CN106692968A - Application of SRPK1 (Serine/Arginine Protein Kinase 1) in diagnosis and treatment of gastric cancer - Google Patents

Abstract

The invention discloses application of an SRPK1 (Serine/Arginine Protein Kinase 1) gene and an expression product thereof in preparation of products for the diagnosis and treatment of gastric cancer. The SRPK1 gene and the expression product thereof, disclosed by the invention, can serve as a specific marker gene for diagnosing the gastric cancer, so that the diagnosis of the gastric cancer is more accurate and rapider; the SRPK1 gene and the expression product thereof, disclosed by the invention, can also serve as a target gene for preparing drugs for treating the gastric cancer, and thus, new ways for treating the gastric cancer are provided.

Description

Applications of the SRPK1 in diagnosing gastric cancer with treatment

Technical field

The present invention relates to oncology.More particularly it relates to SRPK1 genes and its albumen are in stomach The application of cancer context of detection.The invention further relates to application of the SRPK1 inhibitor in curing gastric cancer.

Background technology

China is the High Risk For Gastric Cancer country, and its morbidity and mortality is in the 3rd of whole malignant tumours respectively With the 2nd.Due to the concealment of stomach cancer onset, early stage many non-evident symptons are subtle so that the morning of stomach cancer Phase finds extremely difficult.Particularly in China, the stomach cancer more than 80% has just been in progressive stage when making a definite diagnosis, in advance It is very poor afterwards, seriously threaten people life and health (Leung WK, et al.Lancet Oncol.2008, Yang L.et al.World J Gastroenterol 2006)。

Thus the diagnosis of stomach cancer, especially early diagnoses, and is the key of clinic diagnosis and prognosis.Except image Learn and check that the detection of large biological molecule tumor markers is also a kind of early gastric caacer diagnosis with outside endoscopy Important means.Clinically conventional large biological molecule tumor markers has at present：A. carcinomebryonic antigen (CEA), Glycoprotein antigens (CA19-9, CA125)；B. propepsin (PG)；C. Telomerase；D. osteopontin (OPN)；E. gastric cancer antigen (MGAgs).The application of these macromolecular stomach cancer markers improves patients with gastric cancer Recall rate, but early diagnosis and instruct personalized treatment molecule parting diagnose be still we face greatly choose War, and sensitivity and specificity new tumor markers higher is found, it is to improve early gastric caacer diagnostic level Key.

The treatment of stomach cancer made great progress in past 20 years, at present with chemotherapy aspect progress most Hurry up.Chemotherapeutics includes fluorouracil, mitomycin, anthracycline, platinum class, cytarabine and nitrous Ureas etc..Japanese yew class (Docetaxel and taxol) is used for cancer of the esophagus and stomach cancer determined curative effect, has made difficulty The Survival of the property controlled stomach cancer (including poorly differentiated adenocarcinoma cancer, myxoadenocarcinoma and signet ring cell cancer) patient is notable Improve.Targeted therapy has turned into the popular means for the treatment of cancer in recent years.Targeted therapy refers to specific mesh Target high selectivity is treated, including organ targeting, cell-targeting and molecular targeted.Targeting to stomach cancer at present Therapy study is less, therefore in the urgent need to finding new action target spot, the special new drug of research and development stomach cancer Thing, to improve the specificity and validity of curing gastric cancer.

As can be seen here, at present in the urgent need to finding the early gastric caacer diagnostic markers with high specific and validity Thing, and develop can targeting in stomach cancer, reduce the novel drugs of normal tissue and the injury of human body.

The content of the invention

The invention provides the reagent and the medicine and method for the treatment of stomach cancer of specific diagnosis stomach cancer.

A kind of the first aspect of the present invention, there is provided the purposes of SRPK1 genes or its albumen or its detection reagent, For preparing detection stomach cancer and/or judging the reagent or kit of gastric cancer susceptibility.

In another preference, described kit includes：SRPK1 is carried out quantitative determination reagent and Corresponding label or specification.

In another preference, explanation used below is recorded in described label or specification：To SRPK1 Quantitative determination is carried out, and testing result is used to characterize stomach cancer probability just.

In another preference, described reagent include the specific primer of SRPK1, probe, antibody and Chip.

In another preference, described SRPK1 gene sources in mammal, be preferably derived from people, Mouse, rat.More preferably, from people.

In another preference, described specific primer includes sense primer and anti-sense primer：The upstream Primer sequence has such as SEQ ID NO:Sequence or its complementary series shown in 1, described downstream primer sequence With such as SEQ ID NO:Sequence or its complementary series shown in 2.

In another preference, described chip includes nucleic acid chip and protein-chip.

In another preference, described nucleic acid chip includes that substrate and point sample are cancer-related on substrate The specific oligonucleotide probe of cause, the specific oligonucleotide probe of described cancer related gene include with The probe of the specific binding of SRPK1.

In another preference, described protein-chip includes that stomach cancer of the substrate to point sample on substrate is related The specific antibody of albumen, the specific antibody of described stomach cancer-associated protein includes the specificity of anti-SRPK1 Antibody.

In another preference, when the expression of SRPK1 genes or albumen and/or activity are higher than in the sample of detection Normal sample, then neurological susceptibility of the object with stomach cancer for illustrating the sample is higher than normal subjects.

A kind of the second aspect of the present invention, there is provided diagnostic kit for detecting stomach cancer, described reagent Box contains a container, and the detection reagent of detection SRPK1 is contained in the container；And label or specification, The label or specification indicate the kit for detecting stomach cancer.

In another preference, described detection reagent includes：Specific antibody and/or specific primer.

In another preference, herein below is indicated in described label or specification：

When the SRPK1 of detection object is with respect to the mrna expression amount of beta-actin and the SRPK1 phases of cancer beside organism The ratio between mrna expression amount to beta-actin>1, then the cancered probability of the detection object is pointed out higher than common Crowd.

In another preference, described kit is used to detect people's gastric tissue sample or blood sample.

A kind of the third aspect of the present invention, there is provided purposes of SRPK1 inhibitor, described inhibitor by with In the medicine for preparing suppression Growth of Gastric or propagation, or for preparing the medicine for the treatment of stomach cancer.

In another preference, described inhibitor includes：The antibody of SRPK1, the antisense RNA of SRPK1 nucleic acid, The activity inhibitor of siRNA, shRNA and SRPK1.

In another preference, described inhibitor is siRNA or shRNA.

In another preference, described siRNA positive-sense strands have such as SEQ ID NO:Sequence shown in 5, The antisense strand of the siRNA has such as SEQ ID NO:Sequence shown in 6.

A kind of the fourth aspect of the present invention, there is provided pharmaceutical composition, including SRPK1 inhibitor and pharmacy Upper acceptable carrier.

