CN104001174B

CN104001174B - Applications of the SSX2IP in preventing and treating metastases

Info

Publication number: CN104001174B
Application number: CN201310062585.5A
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: 2018-11-02
Anticipated expiration: 2033-02-27
Also published as: CN104001174A

Abstract

The invention discloses applications of the SSX2IP in preventing and treating metastases.Specifically, the invention discloses SSX2IP inhibitor to prepare the purposes in inhibiting metastases or inhibiting the drug of tumor cell migration.SSX2IP inhibitor can be used as effective ingredient and inhibit metastases or inhibit tumor cell migration, can also increase tumour cell to chemotherapy drug susceptibility or reduce tumour cell to chemotherapeutics drug resistance.In addition, the SSX2IP genes or albumen and its agonist of the present invention can be used for promoting proliferation or the migration of tumour cell, it is used to prepare animal model for tumour.

Description

Applications of the SSX2IP in preventing and treating metastases

Technical field

The present invention relates to oncologies.More particularly it relates to which SSX2IP inhibitor is in prevention and treatment tumour Application in terms of transfer, tumor cell migration and chemotherapy resistance row.The invention further relates to SSX2IP genes, albumen or its swash Dynamic application of the agent in preparing tumor model.

Background technology

Hepatocellular carcinoma (Hepatocellular carcinoma, HCC) is one of most common malignant tumour, occupies the whole world (malignant tumour) incidence the 5th, cancer correlation cause of the death third position, in China, its incidence is only second to lung cancer, occupies second.

In the complex treatment of liver cancer, operation excision and chemotherapy are the important means treated, but due to hepatocarcinoma early diagnosis Difficulty, metastases speed is fast, and not high to chemotherapy medicament sensitive degree, causes various therapeutic effects undesirable, and transfer patient is pre- It is poor afterwards.

In addition in the liver cancer patient of different phase, personalized therapeutic scheme is even more important.But due to lacking specificity The time window of hepatoma Metastasis marker, treatment is often just opened after hepatoma Metastasis, delay prevent metastases it is good when Machine.

Therefore, there is an urgent need in the art to develop the medicine that can inhibit metastases, reduce chemotherapy resistance, and It can predict or the Specific marker of diagnosing tumour transfer especially hepatoma Metastasis, so as to predict hepatoma Metastasis, in turn Basis is provided preferably to prevent or treating metastases.

Invention content

Metastases, tumor cell migration can be prevented and treated the present invention provides one kind and can improve chemosensitivity The SSX2IP inhibitor of property；There are prevention or therapeutic effect drug to metastases or tumor drug resistance the present invention also provides a kind of Screening technique.In addition, the present invention also provides the kits whether a kind of prediction or diagnosing tumour are shifted.

The first aspect of the present invention, provide a kind of SSX2IP (2 interaction protein of synovial sarcoma X breakpoint gene, Synovial Sarcoma X brearkpoint2interacting Protein) inhibitor purposes, be used to prepare inhibition Metastases or the drug for inhibiting tumor cell migration.

In another preferred example, the SSX2IP albumen sources are in mammal；Preferably, from people, mouse or Rat；More preferably, people is derived from.

In another preferred example, the tumour includes liver cancer, gastric cancer, intestinal cancer, lung cancer, prostate cancer, breast cancer, black Plain tumor.

In another preferred example, the tumour is liver cancer.

In another preferred example, the inhibitor includes：The antibody of SSX2IP, the antisense RNA of SSX2IP nucleic acid, The activity inhibitor of siRNA, shRNA and SSX2IP.

In another preferred example, the drug contains pharmaceutically acceptable carrier, SSX2IP inhibitor and optional Chemotherapeutics.

In another preferred example, the drug is administered by application method selected from the group below：Oral, vein note It penetrates, intramuscular injection, hypodermic injection, sublingual administration, rectal perfusion, nasal spray, mouthspray, local skin or whole body are percutaneously used Medicine.

In another preferred example, the preparation of the drug is selected from the group：Tablet, capsule, injection, granule, spray Mist agent.

In another preferred example, the SSX2IP inhibitor is applied (every time or daily) with the dosage of 0.5-5mg/kg weight For mammal.

In another preferred example, it is more preferably people that the mammal, which includes people, mouse, rat,.

In another preferred example, the chemotherapeutics includes amide (CTX), ifosfamide (IFO), mitomycin (MMC), adriamycin (ADM), vincristine (VCR), vinblastine (VBL), etoposide (VP16), brave and fierce (Vumon), cis-platinum (CDDP), fluorouracil (5-FU), carboplatin (CBP) and methotrexate (MTX) (MTX) etc..

The second aspect of the present invention provides a kind of purposes of SSX2IP inhibitor, and (a), which is used to prepare, to be promoted chemotherapy or put The pharmaceutical composition of the Apoptosis of induction is treated, or (b) is used to prepare the promotion for the Apoptosis for promoting chemotherapy or radiotherapy-induced Agent, or (c) it is used to prepare the sensitizer for so that cancer cell is more sensitive to the Apoptosis of chemotherapy or radiotherapy-induced.

In another preferred example, the SSX2IP inhibitor sensitizer use for cancer treatment or promote Apoptosis Accelerating agent.

The SSX2IP albumen includes fusion protein and non pregnant women.

The third aspect of the present invention provides a kind of method of external non-therapeutic inhibition cancer cell migration, including step： In the presence of SSX2IP inhibitor, cancer cell is cultivated, to inhibit cancer cell migration.

In another preferred example, the method includes the addition SSX2IP inhibitor into the cultivating system of cancer cell, from And inhibit cancer cell migration.

In another preferred example, the cancer cell include liver cancer cells, it is stomach cancer cell, colon-cancer cell, lung carcinoma cell, preceding Row gland cell, mammary glandular cell, melanoma cells.

In another preferred example, in the method, a concentration of 0.5-5mg/mL of the SSX2IP inhibitor.

The fourth aspect of the present invention provides the purposes of a kind of SSX2IP genes, SSX2IP albumen or its agonist, is used for The composition (or accelerating agent) for promoting metastases or tumor cell migration is prepared, or as promotion metastases or tumour cell The accelerating agent of migration.

In another preferred example, the SSX2IP genes or albumen source are in mammal；Preferably, from people, Mouse or rat；More preferably, people is derived from.

In another preferred example, the SSX2IP genes or albumen be recombined human SSX2IP genes or albumen or come from people People SSX2IP genes in blood or serum or albumen.

In another preferred example, the drug of the promotion metastases or tumor cell migration is for establishing tumour mould Type.

In another preferred example, the tumour is liver cancer.

In another preferred example, the purposes of the SSX2IP genes, SSX2IP albumen or its agonist further includes：? In the presence of SSX2IP albumen or its agonist, cancer cell is cultivated, to promote growth of cancer cells or proliferation.

The fifth aspect of the present invention provides a kind of screen for inhibiting tumor cell migration or for improving cancer cell medicine The method of the candidate compound of object sensibility, the method includes the steps：

(a) in test group, the addition test compound in the cultivating system of cell, and observe in the cell of the test group The expression quantity and/or activity of SSX2IP；In control group, test compound is not added in the cultivating system of same cell, and Observe the expression quantity and/or activity of SSX2IP in the cell of control group；

Wherein, if the expression quantity of the SSX2IP of cell and/or activity are less than control group in test group, the test is indicated that Compound is expression to SSX2IP and/or activity has the treatment metastases of inhibiting effect or tumor cell migration or can carry The compound of high chemotherapy drug susceptibility；With

(b) for the candidate compound obtained in step (a), the candidate compound is optionally tested to cancer metastasis Migration inhibiting effect or the candidate compound to the influence of cancer cell drug susceptibility.

