CN104630338A - Application of RRM2B gene or protein thereof in metastasis of hepatocellular carcinoma - Google Patents

Abstract

The invention discloses application of RRM2B gene or protein thereof in metastasis of hepatocellular carcinoma, and particularly provides application of detection reagents of ribonucleoside-diphosphate reductase subunit M2B (RRM2B) gene or protein thereof, as well as the RRM2B gene or protein thereof for preparing kits for diagnosing metastasis of hepatocellular carcinoma. Furthermore, the RRM2B has an inhibiting effect on metastasis of hepatocellular carcinoma, and a new approach is provided to diagnosis and treatment of metastasis of hepatocellular carcinoma.

Description

RRM2B gene or the application of its albumen in hepatoma Metastasis

Technical field

The present invention relates to tumour diagnosis and treatment field.Particularly, the present invention relates to RRM2B gene or the application of its albumen in hepatoma Metastasis Diagnosis and Treat.

Background technology

Primary hepatocarcinoma is a kind of malignant tumour of serious harm human health, and the East Asia Region comprising China is the district occurred frequently of liver cancer, because its grade malignancy is high, prognosis mala, has become second fatal tumor in Chinese primary hepatocarcinoma.Wherein hepatocellular carcinoma (hepatocellular carcinoma, HCC) accounts for more than 90% of primary hepatocarcinoma.The generation that research shows liver cancer and viral hepatitis and takes in food that flavacin pollutes etc. and have substantial connection, its generation develop and oncogene unconventionality expression and cancer suppressor gene inactivation closely related.The molecular mechanism that development occurs HCC is very complicated, still imperfectly understands at present.Therefore, the relation of the gene function change that further exploratory development is new and liver cancer genesis and development and malignant characteristics thereof, to disclosing accurate molecular mechanism of its generation development, treatment plan reasonable in design and judging prognosis, the treatment level improving liver cancer is further significant.

Nucleotide is the building block molecule of DNA and RNA, the biosynthetic disorder of Nucleotide extensively affects Normocellular physiological function, finally cause Normocellular vicious transformation, because tumour cell has the ability of infinite multiplication, body is necessary for it to be needed to provide more Nucleotide.Therefore nucleotide metabolism plays very important effect in the generation and development of tumour.Research shows to have the enzyme of a series of key to regulate synthesis and the modification of Nucleotide, and wherein ribonucleotide reductase (RR) is exactly one of them.

In human body, RR is formed by there being three subunits, and a large subunit (RRM1) and two small subunits (RRM2 and RRM2B) are formed.Each subunit role is different, and as RRM1 is extensively admitted as cancer suppressor gene, RRM2 and RRM2B is then considered to affect DNA by the adjustment of p53 and repairs.But, about the concrete function of these two subunits and the research of mechanism of action too controversial, especially RRM2B, the at present not yet clearly mechanism of action of this gene and the concrete effect played in cancer.

Therefore, the generation of these genes and liver cancer, the relation of development, in the urgent need to carrying out the research to tumor-related gene, especially at the genes involved of the very high liver cancer of Chinese sickness rate, and are illustrated in this area, and provide new thinking for the Diagnosis and Treat of liver cancer.

Summary of the invention

The invention provides the mark RRM2B of a kind of diagnosing liver cancer transfer, and the application of RRM2B in Hepatoma therapy transfer.

First aspect present invention, provide a kind of ribonucleotide reductase subunit M2B (Ribonucleoside-diphosphate reductase subunit M2B, RRM2B) purposes of gene or its protein assay reagent, for the preparation of the transcellular test kit of diagnosing liver cancer.

In another preference, described transfer comprises Intrahepatic metastasis and extrahepatic metastases.

In another preference, described extrahepatic metastases comprises Lung metastases, Bone tumour or brain metastes.

In another preference, described RRM2B gene or its albumen from Mammals, preferably, from rodent (mouse or rat) or primate (such as people); More preferably, people is derived from.

In another preference, the encoding sequence of described RRM2B gene is as SEQ ID NO.:42 (Genbank ID:NM_015713.4):

(ATGGGCGACCCGGAAAGGCCGGAAGCGGCCGGGCTGGATCAGGATGAGAGATCATCTTCAGACACCAACGAAAGTGAAATAAAGTCAAATGAAGAGCCACTCCTAAGAAAGAGTTCTCGCCGGTTTGTCATCTTTCCAATCCAGTACCCTGATATTTGGAAAATGTATAAACAGGCACAGGCTTCCTTCTGGACAGCAGAAGAGGTCGACTTATCAAAGGATCTCCCTCACTGGAACAAGCTTAAAGCAGATGAGAAGTACTTCATCTCTCACATCTTAGCCTTTTTTGCAGCCAGTGATGGAATTGTAAATGAAAATTTGGTGGAGCGCTTTAGTCAGGAGGTGCAGGTTCCAGAGGCTCGCTGTTTCTATGGCTTTCAAATTCTCATCGAGAATGTTCACTCAGAGATGTACAGTTTGCTGATAGACACTTACATCAGAGATCCCAAGAAAAGGGAATTTTTATTTAATGCAATTGAAACCATGCCCTATGTTAAGAAAAAAGCAGATTGGGCCTTGCGATGGATAGCAGATAGAAAATCTACTTTTGGGGAAAGAGTGGTGGCCTTTGCTGCTGTAGAAGGAGTTTTCTTCTCAGGATCTTTTGCTGCTATATTCTGGCTAAAGAAGAGAGGTCTTATGCCAGGACTCACTTTTTCCAATGAACTCATCAGCAGAGATGAAGGACTTCACTGTGACTTTGCTTGCCTGATGTTCCAATACTTAGTAAATAAGCCTTCAGAAGAAAGGGTCAGGGAGATCATTGTTGATGCTGTCAAAATTGAGCAGGAGTTTTTAACAGAAGCCTTGCCAGTTGGCCTCATTGGAATGAATTGCATTTTGATGAAACAGTACATTGAGTTTGTAGCTGACAGATTACTTGTGGAACTTGGATTCTCAAAGGTTTTTCAGGCAGAAAATCCTTTTGATTTTATGGAAAACATTTCTTTAGAAGGAAAAACAAATTTCTTTGAGAAACGAGTTTCAGAGTATCAGCGTTTTGCAGTTATGGCAGAAACCACAGATAACGTCTTCACCTTGGATGCAGATTTTTAA)；

The protein sequence of its coding is as SEQ ID NO.:43 (GenbankID:NP_056528.2)

(MGDRAAGDDRSSSDTNSIKSNRKSSRRVIIYDIWKMYKAASWTAVDSKDHWNKKADKYISHIAAASDGIVNNVRSVVARCYGIINVHSMYSIDTYIRDKKRNAITMYVKKKADWARWIADRKSTGRVVAAAVGVSGSAAIWKKRGMGTSNISRDGHCDACMYVNKSRVRIIVDAVKITAVGIGMNCIMKYIVADRVGSKVANDMNISGKTNKRVSYRAVMATTDNVTDAD)。

In another preference, described RRM2B gene or its protein assay reagent comprise the specific antibody of RRM2B, specificity amplification primer, probe or chip.

In another preference, described detection RRM2B albumen or the detection reagent of mRNA comprise:

(a). the specific antibody of anti-RRM2B albumen; And/or

(b). the Auele Specific Primer of mRNA or cDNA of specific amplification RRM2B.

In another preference, shown specific antibody is Anti-p53R2 antibody (ab8105, purchased from Abcam company).

In another preference, the specific primer sequence of mRNA or cDNA of specific amplification RRM2B is as follows:

Upstream primer: AGGAGGTGCAGGTTCCAGAG (SEQ ID NO.:3)

Downstream primer: CTGCTATCCATCGCAAGGC (SEQ ID NO.:24)

In another preference, described detection comprises enzyme linked immunoassay method (ELISA method) and detects or Time-resolved Fluoimmunoassay (TRFIA method) detection.

In another preference, described RRM2B albumen or its specific antibody coupling has or with detectable label.

In another preference, described detectable label is selected from lower group: chromophore, chemiluminescent groups, fluorophore, isotropic substance or enzyme.

In another preference, the specific antibody of described RRM2B is monoclonal antibody or polyclonal antibody.

In another preference, described detection measures liver cancer tissue or normal liver tissue sample.

In another preference, described normal liver tissue comprises cancer beside organism.

Second aspect present invention, provide a kind of diagnostic kit for detecting liver cancer cell transfer, described test kit contains a container, containing the detection reagent detecting RRM2B albumen or mRNA in described container; And label or specification sheets, described label or specification sheets indicate described test kit for detecting liver cancer cell transfer.

In another preference, described detection liver cancer cell transfer refers to judge whether liver cancer cell transfer occurs, and/or judges possibility (susceptibility) size that liver cancer cell transfer occurs.

In another preference, described transfer comprises Intrahepatic metastasis and extrahepatic metastases.

In another preference, described extrahepatic metastases comprises Lung metastases, Bone tumour or brain metastes.

In another preference, described judgement comprises to be prejudged (prediction).

In another preference, described detection RRM2B albumen or the detection reagent of mRNA comprise:

(a). the specific antibody of anti-RRM2B albumen; And/or

(b). the Auele Specific Primer of mRNA or cDNA of specific amplification RRM2B.

