CN104004822A

CN104004822A - Application of SSX2IP in predicating or diagnosing tumor metastasis

Info

Publication number: CN104004822A
Application number: CN201310062583.6A
Authority: CN
Inventors: 霍克克; 李璞
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-02-27
Filing date: 2013-02-27
Publication date: 2014-08-27
Anticipated expiration: 2033-02-27
Also published as: CN104004822B

Abstract

The invention discloses an application of SSX2IP in predicating or diagnosing tumor metastasis. Specifically, the invention provides an application of SSX2IP gene or protein in preparing a reagent or a kit for detecting the tumor metastasis. Studies demonstrate that SSX2IP gene and an expression product thereof can be used as a specific marker gene for diagnosing or predicating tumor (for example, liver cancer) metastasis, and is beneficial to diagnose and predicate the tumor metastasis more accurately and early.

Description

The application of SSX2IP in prediction or diagnosing tumour transfer

Technical field

The present invention relates to oncology.More specifically, the present invention relates to the application in diagnosis of metastasis or prediction of SSX2IP gene or albumen.The invention still further relates to SSX2IP inhibitor in prevention and treatment metastases, tumor cell migration and the application of chemotherapy resistance aspect capable.

Background technology

Hepatocellular carcinoma (Hepatocellular carcinoma, HCC) is one of modal malignant tumour, occupies the 5th of the whole world (malignant tumour) sickness rate, the 3rd of the relevant cause of the death of cancer, and in China, its sickness rate is only second to lung cancer, occupies second.

Hepatic blood supply is abundant, and the rate of transform is high, shifts patient's poor prognosis.The existing biomolecules that can be used as primary liver cancer markers, it has been generally acknowledged that following four classes: cancer embryo and glycoprotein antigen; Enzyme and isozyme; Cytokine; Gene.In the world, the etiologic diagnosis of liver cancer be take and detected Serum AFP (alpha-fetoprotein) as main, but the susceptibility of AFP (40%～65%) and specificity (76%～96%) are all unsatisfactory, and this type of detected result there is no directive significance to patient's prognosis.

In the liver cancer patient of different steps, personalized treatment plan is even more important.But owing to lacking specificity hepatoma Metastasis mark, the time window for the treatment of is often just opened after hepatoma Metastasis, incured loss through delay the good chance that prophylaxis of tumours shifts.

Therefore, this area can be predicted in the urgent need to exploitation or diagnosing tumour shifts the especially Specific marker of hepatoma Metastasis, thereby can predicting liver cancer shift, as prevention or treatment metastases provide basis better.

Summary of the invention

The invention provides the test kit whether a kind of prediction or diagnosing tumour shift, in addition, the present invention also provides a kind of SSX2IP inhibitor that can prevent and treat metastases, tumor cell migration and can improve chemosensitivity; The present invention also provides a kind of screening method that metastases or tumor drug resistance is had to prevention or therapeutic action medicine.

First aspect present invention, a kind of SSX2IP gene (synovial sarcoma X breaking point gene 2 interaction proteins are provided, Synovial Sarcoma X brearkpoint2interacting Protein) or the purposes of SSX2IP albumen, for the preparation of the reagent or the test kit that detect metastases.

In another preference, described SSX2IP dietary protein origin is in Mammals; Preferably, derive from people, mouse or rat; More preferably, derive from people.

In another preference, described SSX2IP dietary protein origin is in people, and its full length amino acid sequence is as shown in SEQ ID NO.:1.

In another preference, described SSX2IP gene nucleotide series is as shown in SEQ ID NO.:2.

In another preference, described tumour comprises: liver cancer, cancer of the stomach, intestinal cancer, lung cancer, prostate cancer, mammary cancer, melanoma.

In another preference, described reagent comprises SSX2IP Auele Specific Primer, specific antibody, probe and/or chip.

In another preference, described detection comprises that enzyme linked immunoassay method (ELISA method) detects or temporal resolution immunofluorescence technique (TRFIA method) detects.

In another preference, described SSX2IP 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 SSX2IP is monoclonal antibody or polyclonal antibody.

In another preference, described detection is to measure tissue sample, blood sample, serum sample or humoral sample.

In another preference, described tissue sample comprises cancerous tissue and cancer beside organism.

A second aspect of the present invention, provides a kind of diagnostic kit for detection of metastases, and described test kit contains a container, contains the detection reagent that detects SSX2IP albumen or mRNA in described container; And label or specification sheets, described label or specification sheets indicate described test kit for detection of metastases.

In another preference, described detection metastases refers to judge whether metastases occurs, and/or possibility (susceptibility) size of metastases occurs in judgement.

In another preference, described judgement comprises to be prejudged.

In another preference, described detection SSX2IP albumen or the detection reagent of mRNA comprise:

(a). the specific antibody of anti-SSX2IP albumen; And/or

(b). the mRNA of specific amplification SSX2IP or the Auele Specific Primer of cDNA.

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

When ratio >=2 of the tumor tissues SSX2IP of detected object expression amount with the SSX2IP expression amount of cancer beside organism, point out the probability of this detected object metastases higher than general population.

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

In another preference, described test kit is also for the predicting tumors survival of patients time.

In another preference, when detected object is tumour patient, if the SSX2IP expression amount Y1 in detected object tumor tissues is significantly higher than the expression amount Y2 of SSX2IP in similar cancer patients's tumor tissues, point out the mean survival time lower than similar cancer patients of this detected object survival time;

If the SSX2IP expression amount Y1 in detected object tumor tissues is significantly lower than the expression amount Y2 of SSX2IP in similar cancer patients's tumor tissues, the mean survival time higher than similar cancer patients of pointing out this detected object survival time.

In another preference, described being significantly higher than refers to Y1/Y2 >=2.

In another preference, described significantly lower than referring to Y1/Y2≤0.5.

A third aspect of the present invention, provides the purposes of a kind of SSX2IP gene or albumen, as the Specific marker that detects metastases for the preparation of detection reagent; Or as the Specific marker that detects metastases.

In another preference, described tumour comprises liver cancer.

A fourth aspect of the present invention, provides a kind of purposes of SSX2IP inhibitor, for the preparation of the medicine that suppresses metastases or inhibition tumor cell migration.

In another preference, described tumour comprises liver cancer, cancer of the stomach, intestinal cancer, lung cancer, prostate cancer, mammary cancer, melanoma.

In another preference, described tumour is liver cancer.

In another preference, described inhibitor comprises: the activity inhibitor of sense-rna, siRNA, shRNA and the SSX2IP of the antibody of SSX2IP, SSX2IP nucleic acid.

