure 7C shows RRM2B gene silenced SMMC-7721 cells transfected with siRNA of Akt1, and their effect on cell migration and invasion was examined by Transwell assay.

FIG. 7D shows LY294002 (10. mu.M) and wortmannin (1. mu.M) acting on SMMC-7721 cells with RRM2B gene silencing for 12 hours, which was examined by Transwell assay for its effect on cell migration and invasion.

FIG. 7E shows SMMC-7721 cells overexpressing the RRM2B gene were infected with sustained activating Akt (Myr-Akt) and immunoblotted to detect the expression levels of p-Akt, Akt1 and RRM 2B.

FIG. 7F shows SMMC-7721 cells overexpressing the RRM2B gene were infected with a persistent activator Akt (Myr-Akt) and their effect on cell migration and invasion was examined by Transwell assay.^*p＜0.05，^**p＜0.01。

Fig. 8 shows that RRM2B inhibits migration and invasion of hepatoma cells by up-regulating Egr-1 expression.

FIG. 8A shows the detection of expression levels of Egr-1 and RRM2B after 48 hours of transfection of pcDNA3.1-Egr-1 plasmid or empty vector pcDNA3.1 into SMMC-7721 cells with RRM2B gene silencing.

FIG. 8B shows that the SMMC-7721 cell with RRM2B gene silenced is transfected with pcDNA3.1-Egr-1 plasmid or empty vector pcDNA3.1, and the effect on cell migration and invasion is detected by Transwell experiment.

FIG. 8C shows SMMC-7721 cells overexpressing the RRM2B gene were transfected with 2 siRNAs to Egr-1 and the expression levels of Egr-1 and RRM2B were measured after 48 hours.

FIG. 8D shows RRM2B baseSince the over-expressed SMMC-7721 cells were transfected with 2 siRNAs to Egr-1, the Transwell assay examined its effect on cell migration and invasion.^*p＜0.05，^**p＜0.01。

Figure 9 shows that E-cadherin is positively correlated with expression of RRM2B in liver cancer.

FIG. 9A shows quantitative PCR detection of E-cadherin expression levels in liver cancer tissues (T) and corresponding paracarcinoma tissues (N).

FIG. 9B shows quantitative PCR detection of the expression level of N-cadherin in liver cancer tissue (T) and corresponding paracarcinoma tissue (N).

FIG. 9C shows immunoblotting for the detection of E-cadherin, N-cadherin, p-Akt, Akt1, and RRM2B expression levels in liver cancer tissues (with concomitant metastasis or non-metastasis).

Figure 9D shows immunohistochemical detection of RRM2B and E-cadherin expression in liver cancer tissues (representative 2 tissues).

Figure 9E shows the analysis of the expression correlation of E-cadherin with RRM2B expression in liver cancer based on the results of immunohistochemistry of RRM2B with E-cadherin.

Figure 10 shows that Egr-1 binds the promoter region of RRM2B and inhibits the expression of RRM 2B.

Figure 10A shows luciferase assays detecting activity of the different truncated fragment RRM2B promoter.

FIG. 10B shows the relative promoter activity detected by luciferase assay in the co-transfection of SMMC-7721 cells with pcDNA3.1-Egr-1 or empty vector pcDNA3.1 and a different truncated fragment of the reporter gene containing the RRM2B promoter.

FIG. 10C shows the relative promoter activity of pcDNA3.1-Egr-1 or empty vector pcDNA3.1 co-transfected with the RRM2B-Luc (-375/-5, containing wild-type or mutated Egr-1 binding sites) plasmid into SMMC-7721 cells tested by luciferase assay.

FIG. 10D shows chromatin co-immunoprecipitation (CHIP) assay detecting the binding of Egr-1 to the RRM2B promoter region.

FIG. 10E shows the expression levels of RRM2B and Egr-1 detected by immunoblotting of cells SMMC-7721 and Huh7 transfected with pcDNA3.1-Egr1 or empty vector pcDNA3.1.

FIG. 10F shows siRNA transfection of SMMC-7721 and Huh7 cells into 2Egr-1 cells and immunoblot detection of the expression levels of RRM2B and Egr-1.

Detailed Description

The inventor of the invention has long and intensive research, and unexpectedly found that the RRM2B gene is low expressed in liver cancer, and the over-expression RRM2B gene has an inhibiting effect on liver cancer metastasis.

Experiments show that the expression of RRM2B in liver cancer is reduced, the expression of RRM2B in liver cancer is in negative correlation with liver cancer metastasis in liver, and the expression of RRM2 affects the process of liver cancer patients, so that whether liver cancer metastasis occurs can be diagnosed by detecting the expression and/or activity of RRM 2B.

The inventor also finds that when the liver cancer cell over-expresses the RRM2B gene, RRM2B can obviously inhibit invasion and migration of the liver cancer cell, and conversely, the RRM2B can obviously promote invasion and migration of the cell after inhibiting expression of the RRM2B through RNA interference.

In addition, in vitro experiment results show that RRM2B can inhibit invasion and migration of hepatoma cells, not through the main pathway p53 of RRM2B, but through regulating the Egr-1/PTEN/Akt1 pathway, the hepatoma metastasis is inhibited. Meanwhile, experiments show that the transcription factor Egr-1 can be combined with a promoter region of the RRM2B gene and inhibit the expression of the RRM2B gene, so that a negative feedback regulation and control loop exists between the Egr-1 and the RRM2B to regulate the expression level of the RRM 2B.

On the basis of this, the present invention has been completed.

As used herein, the terms "liver cancer metastasis", "liver cancer cell metastasis", "invasion or migration of liver cancer cells" are used interchangeably and refer to the primary focus of disease in the liver, and the primary liver cancer undergoes intrahepatic or extrahepatic metastasis through the metastasis pathways such as hematogenous metastasis, lymphatic metastasis, and graft metastasis. Herein, when referring to a cell experiment such as a transwell transmembrane assay, invasion or migration of hepatoma cells refers to a specific migration and proliferation ability exhibited by hepatoma cells after culturing or specific stimulation of hepatoma cells.