In another preference, in described label or specification sheets, indicate following content:

When ratio≤0.5 of the RRM2B expression amount E2 of RRM2B expression amount E1 and normal liver cell or tissue in the liver cancer cell or tissue of detected object, then point out probability that this detected object liver cancer cell shifts higher than general population.

In another preference, described E2 is the normal liver cell of normal population or the RRM2B expression amount of tissue.

In another preference, described normal liver cell or tissue comprise the other liver cell of cancer or tissue.

In another preference, described expression amount is the relative expression quantity relative to crt gene (as beta-actin).

Third aspect present invention, provides the purposes of RRM2B gene, albumen or its promotor, for the preparation of suppressing liver cancer or suppressing the pharmaceutical composition of liver cancer cell transfer.

In another preference, described RRM2B gene or its albumen from Mammals, preferably, from rodent (mouse or rat) or primate (such as people); More preferably, people is derived from.

In another preference, described pharmaceutical composition comprises the treatment RRM2B gene of significant quantity, albumen or its promotor, and pharmaceutically acceptable carrier.

In another preference, described medicine carries out administration by the application method being selected from lower group: oral, intravenous injection, intramuscular injection, subcutaneous injection, sublingual administration, rectal perfusion, nasal spray, mouth spray, local skin or whole body transdermal administration.

In another preference, the preparation of described medicine is selected from lower group: tablet, capsule, injection, granule, sprays.

In another preference, described RRM2B inhibitor is applied to Mammals with the dosage of 0.01-20mg/kg body weight (each or every day).

In another preference, described Mammals comprises people, mouse, rat, more preferably, is people.

Fourth aspect present invention, provides the method for a kind of screening for the candidate compound that suppresses liver cancer cell to shift, comprises step:

In (a) test group, in the culture system of liver cancer cell, add test compounds, and observe expression amount and/or the activity of RRM2B in the cell of described test group; In control group, in isocellular culture system, do not add test compounds, and observe expression amount and/or the activity of RRM2B in the described cell of control group;

Wherein, if the expression amount of the RRM2B of cell and/or activity, higher than control group, just show that this test compounds is the compound having the suppression liver cancer cell of promoter action to shift to expression and/or the activity of RRM2B in test group; And/or

B (), for the candidate compound obtained in step (a), tests the restraining effect that described candidate compound shifts liver cancer cell.

In another preference, comprise step in step (b): in the culture system of test group liver cancer cell, add test compounds, and observe quantity and/or the invasion and attack situation of the distance of liver cancer cell movement; In the culture system of control group liver cancer cell, do not add test compounds, and observe quantity and/or the invasion and attack situation of the distance of liver cancer cell movement; Wherein, if in test group liver cancer cell migration distance or invasion and attack quantity be significantly less than control group, just show this test compounds be to liver cancer cell transfer have inhibiting compound.

In another preference, be also included in described control group and test group in described step (a) and one or many mensuration is carried out to RRM2B and Egr1 expression amount.

In another preference, described repeatedly measure comprise respectively add test compounds simultaneously, add after within 12 hours and after adding 24 hours, RRM2B and Egr1 expression amount and/or active and RRM2B expression amount and/or activity change trend are measured simultaneously.

In another preference, according to measurement result, select Egr1 and RRM2B expression amount and/or activity raise simultaneously and RRM2B expression amount and/or activity change without the candidate compound of downtrending as the compound suppressing liver cancer cell transfer.

Fifth aspect present invention, provides a kind of purposes of RRM2B inhibitor, for the preparation of the composition or the reagent that promote liver cancer cell transfer.

In another preference, described liver cancer cell comprises SMMC-7721, Huh7, HCC-LY10 cell.

Sixth aspect present invention, provide a kind of expression vector of RRM2B gene, described expression vector has following element:

(i) promoter element;

(ii) RRM2B encoding sequence; With

(iii) termination codon sub-element;

Wherein, described promoter element is not combined with Egr1.

In another preference, described promoter element sequence is as shown in SEQ ID NO.:44:

(ggtcatggcaagatcccgtcaatctttcagcttcaaactacttaaacaaatctctcttccattttatttttgttagaccaaactatttttttcaaaatatatttttttcaaaatatttgcctgactttagtaacgtgtccctctccccctcactcacccgcattcacacacacacacacacactttcccccagacacttccccttttcggttgtttacgcacgaaaccagccagccctttccctagcgaagtgtttggcggtgtagctgtcccgtccccggcgcttcccacagcaacagcgcgttctcttcgcctcccgcagtctctcaggacaggccaggctagcgcaaagtaccgtgttcctggaacaccacgagggcgagctcgggaatctcgagccggcctgcaggacacctgccgcaccaagtggctagagcccggggagggcgaggcgaggtgggacgaggcggggtggtgagcgggccgggaaggcgaggccgcgcggactctgggatagctcctcaggagtgggcgcccggagtgggcgagcggaggaggcggggccgatgaggtgaggtggggccagctggagtaggctaagcagtccagagcagggggcgtccctcagtggaaagccgggcgactgggcggtgcaacagagtaaggcggggccagcggaagcagggagatttccttaggccgcaggcggggaaatggggctggccggggtaggcggagccccgaggcaggggaggcgtggctggcagaggtaggcggtgccccaaggcaggcggggcttgccgagacagggataatcccttaggccacaggctgggaggcagggcaggccgaggcggggaggatcccttaggccgcagtaggggaggcgggtcggccgaggcggggagaaacccttaggccgcaggcggggaggcggggctggccgaagttaggcggagccccgaggcgggggaggcggggccgggccggcgcagggagagtcactcaatggacaggcgagaaagcaggaccggcgcggcggggcggggccggccgagtccctagagctgggggcggggcggacccagcggaccagcggaccacctgggtgctgtcgtagttggaggtggcctgaggagctcagttccctcagcgcccgtagcttcggcggagtctg)。

Seventh aspect present invention, provides the method for a kind of diagnosing liver cancer transfer, comprises step:

(i). prepare experimenter and test sample;

(ii). in detection test sample, RRM2B is relative to the mrna expression amount E1 of crt gene (as beta-actin), with the RRM2B expression amount E2 (i.e. reference value) of normal liver cell or tissue, when the probability of its ratio≤0.5 this detected object hepatoma Metastasis of prompting is higher than general population.

In another preference, described sample is liver cancer cell or tissue.

In another preference, described reference value is the expression amount of RRM2B in non-liver cancer tissue sample.

In another preference, described detecting step (ii) comprises the amount detecting RRM2B mRNA, or the amount of RRM2B cDNA; And/or detect the amount of RRM2B albumen.

In another preference, described detecting step (ii) is comprised and being detected by RT-PCR or PCR method.

In another preference, described detecting step (ii) comprises and uses the antibody of anti-RRM2B albumen to detect.

Eighth aspect present invention, provides a kind of method suppressing liver cancer cell to shift, comprises step: the RRM2B gene using safe and effective amount to the object (Mammals) of needs treatment, albumen or its promotor.

In another preference, described transfer comprises Intrahepatic metastasis, extrahepatic metastases.

Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.

Accompanying drawing explanation

Fig. 1 shows the expression of RRM2B in hepatocellular carcinoma and the RRM2B impact on liver cancer cell invasion and attack, migration and transfer.

Figure 1A shows the expression level of Immunohistochemical detection RRM2B and P53 in Tissues of Hepatocellular Carcinoma and corresponding cancer side, and result sees that RRM2B mainly expresses in cell cytosol, and the expression of RRM2B in liver cancer is lower than corresponding cancer beside organism; P53 mainly expresses in nucleus, and the expression of p53 in liver cancer is higher than corresponding cancer beside organism.

Figure 1B shows immunoblotting and detects the expression in liver cancer tissue and cancer beside organism of RRM2B albumen, and result sees that the expression of RRM2B in liver cancer is lower than corresponding cancer beside organism.

Fig. 1 C shows the expression of quantitative PCR detection RRM2B mRNA in liver cancer tissue and cancer beside organism, in 30 pairs of liver cancer tissues and cancer beside organism, the expression of RRM2B in liver cancer lower than corresponding cancer beside organism, p < 0.05.

Fig. 1 D shows Transwell experiment and detects impact on SMMC-7721 and HCC-LY10 cell migration and invasion and attack after RRM2B gene silencing.The migration and invasion of liver cancer cell can be promoted, * p < 0.05, * * p < 0.01 after result sees RRM2B gene silencing.

Fig. 1 E shows Transwell experiment and detects impact on SMMC-7721 and Huh7 cell migration and invasion and attack after RRM2B gene overexpression.The migration and invasion of liver cancer cell can be suppressed, * p < 0.05, * * p < 0.01 after result sees RRM2B gene overexpression.

Fig. 1 F shows by the impact on metastases ability after nude mice liver orthotopic transplantation tumor experiment detection RRM2B gene silencing, can promote after result shows RRM2B gene silencing that liver cancer cell is at the transfer of nude mice liver original position and distant place Lung metastases, * p < 0.05.

Fig. 2 shows the expression level of RRM2B and p53 in hepatoma cell line.

Fig. 3 shows the sudden change of p53 gene in liver cancer.

