CN104004822B - Applications of the SSX2IP in prediction or diagnosing tumour transfer - Google Patents
Applications of the SSX2IP in prediction or diagnosing tumour transfer Download PDFInfo
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Abstract
The invention discloses a kind of applications of SSX2IP in prediction or diagnosing tumour transfer.Specifically, the purposes the present invention provides SSX2IP genes or albumen in the reagent or kit for preparing detection metastases.The research of the present invention confirms that SSX2IP genes and its expression product can be used as diagnosis or predict the specificity marker gene that tumour (such as liver cancer) shifts, and contribute to more acurrate, progress diagnosis of metastasis and prediction more early.
Description
Technical field
The present invention relates to oncologies.More particularly it relates to which SSX2IP genes or albumen are examined in metastases
Application in disconnected or prediction.The invention further relates to SSX2IP inhibitor prevention and treatment metastases, tumor cell migration with
And the application in terms of chemotherapy resistance row.
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.
Hepatic blood supply is abundant, and the rate of transform is high, shifts patient's poor prognosis.The existing life that can be used as primary liver cancer markers
Object molecule, it is generally recognized that have following four classes：Cancer embryo and glycoprotein antigen；Enzyme and isodynamic enzyme；Cell factor；Gene.In global model
In enclosing, to the etiologic diagnosis of liver cancer to detect based on Serum AFP (alpha-fetoprotein), but the sensibility (40%~65%) of AFP and spy
Anisotropic (76%~96%) is unsatisfactory, and such testing result has no directive significance to the prognosis of patient.
In the liver cancer patient of different phase, personalized therapeutic scheme is even more important.But due to lacking specific liver cancer
The time window of Metastatic Marker, treatment is often just opened after hepatoma Metastasis, and the good chance for preventing metastases is delayed.
Therefore, there is an urgent need in the art to develop can predict or diagnosing tumour transfer especially hepatoma Metastasis specificity mark
Will object, it is basic as preferably preventing or treating metastases offer so as to predict hepatoma Metastasis.
Invention content
The present invention provides the kits whether a kind of prediction or diagnosing tumour are shifted, in addition, the present invention also provides one
Kind can prevent and treat metastases, tumor cell migration and the SSX2IP inhibitor that can improve chemosensitivity；This hair
It is bright to additionally provide a kind of screening technique having prevention or therapeutic effect drug to metastases or tumor drug resistance.
First aspect present invention, provide a kind of SSX2IP genes (2 interaction protein of synovial sarcoma X breakpoint gene,
Synovial Sarcoma X brearkpoint2interacting Protein) or SSX2IP albumen purposes, for making
The reagent or kit of standby detection metastases.
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 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 second 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 third 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 fourth aspect of the present invention provides a kind of purposes of SSX2IP inhibitor, be used to prepare inhibit metastases or
Inhibit the drug of 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 fifth 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 sixth 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 adenocarcinoma cell, breast cancer cell, melanoma cells..
In another preferred example, in the method, a concentration of 0.5-5mg/mL of the SSX2IP inhibitor.
The seventh aspect of the present invention provides the purposes of a kind of SSX2IP genes, SSX2IP albumen or its agonist, is used for
Prepare the drug for promoting metastases or tumor cell 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 eighth 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, the tumour includes liver cancer, gastric cancer, intestinal cancer, lung cancer, prostate cancer, breast cancer, black
Plain tumor.
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 chemotherapy drug susceptibility compound.
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 or SSX2IP cDNA for detecting SSX2IP mRNA
Amount；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-7402Vector, BEL-7402SSX2IP
Three 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. statistics is found, be overexpressed the liver cancer cells of the SSX2IP ratio of liver metastasis in nude mouse be 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-7721Vector, SMMC- by cut Healing Experiments
The locomotivity of 7721SSX2IP three classes cells finds the liver cancer cells cell SMMC-7721SSX2IP for being overexpressed SSX2IP,
Locomotivity is significantly higher than non-loaded cells combination blank control groups of cells, and the mean gap distance after 48 hours is：170.83±
36.96 μm, 516.67 ± 25.57 μm, 525.00 ± 31.92 μm, * * P<0.001.
Fig. 3 B are shown detects BEL-7402, BEL-7402Vector, BEL-7402SSX2IP by cut Healing Experiments
The locomotivity of three classes cell finds that the liver cancer cells cell BEL-7402SSX2IP for being overexpressed SSX2IP, locomotivity are aobvious
It writes and combines blank control groups of cells higher than non-loaded cells, the mean gap distance after 48 hours is：183.33 ± 49.07 μm,
312.50 ± 25.00 μm, 329.17 ± 15.96 μm, * * P<0.001.