In another preferred example, include step in step (b)：It adds and surveys in test group, in the cultivating system of cancer cell Compound is tried, and observes the quantity and/or invasion situation of the distance of cancer cell movement；In control group, in the culture of cancer cell Test compound is not added in system, and observes the quantity and/or invasion situation of the distance of cancer cell movement；Wherein, if surveyed The migration distance of cancer cell or invasion quantity indicate that the test compound is to metastases significantly less than control group in examination group Tumor cell migration have an inhibiting effect or can improve the compound of chemotherapy drug susceptibility.

Sixth aspect present invention provides the purposes of a kind of SSX2IP genes or SSX2IP albumen, and it is swollen to be used to prepare detection The reagent or kit of tumor metastasis.

In another preferred example, the SSX2IP albumen sources are in people, full length amino acid sequence such as SEQ ID NO.: Shown in 1.

In another preferred example, the SSX2IP gene nucleotide series such as SEQ ID NO.:Shown in 2.

In another preferred example, the tumour includes：It is liver cancer, gastric cancer, intestinal cancer, lung cancer, prostate cancer, breast cancer, black Melanoma.

In another preferred example, the reagent includes SSX2IP specific primers, specific antibody, probe and/or core Piece.

In another preferred example, the detection includes that enzyme linked immunoassay method (ELISA method) detection or time resolution are exempted from Epidemic disease fluorescence method (TRFIA methods) detects.

In another preferred example, the SSX2IP albumen or the coupling of its specific antibody have or carry detectable label.

In another preferred example, the detectable label is selected from the group：Chromophore, chemiluminescent groups, fluorogen, same to position Element or enzyme.

In another preferred example, the specific antibody of the SSX2IP is monoclonal antibody or polyclonal antibody.

In another preferred example, the detection is to measure tissue sample, blood sample, blood serum sample or humoral sample.

In another preferred example, the tissue sample includes cancerous tissue and cancer beside organism.

The seventh aspect of the present invention provides a kind of diagnostic kit for detecting metastases, the kit Containing a container, the detection reagent containing detection SSX2IP albumen or mRNA in the container；And label or specification, it is described Label or specification indicate the kit for detecting metastases.

In another preferred example, the detection metastases, which refer to, judges whether metastases occur, and/or judges to occur Possibility (neurological susceptibility) size of metastases.

In another preferred example, described to judge to include prejudging.

In another preferred example, the detection reagent of the detection SSX2IP albumen or mRNA include：

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

(b) specific primer of the mRNA or cDNA of specific amplifications SSX2IP.

In another preferred example, the following contents is indicated in the label or specification：

When the ratio between SSX2IP expression quantity of tumor tissues SSX2IP expression quantity and cancer beside organism for detecting object >=2, then carry Show that the probability of the detection object metastases is higher than general population.

In another preferred example, the expression quantity is the relative expression relative to crt gene (such as beta-actin) Amount.

In another preferred example, the kit is additionally operable to prediction tumor patient life span.

In another preferred example, when it is tumor patient to detect object, if the SSX2IP in detection object tumor tissues Expression quantity Y1 is significantly higher than the expression quantity Y2 of SSX2IP in similar cancer patient's tumor tissues, then when prompting the detection object lives Between the mean survival time less than similar cancer patient；

If the SSX2IP expression quantity Y1 in detection object tumor tissues is substantially less than in similar cancer patient's tumor tissues The expression quantity Y2 of SSX2IP then prompts the mean survival time higher than similar cancer patient of detection object lives time.

It is in another preferred example, described that be significantly higher than refer to Y1/Y2 >=2.

In another preferred example, described refers to substantially less than Y1/Y2≤0.5.

The eighth aspect of the present invention provides the purposes of a kind of SSX2IP genes or albumen, as detection metastases Specific marker is used to prepare detection reagent；Or the Specific marker as detection metastases.

In another preferred example, the tumour includes liver cancer.

The present invention provides a kind of methods of prediction or diagnosing tumour transfer, including step：

(a) prepares subject's test sample；

(b) detect test sample in SSX2IP with respect to beta-actin mrna expression amount and compared with reference value, when it Ratio >=2 item prompt the probability of the detection object metastases to be higher than general population.

In another preferred example, the test sample is tissue sample, blood sample, blood serum sample or humoral sample.

In another preferred example, the reference value is the expression quantity of SSX2IP in non-tumor sample.

In another preferred example, the detecting step b includes the amount for detecting SSX2IP mRNA or the amount of SSX2IPcDNA； And/or the amount of detection SSX2IP albumen.

In another preferred example, the detecting step b includes being detected by RT-PCR or PCR method.

In another preferred example, the detecting step b includes being detected using the antibody of anti-SSX2IP albumen.

In addition, the invention also includes a kind of inhibition metastases or the method for tumor cell migration, including step：To needs The object (mammal) for the treatment of applies the SSX2IP albumen or its agonist of safe and effective amount.

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment) It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.

Description of the drawings

Fig. 1 shows that Kaplan-Meier analyses SSX2IP express and the relationship of life cycle in 51 hepatocarcinoma patients, In, SSX2IP high expression patients are substantially less than SSX2IP low expression patient groups (20) (P=0.004) group (31) its life cycle.

Fig. 2 shows the relationship of the abdominal cavity diffusion and liver metastasis that are overexpressed SSX2IP and liver cancer cells：Wherein, Fig. 2A Show that .B photographs to record liver cancer cells in intraperitoneal spread condition.B. statistics find be overexpressed SSX2IP liver cancer cells its Spread in nude mice abdominal cavity and formed tumour ability be significantly higher than transfection non-loaded cells group and untreated cell group (6.2 ± 0.84,2.6 ± 0.89,2.4 ± 1.14, * * P < 0.001).C.BEL-7402,BEL-7402^Vector, BEL-7402^SSX2IPThree classes Cell is entered by tail vein injection in nude mouse respectively, after 10 weeks, the case where putting to death and dissect, photograph to record nude mice liver.D. it unites Meter is found, is overexpressed the liver cancer cells of the SSX2IP ratio of liver metastasis in nude mouse and is significantly higher than unloaded control and blank Control group, transferring ratio are respectively 7/10,2/10 and 1/10, P=0.023.

Fig. 3 shows that SSX2IP can promote the cut healing ability of liver cancer cells.Wherein, cut Healing Experiments react The locomotivity of liver cancer cells.

Fig. 3 A are shown detects SMMC-7721, SMMC-7721 by cut Healing Experiments^Vector, SMMC-7721^SSX2IPThree The locomotivity of class cell finds the liver cancer cells cell SMMC-7721 for being overexpressed SSX2IP^SSX2IP, locomotivity is significantly high Blank control groups of cells is combined in non-loaded cells, the mean gap distance after 48 hours is：170.83 ± 36.96 μm, 516.67 ± 25.57m, 525.00 ± 31.92 μm, * * P < 0.001.

Fig. 3 B are shown detects BEL-7402, BEL-7402 by cut Healing Experiments^Vector, BEL-7402^SSX2IPThree classes The locomotivity of cell finds the liver cancer cells cell BEL-7402 for being overexpressed SSX2IP^SSX2IP, locomotivity is significantly higher than Non-loaded cells combine blank control groups of cells, and the mean gap distance after 48 hours is：183.33 ± 49.07m, 312.50 ± 25.00m, 329.17 ± 15.96 μm, * * P < 0.001.