By sequencing analysis, the catastrophe of p53 gene the 7th exon in 68 routine liver cancer tissues, found that the point mutation occurring AGG → AGT at p53 gene the 7th exon 2 49 bit codon, result in arginine and be mutated into Serine.In 68 routine liver cancer tissues, find that there is 20 examples occur this sudden change, and the liver cancer tissue sequencing analysis p53 gene of the immunohistochemical staining p53 positive is sudden change.

Fig. 4 shows RRM2B and suppresses liver cancer cell EMT sample to change.

Fig. 4 A shows the corresponding morphological change of cell after SMMC-7721 and HCC-LY10 cell RRM2B gene silencing.

After Fig. 4 B shows SMMC-7721 and HCC-LY10 cell RRM2B gene silencing, detection E-calcium is mucoprotein, N-calcium is mucoprotein and the expression level of Slug.

Fig. 4 C shows SMMC-7721 and Huh7 cell RRM2B gene overexpression, and detection E-calcium is mucoprotein, N-calcium is mucoprotein and the expression level of Slug.

Fig. 4 D shows the SMMC-7721 cell nude mice of getting RRM2B gene silencing and becomes tumor tissue, is detected that E-calcium is mucoprotein, N-calcium is mucoprotein and the expression level of RRM2B by immunohistochemical methods.

Fig. 4 E shows SMMC-7721 and the Huh7 cell of RRM2B process LAN or gene silencing, and Zorubicin (2 μ g/ml) processes 24 hours, and MTT experiment detects the vigor of cell.

Fig. 5 shows the expression that RRM2B suppresses liver-cancer stem cell mark CD133 and CD44.

Fig. 5 A shows SMMC-7721 and the Huh7 cell of stable RRM2B process LAN or gene silencing, the expression level of quantitative PCR detection CD133, CD44, notch1, manog and Oct4.

Fig. 5 B shows SMMC-7721 and the Huh7 cell of stable RRM2B process LAN, the expression level of immune-blotting method CD133, CD44 and RRM2B.

Fig. 5 C shows the SMMC-7721 cell of RRM2B gene silencing, the expression level of immune-blotting method CD133, CD44 and RRM2B.

Fig. 6 shows RRM2B affects Akt1 phosphorylation by the expression level of regulation and control Egr-1 and PTEN.

Fig. 6 A shows immunoblotting and detects the expression level that RRM2B stablizes Huh7 and SMMC-7721 cell PTEN, phosphorylation Akt (p-Akt), always Akt and RRM2B of process LAN.

Fig. 6 B shows the expression level of PTEN, p-Akt in SMMC-7721 and the HCC-LY10 cell of immune-blotting method RRM2B gene silencing, total Akt and RRM2B.

After Fig. 6 C to show in Hepatocellular carcinoma cell line, Huh7 and HCC-LY10 process LAN or gene silencing RRM2B, the level of quantitative PCR detection PTEN mRNA.

Fig. 6 D shows transfection in Hepatocellular carcinoma cell line and Huh7 and comprises the reporter gene of PTEN promotor, detects RRM2B to the impact of PTEN promoter activity.

After Fig. 6 E to show in Hepatocellular carcinoma cell line, Huh7 and HCC-LY10 process LAN or gene silencing RRM2B, the level of quantitative PCR detection Egr-1mRNA.

Fig. 6 F shows pcDNA3.1-Egr-1 plasmid or empty carrier pcDNA3.1 and PTEN-Luc plasmid co-transfection SMMC-7721 and Huh7 cell, detects Egr-1 to the impact of PTEN promoter activity.

Fig. 7 show interference Akt1 express or Akt1 phosphorylation inhibitor LY294002 and wortmannin on the impact of fucosylation and invasion and attack.

Fig. 7 A shows the siRNA of the SMMC-7721 cell transfecting Akt1 of RRM2B gene silencing, and immunoblotting detects the expression level that Akt1, E-calcium is mucoprotein and N-calcium is mucoprotein.

Fig. 7 B shows the SMMC-7721 cell 12 hours that LY294002 (10 μMs) and wortmannin (1 μM) act on RRM2B gene silencing, and immunoblotting detects the expression level that Akt1, E-calcium is mucoprotein and N-calcium is mucoprotein.

After Fig. 7 C shows the siRNA of SMMC-7721 cell transfecting Akt1 of RRM2B gene silencing, Transwell experiment detects the impact of its on cell migration and invasion and attack.

Fig. 7 D shows the SMMC-7721 cell 12 hours that LY294002 (10 μMs) and wortmannin (1 μM) act on RRM2B gene silencing, and Transwell experiment detects the impact of its on cell migration and invasion and attack.

The SMMC-7721 cell virus that Fig. 7 E shows RRM2B gene overexpression infects sustained activation Akt (Myr-Akt), and immunoblotting detects the expression level of p-Akt, Akt1 and RRM2B.

The SMMC-7721 cell virus that Fig. 7 F shows RRM2B gene overexpression infects sustained activation Akt (Myr-Akt), and Transwell experiment detects the impact of its on cell migration and invasion and attack. ^*p＜0.05， ^**p＜0.01。

Fig. 8 shows RRM2B by raising migration and the invasion and attack of the expression inhibiting liver cancer cell of Egr-1.

Fig. 8 A shows SMMC-7721 cell transfecting pcDNA3.1-Egr-1 plasmid or the empty carrier pcDNA3.1 of RRM2B gene silencing, detects the expression level of Egr-1 and RRM2B after 48 hours.

Fig. 8 B shows the SMMC-7721 cell transfecting pcDNA3.1-Egr-1 plasmid of RRM2B gene silencing or the impact of empty carrier pcDNA3.1, Transwell experiment its on cell migration of detection and invasion and attack.

Fig. 8 C shows 2 siRNA of the SMMC-7721 cell transfecting Egr-1 of RRM2B gene overexpression, detects the expression level of Egr-1 and RRM2B after 48 hours.

Fig. 8 D shows 2 siRNA, Transwell experiment its on cell migration of detection of the SMMC-7721 cell transfecting Egr-1 of RRM2B gene overexpression and the impact of invasion and attack. ^*p＜0.05， ^**p＜0.01。

Fig. 9 shows the mucoprotein expression positive correlation of expression in liver cancer with RRM2B of E-calcium.

Fig. 9 A shows the mucoprotein expression level in liver cancer tissue (T) and corresponding cancer beside organism (N) of quantitative PCR detection E-calcium.

Fig. 9 B shows the mucoprotein expression level in liver cancer tissue (T) and corresponding cancer beside organism (N) of quantitative PCR detection N-calcium.

Fig. 9 C shows immunoblotting detection liver cancer tissue (occur together transfer or non-diverting), and middle E-calcium is mucoprotein, N-calcium is mucoprotein, the expression level of p-Akt, Akt1 and RRM2B.

Fig. 9 D shows immunohistochemical methods and detects the expression (representational 2 example tissues) that in liver cancer tissue, RRM2B and E-calcium is mucoprotein.

Fig. 9 E shows the result according to the mucoprotein immunohistochemical methods of RRM2B and E-calcium, analyzes the mucoprotein expression correlation of expression in liver cancer with RRM2B of E-calcium.

Figure 10 shows Egr-1 in conjunction with the promoter region of RRM2B and suppresses the expression of RRM2B.

Figure 10 A shows the activity that luciferase assay detects different truncated segment RRM2B promotor.

Figure 10 B shows the reporter gene cotransfection SMMC-7721 cell comprising RRM2B promotor of pcDNA3.1-Egr-1 or empty carrier pcDNA3.1 and different truncated segment, and luciferase assay detects relative promoter activity.

Figure 10 C shows pcDNA3.1-Egr-1 or empty carrier pcDNA3.1 and RRM2B-Luc (-375/-5, comprise the Egr-1 binding site of wild-type or sudden change) plasmid co-transfection SMMC-7721 cell, luciferase assay detects relative promoter activity.

Figure 10 D shows chromatin immune co-precipitation (CHIP) experiment detection Egr-1 and is attached to RRM2B promoter region.

Figure 10 E shows pcDNA3.1-Egr1 or empty carrier pcDNA3.1 transfection SMMC-7721 and Huh7 cell, the expression level of immune-blotting method RRM2B and Egr-1.

Figure 10 F shows the siRNA of SMMC-7721 and Huh7 cell transfecting 2 Egr-1, the expression level of immune-blotting method RRM2B and Egr-1.

Embodiment

The present inventor, through in depth studying for a long time, have unexpectedly discovered that the low expression in liver cancer of RRM2B gene, and the transfer of process LAN RRM2B gene pairs liver cancer is inhibited.

Experiment shows, the expression of RRM2B in liver cancer reduces, and the expression of RRM2B in liver cancer and liver cancer Intrahepatic metastasis are negative correlation, and the expression of RRM2 affects the process of liver cancer patient, therefore, whether expression amount and/or active thus diagnosing liver cancer by detecting RRM2B shift.

The present inventor also finds, when after liver cancer cell process LAN RRM2B gene, RRM2B significantly can suppress invasion and attack and the migration of liver cancer cell, on the contrary, by can significantly promote cell invasion and migration after the expression of RNA AF panel RRM2B.