In another preference, described SRPK1 inhibitor include the antibody of SRPK1, SRPK1 nucleic acid it is anti- The activity inhibitor of adopted RNA, siRNA, shRNA and SRPK1.

The fifth aspect of the present invention, there is provided the suppression Growth of Gastric or propagation of a kind of external non-therapeutic Method, including step：In the presence of SRPK1 inhibitor, cancer cell is cultivated, so as to suppress cancer cell life Long or propagation.

In another preference, described method includes suppressing to addition SRPK1 in the cultivating system of cancer cell Agent, so as to suppress growth of cancer cells or propagation.

A kind of the sixth aspect of the present invention, there is provided method of the candidate compound of screening treatment stomach cancer, it is described Method includes step：

In (a) test group, test compound is added in the cultivating system of cell, and observe the test group Cell in SRPK1 expression quantity and/or activity；In control group, in the cultivating system of same cell not Addition test compound, and observe the expression quantity and/or activity of SRPK1 in the cell of control group；

Wherein, if the expression quantity of the SRPK1 of cell and/or activity are less than control group in test group, indicate that The test compound is the candidate compound of the treatment stomach cancer for having inhibitory action to the expression of SRPK1 and/or activity Thing.

In another preference, the expression quantity of described SRPK1 is detected and drawn by RT-PCR.

In another preference, methods described also includes step：

B () further tests it to Growth of Gastric or increasing for the candidate compound obtained in step (a) The inhibitory action grown；And/or further test whether it plays the role of downward to SRPK1 genes.

In another preference, step is included in step (b)：In test group, the cultivating system of stomach cancer cell Middle addition test compound, and observe the quantity and/or growing state of stomach cancer cell；In control group, in stomach Without test compound in the cultivating system of cancer cell, and observe the quantity and/or growth feelings of stomach cancer cell Condition；Wherein, if the quantity or the speed of growth of stomach cancer cell are less than control group in test group, the survey is indicated that Examination compound is the candidate compound of the treatment stomach cancer for having inhibitory action to the growth of stomach cancer cell or propagation.

The seventh aspect of the present invention, there is provided a kind of method of suppression or treatment stomach cancer, including step：To need The object to be treated (mammal) applies the SRPK1 inhibitor of safe and effective amount.

In another preference, described inhibitor includes：The antibody of SRPK1 albumen, the antisense RNA of SRPK1, The activity inhibitor of siRNA, shRNA and SRPK1.

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and below (such as embodiment) Can be combined with each other between each technical characteristic of middle specific descriptions, so as to constitute new or preferred technical side Case.As space is limited, no longer tire out one by one herein and state.

Brief description of the drawings

Figure 1A be in embodiment 1 western blot detection SRPK1 albumen in Patients with Gastric Cancer cancerous tissue and Expression schematic diagram in cancer beside organism, wherein, " N " refers to cancer beside organism, and " C " refers to stomach organization. Picture shows 12 representative results.SRPK1 gene expression amounts are higher than corresponding cancer in the cancerous tissue of patient Side tissue.The immunohistochemistry means that Figure 1B show detect SRPK1 albumen in human gastric cancer clinical sample Expression.

Fig. 2A shows cDNA sequence (the SEQ ID NO. of SRPK1 genes:1)；Fig. 2 B show SRPK1 Amino acid sequence (the SEQ ID NO. of coded by said gene albumen:2).

Fig. 3 A show that overexpression SRPK1 promotes the cell growth of stomach cancer cell AGS and SGC7901. Western diagram display SRPK1 overexpression situations.Fig. 3 B show that overexpression SRPK1 improves stomach cancer Cell AGS forms the ability of clone in soft agar.

Fig. 4 A show strike low SRPK1 in stomach cancer cell line BGC-803, AGS with RNA perturbation techniques and After expression in SGC7901, vitro growth rates substantially slow down.Under Western diagram display SRPK1 expression Flirt condition.Fig. 4 B show that SRPK1 protein expressions are lowered in stomach cancer cell NCI-N87 reduces the stomach The plate clone Forming ability of JEG-3.Fig. 4 C show and lower SRPK1 in stomach cancer cell BGC-803 Protein expression reduces the ability that the stomach cancer cell line forms clone in soft agar.

Fig. 5 A show the gastric carcinoma cell line SGC-7901 of overexpression SRPK1 nude mice by subcutaneous have it is stronger into Knurl ability.SRPK1 overexpression group is all bigger than control group and heavy in volume and tumor weight, while two Group has statistical significance.Fig. 5 B show that striking the serious stomach cancer cell MGC803 that weakens of low SRPK1 expression exists Nude mice by subcutaneous forms the ability of tumour.SRPK1 expression reduction group group all compares in volume and tumor weight Control group is small and light, while two groups have statistical significance.

Specific embodiment

The present inventor's in-depth study by extensive, first it was unexpectedly observed that SRPK1 is in gastric carcinoma cell lines Middle expression is higher than cancer beside organism or normal structure, and the mark that can be detected as stomach cancer is used to detect or complementary Detection stomach cancer.Additionally, the inhibitor of SRPK1 can suppress the growth of cancer cell.Complete this on this basis Invention.

Experiment also confirms, using the specific inhibitor such as RNA interfering (siRNA) of SRPK1, shRNA can be The external growth for substantially suppressing stomach cancer cell and clonality, and can influence in-vivo tumour into knurl Property.Therefore, the function of SRPK1 is suppressed using SRPK1 inhibitor, the stomach of in vitro and in vivo can be suppressed The growth of cancer cell and one-tenth knurl ability, so that for the treatment of stomach cancer provides new approach.

SRPK1 albumen and polynucleotides

SRPK1 (Serine/Arginine Prote in Kinase 1) is a kind of rich in serine/essence ammonia The splicing factor protein kinase of sour repetitive sequence (Ser/Arg Repeats), it is short positioned at No. 6 chromosomes of people Arm, 655 amino acid of total length, the main alternative splicing by phosphorylation RS protein regulations mRNA.Closely Nian Lai, with deepening continuously for studying SRPK1, it has been found that SRPK1 tables high in kinds of tumors tissue Up to and with promote tumor development effect, including breast cancer, colon cancer, cancer of pancreas, oophoroma, Liver cancer and non-small cell lung cancer etc., but so far, expression and function of the SRPK1 in stomach cancer are still unclear. The present invention elaborates expressions of the SRPK1 in stomach cancer first and the research meanses by cell biology are demonstrate,proved The expression of bright SRPK1 is related with patients clinical pathological parameter, and the increment of stomach cancer cell is promoted in vivo and in vitro, is One potential carcinogen.

In the present invention, " albumen of the present invention ", " polypeptide of the present invention ", " SRPK1 albumen " is interchangeable makes With referring to SRPK1 albumen.It should be understood that the term also active fragment and derivative including SRPK1.