In another preference, described medicine contains pharmaceutically acceptable carrier, SSX2IP inhibitor and optional chemotherapeutics.

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

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

In another preference, described SSX2IP inhibitor is applied to Mammals with the dosage (each or every day) of 0.5-5mg/kg body weight.

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

In another preference, described chemotherapeutics comprises acid amides (CTX), ifosfamide (IFO), mitomycin (MMC), Zorubicin (ADM), vincristine(VCR) (VCR), vincaleucoblastine (VBL), etoposide (VP16), brave and fierce (Vumon), cis-platinum (CDDP), Fluracil (5-FU), carboplatin (CBP) and methotrexate (MTX) etc.

A fifth aspect of the present invention, a kind of purposes of SSX2IP inhibitor is provided, (a) for the preparation of the apoptotic pharmaceutical composition that promotes chemotherapy or radiotherapy-induced, or (b) for the preparation of the apoptotic promotor that promotes chemotherapy or radiotherapy-induced, or (c) for the preparation of making the cancer cells sensitizer more responsive to the apoptosis of chemotherapy or radiotherapy-induced.

In another preference, described SSX2IP inhibitor is used for the sensitizer of cancer therapy or promotes apoptotic promotor.

Described SSX2IP albumen comprises fusion rotein and non-fusion rotein.

A sixth aspect of the present invention, a kind of method that provides external non-therapeutic anticancer to move, comprises step: under SSX2IP inhibitor exists, cultivate cancer cells, thus anticancer migration.

In another preference, described method comprises in the culture system of cancer cells adds SSX2IP inhibitor, thus anticancer migration.

In another preference, described cancer cells comprises liver cancer cell, stomach cancer cell, colon-cancer cell, lung carcinoma cell, prostate cancer cell, breast cancer cell, melanoma cell.。

In another preference, in described method, the concentration of described SSX2IP inhibitor is 0.5-5mg/mL.

A seventh aspect of the present invention, provides the purposes of a kind of SSX2IP gene, SSX2IP albumen or its agonist, for the preparation of the medicine that promotes metastases or tumor cell migration.

In another preference, described SSX2IP gene or dietary protein origin are in Mammals; Preferably, derive from people, mouse or rat; More preferably, derive from people.

In another preference, described SSX2IP gene or albumen are recombinant human SSX2IP gene or albumen or from people SSX2IP gene or albumen in human blood or serum.

In another preference, described promotion metastases or the medicine of tumor cell migration are used for setting up tumor model.

In another preference, described tumour is liver cancer.

In another preference, the purposes of described SSX2IP gene, SSX2IP albumen or its agonist also comprises: under SSX2IP albumen or the existence of its agonist, cultivate cancer cells, thereby promote growth of cancer cells or propagation.

A eighth aspect of the present invention, provides a kind of screening for inhibition tumor cell migration or for improving the method for the candidate compound of cancer cells drug susceptibility, described method comprises step:

(a) in test group, in the culture system of cell, add test compounds, and observe expression amount and/or the activity of SSX2IP 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 SSX2IP in the described cell of control group;

Wherein, if the expression amount of the SSX2IP of cell and/or activity are less than control group in test group, just show that this test compounds is to have inhibiting treatment metastases or tumor cell migration maybe can improve the compound of chemotherapy drug susceptibility to the expression of SSX2IP and/or activity; With

(b) for the candidate compound obtaining in step (a), optionally test described candidate compound to the restraining effect of cancer metastasis or migration or described candidate compound the influence to cancer cells drug susceptibility.

In another preference, in step (b), comprise step: in test group, in the culture system of cancer cells, add test compounds, and observe quantity and/or the invasion and attack situation of the distance that cancer cells moves; In control group, in the culture system of cancer cells, do not add test compounds, and observe quantity and/or the invasion and attack situation of the distance that cancer cells moves; Wherein, if the migration distance of cancer cells or invasion and attack quantity are significantly less than control group in test group, just show that this test compounds is that metastases or tumor cell migration are had and inhibitingly maybe can improve chemotherapy drug susceptibility compound.

The invention provides a kind of method that prediction or diagnosing tumour shift, comprise step:

(a). prepare experimenter and test sample;

(b). detect the mrna expression amount of the relative beta-actin of SSX2IP in test sample and compare with reference value, when its ratio >=2 item point out the probability of this detected object metastases higher than general population.

In another preference, described test sample is tissue sample, blood sample, serum sample or humoral sample.

In another preference, described reference value is the expression amount of SSX2IP in non-tumor sample.

In another preference, described detecting step b comprises the amount that detects SSX2IP mRNA, or the amount of SSX2IP cDNA; And/or the amount of detection SSX2IP albumen.

In another preference, described detecting step b comprises by RT-PCR or PCR method and detecting.

In another preference, described detecting step b comprises and uses the antibody of anti-SSX2IP albumen to detect.

In addition, the present invention also comprises a kind of method that suppresses metastases or tumor cell migration, comprises step: the object (Mammals) for the treatment of to needs is used SSX2IP albumen or its agonist of safe and effective amount.

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

Accompanying drawing explanation

Fig. 1 has shown that Kaplan-Meier analyzes that SSX2IP expresses in 51 routine hepatocarcinoma patients and the relation of lifetime, and wherein, SSX2IP high expression level patient group (31 example) its lifetime significantly lower than the low expression of SSX2IP patient's group (20) (P=0.004).

Fig. 2 had shown and had crossed that spread in the abdominal cavity of expressing SSX2IP and liver cancer cell and the relation of liver metastasis: wherein, Fig. 2 A has shown that .B Taking Pictures recording liver cancer cell is in Intraabdominal spread condition.B. statistics found to express SSX2IP liver cancer cell its in nude mice abdominal cavity, spread and the ability that forms tumour is significantly higher than transfection zero load groups of cells and untreated cell group (6.2 ± 0.84,2.6 ± 0.89,2.4 ± 1.14, * * P<0.001).C.BEL-7402, BEL-7402Vector, BEL-7402SSX2IP tri-class cells enter in nude mouse by tail vein injection respectively, after 10 weeks, put to death and dissect, the situation of Taking Pictures recording nude mice liver.D. statistics is found, crosses liver cancer cell ratio of liver metastasis in nude mouse of expressing SSX2IP and is significantly higher than unloaded contrast and blank group, and transfer ratio is respectively 7/10,2/10 and 1/10, P=0.023.

Fig. 3 has shown that SSX2IP can promote the cut healing ability of liver cancer cell.Wherein, the locomotivity of liver cancer cell has been reacted in cut healing experiment.