Fig. 4 shows that 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, which are shown, is overexpressed SSX2IP Hepatocellular carcinoma cell line SSX2IP, and the ability of invasion and migration is notable
Enhancing.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, which are shown, is overexpressed SSX2IP liver cancer cells BEL-7402SSX2IP, and the ability of invasion and migration significantly increases
By force
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, the quantity statistics for being overexpressed SSX2IP groups comparison control group are：
113.00 ± 6.56,63.33 ± 11.50,60.67 ± 11.02, * P<0.01.
Fig. 5 shows the susceptibility that SSX2IP can reduce liver cancer cells to chemotherapeutics, increases drug resistance
Fig. 5 A show SMMC-7721, SMMC-7721Vector and SMMC-7721SSX2IP to chemotherapeutics 5-Fu's
As a result dose-effect curve and IC50 values show that SMMC-7721SSX2IP is significantly higher than control group to the IC50 values of 5-FU
(24.68 ± 1.17 μM, 14.59 ± 0.77 μM, 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 IC50 values, as a result show BEL-7402SSX2IP to the IC50 values of 5-FU 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
As a result quantitative response curve and IC50 values show that SMMC-7721SSX2IP is significantly higher than control group (11.38 to the IC50 values of CDDP
± 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 IC50 values, as a result show BEL-7402SSX2IP to the IC50 values of CDDP be 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, be handled with CaCl2 methods, 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 cancer are thin
Born of the same parents, breast cancer 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：
(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 have inhibiting effect treatment liver cancer candidate compound.And/or
(b) for the candidate compound obtained in step (a), its inhibition to cancer metastasis or migration is further tested
Effect.Such as, in test group, addition test compound in the cultivating system of cancer cell, and observe the distance of cancer cell movement
Quantity and/or invasion situation；In control group, test compound is not added in the cultivating system of cancer cell, and it is thin to observe cancer
The quantity and/or invasion situation of the distance of born of the same parents' movement；Wherein, if the migration distance of cancer cell or invasion quantity are aobvious in test group
It writes and is less than control group, indicate that the test compound is the treatment liver for having inhibiting effect to hepatoma Metastasis cell or cancer cell migration
The candidate compound of cancer.
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 the anti-SSX2IP of people
(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-7402Vector is non-loaded cells
Group；Group 3：BEL-7402SSX2IP is SSX2IP overexpression groups；
Method：
Respectively above-mentioned three groups of cells are entered to exempt from by the amount of 1 × 106 cell of every mouse 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.
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 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 show in three groups of experimental cell strains that the SMMC-7721SSX2IP cells for being overexpressed SSX2IP pass 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：SSX2IP Hepatocellular carcinoma cell line SSX2IP are overexpressed, invasion and the ability migrated significantly increase, because
This, 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 show in three groups of experimental cell strains that the SMMC-7721SSX2IP cells for being overexpressed SSX2IP pass 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：The ability for being overexpressed its invasion of SSX2IP liver cancer cells BEL-7402SSX2IP and 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 IC50 values, to reflect susceptibility of the tumour cell for medicine.IC50 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%, and IC50 values can
With for weighing the ability of drug-induced apoptosis, i.e. inducibility is stronger, the numerical value is lower, naturally it is also possible to reverse instruction certain
Tolerance degree of the cell to drug.We are by measuring under various concentration, and liver cancer cells are to the apoptosis rate of 5-FU, fitting song
Line, and calculate IC50 values
As a result as shown in Figure 5A：
Fig. 5 A show dose-effect curve and IC50 value of three groups of cells to chemotherapeutics 5-Fu.As a result SMMC- is shown
7721SSX2IP to the IC50 values of 5-FU be significantly higher than control group (24.68 ± 1.17 μM, 14.59 ± 0.77 μM, 13.60 ±
0.88 μM, P<0.001).Thus, it could be seen that SSX2IP can reduce tumour cell SMMC-7721 for the quick of chemotherapeutics 5-FU
Perception.
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 IC50 value of three groups of cells to chemotherapeutics 5-Fu, as a result show BEL-
7402SSX2IP to the IC50 values of 5-FU be significantly higher than control group (28.52 ± 0.65 μM, 12.73 ± 1.81 μM, 14.16 ±
1.20 μM, * * P<0.001).Thus, it could be seen that SSX2IP can reduce tumour cell BEL-7402 for the quick of chemotherapeutics 5-FU
Perception.
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 IC50 value of three groups of cells to chemotherapeutics CDDP, as a result show SMMC-
7721SSX2IP to the IC50 values of CDDP be significantly higher than control group (11.38 ± 1.42 μM, 5.72 ± 0.75 μM, 6.18 ± 0.81 μ
M, 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 IC50 value of three groups of cells to chemotherapeutics CDDP, as a result show BEL-
7402SSX2IP to the IC50 values of CDDP be significantly higher than control group (9.86 ± 1.24 μM, 6.13 ± 0.24 μM and 5.90 ±
0.47 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-7402si-NC is dry for unrelated sequences
Disturb control cell group；Group 3：The siRNA that BEL-7402si-SSX2IP is SSX2IP lowers expression group；
Method：
Respectively above-mentioned three groups of cells are entered to exempt from by the amount of 1 × 106 cell of every mouse 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.