Ribonucleotide Reductase (RR) and ribonucleotide reductase subunit M2B (RRM2B)

In the present invention, "the protein of the present invention", "the polypeptide of the present invention", and "the RRM2B protein" are used interchangeably and refer to ribonucleotide reductase subunit M2B (ribonucleotide-diphosphate reductase subunit M2B, RRM 2B). It is to be understood that the term also includes active fragments and derivatives of RRM 2B.

In the present invention, "gene of the present invention" and "polynucleotide of the present invention" refer to a nucleotide sequence encoding RRM2B protein or active fragment and derivative thereof, including sense and antisense nucleic acids.

In the present invention, the terms "RRM 2B protein" or "RRM 2B polypeptide" are used interchangeably and refer to a protein or polypeptide having the amino acid sequence of the human protein RRM 2B.

Ribonucleotide Reductases (RRs) are widely distributed in various biological cells, and their role is involved in the reduction of ribonucleotides into deoxyribonucleotides, playing a central role in the process of nucleotide metabolism, and being the only enzyme that converts ribonucleotides into deoxyribonucleotides. Deoxyribonucleotides are the raw materials for DNA synthesis and repair, and therefore the enzyme is the rate-limiting enzyme for synthesis and repair in the DNA pathway.

The RR holoenzyme structure comprises a large subunit alpha and a small subunit beta, and the activity can be achieved only by forming an alpha 2 beta 2 heterotetrameric structure. In humans, RR consists of three subunits, a large subunit (RRM1) and two small subunits (RRM2 and RRM 2B).

RRM1 is proved to be a tumor suppressor gene in mouse and human cell lines, and the over-expression of RRM1 can block the cell cycle at the G2 stage, so as to cause apoptosis. RRM1 inhibits lung cancer cell proliferation and metastasis and has a certain relation with PTEN expression.

RRM2B is a homologous gene to RRM2, with about 80% homology to RRM2, and originally served as a target gene for tumor suppressor protein p 53. However, there is a difference between RRM2B and RRM 2. RRM2B acts as a transcriptional target for p53, while cell cycle-related factors regulate transcription of RRM2, such as NF-Y and E2F. The transcription of RRM1 and RRM2 occurs mainly in the S phase of the cell cycle, and since RRM1 has a long half-life and can be stably present in the whole cell cycle, the half-life of RRM2 is relatively short, so the activity of the RR holoenzyme is mainly dependent on the synthesis and degradation of RRM2 protein. However, when the cells are in the dormant period and lack of RRM2, RRM2B binds to RRM1 to form a holoenzyme, providing dntps for DNA damage repair. In the case of DNA damage, cells wild-type at p53 provided dntps for DNA damage repair primarily through expression of RRM 2B. However, if p53 is functionally inactivated, RRM2 supplements the functional repair of RRM2B with the damaged DNA.

Research on human pancreatic cancer cells shows that the over-expression of RRM2 can cause the increase of the invasion capacity of cells and the up-regulation of MMP-9 expression, and the up-regulation of MMP-9 expression is positively correlated with the invasion and metastasis of tumors; the research on the esophageal cancer and the oral cancer finds that the expression of RRM2B promotes the invasion and metastasis of tumors and influences the prognosis; also in small cell lung cancer, RRM2B expression was found to be associated with lung cancer progression. While different reports have concluded how RRM2B affects tumor cell proliferation and invasion, Yanamoto et al believe that RRM2B promotes oral cancer invasion through the E-cadherin/β -catenin pathway, Zhang et al report that RRM2B inhibits oral cancer KB cell proliferation by up-regulating p21 and down-regulating cyclin D1 expression. Furthermore, RRM2B was found to be negatively associated with the proliferation of colon cancer in colon cancer.

In summary, the role of RRM2B in different tumors is different, even though the results of the same tumor study are controversial.

In the present invention, one preferred RRM2B gene has a nucleotide sequence as set forth in SEQ ID No.:42 (Genbank ID: NM-015713.4); the sequence of the encoded RRM2B protein is shown in SEQ ID No.: 43(Genbank ID: NP-056528.2).

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 its native state in a living cell is not isolated and purified, but the same polynucleotide or polypeptide is isolated and purified if it is separated from other substances coexisting in its native state.

As used herein, "isolated RRM2B protein or polypeptide" means RRM2B protein is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. One skilled in the art would be able to purify the RRM2B 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 RRM2B protein includes a fusion protein and a non-fusion protein.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide. The polypeptide 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.

The polynucleotide encoding the mature polypeptide of RRM2B comprises: 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 may be used in amplification techniques of nucleic acids (e.g. PCR) to determine and/or isolate a polynucleotide encoding the RRM2B protein.

The full-length nucleotide sequence or the fragment of the human RRM2B of the invention can be obtained by a PCR amplification method, a recombination method or a synthetic method. For the PCR amplification method, primers are designed based on the disclosed nucleotide sequences, particularly open reading frame sequences, and the sequences are 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 can be obtained by first synthesizing a plurality of small fragments and then performing ligation.

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 produced using the vectors of the invention or the RRM2B protein encoding sequence, 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 RRM2B 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 the human RRM2B 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 expression vectors containing the DNA sequence encoding human RRM2B 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.

In a preferred embodiment, suitable promoters in the expression vectors of the present invention include the wild-type RRM2B gene promoter and the mutant RRM2B gene promoter. Wherein the mutant RRM2B gene promoter does not have binding activity to Egr 1.

A preferred mutant RRM2B gene promoter is as set forth in SEQ ID No.:45, and:

(agggataatcccttaggccacaggctgggaggcagggcaggccgaggcggggaggatcccttaggccgcagtaggggaggcgggtcggccgag gcggggagaaacccttaggccgcaggcgttaaggcttggctggccgaagttaggcggagccccgaggcgggggaggcggggccgggccggcgcagggaga gtcactcaatggacaggcgagaaagcaggaccggcgcggcggggcggggccggccgagtccctagagctgggggcggggcggacccagcggaccagcggaccacctgggtgctgtcgtagttggaggtggcctgaggagctcagttccctcagcgcccgtagcttcggcggagtctg)。

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, the 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 methods, conventional mechanical methods such as microinjection, electroporation, liposome packaging, 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 desired, the physical, chemical and other properties of the recombinant protein can be exploited to isolate and purify the recombinant protein by various separation methods. 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, ultrafiltration, 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.