In the present invention, " gene of the present invention ", " polynucleotides of the present invention ", " SRPK1 genes " refer to volume The nucleotide sequence of code SRPK1 albumen or its active fragment and derivative, including justice and antisensenucleic acids.

In the present invention, term " SRPK1 albumen ", " SRPK1 polypeptides " or " stomach cancer marker SRPK1 " It is used interchangeably, all refers to albumen or polypeptide with SRPK1 amino acid sequences.

The nucleotide sequence of SRPK1 such as SEQ ID NO.:Shown in 1, the nucleotide sequence coded amino acid sequence Row such as SEQ ID NO.:Shown in 2.

As used herein, " separation " refers to that material is separated (if natural from its primal environment Material, primal environment is natural surroundings).Such as polynucleotides and polypeptide under the native state in active somatic cell Do not isolate and purify, but same polynucleotides or polypeptide same other things for existing such as from native state Separated in matter, then isolated and purified.

As used herein, " the SRPK1 albumen or polypeptide of separation " refers to that SRPK1 albumen is substantially free of day Right relative other albumen, lipid, carbohydrate or other materials.Those skilled in the art can use standard Purified technology of protein purifying SRPK1 albumen.Substantially pure polypeptide is in non-reducing polyacrylamide gel It is upper to produce single master tape.

Polypeptide of the invention can be recombinant polypeptide, natural polypeptides, synthesis polypeptide, preferably recombinant polypeptide.

Polynucleotides of the invention can be DNA form or rna form.DNA form includes cDNA, gene Group DNA or artificial synthesized DNA.DNA can be single-stranded or double-strand.DNA can be coding strand or Noncoding strand.

The polynucleotides for encoding the mature polypeptide of SRPK1 include：The coded sequence of encoding mature polypeptide；Into The coded sequence of ripe polypeptide and various additional coding sequences；Coded sequence (and the optional additional volume of mature polypeptide Code sequence) and non-coding sequence.Term " polynucleotides of coded polypeptide " can include encoding this polypeptide Polynucleotides, or also include the polynucleotides of additional code and/or non-coding sequence.

The invention further relates to the variant of above-mentioned polynucleotides, its coding has identical amino acid sequence with the present invention The polypeptide of row or the fragment of polypeptide, analogs and derivatives.The variant of this polynucleotides can be natural hair The variant that raw allelic variant or non-natural occur.These nucleotide variants include substitution variants, Deletion variants and insert variation.As known in the art, allelic variant is replacing for polynucleotides Form is changed, it is probably the substitution of one or more nucleotides, missing or inserts, but will not be from substantially changing Become the function of its coded polypeptide.

The invention further relates to the nucleic acid fragment with above-mentioned sequence hybridization, including the just nucleic acid fragment with antisense. As used herein, the length of " nucleic acid fragment " at least contains 15 nucleotides, preferably at least 30 nucleosides Acid, more preferably at least 50 nucleotides, more than preferably at least 100 nucleotides.Nucleic acid fragment can be used for The amplification technique (such as PCR) of nucleic acid encodes the polynucleotides of SRPK1 albumen to determine and/or separate.

People SRPK1 nucleotides full length sequence of the invention or its fragment can generally use PCR TRAPs, restructuring Method or artificial synthesized method are obtained.For PCR TRAPs, can according to published relevant nucleotide sequence, Especially open reading frame sequence designs primer, and with commercially available cDNA storehouses or by those skilled in the art CDNA storehouses prepared by the conventional method known obtain relevant sequence as template, amplification.When sequence is more long, Usually need to carry out twice or multiple PCR is expanded, the fragment for then again amplifying each time is spelled by proper order It is connected together.

Once obtain relevant sequence, it is possible to obtain relevant sequence in large quantity with recombination method.This leads to It is often to be cloned into carrier, then is transferred to cell, then by conventional method from the host cell after propagation Isolated relevant sequence.

Additionally, can also synthesize relevant sequence with artificial synthesized method, when especially fragment length is shorter. Generally, by first synthesizing multiple small fragments, being then attached again can obtain sequence fragment very long.

It is optimized for obtaining gene of the invention using the method for round pcr DNA amplification/RNA.For PCR Primer can be properly selected according to the sequence information of invention disclosed herein, and available conventional method is closed Into.Can be with conventional method as separated by gel electrophoresis and purifying the DNA/RNA fragments for expanding.

The present invention also relates to the carrier comprising polynucleotides of the invention, and with carrier of the invention or SRPK1 The host cell that albumen coded sequence is produced through genetic engineering, and produced through recombinant technique of the present invention many The method of peptide.

By conventional recombinant DNA technology, can be used to express or raw using polynucleotide sequence of the invention Produce the SRPK1 albumen of restructuring.In general there are following steps：

(1) it is with the polynucleotides (or variant) of encoding human SRPK1 albumen of the invention or many with this is contained The recombinant expression carrier conversion of nucleotides or suitable host cell of transduceing；

(2) host cell cultivated in suitable culture medium；

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

Method well-known to those having ordinary skill in the art can be used for build the DNA sequences encodings of SRPK1 containing people and properly Transcription/translation control signal expression vector.These methods include that recombinant DNA technology in vi, DNA synthesize Technology, In vivo recombination technology etc..Described DNA sequence dna can be effectively connected to the appropriate startup in expression vector On son, to instruct mRNA to synthesize.Ribosome bind site and transcription of the expression vector also including translation initiation Terminator.

Additionally, expression vector preferably includes one or more selected markers, to provide for selecting The dihyrofolate reductase of the phenotypic character of the host cell of conversion, such as eukaryotic culture, neomycin resist Property and green fluorescent protein (GFP), or for the tetracycline or amicillin resistance of Escherichia coli.

Carrier comprising above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence, can be used for The appropriate host cell of conversion, allows it to marking protein.

Host cell can be prokaryotic, such as bacterial cell；Or low eukaryotic, such as yeast cells； Or higher eucaryotic cells, such as mammalian cell.Representative example has：Escherichia coli, streptomyces Bacterial cell；Fungal cell's such as yeast；Plant cell；The insect cell of fruit bat S2 or Sf9；CHO、COS、 Or 293 cell zooblast etc..

Converting host cell with recombinant DNA can be carried out with routine techniques well known to those skilled in the art.Work as place When master is for prokaryotes such as Escherichia coli, the competent cell that can absorb DNA can be harvested after exponential phase of growth, Use CaCl₂Method treatment, step used is generally well-known in the art.Another method is to use MgCl₂.Such as Fruit is needed, and conversion can also be carried out with the method for electroporation.When host is eucaryote, following DNA is can select Transfection method：Calcium phosphate precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging Deng.

The transformant of acquisition can use conventional method culture, express the polypeptide of coded by said gene of the invention.Root According to host cell used, culture medium used may be selected from various conventional mediums in culture.It is being suitable to host Cultivated under conditions of cell growth.After host cell growth is to appropriate cell density, with suitable Method (such as temperature transition or chemical induction) induces the promoter of selection, and cell is further cultured for into a period of time.