In addition, Vitro Experimental Results shows, RRM2B suppresses the invasion and attack of liver cancer cell and the major avenues of approach p53 moved not by RRM2B to work, but is reach by regulation and control Egr-1/PTEN/Akt1 path the effect suppressing hepatoma Metastasis.Meanwhile, experiment shows, Transcription factor Egr-1 can be attached to the promoter region of RRM2B gene and suppress the expression of RRM2B gene, therefore, there is the expression level of a kind of degenerative regulation and control loop regulation and control RRM2B between Egr-1 and RRM2B.

On this basis, the present invention is completed.

As used herein, term " hepatoma Metastasis ", " liver cancer cell transfer ", " invasion and attack of liver cancer cell or migration " can exchange use, all refer to that primary lesion is in liver, by routes of metastasis such as hematogenous metastasis, lymphatic metastasis, Implantation matastasis, make in Primary Hepatic carcinogenesis liver or extrahepatic metastases.In this article, when relating to cell experiment, as transwell wears in film experiment, the invasion and attack of liver cancer cell or migration refer to liver cancer cell after cultivation or particular stimulation, and what liver cancer cell showed specifically divides a word with a hyphen at the end of a line and multiplication capacity.

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

In the present invention, " albumen of the present invention ", " polypeptide of the present invention ", " RRM2B albumen " are used interchangeably, and refer to ribonucleotide reductase subunit M2B (Ribonucleoside-diphosphate reductase subunit M2B, RRM2B).Should be understood that described term also comprises active fragments and the derivative of RRM2B.

In the present invention, " gene of the present invention ", " polynucleotide of the present invention " refer to the nucleotide sequence of coding RRM2B albumen or its active fragments and derivative, comprise justice and antisense nucleic acid.

In the present invention, term " RRM2B albumen " or " RRM2B polypeptide " are used interchangeably, and all refer to albumen or the polypeptide with people's albumen RRM2B aminoacid sequence.

Ribonucleotide reductase (ribonueleotide reductases, RR) be distributed widely in various biomass cells, its effect participates in the process that ribonucleotide is reduced into deoxyribonucleotide, in nucleotide metabolism process, play central role, be the enzyme uniquely making ribonucleotide change deoxyribonucleotide into.And the starting material that deoxyribonucleotide is DNA synthesis and repairs, therefore this enzyme is the rate-limiting enzyme of synthesis and reparation in DNA path.

RR holoenzyme structure comprises large subunit α and small subunit β, forms the different tetramer structure of α 2 β 2 and just can possess activity.In human body, RR is formed by there being three subunits, and a large subunit (RRM1) and two small subunits (RRM2 and RRM2B) are formed.

Prove that RRM1 is tumor suppressor gene mouse and human cell line, process LAN RRM1 can make cell-cycle arrest in the G2 phase, causes apoptosis.RRM1 suppresses the propagation of lung carcinoma cell to have with transfer and the expression of PTEN necessarily to contact.

RRM2B is the homologous gene of RRM2, about has 80% homology with RRM2, at first as the target gene of tumor suppressor protein p53.But RRM2B and RRM2 has any different therebetween.RRM2B transcribes target spot as p53, and cell cycle correlation factor regulates transcribing of RRM2, such as NF-Y and E2F.RRM1 and RRM2 transcribes the S phase mainly occurring in the cell cycle, because the transformation period of RRM1 is very long, can stable existence in the whole cell cycle, and the transformation period of RRM2 is relatively short, and therefore the activity of RR holoenzyme depends primarily on the synthetics and degradation of RRM2 albumen.But when cell is in hibernation, when lacking RRM2, RRM2B and RRM1 combines and forms holoenzyme, for DNA damage reparation provides dNTP.When DNA damage, provide dNTP at the cell of p53 wild-type mainly through the DNA damage reparation that is expressed as of RRM2B.But if p53 functionally inactive, the function that RRM2 then supplements RRM2B repairs impaired DNA.

Find in the research of human pancreatic cancer cell, process LAN RRM2 can cause increase and the MMP-9 up-regulated of cell invasion ability, and invading of the up-regulated of MMP-9 and tumour is moistened and transfer is proportionate; Find that the expression of RRM2B promotes the invasion and attack of tumour, transfer affect prognosis in the research of the esophageal carcinoma and oral carcinoma; The expression of same research discovery RRM2B in small cell lung cancer is relevant to the progress of lung cancer.But in different reports on RRM2B how to affect tumor cell proliferation and invasion and attack have different conclusions, Yanamoto etc. think RRM2B by E-calcium mucoprotein/β-catenin path promotes the invasion and attack of oral carcinoma, but Zhang etc. report that RRM2B is by raising p21 and lowering the propagation of expression inhibiting oral carcinoma KB cell of cyclin D1.In addition, in colorectal carcinoma, find that the propagation of RRM2B and colorectal carcinoma is negative correlation.

In sum, the effect of RRM2B in different tumour is different, even if be also disputable in the result of same tumor research.

In the present invention, a kind of nucleotide sequence of preferred RRM2B gene (Genbank ID:NM_015713.4) as shown in SEQ ID NO.:42; The RRM2B protein sequence (Genbank ID:NP_056528.2) as shown in SEQ ID NO.:43 of its coding.

As used herein, " separation " refers to that material is separated from its primal environment (if natural substance, namely primal environment is natural surroundings).As the polynucleotide under the native state in active somatic cell and polypeptide do not have separation and purification, but same polynucleotide or polypeptide as from native state with in other materials existed separately, then for separation and purification.

As used herein, " the RRM2B albumen of separation or polypeptide " refers to that RRM2B albumen is substantially free of natural other albumen relative, lipid, carbohydrate or other material.Those skilled in the art can purify RRM2B albumen with the purified technology of protein of standard.Substantially pure polypeptide can produce single master tape on non-reducing polyacrylamide gel.In the present invention, RRM2B albumen comprises fusion rotein and non pregnant women.

Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, improvement on synthesis, preferred recombinant polypeptide.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

Polynucleotide of the present invention can be DNA form or rna form.DNA form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-strand.DNA can be coding strand or noncoding strand.

The polynucleotide of the mature polypeptide of coding RRM2B comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional coding sequence; The encoding sequence (with optional additional coding sequence) of mature polypeptide and non-coding sequence.Term " polynucleotide of coded polypeptide " can be the polynucleotide comprising encoding such peptides, also can be the polynucleotide also comprising additional code and/or non-coding sequence.

The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, the sum analogous to general Dedekind sum of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient of non-natural generation.These nucleotide variants comprise and replace varient, Deletion variants and insertion varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be the replacement of one or more Nucleotide, disappearance or insertion, but can not from the function changing in fact its coded polypeptide.

The invention still further relates to the nucleic acid fragment with above-mentioned sequence hybridization, comprise the nucleic acid fragment of justice and antisense.As used herein, the length of " nucleic acid fragment ", at least containing 15 Nucleotide, is better at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or to be separated the polynucleotide of coding RRM2B albumen.

People RRM2B Nucleotide full length sequence of the present invention or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic usually.For pcr amplification method, can according to published relevant nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the cDNA storehouse prepared by ordinary method well known by persons skilled in the art as template, amplification and relevant sequence.When sequence is longer, usually needs to carry out twice or repeatedly pcr amplification, and then the fragment that each time amplifies is stitched together by proper order.

Once obtain relevant sequence, just relevant sequence can be obtained in large quantity with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated from the host cell after propagation by ordinary method and obtains relevant sequence.

In addition, also relevant sequence can be synthesized, when especially fragment length is shorter by the method for synthetic.Usually, by first synthesizing multiple small segment, and then carry out connect can obtain the very long fragment of sequence.

The method of application round pcr DNA amplification/RNA is optimized for and obtains gene of the present invention.Primer for PCR suitably can be selected according to sequence information of the present invention disclosed herein, and using conventional procedures synthesis.Using conventional procedures is as the DNA/RNA fragment increased by gel electrophoresis abstraction and purification.

The present invention also relates to the carrier comprising polynucleotide of the present invention, and with the host cell that carrier of the present invention or RRM2B albumen coded sequence produce through genetically engineered, and the method for polypeptide of the present invention is produced through recombinant technology.

By the recombinant DNA technology of routine, polynucleotide sequence of the present invention can be utilized to can be used to the RRM2B albumen of expression or Restruction.In general following steps are had:

(1). with the polynucleotide (or varient) of encoding human RRM2B albumen of the present invention, or transform or suitable host cell of transduceing with the recombinant expression vector containing these polynucleotide;

(2). the host cell cultivated in suitable substratum;

(3). separation, protein purification from substratum or cell.

Method well-known to those having ordinary skill in the art can be used for building containing people RRM2B DNA sequences encoding and the suitable expression vector of transcribing/translating control signal.These methods comprise recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc.Described DNA sequence dna can be effectively connected in the suitable promotor in expression vector, synthesizes to instruct mRNA.Expression vector also comprises ribosome bind site and the transcription terminator of translation initiation.

In a preference, promotor suitable in expression vector of the present invention comprises wild-type RRM2B gene promoter and saltant type RRM2B gene promoter.Wherein said saltant type RRM2B gene promoter does not have the activity be combined with Egr1.