Fig. 3 A has shown by the cut experiment of healing and has detected SMMC-7721, SMMC-7721Vector, the locomotivity of SMMC-7721SSX2IP tri-class cells, found to express the liver cancer cell cell SMMC-7721SSX2IP of SSX2IP, its locomotivity is significantly higher than unloaded cell combination blank groups of cells, and the mean gap distance after 48 hours is: 170.83 ± 36.96 μ m, 516.67 ± 25.57 μ m, 525.00 ± 31.92 μ m, * * P<0.001.

Fig. 3 B has shown by the cut experiment of healing and has detected BEL-7402, BEL-7402Vector, the locomotivity of BEL-7402SSX2IP tri-class cells, found to express the liver cancer cell cell BEL-7402SSX2IP of SSX2IP, its locomotivity is significantly higher than unloaded cell combination blank groups of cells, and the mean gap distance after 48 hours is: 183.33 ± 49.07 μ m, 312.50 ± 25.00 μ m, 329.17 ± 15.96 μ m, * * P<0.001.

Fig. 4 has shown that SSX2IP can promote invasion and attack and the transfer ability of liver cancer cell.

Fig. 4 A has shown the promotion result of SSX2IP to liver cancer cell SMMC-7721 invasion and attack transfer ability.

Fig. 4 B had shown expression SSX2IP liver cancer cell SMMC-7721SSX2IP, and the ability of its invasion and attack and migration significantly strengthens.In migration experiment, cross the quantity statistics of expressing SSX2IP group contrast control group and be: 123.33 ± 9.45,59.67 ± 4.73,51.33 ± 6.03; In Matrigel, the quantity statistics of excessively expressing SSX2IP group contrast control group is: 92.00 ± 7.00,43.67 ± 4.16, and 38.00 ± 3.61, * * P<0.001.

Fig. 4 C has shown the promotion result of SSX2IP to liver cancer cell BEL-7402 invasion and attack transfer ability

Fig. 4 D had shown expression SSX2IP liver cancer cell BEL-7402SSX2IP, and the ability of its invasion and attack and migration significantly strengthens

In migration experiment, cross the quantity statistics of expressing SSX2IP group contrast control group and be: 154.67 ± 14.05,103.67 ± 10.70,109.00 ± 7.55; * P<0.01.In Matrigel, the quantity statistics of excessively expressing SSX2IP group contrast control group is: 113.00 ± 6.56,63.33 ± 11.50, and 60.67 ± 11.02, * P<0.01.

Fig. 5 has shown that SSX2IP can reduce the susceptibility of liver cancer cell to chemotherapeutics, increases resistance

Fig. 5 A has shown SMMC-7721, SMMC-7721Vector and SMMC-7721SSX2IP are to the dose response curve of chemotherapeutics 5-Fu and IC50 value, result shows that SMMC-7721SSX2IP is significantly higher than control group (24.68 ± 1.17 μ M to the IC50 value of 5-FU, 14.59 ± 0.77 μ M, 13.60 ± 0.88 μ M, * * P<0.001).

Fig. 5 B has shown BEL-7402, BEL-7402Vector and BEL-7402SSX2IP are to the dose response curve of chemotherapeutics 5-Fu and IC50 value, result shows that BEL-7402SSX2IP is significantly higher than control group (28.52 ± 0.65 μ M to the IC50 value of 5-FU, 12.73 ± 1.81 μ M, 14.16 ± 1.20 μ M, * * P<0.001).

Fig. 5 C has shown BEL-7402, SMMC-7721Vector and SMMC-7721SSX2IP are to the dose response curve of chemotherapeutics CDDP and IC50 value, result shows that SMMC-7721SSX2IP is significantly higher than control group (11.38 ± 1.42 μ M to the IC50 value of CDDP, 5.72 ± 0.75 μ M, 6.18 ± 0.81 μ M, * * P<0.001).

Fig. 5 D has shown BEL-7402, BEL-7402Vector and BEL-7402SSX2IP are to the dose response curve of chemotherapeutics CDDP and IC50 value, result shows that BEL-7402SSX2IP is significantly higher than control group (9.86 ± 1.24 μ M to the IC50 value of CDDP, 6.13 ± 0.24 μ M, 5.90 ± 0.47 μ M, * * P<0.001).

Embodiment

The inventor is by extensive and deep research, be surprised to find that first, the inventor, by extensive and deep research, is surprised to find that first, SSX2IP inhibitor can or be treated metastases, tumor cell migration as prevention, and can improve the susceptibility of tumour cell to chemotherapeutics; The inventor has also found that SSX2IP gene or albumen can suppress the medicine of metastases or tumor cell migration for the preparation of improving tumour to chemotherapy drug susceptibility, screening; In addition, the inventor has also found that SSX2IP gene or albumen and agonist thereof can be used as promotion metastases, tumor cell migration, and strengthens the resistance of tumour cell to chemotherapeutics, can be for the preparation of animal resistance, metastasis model.

In addition, the inventor also finds by research, the high expression level of SSX2IP and metastases have very strong dependency, experimental results show that, SSX2IP can be used as the Specific marker that prediction or diagnosing tumour shift, thereby prepares predicting tumors possibility or whether diagnosing tumour the detection kit shifting has occurred.In addition, the inventor has also found that SSX2IP can be for predicting tumors patient's survival time.On this basis, completed the present invention.

SSX2IP albumen and polynucleotide

In the present invention, " albumen of the present invention ", " polypeptide of the present invention ", " SSX2IP albumen " are used interchangeably, and refer to synovial sarcoma X breaking point 2 interaction proteins (referred to as SSX2IP).Should be understood that described term also comprises active fragments and the derivative of SSX2IP.

In the present invention, " gene of the present invention ", " polynucleotide of the present invention " refer to the to encode nucleotide sequence of SSX2IP albumen or its active fragments and derivative, comprises justice and antisense nucleic acid.SSSX2IP is positioned chromosome1p22.3, and full length gene 46kb wherein comprises 14 exons.

In the present invention, term " SSX2IP albumen " or " SSX2IP polypeptide " are used interchangeably, and all refer to have albumen or the polypeptide of people's Protein S SX2IP aminoacid sequence.SSX2IP has been confirmed as the related antigen of acute myeloid leukaemia at present, is the target spot of a potential leukemia immunotherapy.

The Genbank accession number that can be used for 5 kinds of mRNA transcripts of SSX2IP gene nucleotide series of the present invention is NM001166293.1; NM001166294.1; NM001166295.1; NM001166417.1; NM014021.3.