Egr-1/PTEN/Akt1 pathway

PTEN is a very important tumor suppressor protein, is an important negative regulatory factor of a PI3K/Akt signal transduction pathway, and can prevent the generation and development of tumors by effectively antagonizing the PI3K-AKT signal transduction pathway. As a lipid phosphatase, PTEN can dephosphorylate phosphatidylinositol triphosphate (PIP3) on cell membranes to produce PIP2, since PIP3 is a product of PI3K and mediates activation of Akt, and dephosphorylates PIP3 to maintain low levels of PIP3, thereby down-regulating the PI3K/Akt pathway.

Egr-1 can regulate the expression of a plurality of genes as an important transcription factor, and research shows that the promoter region of the PTEN gene contains a binding site of the transcription factor Egr-1, and the Egr-1 can bind to the promoter region of the PTEN gene and can promote the expression level of PTEN. Thus, Egr-1 inhibits the phosphorylation level of Akt, primarily by binding to the promoter region of PTEN and promoting expression of PTEN.

Epithelial-mesenchymal transition (EMT)

Refers to the biological process by which epithelial cells are transformed by a specific process into cells with a mesenchymal phenotype. Plays an important role in embryonic development, chronic inflammation, tissue reconstruction, cancer metastasis and various fibrotic diseases, and is mainly characterized by the reduction of expression of cell adhesion molecules (such as E-cadherin), the transformation of a cytokeratin cytoskeleton into a Vimentin-based cytoskeleton, the morphological characteristics of mesenchymal cells and the like. Through EMT, epithelial cells lose cell polarity, lose epithelial phenotypes such as connection with a basement membrane and the like, and obtain interstitial phenotypes such as higher migration and invasion, apoptosis resistance, extracellular matrix degradation capability and the like.

EMT is an important biological process for obtaining the ability to migrate and invade malignant tumor cells (liver cancer) of epithelial cell origin. The molecular mechanism for regulating and controlling the EMT process of malignant tumor cells is clarified, the pathological significance of the EMT cells in the generation, development and transfer of malignant tumors is determined, and the research on a diagnosis method based on EMT key molecules and a treatment method targeting the EMT key molecules are key scientific problems in the research on the EMT mechanism in the tumor transfer.

Accelerator and pharmaceutical composition

By using the protein of the invention, substances, particularly promoters and the like, which interact with the RRM2B protein can be screened out by various conventional screening methods.

The promoter of the RRM2B protein can promote the expression and/or activity of the RRM2B protein and further promote the expression and/or activity of p53 when being applied (dosed) on treatment, thereby inhibiting liver cancer. In another preferred example, the RRM2B promoter includes an RRM2B gene expression product, a promoting miRNA, a promoting transcriptional regulator, or a promoting targeted small molecule compound.

Generally, these enhancers will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, typically having a pH of about 5 to about 8, preferably a pH of 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: intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.

The invention also provides a pharmaceutical composition, which contains a safe and effective amount of the RRM2B protein or the promoter 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 about 10 milligrams per kilogram of body weight per day.

Inhibitors

In the invention, the RRM2B protein inhibitor can be screened out by various conventional screening methods.

Inhibitors useful in the present invention include: an antibody to RRM2B, an inhibitory mRNA, an antisense RNA to RRM2B nucleic acid, a microRNA (miRNA), an siRNA, an shRNA, and an inhibitor of RRM2B activity. Typical inhibitors of RRM2B are inhibitory miRNA and siRNA.

The RRM2B inhibitor can be used for inducing the metastasis of liver cancer cells in animal models or the invasion and migration of liver cancer cells in cell experiments.

Antibodies

The invention also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, specific for the human RRM2B protein. Herein, "specificity" means that the antibody binds to the human RRM2B gene product or fragment. Preferably, these antibodies bind to the human RRM2B 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. For example, purified human RRM2B gene product or antigenic fragment thereof is injected into animals to produce polyclonal antibodies. Similarly, cells expressing the human RRM2B protein or its antigen may be used to immunize animals to produce antibodies.

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

The RRM2B antibody useful in the present invention may be an anti-human RRM2B protein antibody. The anti-human RRM2B protein antibody can be used in immunohistochemical technique to detect human RRM2B protein in biopsy specimen. One preferred antibody that can be used to detect the RRM2B protein is the Anti-p53R2 antibody (ab8105, available from Abcam).

Detection method and kit

The present invention relates to diagnostic assays for quantitative and in situ measurement of human RRM2B protein levels or mRNA levels. These assays are well known in the art. The level of human RRM2B protein detected in the assay can be used to diagnose liver cancer metastasis or liver cancer cell migration.

One method for detecting the presence or absence of RRM2B protein in a sample is by using antibodies specific for RRM2B protein, which comprises: contacting the sample with an antibody specific for RRM2B protein; observing whether an antibody complex is formed indicates the presence of RRM2B protein in the sample.

The RRM2B protein or its polynucleotide can be used for the diagnosis and treatment of RRM2B protein related diseases. A part or all of the polynucleotide of the present invention can be immobilized as a probe on a microarray or a DNA chip for analysis of differential expression of genes in tissues and gene diagnosis. Antibodies against RRM2B may be immobilized on the protein chip for detection of RRM2B protein in the sample.

The invention also provides a kit for detecting whether liver cancer has metastasis, which comprises a primer pair for specifically amplifying RRM2B and/or an antibody specific to RRM2B, and a label or an instruction.

Wherein the label or instructions recite the following: when the ratio of the mRNA expression quantity of RRM2B relative to actin to the mRNA expression quantity of RRM2B relative to actin of non-cancer tissues of the test object is less than or equal to 0.5, the probability of liver cancer metastasis of the test object is higher than that of the common people.

A typical kit of the invention can be used to detect human liver cancer tissue samples or normal liver tissue samples.

Screening method

The invention also provides a method for screening drugs based on the RRM 2B. One method is to screen compounds which affect (promote) the expression or activity of RRM2B, and then further test the screened compounds for inhibition of liver cancer cell metastasis.