A kind of preferred saltant type RRM2B gene promoter is as shown in SEQ ID NO.:45:

(agggataatcccttaggccacaggctgggaggcagggcaggccgaggcggggaggatcccttaggccgcagtaggggaggcgggtcggccgaggcggggagaaacccttaggccgcaggcgttaaggcttggctggccgaagttaggcggagccccgaggcgggggaggcggggccgggccggcgcagggagagtcactcaatggacaggcgagaaagcaggaccggcgcggcggggcggggccggccgagtccctagagctgggggcggggcggacccagcggaccagcggaccacctgggtgctgtcgtagttggaggtggcctgaggagctcagttccctcagcgcccgtagcttcggcggagtctg)。

In addition, expression vector preferably comprises one or more selected marker, to be provided for the phenotypic character selecting the host cell transformed, as Tetrahydrofolate dehydrogenase, neomycin resistance and green fluorescent protein (GFP) that eukaryotic cell is cultivated, or for colibacillary tsiklomitsin or amicillin resistance.

Comprise the carrier of above-mentioned suitable DNA sequence dna and suitably promotor or control sequence, may be used for transforming suitable host cell, with can marking protein.

Host cell can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, the bacterial cell of streptomyces; Fungal cell is as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast etc. of CHO, COS or 293 cells.

Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.When host be prokaryotic organism as intestinal bacteria time, the competent cell that can absorb DNA can be gathered in the crops at exponential growth after date, uses CaCl ₂method process, step used is well-known in this area.Another kind method uses MgCl ₂.If needed, transform and also can be undertaken by the method for electroporation.When host is eukaryote, can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical methods is as microinjection, electroporation, liposome packaging etc.

The transformant obtained can be cultivated by ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to host cell used, substratum used in cultivation can be selected from various conventional medium.Cultivate under the condition being suitable for host cell growth.When after host cell growth to suitable cell density, the promotor selected with the induction of suitable method (as temperature transition or chemical induction), cultivates for some time again by cell.

Recombinant polypeptide in the above methods can be expressed or be secreted into extracellular in cell or on cytolemma.If needed, can utilize its physics, the albumen of being recombinated by various separation method abstraction and purification with other characteristic of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation process, combination by protein precipitant process (salting-out method), centrifugal, the broken bacterium of infiltration, super process, ultracentrifugation, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.

Egr-1/PTEN/Akt1 path

PTEN is a very important tumor suppressor protein, is the important negative regulators of of PI3K/Akt signal transduction pathway, stops developing of tumour by effective antagonism PI3K-AKT signal transduction pathway.As lipid phosphatase, triphosphoric acid phosphatidylinositols (PIP3) dephosphorylation on cytolemma can be generated PIP2 by PTEN, and be the product of PI3K due to PIP3 and mediate the activation of Akt, PTEN makes PIP3 dephosphorylation, maintain the low-level of PIP3, thus lower PI3K/Akt path.

Egr-1 can regulate and control polygenic expression perhaps as a kind of important transcription factor, research shows that the promoter region at PTEN gene includes the binding site of Transcription factor Egr-1, and Egr-1 can be attached to the promoter region of PTEN gene and can promote the expression level of PTEN.Therefore, Egr-1 mainly through being attached to the promoter region of PTEN and promoting the expression of PTEN, and then suppresses the phosphorylation level of Akt.

Epithelial and stromal transforms (EMT)

Refer to that epithelial cell is converted into the biological procedures with interstitial phenotype cells by specific program.In fetal development, chronic inflammatory diseases, tissue reconstruction, cancer metastasis and multiple fibrotic disease, played vital role, it is the feature etc. main cytoskeleton and form with mesenchymal cell that the minimizing that its main feature has cell adhesion molecule (as mucoprotein in E-calcium) to express, cytokeratin cytoskeleton are converted into vimentin (Vimentin).By EMT, epithelial cell loses cell polarity, loses and the epithelial phenotype such as the connection of basilar membrane, obtains the interstitial phenotype such as ability of higher migration and invasion and attack, anti-apoptotic and degradation of cell epimatrix.

EMT is the important biomolecule process that the malignant cell (liver cancer) of epithelial cell origin obtains migration and invasion ability.Illustrate the molecular mechanism that EMT process occurs regulation and control malignant cell, specify the pathology sense in its generation in malignant tumour, development, transfer, and exploration is the key scientific problems of EMT Mechanism Study in metastases based on the diagnostic method of EMT key molecule and the treatment means of target EMT key molecule.

Promotor and pharmaceutical composition

Utilize albumen of the present invention, by various conventional screening assays, can filter out, with RRM2B albumen, interactional material occur, especially promotor etc.

The promotor of RRM2B albumen of the present invention, when carrying out using (administration) on treating, can promote expression and/or the activity of RRM2B albumen, and then promotes expression and/or the activity of p53, thus suppresses liver cancer.In another preference, the RRM2B gene expression product that described RRM2B promotor comprises, promoted type miRNA, promoted type transcriptional regulator or promoted type target micromolecular compound.

Usually, these promotor can be formulated in nontoxic, inertia with in pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although pH value can with being formulated the character of material and illness to be treated and changing to some extent.The pharmaceutical composition prepared can carry out administration by conventional route, comprising (but being not limited to): in knurl, intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.

Present invention also offers a kind of pharmaceutical composition, it contains the RRM2B albumen of the present invention of safe and effective amount or its promotor and pharmaceutically acceptable carrier or vehicle.This kind of carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should match with administering mode.Pharmaceutical composition of the present invention can be made into injection form, such as, be prepared by ordinary method with physiological saline or the aqueous solution containing glucose and other assistant agents.The pharmaceutical composition of such as Tablet and Capsula and so on, is prepared by ordinary method.Pharmaceutical composition such as injection, solution, Tablet and Capsula should aseptically manufacture.The dosage of activeconstituents is treatment significant quantity, such as every day about 1 microgram-10 mg/kg body weight.

Inhibitor

In the present invention, also by various conventional screening assays, filter out RRM2B protein inhibitor.

Inhibitor used in the present invention comprises: the activity inhibitor of the antibody of RRM2B, the sense-rna of inhibition mRNA, RRM2B nucleic acid, microRNA (miRNA), siRNA, shRNA and RRM2B.Wherein, typical RRM2B inhibitor is inhibition miRNA, siRNA.

RRM2B inhibitor of the present invention can be used for the transfer of liver cancer cell in induced animal model, or the invasion and attack of liver cancer cell and migration in inducing cell experiment.

Antibody

The present invention also comprises and has specific polyclonal antibody and monoclonal antibody to people RRM2B albumen, especially monoclonal antibody.Here, " specificity " refers to that antibody capable is incorporated into people RRM2B gene product or fragment.Preferably, refer to that those can be combined with people RRM2B gene product or fragment but nonrecognition and be incorporated into the antibody of other non related antigen molecule.Antibody of the present invention can be prepared by the various technology that those skilled in that art are known.Such as, the people RRM2B gene product of purification or its antigen fragment are injected in animal body to produce polyclonal antibody.Equally, the cell of expressing people's RRM2B albumen or its antigen also can be used for causing immunity to animal and producing antibody.

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises and have immunocompetent antibody fragment, as Fab ' or (Fab) 2 fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered Single Chain Fv Molecule A; Or chimeric antibody.

RRM2B antibody used in the present invention can be anti-human RRM2B protein antibodies.Anti-human RRM2B protein antibodies of the present invention can be used in immunohistochemistry technology, detects the people RRM2B albumen in biopsy specimen.The antibody that preferably can be used for detecting RRM2B albumen is Anti-p53R2 antibody (ab8105, purchased from Abcam company).

Detection method and test kit

The present invention relates to quantitative and detection and localization people RRM2B protein level or mRNA level in-site diagnostic testing process.These tests are known in the art.The people RRM2B protein level detected in test, may be used for the transfer of diagnosing liver cancer or the migration of liver cancer cell.

A kind of method that whether there is RRM2B albumen in sample that detects utilizes the specific antibody of RRM2B albumen to detect, and it comprises: contacted with RRM2B protein specific antibody by sample; Observe and whether form antibody complex, define antibody complex and just represent in sample to there is RRM2B albumen.

RRM2B albumen or its polynucleotide can be used for the Diagnosis and Treat of RRM2B protein related diseases.Part or all of polynucleotide of the present invention can be used as probe and is fixed in microarray or DNA chip, for analyzing Differential expression analysis and the gene diagnosis of gene in tissue.The antibody of anti-RRM2B can be fixed on protein chip, for detecting the RRM2B albumen in sample.

Present invention also offers and a kind ofly detect the test kit whether liver cancer has transfer, it contains the primer pair of specific amplification RRM2B and/or RRM2B specific antibody and label or specification sheets.

Wherein, described label or specification sheets indicate following content: when the mrna expression amount of the p-Actin muscle of RRM2B phase of detected object and ratio≤0.5 of the mrna expression amount of the p-Actin muscle of RRM2B phase of non-cancerous tissue, then point out the probability of this detected object hepatoma Metastasis higher than general population.

A kind of typical test kit of the present invention can be used for detecting human liver cancer tissue sample or normal liver tissue sample.

Screening method

Present invention also offers the method for carrying out drug screening based on RRM2B.One method first screens the compound of impact (promotion) RRM2B expression or activity, then tests its restraining effect shifted liver cancer cell further to the compound filtered out.