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
It is grouped situation：N=3, group 1：BEL-7402 is liver cancer cells group；Group 2：BEL-7402si-NC is dry for unrelated sequences
Disturb control cell group；Group 3：The siRNA that BEL-7402si-SSX2IP is SSX2IP lowers expression group；
The present embodiment method is CDDP difference lies in chemotherapeutics with embodiment 6.
As a result show that SSX2IP inhibitor siRNABEL-7402si-SSX2IP is below control group to the IC50 values of CDDP
50% or more, therefore, siRNA interfere SSX2IP expression, the BEL-7402 liver cancer cells of low expression SSX2IP, to CDDP's
Sensibility enhances, and therapeutic effect improves.
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-7721miR-NC is unrelated sequences
Interfere control cell group；Group 3：The microRNA that SMMC-7721miR-SSX2IP is SSX2IP lowers expression group；
As a result show that SSX2IP inhibitor siRNASMMC-7721si-SSX2IP is below control group to the IC50 values of CDDP
50% or more, therefore, siRNA disturbs the expression of SSX2IP, the SMMC-7721 liver cancer cells of low expression SSX2IP, to CDDP
Sensibility enhancing, therapeutic effect improve.
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 (13)
1. a kind of reagent of the expression of detection SSX2IP genes or albumen is in the kit for preparing detection hepatoma Metastasis
Purposes.
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 SSX2IP albumen sources are in people, full length amino acid
Sequence such as SEQ ID NO.:Shown in 2.
5. purposes as described in claim 1, which is characterized in that the SSX2IP gene nucleotide series such as SEQ ID
NO.:Shown in 1.
6. purposes as described in claim 1, which is characterized in that the kit is described also comprising a label or specification
Label or specification indicate the kit for detecting hepatoma Metastasis.
7. purposes as described in claim 1, which is characterized in that the reagent includes SSX2IP specific primers, specificity
Antibody, probe and/or chip.
8. purposes as described in claim 1, which is characterized in that the detection hepatoma Metastasis includes the inspection of enzyme linked immunoassay method
It surveys or Time-resolved Fluoimmunoassay detects.
9. purposes as claimed in claim 7, which is characterized in that the specific antibody coupling has or with detectable mark
Note.
10. purposes as claimed in claim 9, which is characterized in that the detectable label is selected from the group：Chromophore, chemistry hair
Light group, fluorogen, isotope or enzyme.
11. purposes as claimed in claim 7, which is characterized in that the specific antibody is monoclonal antibody or polyclonal
Antibody.
12. purposes as described in claim 1, which is characterized in that the kit is for measuring tissue sample or blood sample.
13. purposes as claimed in claim 12, which is characterized in that the tissue sample includes cancerous tissue and cancer beside organism.
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| CN201310062583.6A CN104004822B (en) | 2013-02-27 | 2013-02-27 | Applications of the SSX2IP in prediction or diagnosing tumour transfer |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310062583.6A CN104004822B (en) | 2013-02-27 | 2013-02-27 | Applications of the SSX2IP in prediction or diagnosing tumour transfer |
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| Publication Number | Publication Date |
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| CN104004822B true CN104004822B (en) | 2018-08-07 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1315000A (en) * | 1998-06-26 | 2001-09-26 | 路德维格癌症研究院 | Methods for determining presence of cancer in a sample by determining expression of an ssx gene, peptide derived from said ssx gene and NY-ESO-1 gene, and uses thereof |
-
2013
- 2013-02-27 CN CN201310062583.6A patent/CN104004822B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1315000A (en) * | 1998-06-26 | 2001-09-26 | 路德维格癌症研究院 | Methods for determining presence of cancer in a sample by determining expression of an ssx gene, peptide derived from said ssx gene and NY-ESO-1 gene, and uses thereof |
Non-Patent Citations (3)
| Title |
|---|
| SSX2IP: and emerging role in cancer;Angela Breslin等;《biochemical and biophysical research communications》;20070924;全文 * |
| 人SSX2IP与14-3-3η蛋白相互作用研究;王佳琦等;《中国生物工程杂志》;20101231;摘要、第1页右栏第3段、第4页右栏倒数第2段，第5页右栏倒数第1段 * |
| 大肠癌转移相关分子标签的筛选;齐鲁等;《现代生物医学进展》;20120930;摘要、第5227页右栏最后一段-5228页右栏第1段，具体参见第5228页右栏第6行 * |
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