A preferred method of screening candidate compounds for inhibition of hepatoma cell metastasis comprising the steps of:

(a) in the test group, adding a test compound into a culture system of liver cancer cells, and observing the expression quantity and/or activity of RRM2B in the cells of the test group; in the control group, the test compound is not added to the culture system of the same cells, and the expression amount and/or activity of RRM2B in the cells of the control group are observed;

wherein, if the expression level and/or activity of RRM2B in the cells of the test group is higher than that of the control group, the test compound is a compound which can inhibit the metastasis of the liver cancer cells and has the promotion effect on the expression and/or activity of RRM 2B; and/or

(b) Testing the candidate compound obtained in step (a) for its inhibitory effect on metastasis of hepatoma cells.

In another preferred embodiment, step (b) further comprises the steps of: adding a test compound into a culture system of the liver cancer cells of the test group, and observing the number of moving distances and/or invasion conditions of the liver cancer cells; adding no test compound into the culture system of the liver cancer cells of the control group, and observing the number of moving distances and/or invasion conditions of the liver cancer cells; wherein, if the migration distance or invasion number of the liver cancer cells in the test group is significantly smaller than that of the control group, the test compound is a compound having an inhibitory effect on liver cancer cell metastasis.

In addition, experiments show that when the expression level and/or activity of RRM2B is increased, high expression of Egr1 can be promoted, and after a certain degree of high expression of Egr1, overexpression of RRM2B can be inhibited by combining with the promoter region of RRM2B, so that a negative feedback regulation effect exists between Egr1 and RRM 2B. Based on the discovery, the drug which can promote the expression of RRM2B and antagonize the inhibition effect of Egr1 on RRM2B can be screened, so that the inhibition effect of RRM2B on liver cancer metastasis can be better exerted.

Therefore, step (a) further comprises performing one or more measurements of the expression levels of RRM2B and Egr1 in the control and test groups.

In another preferred embodiment, the plurality of measurements comprises measuring the amount and/or activity of RRM2B and Egr1 and the tendency of RRM2B to change in expression and/or activity at the same time as the test compound is added, at 12 hours after the addition, and at 24 hours after the addition, respectively.

In another preferred example, according to the measurement result, a candidate compound in which the expression level and/or activity of Egr1 and RRM2B are simultaneously increased and the expression level and/or activity of RRM2B is not decreased is selected as the compound for inhibiting the metastasis of hepatoma cells.

The invention also includes a method for inhibiting liver cancer metastasis or liver cancer cell migration, comprising the steps of: administering to a subject (mammal) in need of treatment a safe and effective amount of an RRM2B gene, protein or promoter thereof.

The invention has the advantages of

1. The invention discovers that the expression level of RRM2B is negatively correlated with liver cancer metastasis for the first time, namely the lower the expression level of RRM2B, the higher the possibility of liver cancer metastasis, and accordingly provides a new method for detecting liver cancer metastasis.

2. The invention proves that the over-expression RRM2B can inhibit the transfer of liver cancer, thereby providing a new target point for treating liver cancer.

3. The invention also discovers that RRM2B plays a role in inhibiting invasion and migration of hepatoma cells by influencing an Egr-1/PTEN/Akt1 pathway, specifically by up-regulating the expression of Egr-1, further enabling a transcription factor Egr-1 to be combined with a PTEN promoter region to promote the expression of mRNA and protein levels of PTEN, and finally inhibiting the phosphorylation level of Akt1, rather than playing a role in p 53.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by 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 recommendations. Unless otherwise specified, percentages and parts are percentages and parts by weight.

General procedure

The general methods of the invention are all technical means commonly used in the art, and the preferred materials and methods are as follows:

1. immunohistochemistry

Conventional paraffin sections were xylene dewaxed to water. 1% hydrogen peroxide was incubated at room temperature for 20 minutes to inactivate endogenous peroxidase and washed thoroughly with PBS. And (4) putting the slices into a microwave oven for thermal restoration for 10 minutes, and taking out and naturally cooling. 5-10% normal goat serum is sealed for 20 minutes at room temperature, and then primary antibody is added at 4 ℃ overnight. After washing with PBS, secondary antibody was added thereto at room temperature for 40 minutes. After washing with PBS, DAB was developed and the reaction was terminated. Washing with tap water, and counterstaining with hematoxylin. The slices are dehydrated and dried by gradient alcohol, the xylene is transparent, and the neutral gum is used for sealing the slices.

2. Cells and cell culture

Cells were routinely cultured (37 ℃, 5% CO) in DMEM medium containing 10% fetal bovine serum, 100U/ml penicillin and 100. mu.g/ml streptomycin₂，95％O₂). All cells were analyzed by trypan blue staining during the experiment, and cell viability remained above 90%.

3. Plasmid transfection

The expression of the plasmid was carried out using lipo-2000 transfection reagent from Invitrogen according to the experimental procedures provided by the company. Approximately 6X 10 per well in six well plates one day before transfection⁵And (3) cells are in an 80% confluence state during transfection, 2 mu g of plasmid is dissolved in 250 mu l of opti-MEM culture medium, lipo-2000 transfection reagent is uniformly mixed in 250 mu l of opti-MEM culture medium, the two are uniformly mixed after being cultured at room temperature for 5min, the mixture is cultured at room temperature for 20min, the mixture is reversely and uniformly mixed, the mixture is quickly added into a six-well plate, the culture plate is slightly shaken, the mixture is subjected to static culture after being uniformly mixed, the solution is changed after 4-6 h, and the expression of the transfected plasmid can be detected after 48 h.

4. Immunoblotting (Western Blot)

Collecting 2X 10⁶The cells were washed twice with cold PBS, centrifuged at 2000g for 5 minutes, the supernatant was discarded, 50. mu.l of SDS lysate was boiled at 95 ℃ for 5 minutes, placed on ice for 5 minutes and repeated twice. The cell lysate was centrifuged at 12,000g for 10 minutes at 4 ℃ and the supernatant was total protein extracted. Electrophoresis is carried out by 8-12% SDS-polyacrylamide gel. After the electrophoresis was completed, the membrane was transferred to a nitrocellulose membrane, and the membrane was blocked with 5% skim milk powder (in PBS) at room temperature for 1 hour. Then respectively incubating with primary antibodies at 4 ℃ overnight, fully washing with PBST, then incubating with corresponding horseradish peroxidase (HRP) labeled secondary antibodies for 1 hour at room temperature, fully washing with PBST, and finally detecting protein expression change by using an enhanced chemiluminescence method (ECL).