Fig. 4 .SSX2IP can promote invasion and the transfer ability of liver cancer cells

Fig. 4 A show that SSX2IP invades Hepatocellular carcinoma cell line the promotion result of transfer ability：

Fig. 4 B show that statistical result shows to be overexpressed SSX2IP Hepatocellular carcinoma cell lines^SSX2IP, invade and migrate Ability significantly increase.In migration experiment, the quantity statistics for being overexpressed SSX2IP groups comparison control group are：123.33 ± 9.45, 59.67 ± 4.73,51.33 ± 6.03；In Matrigel, the quantity statistics for being overexpressed SSX2IP groups comparison control group are：92.00 ± 7.00,43.67 ± 4.16,38.00 ± 3.61, * * P < 0.001.

Fig. 4 C show that SSX2IP invades liver cancer cells BEL-7402 the promotion result of transfer ability

Fig. 4 D show statistical result, show to be overexpressed SSX2IP liver cancer cells BEL-7402SSX2IP, invade and move The ability of shifting significantly increases

In migration experiment, the quantity statistics for being overexpressed SSX2IP groups comparison control group are：154.67 ± 14.05,103.67 ± 10.70,109.00 ± 7.55；* P < 0.01.In Matrigel, it is overexpressed the quantity statistics of SSX2IP groups comparison control group For：113.00 ± 6.56,63.33 ± 11.50,60.67 ± 11.02, * P < 0.01.

Fig. 5 .SSX2IP can reduce susceptibility of the liver cancer cells to chemotherapeutics, increase drug resistance

Fig. 5 A show SMMC-7721, SMMC-7721Vector and SMMC-7721SSX2IP to chemotherapeutics 5-Fu's Dose-effect curve and IC₅₀Value, as a result shows ICs of the SMMC-7721SSX2IP to 5-FU₅₀Value is significantly higher than control group (24.68 ± 1.17 μM, 14.59 ± 0.77M, 13.60 ± 0.88 μM, * * P<0.001).

Fig. 5 B show the dosage of BEL-7402, BEL-7402Vector and BEL-7402SSX2IP to chemotherapeutics 5-Fu Response curve and IC₅₀Value, as a result shows ICs of the BEL-7402SSX2IP to 5-FU₅₀Value be significantly higher than control group (28.52 ± 0.65 μM, 12.73 ± 1.81 μM, 14.16 ± 1.20 μM, * * P<0.001).

Fig. 5 C show the agent of BEL-7402, SMMC-7721Vector and SMMC-7721SSX2IP to chemotherapeutics CDDP Quantitative response curve and IC₅₀Value, as a result shows ICs of the SMMC-7721SSX2IP to CDDP₅₀Value be significantly higher than control group (11.38 ± 1.42 μM, 5.72 ± 0.75 μM, 6.18 ± 0.81 μM, * * P<0.001).

Fig. 5 D show the dosage of BEL-7402, BEL-7402Vector and BEL-7402SSX2IP to chemotherapeutics CDDP Response curve and IC₅₀Value, as a result shows ICs of the BEL-7402SSX2IP to CDDP₅₀Value is significantly higher than control group (9.86 ± 1.24 μM, 6.13 ± 0.24 μM, 5.90 ± 0.47 μM, * * P<0.001).

Specific implementation mode

The present inventor's in-depth study by extensive by, for the first time it was unexpectedly observed that the present inventor pass through it is extensive and deep Research, for the first time it was unexpectedly observed that SSX2IP inhibitor may be used as preventing or treating metastases, tumor cell migration, and energy Improve sensibility of the tumour cell to chemotherapeutics；It is carried the present inventors have additionally discovered that SSX2IP genes or albumen can be used for preparing High tumours of chemotherapeutic agents sensibility, screening inhibit the drug of metastases or tumor cell migration；In addition, the present inventor also sends out SSX2IP genes or albumen are showed and its agonist may be used as promoting metastases, tumor cell migration, and it is thin to enhance tumour Born of the same parents can be used for the preparation of animal drug resistance, metastasis model to the drug resistance of chemotherapeutics.

In addition, the present inventor also found by research, the high expression of SSX2IP has very strong correlation with metastases, real The Specific marker bright, SSX2IP can be shifted as prediction or diagnosing tumour is verified, whether may be used to prepare prediction tumour Whether energy or diagnosing tumour have occurred and that the detection kit of transfer.In addition, the present inventors have additionally discovered that SSX2IP can be used for Predict the life span of tumor patient.On this basis, the present invention is completed.

SSX2IP albumen and polynucleotides

In the present invention, " albumen of the present invention ", " polypeptide of the present invention ", " SSX2IP albumen " are used interchangeably, and refer to synovial membrane meat 2 interaction protein of tumor X breakpoint (referred to as SSX2IP).It should be understood that the term further include SSX2IP active fragment and Derivative.

In the present invention, " gene of the present invention ", " polynucleotides of the present invention " refer to coding SSX2IP albumen or its active fragment With the nucleotide sequence of derivative, including justice and antisense nucleic acid.SSSX2IP is positioned at chromosome1p22.3, and gene is complete Long 46kb, wherein including 14 exons.

In the present invention, term " SSX2IP albumen " or " SSX2IP polypeptides " are used interchangeably, and are all referred to people's albumen The albumen or polypeptide of SSX2IP amino acid sequences.SSX2IP has been confirmed as the related antigen of acute myeloid leukaemia at present, is The target spot of one potential leukaemia immunization therapy.

The Genbank accession number of 5 kinds of mRNA transcripts of SSX2IP gene nucleotide series for use in the present invention is NM001166293.1;NM001166294.1;NM001166295.1;NM001166417.1;NM014021.3。

A kind of preferred SSX2IP gene nucleotide series such as SEQ ID NO.:Shown in 1, Gene ID:117178, it compiles The amino acid sequence such as SEQ ID NO. of code:Shown in 2, accession number NP001159889.1.

As used herein, " separation " refers to that substance is separated from its primal environment (if it is crude, original Beginning environment is natural surroundings).If the polynucleotides and polypeptides under the native state in active somatic cell do not isolate and purify, But same polynucleotides or polypeptide are such as separated from other substances with existing in native state, then are isolated and purified.

As used herein, " the SSX2IP albumen or polypeptide of separation " refer to SSX2IP albumen substantially free of naturally with its phase Other albumen, lipid, carbohydrate or the other materials closed.Those skilled in the art can be purified with the purified technology of protein of standard SSX2IP albumen.Substantially pure polypeptide can generate single master tape in non-reducing polyacrylamide gel.In the present invention, SSX2IP albumen includes fusion protein and non pregnant women.

The polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthesis polypeptide, preferably recombinant polypeptide.The present invention's is more Peptide may also include or not include the methionine residues of starting.

The polynucleotides of the present invention can be DNA form or rna form.DNA form includes cDNA, genomic DNA or people The DNA of work synthesis.DNA can be single-stranded or double-strand.DNA can be coding strand or noncoding strand.

The polynucleotides of mature polypeptide for encoding SSX2IP include：The coded sequence of encoding mature polypeptide；Mature polypeptide Coded sequence and various additional coding sequences；The coded sequence (and optional additional coding sequence) of mature polypeptide and non-volume Code sequence.Term " polynucleotides of coding polypeptide " can be the polynucleotides for including this polypeptide of coding, can also be to further include The polynucleotides of additional code and/or non-coding sequence.

