CN103937871B

CN103937871B - The application of SRRP35 gene and expression product in cancer diagnosis and treatment

Info

Publication number: CN103937871B
Application number: CN201310025221.XA
Authority: CN
Inventors: 高勇; 李砚东
Original assignee: Shanghai East Hospital
Current assignee: Shanghai East Hospital
Priority date: 2013-01-23
Filing date: 2013-01-23
Publication date: 2019-07-23
Anticipated expiration: 2033-01-23
Also published as: CN103937871A

Abstract

The invention discloses the applications of a kind of SRRP35 gene and its expression product, are used to prepare the product of cancer diagnosis and treatment.SRRP35 gene of the invention and its expression product can be used as the specificity marker gene of diagnosis cancer especially liver cancer；SRRP35 gene of the invention and its expression product are alternatively arranged as the target gene of preparation treating cancer especially liver-cancer medicine, provide new treatment of cancer approach.

Description

The application of SRRP35 gene and expression product in cancer diagnosis and treatment

Technical field

The present invention relates to oncologies.More particularly it relates to which SRRP35 gene and expression product are examined in cancer Survey the application of aspect, the especially application in the detection of liver cancer.The invention further relates to SRRP35 gene, albumen and its agonists to exist Application in treatment of cancer.

Background technique

Hepatocellular carcinoma (hepatocellularcarcinoma, HCC) is the common malignant tumour in China, death rate position Occupy second.The diagnosis of liver cancer, especially early diagnoses, and is the key that clinic diagnosis and prognosis.It can be used as primary carcinoma of liver mark The biomolecule of object, it is generally recognized that have following four classes: cancer embryo and glycoprotein antigen；Enzyme and isodynamic enzyme；Cell factor；Gene.? In global range, to the etiologic diagnosis of liver cancer to detect based on Serum AFP (alpha-fetoprotein), but AFP sensibility (40%~ 65%) and specific (76%~96%) is unsatisfactory.

In addition, the treatment of liver cancer made great progress in past 20 years, there are some local non-operative treatments Scheme, still, since the chemotherapeutics of current liver cancer continues to use the therapeutic agent of other tumours substantially, specific aim is poor, therefore, always Body unsatisfactory curative effect.

Therefore, thin in order to effectively inhibit liver cancer there is an urgent need in the art to develop the GAP-associated protein GAP that can be used for diagnosing cancer of liver The growth of born of the same parents, there is an urgent need in the art to develop to can be used for inhibiting the drug of liver cancer cell growth, with improve chemotherapy specificity and Validity.

Summary of the invention

The invention discloses the applications of a kind of people SRRP35 gene and its expression product, are used to prepare cancer especially liver cancer Diagnosis and treatment product.

The first aspect of the present invention provides the purposes of a kind of SRRP35 gene or SRRP35 albumen, is used to prepare detection cancer The reagent or kit of disease；

In another preferred example, the cancer is liver cancer.

In another preferred example, the kit includes: the reagent that quantitative detection is carried out to SRRP35 albumen or mRNA And corresponding label or specification.

In another preferred example, the reagent includes SRRP35 specific primer, specific antibody, probe and/or core Piece.

In another preferred example, above-mentioned reagent includes detection chip, including nucleic acid chip and protein-chip.

In another preferred example, the nucleic acid chip includes the spy of the cancer related gene of substrate and point sample on substrate Specific oligonucleotide probe, the specific oligonucleotide probe of the cancer related gene include and SRRP35 gene or mRNA The probe of specific binding.

In another preferred example, the protein-chip includes the cancer-associated proteins of substrate and point sample on substrate Specific antibody, the specific antibody of the cancer-associated proteins include the specific antibody of anti-SRRP35 albumen.

In another preferred example, the SRRP35 albumen includes fusion protein and non pregnant women.

The second aspect of the present invention, provides a kind of for detecting the diagnostic kit of cancer, and the kit contains One container, the detection reagent in the container containing detection SRRP35 albumen or mRNA；And label or specification, the label Or specification indicates the kit for detecting cancer.

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

As the mrna expression amount β-opposite with the SRRP35 of cancer beside organism of the opposite beta-actin of the SRRP35 of test object The ratio between mrna expression amount of actin≤1 then prompts the cancered probability of the test object to be higher than general population.