Antibodies used for immunohistochemistry and immunoblotting are shown in table 1:

TABLE 1

5. Real-time quantitative PCR (realtime PCR)

The real-time quantitative PCR uses double-stranded DNA dye SYBR Premix Ex Taq II kit and ABI7500 system. Reaction system: mu.l SYBR Premix Ex Taq II (2X), 0.2. mu.l forward primer (10. mu.M), 0.2. mu.l reverse primer (10. mu.M), 1. mu.l cDNA, 0.2. mu.l ROX Reference Dye II (50X), 3.4. mu.l deionized water. All reactions were performed in 96-well plates (MicroAmp optical96-well), sealed with optical additive covers (applied biosystems), amplification reactions were performed using triplicate tubes, and standard deviations were calculated to measure experimental errors. The data were analyzed using software attached to the instrument by calculating the threshold cycle number Ct corresponding to when the fluorescence signal exceeded the background fluorescence intensity. Differences in expression of the gene of interest in the case of equal amounts of RNA initiation reactions were reflected by calculating the difference in Ct values (Δ Ct) between the gene of interest and the corresponding internal control GAPDH.

The primers required for quantitative PCR and cloning of the vector are shown in Table 2.

TABLE 2

6. Construction and identification of recombinant vectors

The genomic DNA of SMMC-7721 cells was extracted according to the protocol of the genomic DNA extraction kit of Tianzhuo Bio Inc. Designing primers according to the DNA sequence of the RRM2B gene promoter and the characteristics of the selected pGL3-enhancer vector, designing Mlu I and Bgl II enzyme cutting sites on the upstream primer and the downstream primer respectively, and synthesizing the PCR primers by Shanghai biology. And (3) PCR reaction: the system was as follows, 10. mu.l 5 XPrimeHS DNA Polymerase buffer, 1. mu.l upstream primer, 1. mu.l downstream primer, 5. mu.l genomic DNA, 5. mu.l dNTP, 1. mu.l Mg₂SO₄，1μl Prime HS DNA Polymerase, 26. mu.l deionized water. And (3) PCR reaction conditions: the following cycle was repeated 30 times at 95 ℃ for 5 minutes: 94 ℃ for 30 seconds, 58 ℃ for 45 seconds, and 72 ℃ for 1 minute. Finally 10 minutes at 72 ℃. The target fragment was recovered according to the PCR product recovery kit of Tianzhuo Bio Inc. The fragments are recovered by adopting Nlu I and Bgl II double enzyme digestion, connected by T4DNA ligase, transformed by Escherichia coli E.coli DH5 alpha, and extracted, double enzyme digestion and sequencing identification are carried out on the recombinant plasmid.

7. Synthesis of Egr-1siRNA fragment

According to the design principle of siRNA interference sequence and the Egr-1cDNA sequence provided by GenBank, 2 siRNA interference sequences of Egr-1 are designed and synthesized through Shanghai.

The siRNA interference sequence of Egr-1 is shown in Table 3

TABLE 3

8. Luciferase Activity assay (Luciferase assay)

After the plasmid cotransfection was completed, the culture medium was aspirated, and the cells were washed with PBS. After adding 100. mu.l of cell lysate (PLB) to each well of the cell culture plate, the plate was shaken for 15 minutes in a shaker. The activities of the firefly luciferase and the mulberry luciferase are measured in the same reaction tube in sequence, the interval before the measurement is set to be 2 seconds by a fluorescence photometer, and the measurement time is 10 seconds each time. 100 μ l of LARII was taken to a luciferase assay tube. Add 20. mu.l of cell lysate and mix well by pipetting. Firefly luciferase activity value M1 was read. Then 100 mul of Stop & Glo reagent is added and mixed evenly, and the reading is again the sea-mulberry luciferase activity value M2. M1/M2 is the relative activity of luciferase.

9. Infection of hepatoma cells by lentiviral vectors

The method uses a gene recombination technology to connect RRM2B gene to a lentiviral expression vector pWPXL to construct a lentiviral vector pWPXL-RRM2B, co-transfects HEK293T cells with a lentiviral vector main plasmid pWPXL-RRM2B, a packaging plasmid psPAX2 and pMD2.G, packages lentiviral vectors and determines titer. Collecting virus particles to infect liver cancer cells.

10. Nude mouse transplantation tumor experiment

32 BALB/c nude mice are taken, and the liver of the nude mice is inoculated with the SMMC-7721 of RRM2B gene silence or HCC-LY10 cells 2 x 10 respectively in situ by using a disposable sterile syringe⁶And (4) respectively. Animals were sacrificed after 6 weeks and liver and lung tissues were taken for tissue fixation and embedding.

11. Statistical analysis

Statistical analysis was performed using the SPSS13.0 statistical software package, and all data were expressed as mean. + -. standard deviation. The comparison among the three groups adopts one-factor analysis of variance, the comparison among the two groups adopts t test, the comparison of the rate adopts chi-square test, and p is less than 0.05, which is considered to be statistically significant.

Example 1 the expression level of RRM2B gene in hepatocellular carcinoma and the degree to which the expression level of RRM2B was correlated with intrahepatic metastasis in liver cancer patients were determined.

The expression level of RRM2B in 236 liver cancer tissues is analyzed by an immunohistochemistry method, and the result shows that the expression level of RRM2B protein in liver cancer is obviously lower than that of corresponding paracancerous tissues (figure 1A), and the expression level of RRM2B mRNA and protein in the liver cancer tissues and the paracancerous tissues is detected by quantitative PCR and an immunoblotting method, and the result is consistent with the result of immunohistochemistry, namely the expression level of RRM2B mRNA and protein in the liver cancer tissues is obviously lower than that of the corresponding paracancerous tissues (figures 1B and 1C). Therefore, these results indicate that RRM2B protein expression is reduced in liver cancer tissues.