The invention further relates to the variant of above-mentioned polynucleotides, coding has the more of identical amino acid sequence with the present invention The segment of peptide or polypeptide, analogs and derivatives.The variant of this polynucleotides can be the allelic variant naturally occurred or The variant that non-natural occurs.These nucleotide variants include substitution variants, Deletion variants and insert variation.Such as this Known to field, allelic variant is the alternative forms of a polynucleotides, it may be one or more nucleotide substitution, Missing is inserted into, but not encodes the function of polypeptide from it is substantially changed.

The invention further relates to the nucleic acid fragments hybridized with above-mentioned sequence, include the nucleic acid fragment of justice and antisense.Such as this Used in text, the length of " nucleic acid fragment " at least contains 15 nucleotide, preferably at least 30 nucleotide, more preferably at least 50 cores Thuja acid, preferably at least more than 100 nucleotide.Nucleic acid fragment can be used for the amplification technique (such as PCR) of nucleic acid with determine and/or The polynucleotides of separation coding SSX2IP albumen.

The people SSX2IP nucleotide full length sequences or its segment of the present invention can usually use PCR amplification method, recombination method or people Work synthetic method obtains.It, can be according to published related nucleotide sequence, especially open reading frame for PCR amplification method Sequence carrys out design primer, the commercially available libraries cDNA is used in combination or by the libraries cDNA prepared by conventional method well known by persons skilled in the art As template, expands and obtain related sequence.When sequence is longer, it is often necessary to twice or multiple PCR amplification, then again will Each time the segment amplified is stitched together by proper order.

Once obtaining related sequence, so that it may to obtain related sequence in large quantity with recombination method.This is typically will It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method.

In addition, related sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.In general, logical After first synthesizing multiple small fragments, it is then attached the very long segment of available sequence again.

It is optimized for obtaining the gene of the present invention using the method for round pcr DNA amplification/RNA.Primer for PCR It can be properly selected according to the sequence information of invention disclosed herein, and available conventional method synthesis.Conventional method can be used The DNA/RNA segments of amplification are such as detached and purified by gel electrophoresis.

The present invention also relates to the carriers for the polynucleotides for including the present invention, and with of the invention carrier or SSX2IP albumen The genetically engineered host cell of coded sequence, and the method that generates polypeptide of the present invention through recombinant technique.

By the recombinant dna technology of routine, it can be used to express or produce recombination using the polynucleotide sequence of the present invention SSX2IP albumen.In general there are following steps：

(1) polynucleotides (or variant) of the encoding human SSX2IP albumen of the present invention, or with contain the polynucleotides Recombinant expression carrier conversion or suitable host cell of transduceing；

(2) host cell that is cultivated in suitable culture medium；

(3) be separated from culture medium or cell, protein purification.

Method well-known to those having ordinary skill in the art can be used to build the DNA sequences encodings of SSX2IP containing people and suitable turn The expression vector of record/translation control signal.These methods include recombinant DNA technology in vi, DNA synthetic technologys, In vivo recombination skill Art etc..The DNA sequence dna can be effectively connected in the appropriate promoter in expression vector, to instruct mRNA to synthesize.Expression carries Body further includes the ribosome bind site and transcription terminator of translation initiation.

In addition, expression vector preferably includes one or more selected markers, to provide for selecting conversion The phenotypic character of host cell, such as the dihyrofolate reductase of eukaryotic culture, neomycin resistance and green fluorescence egg (GFP) in vain, or tetracycline or amicillin resistance for Escherichia coli.

The carrier for including above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence can be used for converting suitable When host cell, allow it to expression protein.

Host cell can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells；Or it is high Equal eukaryocytes, such as mammalian cell.Representative example has：Escherichia coli, the bacterial cell of streptomyces；Fungal cell is such as Yeast；Plant cell；The insect cell of drosophila S2 or Sf9；The zooblast etc. of CHO, COS or 293 cells.

It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell.When host is original When core biology such as Escherichia coli, can absorb the competent cell of DNA can harvest after exponential phase of growth, use CaCl₂Method processing, institute With the step of it is generally well-known in the art.Another method is to use MgCl₂.If desired, conversion can also use the side of electroporation Method carries out.When host is eucaryote, following DNA transfection methods can be selected：Calcium phosphate precipitation, conventional mechanical methods are such as Microinjection, electroporation, liposome packaging etc..

The transformant of acquisition can use conventional method culture, express the polypeptide of the coded by said gene of the present invention.According to used Host cell, culture medium used in culture can be selected from various conventional mediums.Under conditions of suitable for host cell growth It is cultivated.After host cell growth is to cell density appropriate, with suitable method (such as temperature transition or chemical induction) Cell is further cultured for a period of time by the promoter for inducing selection.

Recombinant polypeptide in the above methods can be expressed in cells, or on the cell membrane, or secreted outside the cell.Such as Fruit needs, its physics, chemical and other characteristics can be utilized to be separated by various separation methods and purify the albumen of recombination.This A little methods are well-known to those skilled in the art.The example of these methods includes but is not limited to：The renaturation process of routine is used Protein precipitant handles (salting-out method), centrifugation, the broken bacterium of infiltration, super processing, ultracentrifugation, sieve chromatography (gel filtration), inhales The combination of attached chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) and various other liquid chromatography technologies and these methods.

Inhibitor

It can filter out and interact with SSX2IP albumen by various conventional screening assays using albumen of the present invention Substance, especially inhibitor etc..

The inhibitor (including antibody, antisense nucleic acid and other inhibitor) of SSX2IP albumen of the present invention, when in the treatment When being administered (administration), expression and/or the activity of SSX2IP albumen are can inhibit, and then inhibits transfer or the tumour cell of tumour Migration.In general, these substances can be formulated in nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, Middle pH ordinarily is about 5-8, and preferably pH is about 6-8, although pH value can be with the property and illness to be treated for being formulated substance And it is varied from.Prepared pharmaceutical composition can be administered by conventional route, including (but being not limited to)：Tumor It is interior, intramuscular, peritonaeum is interior, intravenous, subcutaneous, intradermal or local administration.

Inhibitor for use in the present invention includes：The antibody of SSX2IP, the antisense RNA of inhibition mRNA, SSX2IP nucleic acid, The activity inhibitor of siRNA, shRNA and SSX2IP.Wherein, typical SSX2IP inhibitor is inhibition miRNA, siRNA.

Typically, using SSX2IP genes as the target for preparing the drug for preventing or treating metastases or tumor cell migration The technical solution of point includes following scheme：

1. chemical synthesis double stranded ribonucleic acid molecule, sequence-specific is directed to SSX2IP gene orders, utilizes liposome The expression of SSX2IP genes, observation soft-agar cloning Forming ability, cell migration ability are interfered in package delivery to tumour cell The change of equal characteristics of cell biology.The core that specificity is directed to SSX2IP can be designed and synthesize using the method for this field routine Acid sequence (such as siRNA).

2. various carriers, including DNA vector, slow virus carrier is utilized to interfere the expression of SSX2IP genes, reach internal The effect for interfering SSX2IP genes detects their therapeutic effects to the diffusion of nude mice knurl abdominal cavity or hepatic metastases, to realize suppression The purpose of tumor proliferation processed.

3. obtaining specific can inhibit the active polypeptide of SSX2IP gene deliveries, monoclonal antibody, reach inhibition The active purposes of SSX2IP, to which reality inhibits the purpose of Nasopharyngeal neoplasms or migration.