In another preferred example, the detection reagent includes: specific primer, specific antibody, probe and/or core Piece；

In another preferred example, the kit is for detecting human tumour tissue sample or blood sample；

In another preferred example, the neoplasmic tissue sample is hepatoma sample.

The third aspect of the present invention provides the purposes of a kind of SRRP35 albumen, SRRP35 gene or its agonist, is used for Preparation inhibits the drug of growth of cancer cells or proliferation, or is used to prepare the drug for the treatment of cancer.

The fourth aspect of the present invention provides a kind of method for inhibiting growth of cancer cells or proliferation of external non-therapeutic, Comprising steps of cancer cell is cultivated, to inhibit growth of cancer cells or proliferation in the presence of SRRP35 albumen or its agonist.

In another preferred example, the method includes the addition SRRP35 agonist into the cultivating system of cancer cell, from And inhibit growth of cancer cells or proliferation.

In another preferred example, the cancer cell is liver cancer cells.

The fifth aspect of the present invention provides a kind of method of candidate compound for screening treating cancer, comprising steps of

(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 SRRP35；In control group, test compound is not added in the cultivating system of same cell, and Observe the expression quantity and/or activity of SRRP35 in the cell of control group；

Wherein, if the expression quantity of the SRRP35 of cell and/or activity are greater than control group in test group, the test is indicated that Compound is the candidate compound of the expression and/or the active treating cancer for having facilitation to SRRP35.

In another preferred example, the cell includes: cancer cell or normal cell；

In another preferred example, the cell is liver cancer cells or liver cell.

In another preferred example, the method also includes steps:

(b) for the candidate compound obtained in step (a), its inhibition to growth of cancer cells or proliferation is further tested Effect.

In another preferred example, comprising steps of being added in the cultivating system of cancer cell in test group in the step (b) Compound is tested, and observes the quantity and/or growing state of cancer cell；In control group, in the cultivating system of cancer cell not Addition test compound, and observe the quantity and/or growing state of cancer cell；Wherein, if in test group cancer cell quantity Or the speed of growth is less than control group, indicates that the test compound is the treatment that growth to cancer cell or proliferation have inhibiting effect The candidate compound of cancer.

The sixth aspect of the present invention additionally provides a kind of method of inhibition or treating cancer, comprising steps of to being needed to treat Object (mammal) application safe and effective amount SRRP35 agonist purposes.

In another preferred example, the cancer includes liver cancer.

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention 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.

Detailed description of the invention

Figure 1A is that real-time quantitative PCR detects SRRP35 gene group by 32 hepatocarcinoma patient cancerous tissues and cancer in embodiment 1 Expression schematic diagram in knitting, wherein " N " refers to cancer beside organism, and " C " refers to liver cancer tissue.20 (62.5%) patients as the result is shown Cancerous tissue in SRRP35 gene expression amount be lower than corresponding cancer beside organism.

Figure 1B shows expression pattern of the real-time quantitative PCR detection SRRP35 gene in human hepatoma cell strain.

Fig. 2A shows overexpression situation of the SRRP35 in hepatoma cell strain Sk-Hep-1 and WRL-68, illustrates SRRP35 The successful expression in carrier for expression of eukaryon.

Fig. 2 B shows that the SRRP35 albumen of overexpression reduces clone's shape of hepatoma cell strain Sk-Hep-1 and WRL-68 At ability.

Fig. 2 C shows that the SRRP35 albumen of overexpression inhibits the increment of Sk-Hep-1 and WRL-68 cell.

Fig. 3 A shows that artificial synthesized siRNA effectively disturbs the expression of SRRP35 gene.

Fig. 3 B shows that the expression of the method silencing SRRP35 by RNA interference promotes liver cancer cells Huh-7 and Hep3B Increment.

Fig. 4 shows that the expression of the method silencing SRRP35 by RNA interference improves liver cancer cells Huh-7 and Hep3B Clonality in soft agar further relates to the pernicious characterization that SRRP35 inhibits liver cancer cells.

Fig. 5 A shows the energy that the SRRP35 gene of overexpression inhibits liver cancer cells WRL-68 to form tumour in nude mice by subcutaneous Power.No matter in volume and tumor weight SRRP35 overexpression group is all smaller than control group and light, while two groups have statistics anticipate Justice.

Fig. 5 B shows the energy that silencing SRRP35 gene expression promotes liver cancer cells Huh-7 to form tumour in nude mice by subcutaneous Power.No matter in volume and tumor weight SRRP35 silencing expression group is all bigger than control group and again, while two groups have statistics Meaning.