Through analysis of the relation between the expression level of the RRM2B protein in 236 liver cancer tissues and the clinical pathology of a liver cancer patient, experiments show that the expression level of the RRM2B protein is obviously negatively related to the intrahepatic metastasis of the liver cancer patient (p is less than 0.05); and the expression level of RRM2B protein is negatively correlated with the tissue grade of liver cancer patients (p < 0.05), but the expression of RRM2B has no obvious correlation with other clinical pathological characteristics of liver cancer patients, such as sex, age, tumor size, AFP level, liver cirrhosis state and HBV infection condition (Table 4).

TABLE 4

Example 2 measurement of inhibitory Effect of RRM2B on migration, invasion and metastasis of liver cancer cells

2.1 in vitro experiments

Firstly, hepatoma cells are infected by lentivirus, and a hepatoma cell line with stable RRM2B gene silencing and overexpression is established. The results show that RRM2B gene can obviously promote the migration and invasion of SMMC-7721 and HCC-LY10 hepatoma carcinoma cells after being silenced (FIG. 1D); on the contrary, the over-expression of RRM2B gene can inhibit SMMC-7721 and Huh7 cell migration and invasion (FIG. 1E).

2.2 in vivo experiments

The experiment of orthotopic transplantation tumor of liver in nude mice proves that RRM2B gene silencing promotes the capacity of SMMC-7721 and HCC-LY10 hepatoma cells to transfer in the liver and transfer in the distant lung of nude mice (FIG. 1F).

In conclusion, the results show that RRM2B can inhibit the migration, invasion and metastasis of liver cancer cells in both cell experiments and in vivo experiments.

Example 3 the status of p53 Gene does not affect the relationship between RRM2B Gene and hepatocellular carcinoma cell metastasis

3.1 determination of RRM2B expression in a hepatoma cell line with known p53 levels in relation to p53

Since RRM2B is the target gene of the wild-type p53 gene, it was analyzed whether the state of the p53 gene affects the effect of RRM2B on the metastasis of hepatoma cells.

Since the status of some liver cancer cell line p53 genes is known, for example, cell line p53 genes such as liver cancer cell line MHCC-97L, MHCC-97H, MHCC-97, SNU-182 are mutant, and cell line p53 genes such as SMMC-7721 are wild-type, and cell p53 genes such as Hep3B are deleted. Therefore, the expression levels of p53 and RRM2B protein in the hepatoma cell line were analyzed, and the results showed that the basal expression level of the basal RRM2B protein was not related to the status of the p53 gene (fig. 2).

3.2 relationship between p53 and RRM2B expression in clinical liver cancer samples

This example analyzed whether the expression level of p53 in 236 liver cancer tissues affected the relationship between RRM2B and intrahepatic metastasis in liver cancer patients. Since wild-type p53 is unstable, liver cancer tissues that were positive for p53 immunohistochemical staining were considered mutant p 53.

In order to confirm whether the p53 gene is mutant or not, 68 cases of mutations of the p53 gene in liver cancer tissues are analyzed, and as a result, a point mutation of AGG → AGT appears at the 249-th exon 7 of the p53 gene, resulting in the mutation of arginine into serine. This mutation was found in 20 out of 68 liver cancer tissues, and sequencing of the liver cancer tissue positive for immunohistochemical staining p53 revealed that the p53 gene was mutated (FIG. 3). It can be seen that the mutation rate of the p53 gene in liver cancer is about 29.4% (20/68), and the p53 gene is proved to be mutant by sequencing analysis of liver cancer tissues with positive p53 immunohistochemical staining. Statistical analysis results showed that expression of p53 did not affect the relationship between RRM2B and intrahepatic metastasis of liver cancer (table 5).

Taken together, these results all indicate that the status of p53 gene does not affect the relationship between RRM2B and hepatocellular carcinoma metastasis.

TABLE 5

Example 4 RRM2B inhibits the influencing pathways of liver cancer cell migration and invasion.

This example studies the pathway of the RRM2B that inhibits the migration and invasion of hepatoma cells.

4.1RRM2B inhibits phosphorylation levels of Akt1

Overexpression of the RRM2B gene in SMMC-7721 and Huh7 cells inhibited the phosphorylation of Akt1, whereas silencing of RRM2B gene up-regulated the phosphorylation of Akt1 (FIGS. 6A and 6B). While PTEN is a prerequisite for antagonism of PI3K-AKT signaling, the results showed that RRM2B overexpression upregulated PTEN expression, whereas PTEN expression was reduced following RRM2B gene silencing (fig. 6A and 6B). Furthermore, it was found by assaying the levels of PTEN mRNA that RRM2B overexpresses the level of PTEN mRNA (6C).

4.2 Effect of RRM2B Gene on PTEN promoter Activity

After constructing a reporter gene vector containing the PTEN promoter, the effect of RRM2B gene on PTEN promoter activity was analyzed, and the results showed that RRM2B was able to up-regulate PTEN promoter activity after overexpression (fig. 6D).

4.3 analysis of whether RRM2B affects PTEN promoter Activity through Egr-1 transcription factor

Since the promoter of PTEN contains the binding site of the transcription factor Egr-1, RRM2B was analyzed for whether it affects PTEN promoter activity via Egr-1 transcription factor.

4.3.1 Effect of RRM2B on Egr-1mRNA expression

Quantitative PCR results showed that RRM2B overexpression upregulated expression of Egr-1mRNA, whereas expression of Egr-1mRNA was reduced following RRM2B gene silencing (fig. 6E).

4.3.2 Effect of Egr-1 on PTEN promoter Activity

The results of luciferase experiments in which SMMC-7721 and Huh7 cells were co-transfected with Egr-1 plasmid and PTEN reporter vector showed that Egr-1 significantly upregulated PTEN promoter activity (FIG. 6F).

In conclusion, RRM2B inhibits the phosphorylation level of Akt1 in hepatoma cells mainly by up-regulating the expression level of Egr-1/PTEN.