A kind of preferred SSX2IP gene nucleotide series as shown in SEQ ID NO.:1, Gene ID:117178, the aminoacid sequence of its coding is as shown in SEQ ID NO.:2, accession number is NP001159889.1.

As used herein, " separated " refers to that material separates (if natural substance, primal environment is natural surroundings) from its primal environment.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 that exist separately, for separation and purification.

As used herein, " separated SSX2IP albumen or polypeptide " refers to that SSX2IP albumen does not basically contain natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can be purified SSX2IP albumen with the purified technology of protein of standard.Substantially pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.In the present invention, SSX2IP albumen comprises fusion rotein and non-fusion rotein.

Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferably 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-stranded.DNA can be coding strand or noncoding strand.

The polynucleotide of the mature polypeptide of coding SSX2IP comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; The encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and can be also the polynucleotide that also comprise 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 the fragment of polypeptide, analogue and derivative with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural occurs.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing in fact the function of 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 ", containing 15 Nucleotide, is at least better at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.The amplification technique (as PCR) that nucleic acid fragment can be used for nucleic acid is to determine and/or the polynucleotide of separated coding SSX2IP albumen.

People SSX2IP Nucleotide full length sequence of the present invention or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic conventionally.For pcr amplification method, can be according to published relevant nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually need to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplified is stitched together by proper order.

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

In addition, also can synthesize relevant sequence by the method for synthetic, especially fragment length more in short-term.Conventionally, by first synthetic a plurality of small segments, and then connect and can obtain the fragment that sequence is very long.

The method of application round pcr DNA amplification/RNA is optimized for and obtains gene of the present invention.Primer for PCR can suitably be selected according to sequence information of the present invention disclosed herein, and available ordinary method is synthetic.Available ordinary method is as the DNA/RNA fragment of and purifying amplification separated by gel electrophoresis.

The present invention also relates to the carrier that comprises polynucleotide of the present invention, and the host cell producing through genetically engineered with carrier of the present invention or SSX2IP albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.

By conventional recombinant DNA technology, can utilize polynucleotide sequence of the present invention to can be used to the SSX2IP albumen of expression or Restruction.In general there are following steps:

(1). with the polynucleotide (or varient) of encoding human SSX2IP albumen of the present invention, or transform or the suitable host cell of transduceing with the recombinant expression vector that contains these polynucleotide;

(2). the host cell of cultivating in suitable substratum;

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

Method well-known to those having ordinary skill in the art can be for building containing people SSX2IP DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described DNA sequence dna can be effectively connected in the suitable promotor in expression vector, to instruct mRNA synthetic.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

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

Comprise above-mentioned suitable DNA sequence dna and the suitable carrier of promotor or control sequence, can be 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 of CHO, COS or 293 cells etc.

With recombinant DNA transformed host cell, can carry out with routine techniques well known to those skilled in the art.When host is prokaryotic organism during as intestinal bacteria, the competent cell that can absorb DNA can be processed by CaCl2 method in exponential growth after date results, and step used is well-known in this area.Another kind method is to use MgCl2.If needed, the also method of available electroporation that transforms is carried out.When host is eukaryote, can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical method is as microinjection, electroporation, liposome packing etc.

The transformant obtaining 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 mediums.Under the condition that is suitable for host cell growth, cultivate.When host cell grows into after suitable cell density, the promotor of selecting with suitable method (as temperature transition or chemical induction) induction, cultivates cell for some time again.

Extracellular can be expressed or be secreted into recombinant polypeptide in the above methods in cell or on cytolemma.If needed, can utilize the albumen of and purification of Recombinant separated by various separation methods with other characteristic its physics, 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 processes, with protein precipitant, process the combination of (salt analysis method), centrifugal, the broken bacterium of infiltration, super processing, 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.

Inhibitor

Utilize albumen of the present invention, by various conventional screening methods, can filter out with SSX2IP albumen interactional material occurs, especially inhibitor etc.

The inhibitor of SSX2IP albumen of the present invention (comprising antibody, antisense nucleic acid and other inhibitor), when using (administration) in treatment, expression and/or the activity that can suppress SSX2IP albumen, and then suppress the transfer of tumour or the migration of tumour cell.Conventionally, but these materials are formulated in to nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is about 5-8 conventionally, preferably pH is about 6-8, although pH value can change to some extent with being formulated the character of material and illness to be treated.The pharmaceutical composition preparing can carry out administration by conventional route, comprising (but being not limited to): in knurl, intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.

Can be used for inhibitor of the present invention comprises: the activity inhibitor of sense-rna, siRNA, shRNA and the SSX2IP of the antibody of SSX2IP, inhibition mRNA, SSX2IP nucleic acid.Wherein, typical SSX2IP inhibitor is inhibition miRNA, siRNA.

Typically, the technical scheme using SSX2IP gene as the target spot of the medicine of preparation prevention or treatment metastases or tumor cell migration comprises following scheme:

1. chemosynthesis double stranded ribonucleic acid molecule, its sequence-specific is for SSX2IP gene order, utilize liposome to be delivered to the expression of tumour cell internal interference SSX2IP gene, observe the change that soft-agar cloning forms the characteristics of cell biology such as ability, cell migration ability.Can utilize the method for this area routine to design and synthetic specificity for the nucleotide sequence (as siRNA) of SSX2IP.

2. utilize various carriers, comprise that DNA vector, lentiviral vectors disturb the expression of SSX2IP gene, reach the effect of body internal interference SSX2IP gene, detect their results for the treatment of to the diffusion of nude mice knurl body abdominal cavity or hepatic metastases, thereby realize the object that suppresses tumor proliferation.

3. obtain the polypeptide, the monoclonal antibody that can specificity suppress SSX2IP gene delivery activity, reach the object that suppresses SSX2IP activity, thereby real inhibition tumor cell shifts or the object of migration.

The present invention also provides a kind of pharmaceutical composition, the SSX2IP inhibitor of the present invention that it contains safe and effective amount (as antibody, antisense sequences (as siRNA) or inhibitor) and pharmaceutically acceptable carrier or vehicle.This class 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, for example, with physiological saline or the aqueous solution that contains glucose and other assistant agents, by ordinary method, be prepared.Pharmaceutical composition such as Tablet and Capsula, can be prepared by ordinary method.Pharmaceutical composition should be manufactured as injection, solution, Tablet and Capsula under aseptic condition.The dosage of activeconstituents be treatment significant quantity, for example every day approximately 1 microgram-10 mg/kg body weight.