The present invention also provides a kind of pharmaceutical compositions, it contains the SSX2IP inhibitor of the present invention of safe and effective amount (such as Antibody, antisense sequences (such as siRNA) or inhibitor) and pharmaceutically acceptable carrier or excipient.This kind of carrier includes (but being not limited to)：Brine, buffer solution, glucose, water, glycerine, ethyl alcohol, and combinations thereof.Pharmaceutical preparation should be with administering mode phase Matching.The pharmaceutical composition of the present invention can be made into injection form, such as with physiological saline or containing glucose and other are auxiliary The aqueous solution of agent is prepared by conventional method.The pharmaceutical composition of such as tablet and capsule etc, can be by conventional method It is prepared.Pharmaceutical composition such as injection, solution, tablet and capsule preferably aseptically manufacture.The dosage of active constituent It is therapeutically effective amount, such as -10 mg/kg weight of about 1 microgram daily.

Antibody

The invention also includes the polyclonal antibodies and monoclonal antibody to people's SSX2IP albumen with specificity, especially singly Clonal antibody.Here, " specificity ", which refers to antibody, can be incorporated into people SSX2IP gene outcomes or segment.Preferably, referring to those energy It is combined with people SSX2IP gene outcomes or segment but nonrecognition and the antibody for being incorporated into other non related antigen molecules.The present invention's Antibody can be prepared by various technologies known to a person skilled in the art.For example, by people's SSX2IP genes of purification Product or its antigen fragment are injected into animal body to generate polyclonal antibody.Equally, express people SSX2IP albumen or it The cell of antigen can also be used to cause immune to animal and generate antibody.

The present invention includes not only complete monoclonal or polyclonal antibody, but also includes having immunocompetent antibody piece Section, such as Fab' or (Fab) 2 segment；Heavy chain of antibody；Antibody light chain；Genetically engineered Single Chain Fv Molecule A；Or chimeric antibody.

The antibody of the present invention includes the antibody that can inhibit SSX2IP functions, can also be not influence people's SSX2IP functions Antibody.It can be caused to generate by being immunized by the segment or functional domain to people's SSX2IP gene outcomes per one kind antibody, and people SSX2IP gene outcomes and its segment can be generated with recombination method or be synthesized with Peptide synthesizer.With non-modified form The antibody that SSX2IP gene outcomes combine, can be using the gene outcome generated in prokaryotic cell (such as E.coli) come immune dynamic Object and obtain.With the posttranslational modification form antibody that such as glycosylation or phosphorylation SSX2IP albumen or polypeptide are combined, can utilize The gene outcome generated in eukaryocyte such as yeast or insect cell obtains animal is immunized.

SSX2IP antibody for use in the present invention can be anti-human SSX2IP protein antibodies.The anti-human SSX2IP eggs of the present invention Bai Kangti can be used in immunohistochemistry technology, detect people's SSX2IP albumen in biopsy specimen.

Pharmaceutical composition and administering mode

The present invention provides contain active constituent (a) SSX2IP inhibitor；(b) pharmaceutically acceptable carrier；And appoint The pharmaceutical composition of (c) chemotherapeutics of choosing.

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

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

In compound preparation, other than containing SSX2IP inhibitor, it also may include other antitumoral compounds, such as change Treat agent.Representative chemotherapeutics includes (but being not limited to)：It is alkylating agent, antimetabolite, folacin, pyrimidine analogue, fast Purine analog and related inhibitors, epipodopyvllotoxins, antibiotic, mono- asparagus fern phthalein amine enzymes of L, are opened up vinca alkaloids Isomerase inhibitors, interferon, platinum coordination complex, the urea of rheum emodin substitution, methyl hydrazine derivatives, adrenal cortex is flutterred to inhibit Agent, adrenocorticotro, progestational hormone, estrogen, antiestrogenic, androgen, antiandrogen and promoting sexual gland hormone-release Hormone analogs.Preferably chemotherapeutics includes：5 one fluorouracils (5-FU), formyl tetrahydrofolic acid, Irinotecan, oxaliplatin, Capecitabine, taxol and Duo Xi taxols.

The dosage form and preparation method of the pharmaceutical composition of the present invention are not particularly limited, and can use the conventional general system in this field The various dosage forms such as legal system piece agent, capsule, granule, sustained release agent, injection.Preferred dosage form be oral preparation (such as tablet) and Injection.

In the present invention, the pharmaceutical preparation of SSX2IP inhibitor or the inhibitor containing SSX2IP can be used for preventing and treating tumour Transfer or tumor cell migration.

It includes (but being not limited to) to be suitable for the invention tumour：Melanoma, non-small cell lung cancer, Small Cell Lung Cancer, Lung cancer, liver cancer, retinoblastoma, astrocytoma, spongioblastoma, hypercytosis, neuroblastoma, squama Shape cell cancer, head cancer, neck cancer, gingival carcinoma, tongue cancer, breast cancer, prostate cancer, kidney, osteocarcinoma, carcinoma of testis, oophoroma, celiothelioma, Sarcoma, cervical carcinoma, human primary gastrointestinal cancers, lymthoma, brain tumor, colon cancer and carcinoma of urinary bladder.

In another preference, the tumour is liver cancer.

In the present invention, administering mode is not particularly limited, can be by oral, intravenous, intramuscular, intraperitoneal or subcutaneous Etc. approach administration.

Invention formulation can be taken or be administered once daily or twice or repeatedly, or is administered with sustained release fashion.It is preferred that Mode be that daily medication is primary because adhering in this way convenient for patient, to significantly improve the compliance of patient's medication.

When taking, accumulated dose that thumping majority case is generally applied daily is everyone 1mg~200g, preferably 10mg~ 100g。

Agonist

It can filter out and interact with SSX2IP albumen by various conventional screening assays using albumen of the present invention Substance, especially agonist etc..

The agonist of SSX2IP albumen of the present invention, when being administered (administration), can promote SSX2IP albumen expression and/ Or activity, and then promote the transfer or migration of cancer cell (including liver cancer).In general, these agonists can be formulated in it is nontoxic, In inert and pharmaceutically acceptable aqueous carrier medium, wherein pH ordinarily is about 5-8, and preferably pH is about 6-8, although pH Value can be varied from the property for being formulated substance.Prepared pharmaceutical composition can pass through external or non-external routine Approach is administered, including (but being not limited to)：Tumor is interior, intramuscular, peritonaeum is interior, intravenous, subcutaneous, intradermal or part is given Medicine.

Culture and tumour of the SSX2IP genes, albumen or its agonist of the present invention especially suitable for tumor cell in vitro The preparation of animal model, especially liver cancer cells Intrahepatic metastasis, DISTANT METASTASES IN or abdominal metastas animal model modeling.

Screening technique

The present invention also provides the methods for carrying out drug screening based on SSX2IP.A kind of method is that first screening influences (inhibition) SSX2IP is expressed or active compound, then further tests its inhibiting effect to cancer cell to the compound filtered out.

Wherein, representative cancer cell includes that liver cancer cells, stomach cancer cell, colon-cancer cell, lung carcinoma cell, prostate are thin Born of the same parents, mammary glandular cell, melanoma cells.