Example 5RRM2B inhibits the development of EMT by inhibiting the phosphorylation level of Akt1 in hepatoma cells

5.1 synthesis of siRNA to Akt1, inhibition of expression level of Akt1 by transfection of siRNA to Akt1 into SMMC-7721 cells, results show that inhibition of Akt1 level is accompanied by increased expression of E-cadherin and decreased expression of N-cadherin, and cell migration and invasion due to silencing of RRM2B gene are reduced (FIGS. 7A and 7C), and the increased expression of E-cadherin and decreased expression of N-cadherin indicate the occurrence of EMT phenomenon.

5.2 action of RRM2B Gene-silenced SMMC-7721 and HCC-LY10 cells with Akt phosphorylation inhibitors LY294002 and wortmannin, the results were consistent with the experimental results of interfering with Akt, i.e., LY294002 and wortmannin, after inhibiting the phosphorylation level of Akt, down-regulated the expression of N-cadherin, up-regulated the expression of E-cadherin, and simultaneously inhibited cell migration and invasion due to RRM2B gene silencing (FIGS. 7B and 7D).

5.3 overexpression of RRM2B consistently activated the phosphorylation level of Akt1

SMMC-7721 cell virus overexpressed by RRM2B gene was infected with sustained activation Akt (Myr-Akt), and it was found that sustained activation of Akt could significantly antagonize cell migration and decrease in migration ability due to overexpression of RRM2B (FIGS. 7E and 7F).

Example 6 Effect of RRM2B on EMT-like changes in hepatoma cells

6.1 the RRM2B gene can enhance the migration, invasion and transfer ability of liver cancer cells after being silenced

After RRM2B gene silencing, the hepatoma cells showed EMT-like changes, including the transition from epithelial to spindle-shaped mesenchymal morphology (fig. 4A), and thus, the hepatoma cells migration, invasion and metastasis capacity was enhanced after RRM2B gene silencing.

The EMT-related molecule was also altered, expression of the epithelial marker E-cadherin decreased, and expression of the mesenchymal marker N-cadherin and the corresponding transcription factor slug increased (fig. 4B).

6.2 over-expression of RRM2B gene can reduce migration, invasion and metastasis of liver cancer cells

After overexpression of the RRM2B gene in hepatoma cells, RRM2B gene overexpression was able to significantly suppress the EMT-like changes in hepatoma cells, including RRM2B up-regulating the expression of E-cadherin, while the expression of N-cadherin and slug was reduced (fig. 4C). Concurrent RRM2B gene silencing nude mouse tumor tissue had decreased E-cadherin and increased N-cadherin expression (fig. 4D).

Therefore, these results indicate that RRM2B gene can finally inhibit the occurrence of EMT in hepatoma cells.

6.3 Effect of RRM2B on cell viability and tumor Stem cells

Since an important feature of EMT is tumor stem cell activity, this example also determined the effect of RRM2B on EMT's characteristics against cell death and against tumor stem cells.

As a result, it was found that RRM2B overexpression increased the drug sensitivity of chemotherapeutic drug doxorubicin, whereas RRM2B gene silencing inhibited the drug sensitivity of doxorubicin (fig. 4E). In addition, RRM2B was found to be able to inhibit the expression of liver cancer stem cell markers CD133 and CD44 (fig. 5).

Therefore, the above results indicate that RRM2B inhibits hepatoma cell EMT-like changes.

Example 7 Effect of increasing Egr-1 expression and/or Activity on RRM 2B-mediated alterations in cell migration and invasion Capacity

The transfection of pcDNA3.1-Egr-1 plasmid into SMMC-7721 cells with RRM2B gene silencing resulted in the finding that Egr-1 overexpression reversed the increase in cell migration and invasion capacity due to RRM2B gene silencing (FIGS. 8A and 8B); in contrast, SMMC-7721 cells overexpressing the RRM2B gene were transfected with 2 sirnas of Egr-1, and were also able to reverse the decrease in cell migration and invasion capacity due to overexpression of the RRM2B gene (fig. 8C and 8D).

Experiments show that RRM2B mainly plays a role in inhibiting migration, invasion and metastasis of liver cancer cells through an Egr-1/PTEN/Akt1 pathway.

Example 8 correlation of E-cadherin and RRM2B in liver cancer tissues

8.1 expression levels of E-cadherin, N-cadherin in liver cancer tissues and their correlation with RRM2B protein expression.

The results showed that in the liver cancer tissue, the expression level of E-cadherin was lower than that in the corresponding paraneoplastic tissue (FIG. 9A), while the expression level of N-cadherin was higher than that in the corresponding paraneoplastic tissue (FIG. 9B).

8.2 changes in liver cancer tissue (with or without metastasis) E-cadherin, N-cadherin, p-Akt1, total Akt, and RRM 2B.

The results showed that E-cadherin and RRM2B were expressed in decreased levels and N-cadherin and p-Akt1 levels in liver cancer tissues with concomitant metastasis (FIG. 9C), which were consistent with in vitro experimental results.

Correlation of expression levels of 3E-cadherin and RRM2B in liver cancer tissues.

The results showed that RRM2B was significantly positively correlated with the expression of E-cadherin protein in liver cancer (fig. 9D and 9E).

Example 9Egr-1 binds the promoter region of RRM2B and inhibits the expression level of RRM2B

9.1 construction of reporter vectors containing different truncated forms of the RRM2B promoter, luciferase assay results showed that RRM2B promoter activity was lower than that of the RRM2B (-234/-5) fragment (-896/-5, -763/-5, -375/-5) (FIGS. 10A and 10B).

The results indicate that at least one repressive transcription factor may exist between RRM2B promoter region-375/-234. By analyzing possible transcription factors between the RRM2B promoter region-375/-234, the results of the study showed that the transcription factor Egr-1 could inhibit the activity of the RRM2B promoter, but that Egr-1 could not inhibit the promoter activity of the-234/-5 fragment, so that the transcription factor Egr-1 could bind to the RRM2B promoter region-375/-234 and inhibit the promoter activity, and that Egr-1 could inhibit the RRM2B promoter activity with dose dependence (FIG. 10C).

9.2 construction of RRM2B reporter gene vectors containing mutant Egr-1 binding sites to demonstrate binding of Egr-1 to the promoter region of RRM 2B.