Antibody

The present invention also comprises that people SSX2IP albumen is had to specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " refers to that antibody capable is incorporated into people SSX2IP gene product or fragment.Preferably, refer to that those can be combined with people SSX2IP gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people SSX2IP gene product of purification or its antigen fragment are injected in animal body to produce polyclonal antibody.Equally, the cell of expression people's SSX2IP albumen or its antigen also can be used for animal to cause immunity and produce antibody.

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab' or (Fab) 2 fragments; Heavy chain of antibody; Light chain of antibody; Genetically engineered scFv molecule; Or chimeric antibody.

Antibody of the present invention comprises the antibody that can suppress SSX2IP function, can be also the antibody that does not affect people SSX2IP function.Each antibody-like can produce by the fragment of people SSX2IP gene product or functional domain are caused to immunity, and people SSX2IP gene product and fragment thereof can produce or synthesize with Peptide synthesizer with recombination method.With the antibody that the SSX2IP of non-modified form gene product is combined, can utilize the gene product producing in prokaryotic cell prokaryocyte (as E.coli) carry out immune animal and obtain.With the antibody that posttranslational modification form is combined as glycosylation or phosphorylation SSX2IP albumen or polypeptide, can utilize the gene product producing in as yeast or insect cell at eukaryotic cell carry out immune animal and obtain.

Can be used for SSX2IP antibody of the present invention can be anti-human SSX2IP protein antibodies.Anti-human SSX2IP protein antibodies of the present invention can be used in immunohistochemistry technology, detects the people SSX2IP albumen in biopsy specimen.

Pharmaceutical composition and administering mode

The invention provides and contain activeconstituents (a) SSX2IP inhibitor; (b) pharmaceutically acceptable carrier; And the pharmaceutical composition of optional (c) chemotherapeutics.

In pharmaceutical composition of the present invention, the content of SSX2IP inhibitor SSX2IP inhibitor is not particularly limited, and is generally 0.01-95wt%, is preferably 0.1-90wt%.

Pharmaceutical composition of the present invention can be single preparations of ephedrine, can be also compound preparation.

In compound preparation, except containing SSX2IP inhibitor, also can comprise other antineoplastic compound, for example chemotherapeutics.Representational chemotherapeutics comprises (but being not limited to): alkylating agent, metabolic antagonist, folacin, pyrimidine analogue, purine analogue and relevant inhibitor, vinca alkaloids, epipodopyvllotoxins, microbiotic, L mono-asparagus fern phthalein amine enzyme, topoisomerase enzyme inhibitor, Interferon, rabbit, platinum coordination complex, the urea that Schuttgelb replaces, methyl hydrazine derivative, adrenal cortex inhibitor, adrenocortical steroid, progestogen, oestrogenic hormon, estrogen antagonist, male sex hormone, androgen antagonist and gonad-stimulating hormone-releasing hormone analog.Preferred chemotherapeutics comprises: 5 one Fluracils (5-FU), formyl tetrahydrofolic acid, irinotecan, oxaliplatin, capecitabine, taxol and Duo Xi taxol.

Formulation and the preparation method of pharmaceutical composition of the present invention are not particularly limited, and the conventional general method for making in available this area is made the various formulations such as tablet, capsule, granule, sustained release dosage, injection.Preferred formulation is oral preparations (as tablet) and injection.

In the present invention, SSX2IP inhibitor or can be used for prevention and treatment metastases or tumor cell migration containing the pharmaceutical preparation of SSX2IP inhibitor.

Be applicable to tumour of the present invention and comprise (but being not limited to): melanoma, nonsmall-cell lung cancer, small cell lung cancer, lung cancer, liver cancer, retinoblastoma, astrocytoma, glioblastoma, hypercytosis, neuroblastoma, squamous cell carcinoma, a cancer, neck cancer, gingival carcinoma, tongue cancer, breast cancer, prostate cancer, kidney, osteocarcinoma, carcinoma of testis, ovarian cancer, mesothelioma, sarcoma, cervical cancer, gastrointestinal cancer, lymphoma, brain tumor, colorectal carcinoma and bladder cancer.

In another preference, described tumour is liver cancer.

In the present invention, administering mode is not particularly limited, can be by oral, intravenously, intramuscular, intraperitoneal or the administration such as subcutaneous.

Preparation of the present invention can be taken or be administered once or twice or repeatedly every day, or with the administration of slowly-releasing mode.Preferred mode is to take medicine once every day, adheres to, thereby significantly improve the conformability that patient takes medicine because be convenient to like this patient.

While taking, the total dose of general application every day of thumping majority case is everyone 1mg～200g, is preferably 10mg～100g.

Agonist

Utilize albumen of the present invention, by various conventional screening methods, can filter out with SSX2IP albumen interactional material occurs, especially agonist etc.

The agonist of SSX2IP albumen of the present invention, when using (administration), can promote expression and/or the activity of SSX2IP albumen, and then promotes transfer or the migration of cancer cells (comprising liver cancer).Conventionally, but these agonists are formulated in to nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is about 5-8 conventionally, preferably pH is about 6-8, although pH value can change to some extent with being formulated the character of material.The pharmaceutical composition preparing can carry out administration by external or non-external conventional route, comprising (but being not limited to): in knurl, intramuscular, intraperitoneal, intravenously, subcutaneous, intracutaneous or topical.

SSX2IP gene of the present invention, albumen or its agonist are specially adapted to the cultivation of tumor cell in vitro and the preparation of animal model for tumour, and especially animal model modeling is shifted in liver cancer cell Intrahepatic metastasis, distant metastasis or abdominal cavity.

Screening method

The present invention also provides the method for carrying out drug screening based on SSX2IP.Method is first to screen impact (inhibition) SSX2IP expression or an active compound, then the compound filtering out is further tested to its restraining effect to cancer cells.

Wherein, representational cancer cells comprises liver cancer cell, stomach cancer cell, colon-cancer cell, lung carcinoma cell, prostate cancer cell, breast cancer cell, melanoma cell.

The method of the candidate compound of screening prevention provided by the invention or treatment metastases, tumor cell migration or raising chemotherapy drug susceptibility, based on this compound, on the expression amount of SSX2IP and/or active impact, a kind of typical screening method comprises step:

Wherein, if the expression amount of the SSX2IP of cell and/or activity are less than control group in test group, just show that this test compounds is to the expression of SSX2IP and/or the active candidate compound that has inhibiting Hepatoma therapy.And/or

(b) for the candidate compound obtaining in step (a), further test its restraining effect to cancer metastasis or migration.As, in test group, in the culture system of cancer cells, add test compounds, and observe quantity and/or the invasion and attack situation of the distance that cancer cells moves; In control group, in the culture system of cancer cells, do not add test compounds, and observe quantity and/or the invasion and attack situation of the distance that cancer cells moves; Wherein, if the migration distance of cancer cells or invasion and attack quantity are significantly less than control group in test group, just show that this test compounds is the candidate compound that migration has inhibiting Hepatoma therapy to hepatoma Metastasis cell or cancer cells.