Recombinant polypeptide in the above methods can be expressed or be secreted into thin in the cell or on cell membrane It is extracellular.If desired, its physics, chemistry and other characteristics can be utilized to be separated by various separation methods With the albumen of purification of Recombinant.These methods are well-known to those skilled in the art.The example bag of these methods Include but be not limited to：Conventional renaturation process, (salting-out method), centrifugation, infiltration are processed with protein precipitant Broken bacterium, super treatment, ultracentrifugation, sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, The combination of high performance liquid chroma- tography (HPLC) and other various LC technologies and these methods.

Antibody

There is specific polyclonal antibody and monoclonal antibody present invention additionally comprises to people's SRPK1 albumen, especially It is monoclonal antibody.Here, " specificity " refers to that antibody can be incorporated into people SRPK1 gene outcomes or piece Section.It is preferred that referring to that those can be combined with people SRPK1 gene outcomes or fragment but nonrecognition and be incorporated into other The antibody of non related antigen molecule.Antibody of the invention can be by known to a person skilled in the art various Technology is prepared.For example, the people SRPK1 gene outcomes or its antigen fragment of purification are injected into animal In vivo producing polyclonal antibody.Equally, the cell of expression people SRPK1 albumen or its antigen can also be used Antibody is produced come immune to animal cause.

The present invention not only includes complete monoclonal or polyclonal antibody, but also including with immunocompetent Antibody fragment, such as Fab ' or (Fab)₂Fragment；Heavy chain of antibody；Antibody light chain；Genetically engineered is single-stranded Fv molecules；Or chimeric antibody.

Antibody of the invention includes that the antibody of SRPK1 functions can be suppressed, or does not influence people SRPK1 The antibody of function.Each antibody-like can cause to exempt from by the fragment to people's SRPK1 gene outcomes or functional domain Epidemic disease and produce, and people SRPK1 gene outcomes and its fragment can be produced with recombination method or use Peptide synthesizer Synthesized.The antibody combined with the SRPK1 gene outcomes of non-modified form, it is possible to use in prokaryotic The gene outcome produced in (such as E.coli) is obtained animal is immunized.With posttranslational modification form such as glycosyl Change or phosphorylation SRPK1 albumen or the antibody of polypeptide combination, it is possible to use in eukaryotic such as yeast or insect The gene outcome produced in cell is obtained animal is immunized.

Can be used for SRPK1 antibody of the invention can be anti-human SRPK1 protein antibodies.It is of the invention anti-human SRPK1 protein antibodies can be used in immunohistochemistry technology, the people's SRPK1 albumen in detection biopsy specimen.

Inhibitor and pharmaceutical composition

Using albumen of the present invention, by various conventional screening assays, can filter out and phase occurs with SRPK1 albumen The material of interaction, especially inhibitor etc..

The inhibitor (including antibody, antisensenucleic acids and other inhibitor) of SRPK1 albumen of the present invention, when When (administration) is administered in treatment, expression and/or the activity of SRPK1 albumen can be suppressed, and then suppress stomach cancer The growth of cell or propagation.Generally, can these materials be formulated in nontoxic, inert and can be pharmaceutically connect In the aqueous carrier medium received, wherein pH ordinarily be about 5-8, and preferably pH is about 6-8, although pH value Can be varied from the property for being formulated material and illness to be treated.The pharmaceutical composition for preparing can It is administered with by conventional route, including (but being not limited to)：Stomach enteral, knurl are interior, intramuscular, abdomen Film is interior, intravenous, subcutaneous, intracutaneous or part are administered.

Can be used for inhibitor of the invention includes：The antibody of SRPK1, the antisense RNA of SRPK1 nucleic acid, siRNA, The activity inhibitor of shRNA and SRPK1.Wherein, typical SRPK1 inhibitor is siRNA and shRNA.

Can be used for siRNA of the invention includes various siRNA with SRPK1 as target spot, those skilled in the art Conventional screening can be carried out according to the target spot, wherein, a kind of preferred siRNA includes such as SEQ ID NO:5 (just Adopted chain) and SEQ ID NO.:Sequence shown in 6 (antisense strands).

Typically, SRPK1 genes are included as the technical scheme of the target spot for preparing curing gastric cancer medicine following Scheme：

1. chemical synthesis double stranded ribonucleic acid molecule, its sequence-specific is directed to SRPK1 gene orders, utilizes Liposome is delivered to the expression of interference SRPK1 genes in stomach cancer cell, and observation soft-agar cloning forms energy The change of the characteristics of cell biology such as power, cell propagation.Can design and close using the conventional method in this area The nucleotide sequence (such as siRNA) of SRPK1 is directed into specificity.

2. utilize various carriers, including DNA vector, slow virus carrier to disturb the expression of SRPK1 genes, Reach in vivo interference SRPK1 genes effect, detect they to nude mice by subcutaneous into knurl body therapeutic effect, So as to realize suppressing the purpose of stomach cancer propagation.

3. polypeptide, the monoclonal antibody for being capable of specificity suppression SRPK1 gene delivery activity are obtained, suppression is reached The purpose of SRPK1 activity processed, so that reality suppresses the purpose of propagation in stomach cancer cell body.

Present invention also offers a kind of pharmaceutical composition, the SRPK1 of the present invention that it contains safe and effective amount suppresses Agent (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)：Salt solution, buffer solution, glucose, water, glycerine, ethanol and its Combination.Pharmaceutical preparation should match with administering mode.Pharmaceutical composition of the invention can be made into injection shape Formula, such as aqueous solution with physiological saline or containing glucose and other assistant agents are prepared by conventional method. The pharmaceutical composition of such as tablet and capsule etc, can be prepared by conventional method.Pharmaceutical composition is such as Injection, solution, tablet and capsule are preferably aseptically manufactured.The dosage of active component is effective treatment Amount, such as daily mg/kg body weight of about 1 microgram -10.

Detection method and kit

The invention further relates to the diagnostic test side of quantitative and detection and localization people SRPK1 protein levels or mRNA level in-site Method.These experiments are known in the art.The people's SRPK1 protein levels detected in experiment, Ke Yiyong In diagnosis of gastric cancer.

A kind of method in detection sample with the presence or absence of SRPK1 albumen is resisted using the specificity of SRPK1 albumen Body is detected that it includes：Sample is contacted with SRPK1 protein specific antibodies；See whether to form anti- Nanocrystal composition, forms antibody complex and means that in sample there is SRPK1 albumen.