Specific embodiment

The present inventor after extensive and in-depth study, for the first time it was unexpectedly observed that SRRP35 low expression in cancerous tissue, and The high expression in cancer beside organism and normal tissue, therefore SRRP35 can be used as the marker of cancer detection for detecting or complementary Detect cancer.In addition, the agonist of SRRP35 can inhibit the growth of cancer cell (especially liver cancer cells).It completes on this basis The present invention.

The present inventor is also using carrier for expression of eukaryon system as mediated system, the mistake in liver cancer cells Sk-hep-1 and WRL-68 Express SRRP35 gene (identified overexpression multiple is greater than 20 times).It is found by cell growth assay: SRRP35 gene It is overexpressed the obvious growth for inhibiting liver cancer cells Sk-hep-1 and WRL-68.Therefore, SRRP35 gene can be used for the base of liver cancer Because for the treatment of.

SRRP35 albumen and polynucleotides

In the present invention, " albumen of the present invention ", " polypeptide of the present invention ", " SRRP35 albumen " are used interchangeably, and are referred to referred to as SRRP35).It should be understood that the term further includes the active fragment and derivative of SRRP35.

In the present invention, " gene of the present invention ", " polynucleotides of the present invention " refer to coding SRRP35 albumen or its active fragment With the nucleotide sequence of derivative, including justice and antisense nucleic acid.In cell chromosome 6q15, cDNA is complete for the SRRP35 assignment of genes gene mapping A length of 786bp, the albumen of 261 amino acid of encoding full leng.

In the present invention, term " SRRP35 albumen ", " SRRP35 polypeptide " or " cancer markers SRRP35 " is interchangeable makes With all referring to albumen or polypeptide with people's Protein S RRP35 amino acid sequence.

SRRP35 also known as SRSF12(serine/arginine-rich splicing factor12, rich in serine and Arginic shear factor 12), it gains the name because being originally found inhibiting factor and molecular weight as SR and being 35kD.SR albumen is one Class is rich in serine and the arginic shear factor with RRM RNA binding structural domain, has regulation eukaryocyte pre- MRNA transcript alternative splicing falls introne and splices the function of exon.In vivo study discovery, SRRP35 can antagonism its Its SR albumen and activate adenovirus E 1 A pre-mRNA5 ' least significant end alternative splicing (Alison E, et al.2001.THE JOURNALOF BIOLOGICAL CHEMISTRY).

The cDNA sequence of SRRP35 gene is as shown in SEQ IDNO.:1；Genbank accession number 135295, SRRP35's The amino acid sequence NP542781.3 of cDNA sequence CCDS47459.1 and its coding.

The amino acid sequence of SRRP35 coded by said gene albumen is as shown in SEQ ID NO.:2.

As used herein, " separation " it is (former if it is crude to refer to that substance is separated from its primal environment 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 such as from separating in other substances with existing in native state, then isolate and purify.

As used herein, " isolated SRRP35 albumen or polypeptide " refer to SRRP35 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 SRRP35 albumen.Substantially pure polypeptide can generate single master tape in non-reducing polyacrylamide gel.In the present invention, SRRP35 albumen includes fusion protein and non pregnant women.

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

Polynucleotides of the 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 for encoding the mature polypeptide of SRRP35 include: the coded sequence of an 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.The term polynucleotides of polypeptide " coding " can be the polynucleotides including encoding this polypeptide, be also possible 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 insertion variant.Such as this Known to field, allelic variant is the alternative forms of a polynucleotides, it may be one or more nucleotide substitution, Missing or insertion, but not from substantially change its encode polypeptide function.

The invention further relates to the nucleic acid fragments hybridized with above-mentioned sequence, the nucleic acid fragment including 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 SRRP35 albumen.

People SRRP35 nucleotide full length sequence or its segment of the 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, and with the commercially available library cDNA or by the library 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 carry out twice or repeatedly PCR amplification, then again will Each time the segment amplified is stitched together by proper order.

Once obtaining related sequence, so that it may obtain related sequence in large quantity with recombination method.This is usually 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 gene of the invention using round pcr DNA amplification/RNA method.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 segment of amplification is such as separated and purified by gel electrophoresis.