Detection method and test kit

The present invention relates to the diagnostic testing process of quantitative and detection and localization people SSX2IP protein level or mRNA level.These tests are known in the art.The people SSX2IP protein level detecting in test, can be for the transfer of diagnosing tumour or the migration of tumour cell.

A kind ofly detect that in sample, whether to have the method for SSX2IP albumen be to utilize the specific antibody of SSX2IP albumen to detect, it comprises: sample is contacted with SSX2IP protein specific antibody; Observe whether to form antibody complex, formed antibody complex and just represented to exist in sample SSX2IP albumen.

SSX2IP albumen or its polynucleotide can be used for diagnosis and the treatment of SSX2IP protein related diseases.Part or all of polynucleotide of the present invention can be used as probe and is fixed on microarray or DNA chip, for analyzing Differential expression analysis and the gene diagnosis of tissue gene.The antibody of anti-SSX2IP can be fixed on protein chip, for detection of the SSX2IP albumen in sample.

The present invention also provides a kind of test kit that detects liver cancer, the primer pair that it contains specific amplification SSX2IP and/or SSX2IP specific antibody and label or specification sheets.

Wherein, described label or specification sheets indicate following content: the ratio >2 when the mrna expression amount of the mutually p-Actin muscle of SSX2IP of the mrna expression Liang Yu of the mutually p-Actin muscle of the SSX2IP of detected object cancer beside organism, points out the probability of this detected object metastases higher than general population.

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

The present invention also comprises a kind of method that suppresses metastases or tumor cell migration, comprises step: the object (Mammals) for the treatment of to needs is used the SSX2IP inhibitor of safe and effective amount.

Beneficial effect of the present invention

1. SSX2IP inhibitor of the present invention can suppress the transfer of tumour, particularly liver cancer or the migration of liver cancer cell effectively, can be used as the medicine of prevention or treatment metastases or tumor cell migration.

2. SSX2IP inhibitor of the present invention can significantly reduce the resistance of tumour cell to chemotherapeutics, thereby improves the susceptibility to chemotherapeutics, to improve the effect of chemotherapeutics.

3. SSX2IP inhibitor of the present invention can also be used as the screening of medicine, thereby filters out can effectively suppressing the medicine of metastases or tumor cell migration, or the medicine to chemotherapeutic sensitivity.

4. SSX2IP gene of the present invention or albumen and agonist thereof can be used for promoting transfer or the migration of tumour, can be used for the cultivation of in vitro cancer cells or the modeling of animal model for tumour.

5. SSX2IP gene of the present invention or albumen can be for the Specific markers as diagnosis or predicting tumors, especially hepatoma Metastasis, and the probability of SSX2IP high expression level person metastases is larger.

6. SSX2IP gene of the present invention or albumen can also be for predicting tumors patient's survival times, and SSX2IP expression amount is higher, and the survival time of tumour patient is shorter.

Embodiment 1

Whether preparation prediction or diagnosing tumour shift/predict the test kit of survival of patients phase

Whether preparation one shifts/predicts the test kit of survival of patients phase for histology prediction or diagnosing tumour, and described test kit comprises:

(a) container, and be positioned at the specificity of container for the following antibody of SSX2IP: the antibody of the anti-SSX2IP of people (purchased from Abcam and Sigma company); With

(b) and label or specification sheets, whether described label or specification sheets indicate described test kit and shift and detect or the survival of patients phase for predicting tumors.

The dependency of embodiment 2SSX2IP protein expression and tumour Tumor thrombus and tumour coating integrity

Material: 53 hepatocarcinoma patient samples, are all collected in Zhongshan Hospital Attached to Fudan Univ.Obtain the approval of experimenter's informed consent and the Ethic review council of hospital.

Method: adopt the test kit of embodiment 1 preparation, grouping situation is as shown in table 1; Observation index: tumor size, Tumor thrombus and tumour coating integrity; Measure the expression level of SSX2IP in tumour knurl body, and it is meaningless for having of cancer patients's complication to observe its expression level.

Result: as shown in table 1:

Table 1

In malignant tumour, the volume size of tumour has represented the height of grade malignancy to a certain extent; And the formation of cancer embolus explanation tumour has possessed the ability that starts migration and invasion.

From table 1, patient's group of high expression level SSX2IP, its tumour is larger, and the probability of generation cancer embolus is higher.Therefore, the SSX2IP level recording in patient's knurl is high, can illustrate that the grade malignancy of this patient tumors may be relatively high, and this tumour possessed the ability of migration and invasion, and the probability of transfer is greater than contrast crowd.Wherein, P represents that SSX2IP high expression level has not statistically significant (P<0.05 is for there being statistical significance) for observation index.

In addition, the integrity of the high expression level of SSX2IP and tumour coating has certain dependency, and in the sample of the high expression level of SSX2IP, coating integrity declines.

Therefore, the transfer of SSX2IP expression amount and tumour has closely related, can be used as the Specific marker whether tumour shifts.

The dependency of embodiment 3SSX2IP protein expression and cancer patients's survival time

Material: 51 hepatocarcinoma patients are carried out to postoperative tracking and follow up a case by regular visits to, obtain the approval of experimenter's informed consent and the Ethic review council of hospital,

Method: according to patient information, postoperative phone is followed the tracks of and followed up a case by regular visits to, and the time length is more than 5 years

Result is as shown in Figure 1:

The cancer patients's of the relative high expression level of SSX2IP survival time is shorter than the low expresser of SSX2IP.Wherein therefore, the expression amount of SSX2IP can be for prediction cancer patients's survival time for high expression level.

The abdominal cavity diffusion of embodiment 4SSX2IP and liver cancer cell BEL-7402 and the relation of liver metastasis

Material: liver cancer cell is BEL-7402 (can purchased from Shanghai Inst. of Life Science, CAS cell resource center)

Grouping situation: n=3, group 1:BEL-7402 is liver cancer cell group; Group 2:BEL-7402Vector is unloaded groups of cells; Group 3:BEL-7402SSX2IP is that SSX2IP crosses expression group;

Method:

Respectively above-mentioned three groups of cells are entered in immunodeficient mouse body by abdominal cavity and tail vein injection by the amount of 1 * 106 cell of every mouse, after six weeks, put to death and dissect, Taking Pictures recording liver cancer cell is in Intraabdominal spread condition.Same procedure is applied to another three groups of mouse, after ten weeks, puts to death and dissects, and takes pictures and records Intrahepatic metastasis situation.