SRPK1 albumen or its polynucleotides can be used for the diagnosis and treatment of SRPK1 protein related diseases.The present invention Part or all of polynucleotides can be fixed on microarray or DNA chip as probe, for analyzing The Differential expression analysis of gene and gene diagnosis in tissue.The antibody of anti-SRPK1 can be fixed on protein core On piece, for detecting the SRPK1 albumen in sample.

Present invention also offers a kind of kit for detecting stomach cancer, it contains the primer of specific amplification SRPK1 Pair and/or SRPK1 specific antibodies and label or specification.

Wherein, described label or specification indicate herein below：When the SRPK1 of detection object is with respect to β-flesh The ratio between mrna expression amount of the mrna expression amount of filamentous actin beta-actin relative with the SRPK1 of cancer beside organism >1, then it is higher than general population to point out the cancered probability of the detection object.

Described kit can be used to detect people's gastric tissue sample or blood sample.

Screening technique

Method present invention also offers drug screening is carried out based on SRPK1.A kind of method is first screening influence (suppression) SRPK1 expression or the compound of activity, then further test it thin to cancer to the compound for filtering out The inhibitory action of born of the same parents.

Wherein, representational cancer cell includes being stomach cancer cell.

A kind of preferred screening technique can be based on the expression of the mRNA of SRPK1.Comprise the following steps that：

In (a) test group, test compound is added in the cultivating system of cell, and observe the test group Cell in SRPK1 expression quantity and/or activity；In control group, in the cultivating system of same cell not Addition test compound, and observe the expression quantity and/or activity of SRPK1 in the cell of control group；

If the expression quantity of the SRPK1 of cell and/or activity are less than control group in test group, the test is indicated that Compound is the candidate compound of the treatment stomach cancer for having inhibitory action to the expression of SRPK1 and/or activity.

Wherein, the expression quantity of described SRPK1 is detected and drawn by RT-PCR.

This screening technique may also include step：

B () further tests it to Growth of Gastric or increasing for the candidate compound obtained in step (a) The inhibitory action grown；And/or further test whether it plays the role of downward to SRPK1 genes.

In test group, test compound is added in the cultivating system of stomach cancer cell, and observe the number of stomach cancer cell Amount and/or growing state；In control group, without test compound in the cultivating system of stomach cancer cell, And observe the quantity and/or growing state of stomach cancer cell；Wherein, if in test group the quantity of stomach cancer cell or The speed of growth is less than control group, indicates that the test compound is that have suppression to the growth of stomach cancer cell or propagation The candidate compound of the treatment stomach cancer of effect.

The results show specificity can suppress for the small ribonucleic acid molecules of SRPK1 genes in the present invention The external Tumor formation energy bred with pernicious and suppression stomach cancer cell in nude mice by subcutaneous of stomach cancer cell, explanation SRPK1 genes can be used as preparing the target gene of curing gastric cancer medicine.

Beneficial effects of the present invention

The expression and activity level of 1.SRPK1 can be used as the mark of stomach cancer detection：SRPK1 is in stomach cancer Tissue or intraserous expression high can be as the marks of diagnosis of gastric cancer, and its specificity, sensitivity are high, behaviour Make easy.

2.SRPK1 inhibitor can effectively suppress proliferation of human gastric cancer cell, or as the medicine for the treatment of stomach cancer.

Following examples are only illustrative of the invention and is not intended to limit the scope of the invention.Do not noted in embodiment The experimental technique of bright actual conditions, generally according to normal condition, such as Sambrook et al., molecular cloning： Laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) in institute The condition stated, or according to the condition proposed by manufacturer.

Universal method：

The acquisition of 1 clinical tissue sample

Stomach cancer and cancer beside organism take from the patients with gastric cancer of operative treatment, are signed with patient before sample is obtained Informed Consent Form.The liver of surgery excision once in vitro, beyond rapid excised tumor primary tumor and surrounding 5cm Cancer beside organism, it is quick-frozen and move to -80 DEG C of Refrigerator stores in input liquid nitrogen, be stored in liquid nitrogen during transport. Cancer makes last diagnostic with cancer beside organism by pathologist.

2 tissues and cell RNA extracting

Using TRIzol Reagent (Invitrogen) reagent extracting RNA, concrete operations are as follows：

(1) vessel such as mortar, stone roller pestle and homogenizer are cleaned, and are rinsed with ddH2O and DEPC H2O again respectively, Then dried in 180 DEG C of baking ovens about 4 hours, to remove RNase；

(2) add appropriate liquid nitrogen to be allowed to precooling in mortar, tissue is taken out rapidly from liquid nitrogen, cut about 50-100mg sizes, the grind into powder in mortar；

(3) in ground tissue powder completely being moved into the EP pipes without RNase as far as possible with curet, EP Pipe is added in advance appropriate volume (1ml) TRIzol reagents, fully homogenate；

(4) room temperature is placed 5 minutes, in proportion to addition chloroform (200 μ l/1ml TRIzol) in centrifuge tube, Rapid acutely vibration 15 seconds, are stored at room temperature 2-3 minutes, 4 DEG C, are centrifuged 15 minutes under the conditions of 12000 × g；

(5) upper strata aqueous phase is transferred to as much as possible in the new EP pipes without RNase, is added isometric different Propyl alcohol, overturns and mixes 5 times, is stored at room temperature 10 minutes, 4 DEG C, is centrifuged 10 minutes under the conditions of 12000 × g, Now visible RNA precipitate；

(6) supernatant is outwelled, adds 75% ethanol (1ml/1ml TRIzol), mixed, wash RNA, 4 DEG C of centrifugation, is centrifuged 5 minutes under the conditions of 7500 × g；

(7) supernatant is abandoned, residual ethanol is eliminated as far as possible, precipitation spontaneously dries 5-10min, and (attention is sure not completely Dry)；30-50 μ l DEPC H2O, pressure-vaccum is added several times, to dissolve RNA precipitate；

(8) ELIASA determines RNA concentration and purity OD 260/280 (1.8-2.0)；Gel electrophoresis observation has Without degraded, -80 DEG C of preservations.

(9) cell line RNA extractings, the cell in growth period of taking the logarithm draws nutrient solution, according to the face of culture dish Product adds TRIzol reagents (1ml TRIzol/10cm2) cell lysis of respective amount, blows and beats several times, The cell that will be cleaved is collected into the EP pipes without RNase, and remaining is according to above-mentioned steps 4) -8) complete chlorine Imitative-isopropanol method isolates and purifies RNA.

The reverse transcription of 3RNA

With M-MLV Reverse Transcriptase (Promega) reverse transcription, operate as follows：

(1) following components is added in the EP pipes of nuclease free：

It is placed in PCR instrument, 70 DEG C, 5 minutes, immediately after in cooled on ice 5min.

(2) following components is added in above-mentioned system：

After gently mixing, it is placed in PCR instrument, 37 DEG C, 60min.

The cDNA that reverse is obtained is placed in 4 DEG C of preservations.