The present invention also relates to the carriers comprising polynucleotides of the invention, and with carrier of the invention or SRRP35 albumen The genetically engineered host cell of coded sequence, and the method for generating 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 polynucleotide sequence of the invention SRRP35 albumen.In general there are following steps:

(1) polynucleotides (or variant) of encoding human SRRP35 albumen of the 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 construct the DNA sequences encoding of SRRP35 containing people and suitable turn Record/translation control signal expression vector.These methods include recombinant DNA technology in vi, DNA synthetic technology, 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 White (GFP), or tetracycline or amicillin resistance for Escherichia coli.

Carrier comprising 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；CHO, COS or the zooblast of 293 cells etc..

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, the competent cell that can absorb DNA can harvest after exponential phase of growth, use CaCl₂Method processing, institute With the step of it is generally well-known in the art.Another method is using MgCl₂.If desired, conversion can also use the side of electroporation Method carries out.When host is eucaryote, following DNA transfection method 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 coded by said gene of the invention.According to used Host cell, culture medium used in culture can be selected from various conventional mediums.Under conditions of being 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, and can be separated by various separation methods and purify the albumen of recombination using its physics, chemical and other characteristics.This A little methods are well-known to those skilled in the art.The example of these methods includes but is not limited to: conventional renaturation process is used Protein precipitant handles (salting-out method), centrifugation, permeates broken bacterium, 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.

Antibody

The invention also includes the polyclonal antibodies and monoclonal antibody to people's SRRP35 albumen with specificity, especially singly Clonal antibody.Here, " specificity " refers to that antibody can be incorporated into people SRRP35 gene product or segment.Preferably, referring to those energy In conjunction with people SRRP35 gene product or segment but nonrecognition and the antibody for being incorporated into other non related antigen molecules.Of the invention Antibody can be prepared by various technologies known to those skilled in the art.

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

The antibody of anti-human SRRP35 albumen can be used in immunohistochemistry technology, detect the people SRRP35 in biopsy specimen Albumen.

Agonist and pharmaceutical composition

It can filter out and interact with SRRP35 albumen by various conventional screening assays using albumen of the present invention Substance, especially agonist etc..

The agonist of SRRP35 albumen of the present invention can promote SRRP35 albumen when being administered (administration) in the treatment Expression and/or activity, and then inhibit cancer cell (including liver cancer) growth or proliferation.In general, these agonists can be prepared In nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein pH is usually about 5-8, and preferably pH is about 6-8, although pH value can be varied with the property and illness to be treated for being formulated substance.Prepared pharmaceutical composition It can be administered by conventional route, including (but being not limited to): tumor is interior, intramuscular, peritonaeum is interior, intravenous, subcutaneous, skin Interior or local administration.

The present invention also provides a kind of pharmaceutical composition, SRRP35 albumen of the present invention that it contains safe and effective amount or its swash Dynamic agent and pharmaceutically acceptable carrier or excipient.This kind of carrier includes (but being not limited to): salt water, buffer, grape Sugar, water, glycerol, ethyl alcohol, and combinations thereof.Pharmaceutical preparation should match with administration mode.Pharmaceutical composition of the invention can be by Injection form is made, such as the aqueous solution with physiological saline or containing glucose and other adjuvants is made by conventional method It is standby.The pharmaceutical composition of such as tablet and capsule etc can be prepared by conventional method.Pharmaceutical composition such as injection, molten Liquid, tablet and capsule preferably aseptically manufacture.The dosage of active constituent is therapeutically effective amount, such as about 1 microgram-daily 10 mg/kg weight.

Detection method and kit

The invention further relates to quantitative and detection and localization people SRRP35 protein level or mRNA level in-site diagnostic testing process.This A little tests are known in the art.People SRRP35 protein level detected, can be used for diagnosing liver cancer in test.

A kind of method in test sample with the presence or absence of SRRP35 albumen be using SRRP35 albumen specific antibody into Row detection, it includes: to contact sample with SRRP35 protein specific antibody；It sees whether to form antibody complex, form Antibody complex means that there are SRRP35 albumen in sample.

SRRP35 albumen or its polynucleotides can be used for the diagnosing and treating of SRRP35 protein related diseases.Of the invention is more Part or all of nucleotide can be used as probe and be fixed in microarray or DNA chip, for analyzing the difference of gene in tissue Different expression analysis and gene diagnosis.The antibody of anti-SRRP35 can be fixed on protein-chip, in test sample SRRP35 albumen.