Preferred screening, for a method for the candidate compound that suppresses liver cancer cell to shift, comprises step:

In (a) test group, in the culture system of liver cancer cell, add test compounds, and observe expression amount and/or the activity of RRM2B in the cell of described test group; In control group, in isocellular culture system, do not add test compounds, and observe expression amount and/or the activity of RRM2B in the described cell of control group;

Wherein, if the expression amount of the RRM2B of cell and/or activity, higher than control group, just show that this test compounds is the compound having the suppression liver cancer cell of promoter action to shift to expression and/or the activity of RRM2B in test group; And/or

B (), for the candidate compound obtained in step (a), tests the restraining effect that described candidate compound shifts liver cancer cell.

In another preference, in step (b), also comprise step: in the culture system of test group liver cancer cell, add test compounds, and observe quantity and/or the invasion and attack situation of the distance of liver cancer cell movement; In the culture system of control group liver cancer cell, do not add test compounds, and observe quantity and/or the invasion and attack situation of the distance of liver cancer cell movement; Wherein, if in test group liver cancer cell migration distance or invasion and attack quantity be significantly less than control group, just show this test compounds be to liver cancer cell transfer have inhibiting compound.

In addition, experiment finds, after RRM2B expression amount and/or activity increase, the high expression level of Egr1 can be promoted, and after Egr1 high expression level to a certain degree, again by suppressing the process LAN of RRM2B in conjunction with RRM2B promoter region, therefore, between Egr1 and RRM2B, there is degenerative regulating effect.Find based on this, can screen RRM2B can be promoted to express and can antagonism Egr1 to the inhibiting medicine of RRM2B, thus more preferably play RRM2B to the restraining effect of hepatoma Metastasis.

Therefore, be also included in described control group and test group in step (a) one or many mensuration is carried out to RRM2B and Egr1 expression amount.

In another preference, described repeatedly measure comprise respectively add test compounds simultaneously, add after within 12 hours and after adding 24 hours, RRM2B and Egr1 expression amount and/or active and RRM2B expression amount and/or activity change trend are measured simultaneously.

In another preference, according to measurement result, select Egr1 and RRM2B expression amount and/or activity raise simultaneously and RRM2B expression amount and/or activity change without the candidate compound of downtrending as the compound suppressing liver cancer cell transfer.

The present invention also comprises a kind of method suppressing hepatoma Metastasis or fucosylation, comprises step: the RRM2B gene using safe and effective amount to the object (Mammals) of needs treatment, albumen or its promotor.

Beneficial effect of the present invention

1. the Late Cambrian of the present invention expression amount of RRM2B and the transfer of liver cancer are negative correlation, and namely RRM2B expression amount is lower, and the possibility of hepatoma Metastasis is larger, provide a kind of novel method detecting hepatoma Metastasis accordingly.

2. present invention demonstrates that process LAN RRM2B can suppress the transfer of liver cancer, thus provide the new target spot of liver cancer treatment.

3. the present invention also finds, RRM2B is by affecting Egr-1/PTEN/Akt1 path, specifically by the expression of raising Egr-1, and then make Transcription factor Egr-1 be attached to the PTEN promoter region promotion mRNA of PTEN and the expression of protein level, finally suppress the phosphorylation level of Akt1, play and suppress the invasion and attack of liver cancer cell and the function of migration, instead of worked by p53.

Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number are weight percent and parts by weight.

Universal method

Universal method of the present invention is this area common technology means, and preferred materials and methods is as follows:

1, immunohistochemical methods

The dewaxing of routine paraffin wax section dimethylbenzene is to water.1% hydrogen peroxide at room temperature hatches 20 minutes, to eliminate the activity of endogenous peroxydase, and PBS cleaning down.Slice, thin piece is placed in microwave oven hot repair multiple 10 minutes, takes out naturally cooling.5 ~ 10% Normal Goat Serum room temperatures are closed and are added primary antibodie 4 DEG C after 20 minutes and spend the night.PBS adds two and resists after rinsing, room temperature 40 minutes.After PBS rinses, termination reaction after DAB colour developing.Tap water, Hematorylin is redyed.Cut into slices dry through gradient alcohol dehydration, dimethylbenzene is transparent, neutral gum mounting.

2, cell and cell cultures

Cell cellar culture in the DMEM substratum containing 10% foetal calf serum, 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates (37 DEG C, 5%CO ₂, 95%O ₂).All cells is at experimental session, and expect blue staining analysis through platform, cell viability remains on more than 90%.

3, plasmid transfection

The expression of plasmid adopts the lipo-2000 transfection reagent of Invitrogen company, and the experimental procedure provided according to company is carried out.Day before transfection is every hole kind about 6 × 10 in six orifice plates ⁵individual cell, during transfection, cell should be in 80% remittance sheet state, in 250 μ l opti-MEM substratum, dissolve 2 μ g plasmids, mixes lipo-2000 transfection reagent in 250 μ l opti-MEM substratum, the latter two mixings of incubate at room temperature 5min, incubate at room temperature 20min, puts upside down mixing, is added in six orifice plates fast, jog culture plate, quiescent culture after mixing, changed liquid after 4 ~ 6 hours, can detect the expression of plasmid after transfection after 48 hours.

4, immunoblotting (Western Blot)

Collect 2 × 10 ⁶cell, washes twice, 2000g centrifugal 5 minutes with cold PBS, abandons supernatant, and 50 μ l SDS lysates, 95 DEG C are boiled 5 minutes, put 5 minutes on ice, repeat twice.By centrifugal 10 minutes for cell pyrolysis liquid 4 DEG C 12,000g, supernatant liquor is the total protein extracted.With 8% ~ 12%SDS-polyacrylamide gel electrophoresis.Transfer to nitrocellulose filter after electrophoresis terminates, film 5% skim-milk (PBS preparation) room temperature closes 1 hour.Then respectively with primary antibodie 4 DEG C of overnight incubation, after fully washing with PBST, the two anti-incubated at room marked with corresponding horseradish peroxidase (HRP) 1 hour, after fully washing, finally uses Enhanced chemiluminescence (ECL) to detect protein expression change with PBST.

The antibody that immunohistochemical methods and immunoblotting use is as shown in table 1:

Table 1

5, real-time quantitative PCR (realtime PCR)

What real-time quantitative PCR adopted is double-stranded DNA dyestuff SYBR Premix Ex Taq II test kit and ABI7500 system.Reaction system: 5 μ l SYBR Premix Ex Taq II (2 ×), 0.2 μ l upstream primer (10 μMs), 0.2 μ l downstream primer (10 μMs), 1 μ l cDNA, 0.2 μ l ROX Reference Dye II (50 ×), 3.4 μ l deionized waters.Respond is carried out in 96 orifice plates (MicroAmp optical96-well), seal with optical adhesive covers (Applied Biosystems), amplified reaction adopts three multiple pipes, and calculates standard deviation to weigh experimental error.The software analysis that data acquisition instrument is subsidiary, by calculating when fluorescent signal exceedes threshold cycle number Ct corresponding to background fluorescence intensity.By calculating the difference (Δ Ct) of the Ct value of goal gene and corresponding internal reference GAPDH, be reflected in the difference of destination gene expression when equivalent RNA initial action.

Quantitative PCR and the primer needed for cloning vector as shown in table 2.

Table 2

6, the structure of recombinant vectors and qualification

The genome DNA extracting reagent kit specification sheets of biotech firm is followed to carry out operation extracting SMMC-7721 cell genomic dna according to sky.According to DNA sequence dna and the selected pGL3-enhancer carrier feature of RRM2B gene promoter, design primer, and respectively at upstream and downstream primer designing the restriction enzyme site of Mlu I and Bgl II, PCR primer is raw work synthesis through Shanghai.PCR reacts: system is as follows, 10 μ l5 × Prime hS DNA Polymerase buffer, 1 μ l upstream primer, 1 μ l downstream primer, 5 μ l genomic dnas, 5 μ l dNTP, 1 μ l Mg ₂sO ₄, 1 μ l Prime hS DNA Polymerase, 26 μ l deionized waters.PCR reaction conditions: 95 DEG C 5 minutes, be repeated below circulation 30 times: 94 DEG C 30 seconds, 58 DEG C 45 seconds, 72 DEG C 1 minute.Last 72 DEG C 10 minutes.Object fragment is reclaimed with the PCR primer recovery test kit operation of biotech firm according to sky.Adopt Nlu I and Bgl II double digestion to reclaim fragment, T4DNA ligase enzyme connects, and E. coli DH5 α transforms, and carries out the extracting of recombinant plasmid, double digestion and order-checking qualification.

7, the synthesis of Egr-1siRNA fragment

According to the Egr-1cDNA sequence that principle of design and the GenBank of siRNA interference sequence provide, the sequence of 2 siRNA interference of design Egr-1, the raw work synthesis through Shanghai.

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

Table 3

8, uciferase activity measures (Luciferase assay)

After plasmid co-transfection terminates, sucking-off nutrient solution, uses PBS washed cell.In each porocyte culture plate adds after 100 μ l cell pyrolysis liquids (PLB), be placed in shaking table shake 15 minutes.The determination of activity of Photinus pyralis LUC and extra large Shen luciferase is successively carried out in same reaction tubes, fluorophotometer is set 2 seconds and measure space before, each 10 seconds of minute.Get 100 μ l LARII to luciferase assay test tube.Add the cell pyrolysis liquid of 20 μ l, be mixed with suction pipe pressure-vaccum.Read Photinus pyralis LUC activity value M1.Add the Stop & Glo reagent Homogeneous phase mixing of 100 μ l again, reading is extra large Shen uciferase activity value M2 again.M1/M2 is the relative reactivity of luciferase.