4 real-time quantitative PCRs

Real-time quantitative PCR reaction is usedPremix Ex Taq^TM(Perfect Real Time) is tried The reaction system of agent box (TaKaRa Biotechnology Co., Ltd.s Dal ian, China), utilizes Thermal Cycler Dice^TMReal Time System (TP800 real-time fluorescence quantitative PCR instrument, TaKaRa) Operated.The amplified production length of quantitative PCR the most properly (can extend to 300 with 80bp-150bp bp)。

Reaction system is as follows：

Reaction condition：

Solubility curve analytical procedure：

95℃ 15sec

60℃ 30sec

95℃ 15sec

Dissociation time is 4sec.

The default value that autofluorescent background signal and threshold value are set using instrument, each PCR reactions can be automatic after terminating Generation, Ct values represent that the fluorescence signal in each reaction tube reaches given threshold (10 times of Baseline fluorescence intensity) When the period that is experienced；Genes of interest SRPK1 each template does 3 multiple pipes, and the Ct values for obtaining are averaged Value；The Ct average values of SRPK1 genes subtract the Ct average values of the reference gene (β-actin) of corresponding template, Obtain Δ Ct.The Δ Ct of stomach cancer group subtracts the Δ Ct of corresponding adjacent tissues, obtains Δ Δ Ct values, stomach cancer group and The multiple proportion of the SRPK1 genes in the group of cancer side uses 2^-ΔΔCtRepresent.

5 construction of eukaryotic expression vector

(1) template：The cDNA library of stomach cancer cell AGS.

(2) selection of carrier for expression of eukaryon：pcDNA^TM3.1/myc-His (-) A, 5522nucleotides.

(3) according to SRPK1mRNA (NM_003137.4) sequence, with reference to expression vector pcDNA^TM The restriction enzyme site design primer of 3.1/myc-His (-) A, primer sequence is Forward:5- actggaattcCCACCATGGAGCGGAAAGTGCTTGCG-3；Reverse:5- tccaAAGCTTGGAGTTAAGCCAAGGGTG-3.The terminator codon of SRPK1 is gone wherein in reverse primer Remove so that c-myc and 6xHis labels on the C-terminal band of SRPK1.Using high fidelity DNA polymerase PrimeSTAR^TMHS DNA Polymerase (TaKaRa), are template amplification base with ags cell cDNA Because of SRPK1 total length opening code-reading frames, 50 μ l overall reaction system compositions are as follows：

Using two-step PCR (98 DEG C, 10sec；60 DEG C, 90sec), expand 35 circulations.PCR (gel purification kit is reclaimed in primer size about 1.9kb, 1% agarose gel electrophoresis identification size, rubber tapping： MACHEREY-NAGEL the PCR primer of clip size) is met.

(1) EcoRI, Hind III (TaKaRa Biotechnology Inc.Dal ian, China) double digestion Reclaim PCR primer and vector plasmid pcDNA^TM3.1/myc-His (-) A, endonuclease reaction system is as follows：

37 DEG C of endonuclease reactions 1 hour；Digestion products are reclaimed in rubber tapping.

(2) connect：The PCR primer that digestion is reclaimed is with carrier according to mole ratio (4:1) ratio mixing, DNA Connection enzyme system link, also includes (the TaKaRa Code of 2.5 4 × Solution of μ l I in system：D102A), DdH2O polishings are to 10 μ l, 16 DEG C of connection 2h or even overnight；

(3) convert：Take 10 μ l connection products and 100 μ l competence bacterium (TOP10 or DH5 α) are mixed Close, 30min, 42 DEG C of heat shock 90sec are placed on ice, be immediately placed on 5min on ice, add 800 μ l LB nutrient solutions without antibiotic, 37 DEG C, 200rpm shaken cultivation 30min make thalline recover and expand A generation, 3000rpm centrifugation 2min, the most of supernatant of removal stays 50-100 μ l bacterium solutions, gently blow Beat precipitation to mix, be then uniformly applied on the LB flat boards of amicillin resistance (Amp+), 37 DEG C of cultures 12-16 hours.

(4) clone identification：Picking by after ammonia benzyl resistance screening grow bacterium colony plus ampicillin liquid Amplification Culture in culture medium, extracting plasmid carries out digestion identification：Take that 1-2 μ g are small to take out plasmid EcoRI, Hind III double digestion, agarose gel electrophoresis identification endonuclease bamhi size, carrier pcDNA^TM 3.1/myc-His(-)A Clip size about 5.5kb, SRPK1 reading frames clip size about 1968bp, the clone for meeting size send sequencing Confirm the correctness of Insert Fragment sequence.

The measure of 6 cell growth curves

(1) different types of stomach cancer cell is pressed into 3-5 × 10 according to its growth characteristics³/ 100 μ l/ holes calculate Cell total amount, after abundant vitellophag, is diluted to required concentration, is inoculated in 96 orifice plates.Every group three daily Multiple holes, by 5-7 days inoculating cells；

(2) observation of cell state and number after cell is substantially adherent.With CCK-8 developers (Cell Counting Kit-8, DOJINDO, Japan) chromogenic reaction is carried out, every 100 μ l nutrient solutions add 10 μ L CCK-8,37 DEG C, 5%CO2 incubators are placed and are incubated 1h, and ELIASA determines the absorbance at 450nm, Record, determines the actual initial density of cell, used as growth relative zero.

(3) half amount changes liquid daily or every other day, specific depending on requirement of experiment；

(4) basis of microscopic observation cellular morphology, Fixed Time Interval measurement, records cell growth condition；

It is general to survey 5 to 7 days.After end to be determined, collect data and processed, chart is drawn with Excel.

7 cell clonal formations are tested

(1) transfect：Using Lipofectamine^TM2000 (Invitrogen) transfectional cells, overexpression or The expression of SRPK1 genes in silenced cell；

(2) cell after transfecting, in 6 orifice plates or 35mm culture dishes with disappearing after normal nutrient solution culture 24h Change and count, 100mm culture dishes (different cell line numbers are different) are seeded to by certain amount, continue to cultivate 24h, then adds the G418 (600-1000 μ g/ml) of debita spissitudo according to cell category, is turned with screening Contaminate positive cell clone；

(3) cultivate 2-3 weeks, changed fresh medium every 3-5 days therebetween and add G418 screenings, until having Macroscopic cell clonal formation；

(4) the training liquid in culture dish is sucked, 1 × PBS is washed twice, coomassie brilliant blue R_250 dyeing 2h is used After water is gently rinsed, then with coomassie brilliant blue staining destainer decolouring 30-60min；

(5) Clone formation coloration result is taken pictures, and each is cultivated according to identical standard (cell clone size) Cell clone on ware is counted.