As a result, it was found that Egr-1 could not inhibit the promoter activity of RRM2B containing an Egr-1 binding site mutant.

Binding of Egr-1 to the promoter of RRM2B was then demonstrated by chromatin immunoprecipitation experiments (fig. 10D). These results indicate that the transcription factor Egr-1 binds to and inhibits the expression of the RRM2B promoter region.

9.3 transfection of Egr-1 overexpression and interference vectors in SMMC-7721 and Huh7 cells, respectively, demonstrated that Egr-1 inhibits the expression of RRM 2B.

The results show that Egr-1 is able to suppress the expression of RRM2B (fig. 10E), whereas interfering with Egr-1 up-regulates the expression of RRM2B (fig. 10F). Thus, all of these results indicate that there is a negative feedback loop between RRM2B and Egr-1 that regulates the expression level of RRM2B from liver cancer.

All documents referred to herein are incorporated by reference into this application as if each had been 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 (26)

1. The application of ribonucleotide reductase subunit M2B (Ribonucleoside-diphosphate reductasexunit M2B, RRM2B) gene or protein detection reagent thereof is characterized in that the kit is used for preparing a kit for diagnosing liver cancer cell metastasis.

2. The use of claim 1, wherein the metastasis includes intrahepatic and extrahepatic metastasis.

3. The use according to claim 2, wherein the extrahepatic metastasis comprises lung metastasis, bone metastasis, or brain metastasis.

4. The use according to claim 1, wherein the RRM2B gene or protein thereof is from a mammal.

5. The use according to claim 1, wherein the coding sequence of the RRM2B gene is as shown in SEQ ID No. 42.

6. The use according to claim 1, wherein the RRM2B gene or its protein detection reagent comprises an antibody specific for RRM2B, a specific amplification primer, a probe or a chip.

7. The use according to claim 6, wherein the detection reagent for detecting RRM2B protein or mRNA comprises:

(a) an antibody specific against RRM2B protein; and/or

(b) A specific primer that specifically amplifies mRNA or cDNA of RRM 2B.

8. The use according to claim 7, wherein the specific antibody is an Anti-p53R2 antibody.

9. The use according to claim 7, wherein the primer sequences specific for the amplification of mRNA or cDNA of RRM2B are as follows:

an upstream primer: AGGAGGTGCAGGTTCCAGAG (SEQ ID NO. 3)

A downstream primer: CTGCTATCCATCGCAAGGC (SEQ ID NO: 24).

10. The use of claim 6, wherein said detection comprises enzyme-linked immunosorbent assay or time-resolved immunofluorescence assay.

11. The use according to claim 6, wherein the RRM2B protein or antibody specific therefor is conjugated to or carries a detectable label.

12. The use of claim 11, wherein the detectable label is selected from the group consisting of: a chromophore, a chemiluminescent group, a fluorophore, an isotope, or an enzyme.

13. The use according to claim 6, wherein the antibody specific for RRM2B is a monoclonal or polyclonal antibody.

14. The use of claim 1, wherein said detection is of a liver cancer tissue or a normal liver tissue sample.

15. The use of claim 14, wherein the normal liver tissue comprises para-cancerous tissue.

The application of RRM2B gene and protein is characterized in that the RRM2B gene and protein are used for preparing a pharmaceutical composition for inhibiting liver cancer.

17. The use according to claim 16, for the preparation of a pharmaceutical composition for inhibiting metastasis of hepatoma cells.

18. The use of claim 16, wherein the pharmaceutical composition comprises a therapeutically effective amount of the RRM2B gene or protein and a pharmaceutically acceptable carrier.

19. The use of claim 16, wherein the medicament is administered by a mode of administration selected from the group consisting of: oral, intravenous, intramuscular, subcutaneous, sublingual, rectal, nasal spray, oral spray, topical or systemic transdermal administration of the skin.

20. The use of claim 16, wherein the pharmaceutical formulation is selected from the group consisting of: tablet, capsule, injection, granule, and spray.

21. A method of screening a candidate compound for inhibiting metastasis of hepatoma cells, comprising the steps of:

(a) in the test group, adding a test compound into a culture system of liver cancer cells, and observing the expression quantity and/or activity of RRM2B in the cells of the test group; in the control group, the test compound is not added to the culture system of the same cells, and the expression amount and/or activity of the RRM2B in the cells of the control group are observed;

wherein, if the expression level and/or activity of RRM2B in the cells of the test group is higher than that of the control group, the test compound is a compound which can inhibit the metastasis of the liver cancer cells and has the promotion effect on the expression and/or activity of RRM 2B; and/or

(b) Testing the candidate compound obtained in step (a) for its inhibitory effect on metastasis of hepatoma cells.

22. The method of claim 21, wherein the step (b) comprises the steps of: adding a test compound into a culture system of the liver cancer cells of the test group, and observing the number of moving distances and/or invasion conditions of the liver cancer cells; adding no test compound into the culture system of the liver cancer cells of the control group, and observing the number of moving distances and/or invasion conditions of the liver cancer cells; wherein, if the migration distance or the invasion number of the liver cancer cells in the test group is obviously smaller than that of the control group, the test compound is a compound which has an inhibitory effect on liver cancer cell metastasis.

23. The method of claim 21, wherein step (a) further comprises one or more assays for the expression levels of RRM2B and Egr1 in said control and test groups.

24. The method of claim 23, wherein said plurality of measurements comprises measurements of the amount and/or activity of RRM2B and Egr1 and the trend of RRM2B expression and/or activity at the same time as the test compound is added, 12 hours after the addition, and 24 hours after the addition, respectively.

25. The method according to claim 24, wherein the candidate compound having both increased expression level and/or activity of Egr1 and RRM2B and no decreased change in expression level and/or activity of RRM2B is selected as the compound for inhibiting metastasis of hepatoma cells based on the results of the assay.

26. An expression vector of RRM2B gene, which is characterized by comprising the following elements:

(i) a promoter element;

(ii) RRM2B coding sequence; and

(iii) a stop codon element;

wherein the promoter element does not bind to Egr1, and the sequence of the promoter element is shown in SEQ ID No. 45.