Screening provided by the invention prevents or treats metastases, tumor cell migration or improves chemotherapy drug susceptibility A kind of method of candidate compound, based on the compound on the expression quantity of SSX2IP and/or active influence, typical screening side Method includes step：

Wherein, if the expression quantity of the SSX2IP of cell and/or activity are less than control group in test group, the test is indicated that Compound is expression to SSX2IP and/or activity have inhibiting effect treatment liver cancer candidate compound.And/or

(b) for the candidate compound obtained in step (a), its suppression shifted or migrated to liver cancer cells is further tested It makes and uses.Such as, in test group, addition test compound in the cultivating system of liver cancer cells, and observe liver cancer cells movement The quantity and/or invasion situation of distance；In control group, test compound is not added in the cultivating system of liver cancer cells, and Observe the quantity and/or invasion situation of the distance of liver cancer cells movement；Wherein, if in test group liver cancer cells migration distance Or invasion quantity indicates that the test compound is that have suppression to hepatoma Metastasis cell or fucosylation significantly less than control group The candidate compound of the treatment liver cancer of making.

Detection method and kit

The present invention relates to quantitative and detection and localization people SSX2IP protein levels or mRNA level in-site diagnostic testing process.These Experiment is known in the art.The people's SSX2IP protein levels detected in experiment, can be used for diagnosing tumour transfer or The migration of tumour cell.

In a kind of detection sample with the presence or absence of the method for SSX2IP albumen be using SSX2IP albumen specific antibody into Row detection, it includes：Sample is contacted with SSX2IP protein specific antibodies；It sees whether to form antibody complex, form Antibody complex means that there are SSX2IP albumen in sample.

SSX2IP albumen or its polynucleotides can be used for the diagnosing and treating of SSX2IP protein related diseases.The present invention's is more Part or all of nucleotide can be used as probe and be fixed in microarray or DNA chip, the difference for analyzing gene in tissue Different expression analysis and gene diagnosis.The antibody of anti-SSX2IP can be fixed on protein-chip, for detecting in sample SSX2IP albumen.

The present invention also provides it is a kind of detection liver cancer kit, it contain specific amplification SSX2IP primer pair and/ Or SSX2IP specific antibodies and label or specification.

Wherein, the label or specification indicate the following contents：When the SSX2IP of detection object it is opposite-actin Mrna expression amount is opposite with the SSX2IP of cancer beside organism-the ratio between the mrna expression amount of actin>2, then prompt the detection object The probability of metastases is higher than general population.

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

The invention also includes a kind of inhibition metastases or the methods of tumor cell migration, including step：To needing to treat Object (mammal) apply safe and effective amount SSX2IP inhibitor.

Beneficial effects of the present invention

1. SSX2IP inhibitor of the present invention can effectively inhibit the transfer of tumour, especially liver cancer or moving for liver cancer cells It moves, can be used as prevention or treats the drug of metastases or tumor cell migration.

2. SSX2IP inhibitor of the present invention can significantly reduce drug resistance of the tumour cell to chemotherapeutics, to raising pair The susceptibility of chemotherapeutics, to improve the effect of chemotherapeutics.

3. the SSX2IP inhibitor of the present invention is also used as the screening of drug, to filter out to can effectively inhibit swollen The drug of tumor metastasis or tumor cell migration, or the drug sensitive to chemotherapeutic.

4. SSX2IP genes of the present invention or albumen and its agonist can be used for promote tumour transfer or migration, can be used for from The culture of body cancer cell or the modeling of animal model for tumour.

5. SSX2IP genes of the present invention or albumen are used as diagnosing or predicting the spy of tumour, especially hepatoma Metastasis Anisotropic marker, i.e. the probability bigger of SSX2IP high expressors metastases.

6. SSX2IP genes of the present invention or albumen can be also used for the life span of prediction tumor patient, i.e. SSX2IP expression Amount is higher, and the life span of tumor patient is shorter.

Embodiment 1

It prepares prediction or whether diagnosing tumour shifts/predict the kit of patient survival

Prepare a kit for whether shifting/predicting patient survival for histology prediction or diagnosing tumour, institute Stating kit includes:

(a) container, and specificity in container are directed to the following antibody of SSX2IP：The antibody of anti-human SSX2IP (being purchased from Abcam and Sigma companies)；With

(b) and label or specification, the label or specification indicate the kit for predicting whether tumour turns Move detection or patient survival.

The correlation of embodiment 2SSX2IP protein expressions and tumour Tumor thrombus and tumor capsule integrality

Material：53 hepatocarcinoma patient samples, are collected in Zhongshan Hospital Attached to Fudan Univ.Obtain the informed same of subject The approval of the Institutional Review Board of meaning and hospital.

Method：The kit prepared using embodiment 1, grouping situation are as shown in table 1；Observation index：Tumor size, cancer embolus Formation and tumor capsule integrality；The expression of SSX2IP in tumour knurl is measured, and observes its expression for cancer Complication for patients has meaningless.

As a result：As shown in table 1：

Table 1

In malignant tumour, the volume size of tumour represents the height of grade malignancy to a certain extent；And cancer embolus It is formed and illustrates that tumour has been provided with the ability for starting to migrate and invade.

By table 1 as it can be seen that patient's group of high expression SSX2IP, tumour bigger, and the probability higher of generation cancer embolus.Therefore, The SSX2IP measured in patient's tumor is horizontal high, it may be said that the grade malignancy of the bright patient tumors may be relatively high, and the tumour Has the ability of migration and invasion, the probability of transfer is more than control crowd.Wherein, P indicates SSX2IP high expression for observation Index has not statistically significant (P<0.05 is statistically significant).

In addition, the high expression of SSX2IP and the integrality of tumor capsule have certain correlation, the sample of the high expression of SSX2IP In this, coating integrality declines.

Therefore, the transfer of SSX2IP expression quantity and tumour has closely related, the specificity that whether can be shifted as tumour Marker.

The correlation of embodiment 3SSX2IP protein expressions and cancer patient's life span

Material：Postoperative tracking follow-up is carried out to 51 hepatocarcinoma patients, obtains the informed consent of subject and the ethics of hospital The approval of examination board,

Method：According to patient information, postoperative phone tracks follow-up, 5 years duration or more

The results are shown in Figure 1：

The life span of the cancer patient of the relatively high expression of SSX2IP is shorter than SSX2IP low expression persons.Wherein Gao Biaodayin This, the expression quantity of SSX2IP can be used for predicting the life span of cancer patient.

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

Material：Liver cancer cell lines are that BEL-7402 (is purchased from Shanghai Inst. of Life Science, CAS cellular resources Center)

It is grouped situation：N=3, group 1：BEL-7402 is liver cancer cells group；Group 2：BEL-7402^VectorFor non-loaded cells group； Group 3：BEL-7402^SSX2IPFor SSX2IP overexpression groups；

Method：

Above-mentioned three groups of cells are pressed into every mouse 1 × 10 respectively⁶The amount of a cell enters to exempt from by abdominal cavity and tail vein injection It in epidemic disease deficient mice body, after six weeks, puts to death and dissects, photograph to record liver cancer cells in intraperitoneal spread condition.Same procedure It applied to another three groups of mouse, puts to death and dissects after ten weeks, take pictures and record Intrahepatic metastasis situation.

The results are shown in Figure 2：

Fig. 2A shows that mouse peritoneal transfer naked eyes are seen, it is seen that is overexpressed the mouse peritoneal transfer of the liver cancer cells of SSX2IP Tubercle number is significantly more than non-loaded cells group and untreated cell group.

Fig. 2 B show that the mouse peritoneal metastatic nodules for the liver cancer cells that SSX2IP is overexpressed through pathological statistics are 6.2 ± 0.84, significantly more than non-loaded cells group (2.4 ± 1.14) and untreated cell group (2.6 ± 0.89)

The case where Fig. 2 C are shown after tail vein injection tumour carrier cell 10 weeks, are put to death and are dissected, observation nude mice liver, can See that macroscopic Nodules occurs in the liver portion of part nude mice.