8 Western blottings (Western Blot)

(1) prepared by protein sample：After the cell of culture sucks culture supernatant, washed twice with the 1XPBS of precooling, Add 2 × SDS lysates (100mM Tris-Cl, pH=6.8,4%SDS, 20% glycerine), fully cracking Afterwards, boiling water bath heating 10min, 12000 × g centrifugation 10min, supernatant is transferred in new pipe,BCA Protein Assay Kit are quantified to the albumen for obtaining, -80 DEG C of preservations；

(2) protein electrophoresis is separated：The appropriate sample-loading buffer containing 200mM DTT is added in protein sample (loading buffer), boiling water bath heating 10min, is slightly centrifuged, SDS-PAGE proteins gel electrophoresis Separate sample；

(3) transferring film：Running gel, nitrocellulose membrane, thickness (thin) filter paper backing plate are dipped in (24 in transferring film buffer solution MM Tris, 192mM glycine, 20% methyl alcohol) balance 15-20min.By the thickness filter paper backing plate of positive pole -1 The order of the thin filter paper backing plate-negative pole of-nitrocellulose membrane--2 layers of running gel is put well, wet to turn instrument (XCell SureLock^TM, invitrogen) and 30 volts of transferring film 30-40min；

(4) close：5% skimmed milk power/0.1%PBST is used as confining liquid, horizontal shaker, room temperature closing 30min-2h；

(5) primary antibody：Primary antibody dilutes (reference antibody specification recommended density) with confining liquid, is incubated at room temperature 2h Or 4 DEG C of overnight incubations, 0.1%PBST washes three times, each 5min；

(6) secondary antibody：Fluorescence secondary antibody dilutes (1 with confining liquid:1000) 30min, 0.1%PBST, are incubated at room temperature Wash three times, each 5min；

(7) film is swept：ODYSSEY infrared imaging systems scan nitrocellulose filter, preserve image.

9 soft-agar clonings form experiment

(1) 1% and 2% low melting point Agarose (TaKaRa companies), autoclave sterilization are prepared respectively；

(2) 2 × DMEM nutrient solutions (2.5 × DMEM, containing 20%FBS) is prepared；

(3) 2%Agarose and 2 × DMEM nutrient solutions by 37 DEG C of insulations are mixed by same volume, with every hole 0.5ml is added in 24 orifice plates, is placed in 4 DEG C of refrigerators, it is to be solidified after use；

(4) fully the cell of digestion culture, into individual cells, is counted, and is diluted to same concentrations (3000-5000 Individual/0.5ml)；

(5) cell suspension is mixed with the 1%Agarose of 37 DEG C of insulations with same volume, is added to and has completed In 24 orifice plates of lower floor's glue, per hole 0.5ml, 4 DEG C of refrigerator 10min are placed in；

(6) 0.2ml nutrient solutions are added on the soft agar of solidification, 37 DEG C, in 5%CO2 incubators are placed in, Continue to cultivate 2-3 weeks；

(7) growing state of each hole of basis of microscopic observation the inside cell clone, counts, and is analyzed.

10 tumor formation in nude mice

Mouse used is the 5-6 weeks male BLAB/c nu nude mice of size, is had by Shanghai Si Laike experimental animals Limit company provides, and raises in southern model animal Culture Center；

(1) cell after treatment is taken, subcutaneous (the different cell categories inoculation numbers of mouse is inoculated in equal number It is different), to avoid error caused by individual difference, allogenic cell different disposal from symmetrical being inoculated in together One mouse；

(2) after visual tumors to appear, every 3 days monitoring tumor sizes, slide measure reads tumour major diameter and short Footpath, calculates gross tumor volume as follows：Volume=major diameter × minor axis²；

(3) after continuing to monitor about 7-8 times, disposal data, statistics.

11 antibody are obtained and immune detection

(1) antigen protein is obtained

The cDNA sequence of people's SRPK1 genes is obtained from Genebank databases, is expanded by PCR and obtained Encoder block, in insertion prokaryotes or eukaryotic expression vector, expresses SRPK1 albumen, and by gene work The purification system purifying protein of journey expression product.

(2) Antibody preparation

Antibody can be prepared using following several method：

A cell fusion methods：With the SRPK1 protein immune animals (including rabbit, goat etc.) of above-mentioned preparation, Spleen cell is obtained, then is merged with myeloma cell, and routinely monoclonal antibody technology of preparing prepares Dan Ke Grand antibody.

B utilizes phage display storehouse, the spleen IgG variable regions of the immune animal of clone to be simultaneously expressed as gene work Journey monoclonal antibody.

C prepares polyvalent antibody using the protein immune animal of purifying.

(3) detect

Antibody (resisting more or monoclonal antibody) prepared by a, the pathological examination of stomach cancer is carried out with histochemical method, cloudy Property signal be stomach cancer.

B takes patients serum, is detected with ELISA method, and negative reaction is the suspicious patient of stomach cancer.

C using SRPK1 antibody as protein-chip one of probe, for kinds of tumors diagnosis.

Embodiment 1：Expression patterns of the SRPK1 in clinical sample

By lot of experiments, it is found that shear factor kinases SRPK1 is substantially raised in stomach organization. In 89 pairs of clinical samples, the expression for finding there is SRPK1 in 66 pairs of sample stomach organizations is detected by SABC Apparently higher than corresponding cancer beside organism, rise rate reaches 74.2%, and statistical analysis show p<0.01, explanation The reliability of result.And 12 pairs of clinical sample extracting histones are have chosen, row Western blot experiments, Result display SRPK1 is significantly higher than corresponding cancer beside organism (Figure 1A) in 8 pairs of sample cancerous tissues.Figure 1B Show the expression of SRPK1 in 1 couple representativeness clinical sample of selection.

Embodiment 2：Overexpression SRPK1 genes suppress the propagation of stomach cancer cell

By NCBI internet retrievals, cDNA sequence CCDS47415.1 (the SEQ ID NO. of SRPK1 are retrieved: 1) amino acid sequence NP_003128.3 (the SEQ ID NO. of (Fig. 2A) and its coding:2) (Fig. 2 B). In order to verify functions of the SRPK1 in stomach carcinogenesis, its carrier for expression of eukaryon is constructed first, by SRPK1 CDNA clone enter pcDNA^TM3.1/myc-His (-) A, transfects the plasmid for building laggard into stomach cancer cell Row western blot detect that discovery SRPK1 is successfully expressed (Fig. 3 A).Ensuing functional experiment life Curve determination result long shows that the overexpression of SRPK1 promotes the propagation energy of stomach cancer cell AGS and SGC7901 Power (Fig. 3 A).Primer sequence for building SRPK1 carrier for expression of eukaryon is Forward: actggaattcCCACCATGGAGCGGAAAGTGCTTGCG-3(SEQ ID NO.:3)；Reverse:5- tccaAAGCTTGGAGTTAAGCCAAGGGTG-3(SEQ ID NO.:4)。