Result is as shown in Figure 2:

Fig. 2 A has shown that mouse peritoneal shifts naked eyes and sees, and can meet the mouse peritoneal metastatic nodules of the liver cancer cell of expressing SSX2IP significantly more than unloaded groups of cells and untreated cell group.

Fig. 2 B shown through pathological statistics, and the mouse peritoneal metastatic nodules of crossing the liver cancer cell of expressing SSX2IP is 6.2 ± 0.84, significantly more than unloaded groups of cells (2.4 ± 1.14) and untreated cell group (2.6 ± 0.89)

Fig. 2 C has shown after tail vein injection tumour carrier cell 10 weeks, puts to death and dissects, and observes the situation of nude mice liver, and macroscopic Nodules appears in the liver portion of visible part nude mice.

Fig. 2 D has shown in three groups of mouse, and the mouse and the ratio that does not shift mouse of distant metastasis occurs, and wherein, SSX2IP crosses in 10 mouse of expression group has 7 abdominal cavity transfer has occurred; In 10 mouse of unloaded groups of cells, there is 1 abdominal cavity transfer has occurred; In 10 mouse of liver cancer cell group, there is 1 abdominal cavity transfer has occurred.

Abdominal cavity transfer and Intrahepatic metastasis incidence that conclusion: SSX2IP crosses expression group mouse are significantly better than other groups, and visible SSX2IP gene or albumen can promote the transfer of tumour.

The relation of the locomotivity of embodiment 5SSX2IP and liver cancer cell SMMC-7721

Material: liver cancer cell is SMMC-7721 (can purchased from Shanghai Inst. of Life Science, CAS cell resource center).

Grouping situation: with embodiment 1, wherein clone changes SMMC-7721 into.

Method: the present embodiment adopts the locomotivity of conventional cut healing experiment reaction liver cancer cell, and wherein mean gap is apart from the locomotivity that represents cell, and mean gap distance is longer, and signaling ability is stronger.

Result is as shown in Figure 3:

Fig. 3 A has shown three groups of different mean gap distances after 48 hours, wherein, crosses the liver cancer cell group of expressing SSX2IP: 170.83 ± 36.96 μ m; Unloaded groups of cells: 516.67 ± 25.57 μ m; Blank groups of cells: 525.00 ± 31.92 μ m.

Conclusion: SSX2IP crosses expression group cancer cells migration distance and is significantly distal to other groups, and visible SSX2IP gene or albumen can promote the migration of tumour cell.

The relation of the locomotivity of embodiment 6SSX2IP and liver cancer cell BEL-7402

Material and grouping situation are with embodiment 1

Method: with embodiment 2

Result is as shown in Figure 3:

Fig. 3 B has shown three groups of different mean gap distances after 48 hours, wherein, crosses the liver cancer cell group of expressing SSX2IP: 183.33 ± 49.07 μ m; Unloaded groups of cells: 312.50 ± 25.00 μ m; Blank groups of cells: 329.17 ± 15.96 μ m.

The relation of the migration invasive ability of embodiment 7SSX2IP and liver cancer cell SMMC-7721

Material grouping situation: with embodiment 2

Method: the present embodiment adopts the Matrigel reaction transfer ability of liver cancer cell and the invasive ability of penetration cell epimatrix: Transwell cell, can think that this is a kind of membrane filter (Membrane filters), also can think a kind of support (permeable supports) that has permeability.By adding up the cell quantity through this metafiltration film, can reflect the transfer ability of cell; On film, add further the matrigel that imitates extracellular matrix components, can detect the ability that cell-penetrating cell matrix completes Invasion and Metastasis

Result is as shown in Figure 4:

Fig. 4 A shown in three groups of experimental cell strains, cross the SMMC-7721SSX2IP cell of expressing SSX2IP through the amount of Transwell filter membrane significantly more than two other control group.

In the migration experiment of Fig. 4 B, the quantity statistics of excessively expressing SSX2IP group contrast control group is: 123.33 ± 9.45,59.67 ± 4.73,51.33 ± 6.03; In Matrigel, the quantity statistics of excessively expressing SSX2IP group contrast control group is: 92.00 ± 7.00,43.67 ± 4.16, and 38.00 ± 3.61, P<0.001.

Conclusion: cross expression SSX2IP liver cancer cell SMMC-7721SSX2IP, the ability of its invasion and attack and migration significantly increases, and therefore, SSX2IP has promoter action to the invasion and attack transfer ability of liver cancer cell.

The invasive ability of embodiment 8SSX2IP and liver cancer cell BEL-7402

Material grouping situation is with embodiment 1.

Method is with embodiment 4

Result is as shown in Fig. 4 C and 4D:

Fig. 4 C shown in three groups of experimental cell strains, cross the SMMC-7721SSX2IP cell of expressing SSX2IP through the amount of Transwell filter membrane significantly more than two other control group.

In the migration experiment of Fig. 4 D, the quantity statistics of excessively expressing SSX2IP group contrast control group is: 154.67 ± 14.05,103.67 ± 10.70,109.00 ± 7.55; * P<0.01.In Matrigel, the quantity statistics of excessively expressing SSX2IP group contrast control group is: 113.00 ± 6.56,63.33 ± 11.50, and 60.67 ± 11.02, P<0.01.

Conclusion: the ability of excessively expressing its invasion and attack of SSX2IP liver cancer cell BEL-7402SSX2IP and migration significantly increases, and therefore, SSX2IP has promoter action to the invasion and attack transfer ability of liver cancer cell.

Embodiment 9SSX2IP and the relation of liver cancer cell SMMC-7721 to the susceptibility of chemotherapeutics 5-Fu

Material, grouping situation: with embodiment 2

Method: the present embodiment, by measuring IC50 value, reflects that tumour cell is for the susceptibility of medicine.IC50 refers to the concentration of a suppressed half inhibitor; Can be understood as certain density certain drug-induced apoptosis of tumor cells 50%, this concentration is called 50% inhibition concentration, corresponding concentration when to be apoptotic cell equal 50% with the ratio of whole cell count, IC50 value can be used for weighing the ability of drug-induced apoptosis, be that inducibility is stronger, this numerical value is lower, can certainly the tolerance degree of certain cell of reverse instruction to medicine.We are by being determined under different concns, the apoptosis rate of liver cancer cell to 5-FU, and matched curve, and calculate IC50 value.