The present invention also provides it is a kind of detect liver cancer kit, it contain specific amplification SRRP35 primer pair and/ Or SRRP35 specific antibody.

Screening technique

The present invention also provides the methods for carrying out drug screening based on SRRP35.A kind of method is that first screening influences (promotion) SRRP35 expression or active compound, then further test it to cancer cell to the compound filtered out.A kind of screening side Method can be based on the expression of the mRNA of SRRP35.

Wherein, representative cancer cell includes (but being not limited to): liver cancer cells.

Universal method:

(1) acquisition of clinical tissue sample

Liver cancer and cancer beside organism are derived from the liver cancer patient of operative treatment, have signed and have known together with patient before obtaining sample Meaning book.Perform the operation excision liver once in vitro, cancer beside organism other than rapid excised tumor primary tumor and surrounding 5cm puts into liquid Quick-frozen and move to -80 DEG C of refrigerators and save in nitrogen, when transport, is stored in liquid nitrogen.Cancer is done by pathologist with cancer beside organism Last diagnostic out.Sample is divided into I grades of I-II according to Edmondson grade scale.

(2) tissue and cell RNA extracting

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

1) mortar, the stone roller vessel such as pestle and homogenizer are cleaned, and use ddH again respectively₂O and DEPC H₂O is rinsed, then at 180 DEG C It is dried in baking oven about 4 hours, to remove RNA enzyme；

2) appropriate liquid nitrogen is added in mortar to be allowed to be pre-chilled, tissue is taken out rapidly from liquid nitrogen, cuts about 50-100mg Size, the grind into powder in mortar；

3) ground tissue powder is completely moved to as far as possible in the EP pipe of no RNA enzyme with curet, EP pipe is preparatory Appropriate volume (1ml) TRIzol reagent is added, is sufficiently homogenized；

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

5) upper strata aqueous phase is transferred in the EP pipe of new no RNA enzyme as much as possible, isometric isopropanol is added, overturned It mixes 5 times, is stored at room temperature 10 minutes, 4 DEG C, be centrifuged 10 minutes under the conditions of 12000 × g, at this time visible RNA precipitate；

6) supernatant is outwelled, is added 75% ethyl alcohol (1ml/1ml TRIzol), mixed, wash RNA, be centrifuged 4 DEG C, 7500 × g Under the conditions of be centrifuged 5 minutes；

7) supernatant is abandoned, eliminates residual ethanol as far as possible, precipitating spontaneously dries 5-10min(and pays attention to being sure not to be completely dried)；Add Enter 30-50 μ l DEPC H₂O, pressure-vaccum several times, dissolve RNA precipitate；

8) microplate reader measurement RNA concentration and purity OD260/280(1.8-2.0)；Gel electrophoresis observation is whether there is or not degradation, and -80 DEG C save.

Cell strain RNA extracting, the cell of logarithmic growth phase draw culture solution, are added according to the area of culture dish corresponding TRIzol reagent (the 1ml TRIzol/10cm of amount²) lytic cell, it blows and beats several times, the cell being cleaved is collected to no RNA In the EP pipe of enzyme, remaining is according to above-mentioned steps 4) -8) complete chloroform-isopropanol method isolate and purify RNA.

(3) reverse transcription of RNA

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

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

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

2) following components is added in the above system:

It after mixing gently, is placed in PCR instrument, 37 DEG C, 60min.

Obtained cDNA is reversed to be placed in 4 DEG C of preservations.

(4) real-time quantitative PCR

Real-time quantitative PCR reaction usesPremix ExTaq^TM(Perfect RealTime) kit The reaction system of (TaKaRaBiotechnology Co., Ltd. Dalian, China), utilizes Thermal CyclerDice^TM RealTime System(TP800 real-time fluorescence quantitative PCR instrument, TaKaRa) it is operated.The amplified production length of quantitative PCR It is the most suitable (300bp can be extended to) with 80bp -150bp.

Reaction system is as follows:

Reaction condition:

Solubility curve analytical procedure:

95℃ 15sec

60℃ 30sec

95℃ 15sec

Dissociation time is 4sec.