9, lentiviral vectors infects liver cancer cell

Present method uses gene recombination technology, RRM2B gene is connected in Lentiviral pWPXL, build lentiviral vectors pWPXL-RRM2B, by lentiviral vectors main body plasmid pWPXL-RRM2B, packaging plasmid psPAX2 and pMD2.G cotransfection HEK293T cell, packaging lentiviral vectors also measures titre.Collect infestation with virus particles liver cancer cell.

10, transplanted tumor in nude mice experiment

Get BALB/c nude mice 32, use disposable sterilized injector at SMMC-7721 or the HCC-LY10 cell 2 × 10 of the liver in-situ inoculating RRM2B gene silencing of nude mice respectively ⁶individual.Put to death animal after 6 weeks, getting liver and lung tissue, to carry out tissue fixing with embedding.

11, statistical analysis

Adopt SPSS13.0 statistical package to carry out statistical analysis, all data all mean ± standard deviation represent.Compare employing one-way analysis of variance between three groups of groups, compare and adopt t inspection between two groups of groups, the comparison of rate adopts chi square test, and p < 0.05 thinks there is statistical significance.

Embodiment 1 measures expression level and the liver cancer patient Intrahepatic metastasis degree of correlation of the expression level of RRM2B gene in hepatocellular carcinoma and RRM2B.

First the expression level of 236 routine liver cancer tissue RRM2B albumen is analyzed by Immunohistochemical Method, found that the expression of RRM2B albumen in liver cancer is starkly lower than corresponding cancer beside organism (Figure 1A), in addition the expression level of RRM2B mRNA and albumen in liver cancer tissue and cancer beside organism is have detected by quantitative PCR and immunoblotting, result is consistent with the result of immunohistochemical methods, and namely in liver cancer tissue, the expression of RRM2B mRNA and albumen is starkly lower than corresponding cancer beside organism (Figure 1B and 1C).Therefore, these results illustrate that the expression of RRM2B albumen in liver cancer tissue reduces.

Process is to the relationship analysis between the expression level of 236 routine liver cancer tissue RRM2B albumen and liver cancer patient clinical pathology, and experiment shows that the expression level of RRM2B albumen and the Intrahepatic metastasis of liver cancer patient are obvious negativity relevant (p < 0.05); And the expression level of RRM2B albumen is negative correlation (p < 0.05) with the organizational hierarchy with liver cancer patient, but the clinical pathologic characteristic of the expression of RRM2B and other liver cancer patients does not have obvious dependency, as the situation (table 4) of the level of sex, age, tumor size, AFP, the state of liver cirrhosis and HBV infection.

Table 4

The migration of embodiment 2RRM2B liver cancer cell, the inhibition of Infiltration and metastasis measure

2.1 experiment in vitro

First by slow virus infection liver cancer cell, the hepatoma cell line stablizing RRM2B gene silencing and process LAN is set up.Result shows, obviously can promote the migration and invasion (Fig. 1 D) of SMMC-7721 and HCC-LY10 liver cancer cell after RRM2B gene silencing; Otherwise, SMMC-7721 and Huh7 cell migration and invasion and attack (Fig. 1 E) after RRM2B gene overexpression, can be suppressed.

2.2 experiment in vivo

SMMC-7721 and HCC-LY10 liver cancer cell is facilitated in the ability (Fig. 1 F) of nude mice Intrahepatic metastasis with distant place Lung metastases after the experiment of nude mice liver orthotopic transplantation tumor proves RRM2B gene silencing.

To sum up, these results show that RRM2B all can suppress migration, the Infiltration and metastasis of liver cancer cell in cell experiment and experiment in vivo.

The state of embodiment 3p53 gene do not affect RRM2B gene and liver cancer cell shift between relation

3.1 measure RRM2B in the hepatoma cell line of known p53 level expresses the relation with p53

Due to the target gene that RRM2B is wild-type p 53 gene, whether the state therefore analyzing p53 gene affects the impact of RRM2B on the transfer of liver cancer cell.

Because the state of some hepatoma cell line p53 genes is known, such as saltant type as clone p53 genes such as liver cancer cell born of the same parents system MHCC-97L, MHCC-97H, MHCC-97, SNU-182, be wild-type as SMMC-7721 cell p53 gene, Hep3B cell p53 gene is absence type.Therefore analyze the expression level of p53 and RRM2B albumen in hepatoma cell line, result shows the basal level expression of basic RRM2B albumen and the state uncorrelated (Fig. 2) of p53 gene.

The relation that in 3.2 clinical liver cancer samples, p53 and RRM2B expresses

Whether the expression level that the present embodiment analyzes p53 in 236 routine liver cancer tissues affects the relation between RRM2B and liver cancer patient Intrahepatic metastasis.Because the p53 of wild-type is unstable, think that the liver cancer tissue of the p53 immunohistochemical staining positive is the p53 of saltant type.

In order to confirm whether p53 gene is saltant type, analyzing the situation of p53 transgenation in 68 routine liver cancer tissues, found that the point mutation occurring AGG → AGT at p53 gene the 7th exon 2 49 bit codon, result in arginine and be mutated into Serine.In 68 routine liver cancer tissues, find that there is 20 examples occur this sudden change, and the liver cancer tissue sequencing analysis p53 gene of the immunohistochemical staining p53 positive is sudden change (Fig. 3).The mutation rate of visible p53 gene in liver cancer is approximately 29.4% (20/68), and the liver cancer tissue sequencing analysis p53 gene confirming the p53 immunohistochemical staining positive is saltant type.Statistic analysis result shows that the expression of p53 does not affect the relation (table 5) between RRM2B and liver cancer Intrahepatic metastasis.

To sum up, these results all show the state of p53 gene do not affect RRM2B and liver cancer cell shift between relation.

Table 5

What have studied that RRM2B suppresses the migration of liver cancer cell and invasion and attack affects path.

4.1RRM2B suppresses the phosphorylation level of Akt1

The phosphorylation level of Akt1 can be suppressed after SMMC-7721 and Huh7 cell RRM2B gene overexpression, otherwise, raise the phosphorylation level (Fig. 6 A and 6B) of Akt1 after RRM2B gene silencing.And PTEN is the prerequisite of antagonism PI3K-AKT signal, result display RRM2B process LAN can raise the expression of PTEN, otherwise after RRM2B gene silencing, the expression of PTEN reduces (Fig. 6 A and 6B).In addition, can be found by the level determination of PTEN mRNA, RRM2B process LAN has raised the level (6C) of PTEN mRNA.

4.2RRM2B the impact of gene pairs PTEN promoter activity

After structure comprises the Reporter gene vector of PTEN promotor, analyze the impact of RRM2B gene pairs PTEN promoter activity, the activity (Fig. 6 D) of PTEN promotor after result shows RRM2B process LAN, can be raised.

Whether 4.3 analyze RRM2B passes through the promoter activity that Egr-1 transcription factor affects PTEN

Due to the binding site containing Transcription factor Egr-1 in the promotor of PTEN, therefore analyze RRM2B and whether pass through the promoter activity that Egr-1 transcription factor affects PTEN.

4.3.1RRM2B on the impact that Egr-1mRNA expresses

Quantitative PCR result display RRM2B process LAN can raise the expression of Egr-1mRNA, otherwise after RRM2B gene silencing, the expression of Egr-1mRNA reduces (Fig. 6 E).

4.3.2Egr-1 on the impact of PTEN promoter activity

SMMC-7721 and Huh7 cell common transfection Egr-1 plasmid and PTEN Reporter gene vector, the result display of luciferase assay, Egr-1 obviously raises PTEN promoter activity (Fig. 6 F).

To sum up, RRM2B mainly suppresses the phosphorylation level of liver cancer cell Akt1 by the expression level of rise Egr-1/PTEN.

Embodiment 5RRM2B suppresses the generation of EMT by suppressing the phosphorylation level of liver cancer cell Akt1

The siRNA of 5.1 synthesis Akt1, at the siRNA of SMMC-7721 cell transfecting Akt1, after suppressing the expression level of Akt1, found that and decline with the expression that expression is risen, N-calcium is mucoprotein that E-calcium is mucoprotein after suppressing the level of Akt1 simultaneously, reduce the cell migration because RRM2B gene silencing causes and invasion and attack (Fig. 7 A and 7C) simultaneously, the mucoprotein expression of E-calcium is risen, expression that N-calcium is mucoprotein declines and then illustrate and occurred EMT phenomenon.

SMMC-7721 and the HCC-LY10 cell of 5.2 employing Akt phosphorylation inhibitor LY294002 and wortmannin effect RRM2B gene silencing, result is consistent with the experimental result of interference Akt, namely after LY294002 and wortmannin suppresses the phosphorylation level of Akt, lower the expression that N-calcium is mucoprotein, raise the expression that E-calcium is mucoprotein, inhibit the cell migration because RRM2B gene silencing causes and invasion and attack (Fig. 7 B and 7D) simultaneously.