Result is as shown in Figure 5A:

Fig. 5 A has shown that three groups of cells are to the dose response curve of chemotherapeutics 5-Fu and IC50 value.Result shows that SMMC-7721SSX2IP is significantly higher than control group (24.68 ± 1.17 μ M, 14.59 ± 0.77 μ M, 13.60 ± 0.88 μ M, P<0.001) to the IC50 value of 5-FU.Therefore visible, SSX2IP can reduce tumour cell SMMC-7721 for the susceptibility of chemotherapeutics 5-FU.

Embodiment 10SSX2IP and the relation of liver cancer cell BEL-7402 to the susceptibility of chemotherapeutics 5-Fu

Material, grouping situation: with embodiment 1

Method: with embodiment 6

Result is as shown in Figure 5 B:

Fig. 5 B has shown that three groups of cells are to the dose response curve of chemotherapeutics 5-Fu and IC50 value, result shows that BEL-7402SSX2IP is significantly higher than control group (28.52 ± 0.65 μ M to the IC50 value of 5-FU, 12.73 ± 1.81 μ M, 14.16 ± 1.20 μ M, * * P<0.001).Therefore visible, SSX2IP can reduce tumour cell BEL-7402 for the susceptibility of chemotherapeutics 5-FU.

Embodiment 11SSX2IP and the relation of liver cancer cell SMMC-7721 to the susceptibility of chemotherapeutics CDDP

Material, grouping situation: with embodiment 2

Method: with embodiment 6, difference is that chemotherapeutics changes CDDP (cis-platinum) into.

Result: as shown in Figure 5 C:

Fig. 5 C has shown that three groups of cells are to the dose response curve of chemotherapeutics CDDP and IC50 value, result shows that SMMC-7721SSX2IP is significantly higher than control group (11.38 ± 1.42 μ M to the IC50 value of CDDP, 5.72 ± 0.75 μ M, 6.18 ± 0.81 μ M, P<0.001).Therefore visible, SSX2IP can reduce tumour cell SMMC-7721 for the susceptibility of chemotherapeutics CDDP.

Embodiment 12SSX2IP and the relation of liver cancer cell BEL-7402 to the susceptibility of chemotherapeutics CDDP

Material, grouping situation are with embodiment 1

Method: with embodiment 8.

Result: as shown in Figure 5 D:

Fig. 5 D. has shown that three groups of cells are to the dose response curve of chemotherapeutics CDDP and IC50 value, result shows that BEL-7402SSX2IP is significantly higher than control group (9.86 ± 1.24 μ M to the IC50 value of CDDP, 6.13 ± 0.24 μ M and 5.90 ± 0.47 μ M, * * P<0.001)

The abdominal cavity diffusion of embodiment 13SSX2IP inhibitor siRNA liver cancer cell BEL-7402 and the relation of liver metastasis

Material: with embodiment 1

Grouping situation: n=3, group 1:BEL-7402 is liver cancer cell group; Group 2:BEL-7402si-NC is irrelevant sequence interference control cells group; The siRNA that group 3:BEL-7402si-SSX2IP is SSX2IP lowers expression group;

Method:

Conclusion: siRNA disturbs the cell of SSX2IP expression group, in the abdominal cavity of mouse, shift and Intrahepatic metastasis incidence significantly lower than other groups (the equal <40% of metastatic tumor quantity), by inhibition SSX2IP gene or albumen, can suppress as seen propagation and the transfer of tumour.

Embodiment 14SSX2IP inhibitor siRNA improves the susceptibility of liver cancer cell BEL-7402 to chemotherapeutics CDDP

Material: with embodiment 1

The present embodiment method is with embodiment 6, and difference is that chemotherapeutics is CDDP.

Result show SSX2IP inhibitor siRNABEL-7402si-SSX2IP to the IC50 value of CDDP all lower than the more than 50% of control group, therefore, siRNA disturbs the expression of SSX2IP, the BEL-7402 liver cancer cell of low expression SSX2IP, the susceptibility of CDDP is strengthened, and result for the treatment of improves.

Embodiment 15SSX2IP inhibitor microRNA improves the susceptibility of liver cancer cell SMMC-7721 to chemotherapeutics 5-FU

The present embodiment method is with embodiment 10, and difference is that liver cancer cell group uses SMMC-7721 instead, and chemotherapeutics is 5-FU.

Grouping situation: n=3, group 1:SMMC-7721 is liver cancer cell group; Group 2:SMMC-7721miR-NC is irrelevant sequence interference control cells group; The microRNA that group 3:SMMC-7721miR-SSX2IP is SSX2IP lowers expression group;

Result show SSX2IP inhibitor siRNASMMC-7721si-SSX2IP to the IC50 value of CDDP all lower than the more than 50% of control group, therefore, siRNA has disturbed the expression of SSX2IP, the SMMC-7721 liver cancer cell of low expression SSX2IP, the susceptibility of CDDP is strengthened, and result for the treatment of improves.

All documents of mentioning in the present invention are all quoted as a reference in this application, just as each piece of document, are quoted as a reference separately.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

1. synovial sarcoma X breaking point gene 2 interaction proteins are a purposes for SSX2IP gene or albumen, it is characterized in that, for the preparation of the reagent or the test kit that detect metastases.

2. purposes as claimed in claim 1, is characterized in that, described SSX2IP dietary protein origin is in Mammals; Preferably, derive from people, mouse or rat.

3. purposes as claimed in claim 1, is characterized in that, described tumour comprises: liver cancer, cancer of the stomach, intestinal cancer, lung cancer, prostate cancer, mammary cancer, melanoma.

4. purposes as claimed in claim 1, is characterized in that, described reagent comprises SSX2IP Auele Specific Primer, specific antibody, probe and/or chip.

5. purposes as claimed in claim 1, is characterized in that, described detection is to measure tissue sample, blood sample, serum sample or humoral sample.

6. for detection of a diagnostic kit for metastases, it is characterized in that, described test kit contains a container, contains the detection reagent that detects SSX2IP albumen or mRNA in described container; And label or specification sheets, described label or specification sheets indicate described test kit for detection of metastases.

7. test kit as claimed in claim 6, is characterized in that, described detection SSX2IP albumen or the detection reagent of mRNA comprise:

(a). the specific antibody of anti-SSX2IP albumen; And/or

8. test kit as claimed in claim 6, is characterized in that, indicates following content in described label or specification sheets:

9. diagnostic kit as claimed in claim 6, is characterized in that, described test kit is also for the predicting tumors survival of patients time.

10. a purposes for SSX2IP gene or albumen, is characterized in that, as the Specific marker that detects metastases for the preparation of detection reagent; Or as the Specific marker that detects metastases.