Fig. 2 D are shown in three groups of mouse, and the mouse of DISTANT METASTASES IN occurs and does not shift the ratio of mouse, wherein SSX2IP There are 7 abdominal metastas has occurred in 10 mouse of overexpression group；There is 1 abdominal cavity has occurred in 10 mouse of non-loaded cells group to turn It moves；There is 1 abdominal metastas has occurred in 10 mouse of liver cancer cells group.

Conclusion：The abdominal metastas and Intrahepatic metastasis incidence of SSX2IP overexpression group mouse are significantly stronger than other groups, it is seen that SSX2IP genes or albumen can promote the transfer of tumour.

The relationship of the locomotivity of embodiment 5SSX2IP and Hepatocellular carcinoma cell line

Material：Liver cancer cell lines are that SMMC-7721 (is purchased from Shanghai Inst. of Life Science, CAS cellular resources Center).

It is grouped situation：With embodiment 1, wherein cell line is changed to SMMC-7721.

Method：The present embodiment using conventional cut Healing Experiments reaction liver cancer cells locomotivity wherein mean gap away from From the locomotivity for indicating cell, mean gap distance is longer, and cell motility is stronger.

The results are shown in Figure 3：

Fig. 3 A show three groups of different mean gap distances after 48 hours, wherein are overexpressed the liver cancer cells of SSX2IP Group：170.83±36.96μm；Non-loaded cells group：516.67±25.57μm；Blank control groups of cells：525.00±31.92μm.

Conclusion：SSX2IP overexpression groups cancer cell migration distance is significantly distal to other groups, it is seen that SSX2IP genes or albumen It can promote the migration of tumour cell.

The relationship of the locomotivity of embodiment 6SSX2IP and liver cancer cells BEL-7402

Material and grouping situation are the same as embodiment 1

Method：With embodiment 2

The results are shown in Figure 3：

Fig. 3 B show three groups of different mean gap distances after 48 hours, wherein are overexpressed the liver cancer cells of SSX2IP Group：183.33±49.07μm；Non-loaded cells group：312.50±25.00μm；Blank control groups of cells：329.17±15.96μm.

The relationship of the migration invasive ability of embodiment 7SSX2IP and Hepatocellular carcinoma cell line

Material is grouped situation：With embodiment 2

Method：The present embodiment reacts the invasion of the transfer ability and penetration cell epimatrix of liver cancer cells using Matrigel Ability：The cells Transwell, it is believed that this is a kind of molecular filter (Membrane filters), and being also believed to one kind has The holder (permeable supports) of permeability.By counting the cell quantity across this layer of filter membrane, can reflect thin The transfer ability of born of the same parents；It is further added to the matrigel for imitating extracellular matrix components on film, can detect that cell is worn Saturating cellular matrix completes the ability of invasion transfer

The results are shown in Figure 4：

Fig. 4 A, it shows in three groups of experimental cell strains, is overexpressed the SMMC-7721 of SSX2IP^SSX2IPCell passes through The amount of Transwell filter membranes is significantly more than other two control group.

In the migration experiment of Fig. 4 B, the quantity statistics for being overexpressed SSX2IP groups comparison control group are：123.33 ± 9.45, 59.67 ± 4.73,51.33 ± 6.03；In Matrigel, the quantity statistics for being overexpressed SSX2IP groups comparison control group are：92.00 ± 7.00,43.67 ± 4.16,38.00 ± 3.61, P < 0.001.

Conclusion：It is overexpressed SSX2IP Hepatocellular carcinoma cell lines^SSX2IP, it invades and the ability of migration significantly increases, therefore, SSX2IP has facilitation to the invasion transfer ability of liver cancer cells.

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

Material is grouped situation with embodiment 1.

Method is the same as embodiment 4

As a result as shown in figures 4 c and 4d：

Fig. 4 C are shown in three groups of experimental cell strains, are overexpressed the SMMC-7721 of SSX2IP^SSX2IPCell passes through The amount of Transwell filter membranes is significantly more than other two control group.

In the migration experiment of Fig. 4 D, the quantity statistics for being overexpressed SSX2IP groups comparison control group are：154.67 ± 14.05, 103.67 ± 10.70,109.00 ± 7.55；* P < 0.01.In Matrigel, it is overexpressed the quantity of SSX2IP groups comparison control group Statistics is：113.00 ± 6.56,63.33 ± 11.50,60.67 ± 11.02, P < 0.01.

Conclusion：It is overexpressed SSX2IP liver cancer cells BEL-7402^SSX2IPIt is invaded and the ability of migration significantly increases, therefore, SSX2IP has facilitation to the invasion transfer ability of liver cancer cells.

Embodiment 9SSX2IP is with Hepatocellular carcinoma cell line to the relationship of the susceptibility of chemotherapeutics 5-Fu

Material, grouping situation：With embodiment 2

Method：The present embodiment is by measuring IC₅₀Value, to reflect susceptibility of the tumour cell for medicine.IC₅₀It is Refer to the concentration of inhibitor when being suppressed half；Certain certain density drug-induced apoptosis of tumor cells 50% is can be understood as, it should Concentration is known as 50% inhibition concentration, i.e. the ratio between apoptotic cell and whole cell numbers is equal to concentration corresponding when 50%, IC₅₀Value can be with For weighing the ability of drug-induced apoptosis, i.e. inducibility is stronger, and the numerical value is lower, naturally it is also possible to which certain is thin for reverse instruction Tolerance degree of the born of the same parents to drug.We are by measuring under various concentration, and liver cancer cells are to the apoptosis rate of 5-FU, matched curve, And calculate IC₅₀Value

As a result as shown in Figure 5A：

Fig. 5 A show dose-effect curve and IC of three groups of cells to chemotherapeutics 5-Fu₅₀Value.As a result SMMC- is shown 7721^SSX2IPTo the IC of 5-FU₅₀Value be significantly higher than control group (24.68 ± 1.17 μM, 14.59 ± 0.77 μM, 13.60 ± 0.88 μ M, P<0.001).Thus, it could be seen that SSX2IP can reduce sensibility of the tumour cell SMMC-7721 for chemotherapeutics 5-FU.

Relationships of the embodiment 10SSX2IP and liver cancer cells BEL-7402 to the susceptibility of chemotherapeutics 5-Fu

Material, grouping situation：With embodiment 1

Method：With embodiment 6

As a result as shown in Figure 5 B：

Fig. 5 B show dose-effect curve and IC of three groups of cells to chemotherapeutics 5-Fu₅₀Value, as a result shows BEL- 7402^SSX2IPTo the IC of 5-FU₅₀Value be significantly higher than control group (28.52 ± 0.65 μM, 12.73 ± 1.81 μM, 14.16 ± 1.20 μ M, * * P<0.001).Thus, it could be seen that SSX2IP can reduce sensibility of the tumour cell BEL-7402 for chemotherapeutics 5-FU.

Embodiment 11SSX2IP is with Hepatocellular carcinoma cell line to the relationship of the susceptibility of chemotherapeutics CDDP

Material, grouping situation：With embodiment 2

Method：With embodiment 6, difference lies in chemotherapeutics to be changed to CDDP (cis-platinum).