The phosphorylation level of 5.3RRM2B process LAN energy sustained activation Akt1

Sustained activation Akt (Myr-Akt) is infected at the SMMC-7721 cell virus of RRM2B gene overexpression, can the reduction (Fig. 7 E and 7F) of the obviously cell migration that causes due to RRM2B process LAN of antagonism and transfer ability after found that Akt sustained activation.

The impact that embodiment 6RRM2B changes liver cancer cell EMT sample

Fucosylation, Infiltration and metastasis ability can be strengthened after 6.1RRM2B gene silencing

After RRM2B gene silencing, liver cancer cell occurs that EMT sample changes, and comprises and occurs that cell is transformed into fusiform interstitial form (Fig. 4 A) by Epithelial morphology, and therefore, after RRM2B gene silencing, fucosylation, Infiltration and metastasis ability strengthen.

The molecule that EMT is relevant also changes, and the mucoprotein expression of epithelial mark E-calcium reduces, and the expression that is mucoprotein and corresponding transcription factor slug of the mark N-calcium of mesenchymal cell increases (Fig. 4 B).

Fucosylation, Infiltration and metastasis ability can be reduced after 6.2RRM2B gene overexpression

In liver cancer cell after process LAN RRM2B gene, RRM2B gene overexpression significantly can suppress the change of liver cancer cell EMT sample, comprises RRM2B and raises the mucoprotein expression of E-calcium, and N-calcium is mucoprotein reduces (Fig. 4 C) with the expression of slug.The mucoprotein reduction of E-calcium in the nude mice tumor tissue of simultaneously RRM2B gene silencing, and the mucoprotein expression of N-calcium increases (Fig. 4 D).

Therefore, these results show that RRM2B gene finally can suppress the generation of the EMT of liver cancer cell.

6.3RRM2B on cell viability and tumor stem cell impact

Because a key character of EMT has tumor stem cell activity exactly, therefore the present embodiment also measured were RRM2B on EMT on opposing necrocytosis and opposing tumor stem cell characteristic and affect.

Found that RRM2B process LAN adds the drug susceptibility of chemotherapeutic drugs Doxorubicin, otherwise RRM2B gene silencing suppresses the drug susceptibility (Fig. 4 E) of Zorubicin.In addition, found that RRM2B can suppress the expression (Fig. 5) of liver-cancer stem cell mark CD133 and CD44.

Therefore, above result shows that RRM2B suppresses liver cancer cell EMT sample to change.

Embodiment 7 improves cell migration that the expression amount of Egr-1 and/or activity mediate RRM2B and the impact that invasive ability changes

At the SMMC-7721 cell transfecting pcDNA3.1-Egr-1 plasmid of RRM2B gene silencing, the increase (Fig. 8 A and 8B) of cell migration because RRM2B gene silencing causes and invasive ability after found that Egr-1 process LAN, can be reversed; Otherwise, at 2 siRNA of the SMMC-7721 cell transfecting Egr-1 of RRM2B gene overexpression, the decline (Fig. 8 C and 8D) of cell migration because RRM2B gene overexpression causes and invasive ability can be reversed equally.

Experiment shows, RRM2B suppresses the migration of liver cancer cell, Infiltration and metastasis is mainly played a role by Egr-1/PTEN/Akt1 path.

Mucoprotein and the dependency of RRM2B in liver cancer tissue of embodiment 8E-calcium

The expression level that in 8.1 liver cancer tissues, E-calcium is mucoprotein, N-calcium is mucoprotein and the dependency with RRM2B protein expression thereof.

Result is presented in liver cancer tissue, and the mucoprotein expression level of E-calcium is lower than corresponding cancer beside organism (Fig. 9 A), and the mucoprotein expression level of N-calcium is higher than corresponding cancer beside organism (Fig. 9 B).

(occurring together and shift or the transfer that do not occur together) in 8.2 liver cancer tissues, E-calcium is mucoprotein, N-calcium is mucoprotein, the change of p-Akt1, total Akt and RRM2B.

Result shows, and in the liver cancer tissue occurring together transfer, E-calcium is mucoprotein to be reduced with the expression of RRM2B, and N-calcium is mucoprotein increases (Fig. 9 C) with level that is p-Akt1, and these all want external experimental result consistent.

The dependency of expression level in liver cancer tissue of the mucoprotein and RRM2B of 8.3E-calcium.

Result shows that the mucoprotein expression of protein expression level in liver cancer of RRM2B and E-calcium is obvious positive correlation (Fig. 9 D and 9E).

Embodiment 9Egr-1 in conjunction with RRM2B promoter region and suppress the expression level of RRM2B

9.1 build the Reporter gene vector comprising the RRM2B promotor of different clipped form, luciferase assay result is presented at (-896/-5,-763/-5 ,-375/-5) RRM2B promoter activity is lower than the promoter activity (Figure 10 A and 10B) of RRM2B (-234/-5) fragment.

Result shows at least may there is an inhibition transcription factor between the-375/-234 of RRM2B promoter region.By analyzing transcription factor possible between the-375/-234 of RRM2B promoter region, result of study shows that Transcription factor Egr-1 can suppress the activity of RRM2B promotor, but Egr-1 can not suppress the promoter activity of-234/-5 fragment, therefore Transcription factor Egr-1 can be incorporated into RRM2B promoter region-375/-234 and suppress the activity of promotor, and Egr-1 can suppress RRM2B promoter activity to have dose-dependently (Figure 10 C) in addition.

9.2 structures comprise the RRM2B Reporter gene vector of Egr-1 binding site saltant type to prove that Egr-1 is attached to the promoter region of RRM2B.

Found that Egr-1 can not suppress the promoter activity of the RRM2B containing Egr-1 binding site saltant type.

Prove that Egr-1 is attached to (Figure 10 D) in the promotor of RRM2B by chromatin imrnunoprecipitation experiment again.These results show that Transcription factor Egr-1 is attached to RRM2B promoter region and suppresses it to express.

9.3 respectively at the over-express vector of SMMC-7721 and Huh7 cell transfecting Egr-1 and interference carrier to prove that Egr-1 suppresses the expression of RRM2B.

Result display Egr-1 can suppress the expression (Figure 10 E) of RRM2B, otherwise can raise the expression (Figure 10 F) of RRM2B after the expression of interference Egr-1.Therefore, these all results show the expression level between RRM2B and Egr-1 with a kind of negative feedback loop regulation and control liver cancer RRM2B.

The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims (10)

1. ribonucleotide reductase subunit M2B (Ribonucleoside-diphosphate reductase subunit M2B, RRM2B) purposes of gene or its protein assay reagent, it is characterized in that, for the preparation of the transcellular test kit of diagnosing liver cancer.

2. purposes as claimed in claim 1, is characterized in that, described RRM2B gene or its protein assay reagent comprise the specific antibody of RRM2B, specificity amplification primer, probe or chip.

3. purposes as claimed in claim 1, is characterized in that, described detection measures liver cancer tissue or normal liver tissue sample.

4. for detecting a diagnostic kit for liver cancer cell transfer, it is characterized in that, described test kit contains a container, containing the detection reagent detecting RRM2B albumen or mRNA in described container; And label or specification sheets, described label or specification sheets indicate described test kit for detecting liver cancer cell transfer.

The purposes of 5.RRM2B gene, albumen or its promotor, is characterized in that, for the preparation of suppressing liver cancer or suppressing the pharmaceutical composition of liver cancer cell transfer.

6. purposes as claimed in claim 5, is characterized in that, described pharmaceutical composition comprises the RRM2B gene for the treatment of significant quantity, albumen or its promotor, and pharmaceutically acceptable carrier.

7. screen the method for the candidate compound suppressing liver cancer cell to shift, it is characterized in that, comprise step:

In (a) test group, in the culture system of liver cancer cell, add test compounds, and observe expression amount and/or the activity of RRM2B in the cell of described test group; In control group, in isocellular culture system, do not add test compounds, and observe expression amount and/or the activity of RRM2B in the described cell of control group;

Wherein, if the expression amount of the RRM2B of cell and/or activity, higher than control group, just show that this test compounds is the compound having the suppression liver cancer cell of promoter action to shift to expression and/or the activity of RRM2B in test group; And/or

B (), for the candidate compound obtained in step (a), tests the restraining effect that described candidate compound shifts liver cancer cell.

8. method as claimed in claim 7, is characterized in that, comprise step in step (b): in the culture system of test group liver cancer cell, add test compounds, and observe quantity and/or the invasion and attack situation of the distance of liver cancer cell movement; In the culture system of control group liver cancer cell, do not add test compounds, and observe quantity and/or the invasion and attack situation of the distance of liver cancer cell movement; Wherein, if in test group liver cancer cell migration distance or invasion and attack quantity be significantly less than control group, just show this test compounds be to liver cancer cell transfer have inhibiting compound.

9. a purposes for RRM2B inhibitor, is characterized in that, for the preparation of the composition or the reagent that promote liver cancer cell transfer.

10. an expression vector for RRM2B gene, is characterized in that, described expression vector has following element:

(i) promoter element;

(ii) RRM2B encoding sequence; With

(iii) termination codon sub-element;

Wherein, described promoter element is not combined with Egr1.