Embodiment 3：The expression of silence SRPK1 promotes cell growth

In order to further verify the cell growth promotion functions of SRPK1, we take RNA to disturb silence its table The mode for reaching detects the change of cell growth status.For disturbing the siRNA of SRPK1 expression by Shanghai Ji code Pharmaceutical Co. Ltd synthesizes, and sequence is：Positive-sense strand 5-GGUCAGUCAUUCAGUGAACAAdTdT-3 (SEQ ID NO.:5)；Antisense strand 5-UUGUUCACUGAAUGACUGACCdTdT-3 (SEQ ID NO.:6).As dry The NC non-specificity nucleotides sequences for disturbing control are classified as：Positive-sense strand 5-UUCUCCGAACGUGUCACGUdTdT-3 (SEQ ID NO.:7)；Antisense strand 5-ACGUGACACGUUCGGAGAAdTdT-3 (SEQ ID NO.:8).Through Western Blot detection protein expressions show that artificial synthesized interference siRNA can effectively lower the expression of SRPK1 The downward that level (Fig. 4 A), growth curve experiment and colony formation also demonstrate SRPK1 can suppress The growth of stomach cancer cell BGC-803, AGS, SGC7901 and NCI-N87 and formed clone ability (Fig. 4 A, B), strong support SRPK1 promotes the conclusion of growth of tumour cell.

Embodiment 4：SRPK1 suppresses the pernicious sign of stomach cancer cell

Soft-agar cloning Forming ability is a strong In vitroindex for reflecting degree of malignancy of tumor cell. In order to detect influences of the SRPK1 to stomach cancer cell grade malignancy, according to experimental result above, this experiment choosing Research objects of the WRL-68 as SRPK1 overexpression has been taken, ags cell has been have chosen as SRPK1 overexpression Research object, have chosen the research object that MGC803 strikes low expression for SRPK1.By to being grown in soft fine jade Number of cell clones analysis (Fig. 3 B and Fig. 4 C) in fat, it is final to determine that overexpression SRPK1 be effectively facilitated Ags cell and silence expression SRPK1 can effectively suppress the grade malignancy of MGC803 cells, show SRPK1 Influence the pernicious of stomach cancer cell.

Embodiment 5：SRPK1 influences stomach cancer cell in the one-tenth knurl ability of nude mice by subcutaneous

Experiment in vitro has clearly indicated that SRPK1 promotes the growth of stomach cancer cell, next using tumor bearing nude mice Model studies influence of the expression of SRPK1 to stomach cancer cell nude mice by subcutaneous one-tenth knurl ability.

By 1 × 10⁶The SGC7901 cells and compared with control cells of individual overexpression SRPK1 are symmetrically injected into mice belly It is subcutaneous, the growing state of knurl body is observed, and the knurl body length and width every 3 days are recorded with slide measure. After 1 month, kill nude mice taking-up knurl body and weigh.Result shows that the overexpression of SRPK1 effectively facilitates SGC7901 One-tenth knurl ability (Fig. 5 A) of the cell in nude mice by subcutaneous.Equally, by 2 × 10⁶Individual silence expresses the MGC803 of SRPK1 It is subcutaneous that cell and compared with control cells are symmetrically injected into mice belly, and observation knurl body growing state is simultaneously recorded.Final Result shows that the downward of SRPK1 effectively inhibits MGC803 cells nude mice by subcutaneous into the ability (Fig. 5 B) of knurl.

Present invention experiment confirms expression of the SRPK1 genes in stomach organization apparently higher than cancer beside organism, and The growth of stomach cancer cell can be remarkably promoted by external source SRPK1 gene overexpressions, disturbs interior by RNAi SRPK1 expression in source can substantially suppress Growth of Gastric and clone is formed in soft agar；Zoopery Show that SRPK1 expression promotes the one-tenth knurl ability of stomach cancer cell.Therefore SRPK1 genes and its expression product can be made Mark for diagnosis of gastric cancer and the drug target for curing gastric cancer, make diagnosing gastric cancer more accurate, quick. In a word, SRPK1 genes of the present invention and application thereof provide new therapy target and effective new drug to prevent and treat stomach cancer.

The all documents referred in the present invention are all incorporated as reference in this application, just as each document It is individually recited as with reference to such.In addition, it is to be understood that after above-mentioned instruction content of the invention has been read, Those skilled in the art can make various changes or modifications to the present invention, and these equivalent form of values equally fall within this Shen Please appended claims limited range.

Claims (10)

1. a kind of serine/Arginine Protein Kinase 1 (Serine/Arginine Protein Kinase 1, SRPK1) the purposes of gene or its albumen or its detection reagent, it is characterised in that for prepare detection stomach cancer and/ Or judge the reagent or kit of gastric cancer susceptibility.

2. purposes as claimed in claim 1, it is characterised in that described kit includes：To SRPK1 Carry out quantitative detecting reagent and corresponding label or specification.

3. purposes as claimed in claim 1, it is characterised in that described detection reagent includes SRPK1's Specific primer, probe, antibody and chip.

4. purposes as claimed in claim 1, it is characterised in that described specific primer such as SEQ ID NO.: Shown in 1 and 2.

5. a kind of purposes of SRPK1 inhibitor, it is characterised in that described inhibitor be used to prepare and suppress The medicine of Growth of Gastric or propagation, or for preparing the medicine for the treatment of stomach cancer.

6. purposes as claimed in claim 5, it is characterised in that described inhibitor includes：SRPK1's Antibody, the antisense RNA of SRPK1 nucleic acid, the activity inhibitor of siRNA, shRNA and SRPK1.

7. a kind of pharmaceutical composition, it is characterised in that described pharmaceutical composition include SRPK1 inhibitor with And pharmaceutically acceptable carrier.

8. a kind of method for suppressing Growth of Gastric or propagation of external non-therapeutic, it is characterised in that bag Include step：In the presence of SRPK1 inhibitor, cancer cell is cultivated, so as to suppress growth of cancer cells or propagation.

9. a kind of method that the candidate compound of stomach cancer is treated in screening, it is characterised in that methods described includes step Suddenly：

In (a) test group, test compound is added in the cultivating system of cell, and observe the test group Cell in SRPK1 expression quantity and/or activity；In control group, in the cultivating system of same cell not Addition test compound, and observe the expression quantity and/or activity of SRPK1 in the cell of control group；

Wherein, if the expression quantity of the SRPK1 of cell and/or activity are less than control group in test group, indicate that The test compound is the candidate compound of the treatment stomach cancer for having inhibitory action to the expression of SRPK1 and/or activity Thing.

10. method as claimed in claim 9, it is characterised in that methods described also includes step：

B () further tests it to Growth of Gastric or increasing for the candidate compound obtained in step (a) The inhibitory action grown；And/or further test whether it plays the role of downward to SRPK1 genes.