As a result：As shown in Figure 5 C：

Fig. 5 C show dose-effect curve and IC of three groups of cells to chemotherapeutics CDDP₅₀Value, as a result shows SMMC- 7721^SSX2IPTo the IC of CDDP₅₀Value be significantly higher than control group (11.38 ± 1.42 μM, 5.72 ± 0.75 μM, 6.18 ± 0.81 μM, P <0.001).Thus, it could be seen that SSX2IP can reduce sensibility of the tumour cell SMMC-7721 for chemotherapeutics CDDP.

Relationships of the embodiment 12SSX2IP and liver cancer cells BEL-7402 to the susceptibility of chemotherapeutics CDDP

Material, grouping situation are the same as embodiment 1

Method：With embodiment 8.

As a result：As shown in Figure 5 D：

Fig. 5 D. show dose-effect curve and IC of three groups of cells to chemotherapeutics CDDP₅₀Value, as a result shows BEL- ICs of the 7402SSX2IP to CDDP₅₀Value is significantly higher than (9.86 ± 1.24 μM, 6.13 ± 0.24 μM and 5.90 ± 0.47 of control group μM, * * P<0.001)

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

Material：With embodiment 1

It is grouped situation：N=3, group 1：BEL-7402 is liver cancer cells group；Group 2：BEL-7402^si-NCIt is interfered for unrelated sequences Control cell group；Group 3：BEL-7402^si-SSX2IPExpression group is lowered for the siRNA of SSX2IP；

Method：

Conclusion：SiRNA interferes the cell of SSX2IP expression groups, notable in the abdominal metastas and Intrahepatic metastasis incidence of mouse Less than other groups, (metastatic tumor quantity is equal<40%), it is seen that by inhibit SSX2IP genes or albumen can inhibit tumour proliferation and Transfer.

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

Material：With embodiment 1

The present embodiment method is CDDP difference lies in chemotherapeutics with embodiment 6.

As a result SSX2IP inhibitor siRNABEL-7402 is shown^si-SSX2IPTo the IC of CDDP₅₀Value is below the 50% of control group More than, therefore, siRNA interferes the expression of SSX2IP, the BEL-7402 liver cancer cells of low expression SSX2IP, to the sensibility of CDDP Enhancing, therapeutic effect improve.

Embodiment 15SSX2IP inhibitor microRNA improves sensitivity of the Hepatocellular carcinoma cell line to chemotherapeutics 5-FU Degree

For the present embodiment method with embodiment 10, difference lies in liver cancer cells groups to use SMMC-7721, chemotherapeutics 5- instead FU。

It is grouped situation：N=3, group 1：SMMC-7721 is liver cancer cells group；Group 2：SMMC-7721^miR-NCIt is dry for unrelated sequences Disturb control cell group；Group 3：SMMC-7721^miR-SSX2IPExpression group is lowered for the microRNA of SSX2IP；

As a result SSX2IP inhibitor siRNASMMC-7721 is shown^si-SSX2IPTo the IC of CDDP₅₀Value is below control group 50% or more, therefore, siRNA disturbs the expression of SSX2IP, the SMMC-7721 liver cancer cells of low expression SSX2IP, to CDDP's Sensibility enhances, and therapeutic effect improves.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Claims

1. a kind of purposes of the inhibitor of 2 interaction proteins of synovial sarcoma X breakpoint gene, that is, SSX2IP, which is characterized in that It is used to prepare the drug for inhibiting hepatoma Metastasis or inhibiting fucosylation.

2. purposes as described in claim 1, which is characterized in that the SSX2IP albumen sources are in mammal.

3. purposes as claimed in claim 2, which is characterized in that the SSX2IP albumen sources are in people, mouse or rat.

4. purposes as described in claim 1, which is characterized in that the inhibitor includes：The antisense RNA of SSX2IP nucleic acid, The activity inhibitor of siRNA, shRNA and SSX2IP.

5. purposes as described in claim 1, which is characterized in that the activity inhibitor of the SSX2IP includes the anti-of SSX2IP Body.

6. purposes as described in claim 1, which is characterized in that the drug contain pharmaceutically acceptable carrier, SSX2IP inhibitor and optional chemotherapeutics.

7. purposes as described in claim 1, which is characterized in that the preparation of the drug is selected from the group：Tablet, capsule, Injection, granule, spray.

8. purposes as described in claim 1, which is characterized in that the SSX2IP inhibitor is with the agent of 0.5-5mg/kg weight Amount is applied to mammal.

9. purposes as claimed in claim 8, which is characterized in that the mammal includes people, mouse and rat.

10. purposes as claimed in claim 6, which is characterized in that the chemotherapeutics include cyclophosphamide, ifosfamide, Mitomycin, adriamycin, vincristine, vinblastine, etoposide, brave and fierce, cis-platinum, fluorouracil, carboplatin and methotrexate (MTX).

11. a kind of purposes of SSX2IP inhibitor, which is characterized in that (a) is used to prepare the liver cancer for promoting chemotherapy or radiotherapy-induced The pharmaceutical composition of Apoptosis, or it (b) is used to prepare the accelerating agent for the hepatoma cell apoptosis for promoting chemotherapy or radiotherapy-induced, or (c) it is used to prepare the sensitizer for so that liver cancer cells are more sensitive to the Apoptosis of chemotherapy or radiotherapy-induced.

12. a kind of method that external non-therapeutic inhibits fucosylation, which is characterized in that including step：Press down in SSX2IP In the presence of preparation, liver cancer cells are cultivated, to inhibit fucosylation.

13. method as claimed in claim 12, which is characterized in that a concentration of 0.5-5mg/ of the SSX2IP inhibitor mL。

14. the purposes of a kind of SSX2IP genes, SSX2IP albumen or its agonist, which is characterized in that be used to prepare promotion liver cancer The composition of transfer or fucosylation.

15. purposes as claimed in claim 14, which is characterized in that the SSX2IP genes or albumen are recombined human SSX2IP Gene or albumen or the people SSX2IP genes in human blood or serum or albumen.

16. purposes as claimed in claim 14, which is characterized in that the group of the promotion hepatoma Metastasis or fucosylation Object is closed for establishing Hepatic neoplasm model.

17. a kind of candidate compound of screening for inhibiting fucosylation or for improving liver cancer cells drug susceptibility Method, which is characterized in that the method includes the steps：

(a) in test group, the addition test compound in the cultivating system of liver cancer cells, and the liver cancer for observing the test group is thin The expression quantity and/or activity of SSX2IP in born of the same parents；In control group, test chemical combination is not added in the cultivating system of same cell Object, and observe the expression quantity and/or activity of SSX2IP in the cell of control group；

Wherein, if the expression quantity of the SSX2IP of liver cancer cells and/or activity are less than control group in test group, the test is indicated that Compound is expression to SSX2IP and/or activity has the treatment hepatoma Metastasis of inhibiting effect or fucosylation or can carry The compound of high chemotherapy drug susceptibility；With

(b) for the candidate compound obtained in step (a), optionally test the candidate compound to liver cancer cells transfer or The influence of the inhibiting effect of migration or the candidate compound to liver cancer cells drug susceptibility.

18. method as claimed in claim 17, which is characterized in that in step (b) include step：In test group, liver cancer is thin Addition test compound in the cultivating system of born of the same parents, and observe the quantity and/or invasion situation of the distance of liver cancer cells movement；Right According in group, test compound is not added in the cultivating system of liver cancer cells, and observe the quantity of the distance of liver cancer cells movement And/or invasion situation；Wherein, if the migration distance of liver cancer cells or invasion quantity are significantly less than control group in test group, just Show that the test compound is that have inhibiting effect to hepatoma Metastasis or fucosylation or can improve chemotherapy medicament sensitive The compound of property.