Autofluorescent background signal and threshold value use the default value of instrument setting, and each PCR can be automatically generated after reaction, Ct Value indicates that the fluorescence signal in each reaction tube reaches recurring number experienced when given threshold (10 times of Baseline fluorescence intensity)； The each template of target gene SRRP35 does 3 multiple pipes, and obtained Ct value is averaged；The Ct average value of SRRP35 gene subtracts phase The Ct average value for answering the reference gene (β-actin) of template, obtains Δ Ct.The Δ Ct of liver cancer group subtracts the Δ of corresponding adjacent tissues Ct, obtains Δ Δ Ct value, and the multiple proportion of the SRRP35 gene by liver cancer group and cancer in group is with 2^-ΔΔCtIt indicates.

(5) construction of eukaryotic expression vector

1) template: the cDNA library of people's liver immortalized cells L02.

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

3) according to SRRP35mRNA(NM080743.4) sequence, in conjunction with expression vector pcDNA^TM3.1/myc-His (-) A's Restriction enzyme site design primer, primer sequence such as SEQ ID NO.:3(are positive) and SEQIDNO.:4(it is reversed) shown in.It is wherein anti- Into primer, the terminator codon of SRRP35 is removed, so that c-myc and 6xHi s label on the C-terminal band of SRRP35.It utilizes High fidelity DNA polymerase PrimeSTAR^TMHS DNA Polymerase(TaKaRa), using L02cDNA as template amplification gene SRRP35 overall length opening code-reading frame, 50 μ l overall reaction system ingredients are as follows:

Using two-step PCR (98 DEG C, 10sec；60 DEG C, 90sec), expand 35 circulations.PCR product size about 0.8kb, 1% agarose gel electrophoresis identifies size, is tapped and recovered (gel purification kit: MACHEREY-NAGEL) and meets clip size PCR product.

(TaKaRaBiotechnology Inc.Dalian, the China) double digestion of EcoRV, Hind III recycle PCR product and Vector plasmid pcDNA^TM3.1/myc-His (-) A, endonuclease reaction system are as follows:

37 DEG C endonuclease reaction 1 hour；It is tapped and recovered digestion products.

4) connect: the PCR product and carrier of digestion recycling are mixed according to the ratio of mole ratio (4:1), DNA ligase body Tethers connects, and further includes 2.5 μ l4 × Solution I (TaKaRa Code:D102A), ddH in system₂O polishing is to 10 μ l, and 16 DEG C It connects 2h or even stays overnight；

5) it converts: taking 10 μ l connection products to mix with 100 μ l competent bacterias (TOP10 or DH5 α), place on ice 30min, 42 DEG C of heat shock 90sec, are immediately placed on 5min on ice, are added the not antibiotic LB culture solution of 800 μ l, 37 DEG C, 200rpm shaken cultivation 30min makes thallus recover and expands a generation, and 3000rpm is centrifuged 2min, removes most of supernatant, stays 50- 100 μ l bacterium solutions, gently piping and druming precipitating mixes, and has then uniformly been applied to amicillin resistance (Amp⁺) LB plate on, 37 DEG C Culture 12-16 hours.

6) clone identification: fluid nutrient medium of the bacterium colony that picking is grown after ammonia benzyl resistance screening in plus ampicillin Middle expansion culture, extracts plasmid and carries out digestion identification: taking small III double digestion of pumping plasmid EcoRV, Hind of 1-2 μ g, agarose is solidifying Gel electrophoresis identifies endonuclease bamhi size, carrier pcDNA^TM3.1/myc-His (-) A clip size about 5.5kb, SRRP35 reading frame Clip size about 800bp, the clone for meeting size send the correctness of sequencing confirmation Insert Fragment sequence.

(6) measurement of cell growth curve

1) different types of HCC cell is pressed into 3-5 × 10 according to its growth characteristics³/ 100 holes μ l/ calculate cell total amount, fill After dividing vitellophag, it is diluted to required concentration, is inoculated in 96 orifice plates.Daily every group of three wells, by 5-7 days inoculating cells；

2) cell state and number are observed after cell is substantially adherent.With CCK-8 color developing agent (Cell CountingKit- 8, DOJINDO, Japan) carry out chromogenic reaction, every 100 μ l culture solution adds 10 μ l CCK-8, and 37 DEG C, 5%CO₂Incubator placement is incubated 1h is educated, microplate reader measures the absorbance at 450nm, and record determines the actual initial density of cell, as growth relative zero.

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

4) microscopically observation cellular morphology, Fixed Time Interval measurement, records cell growth condition；

5) general to survey 5 to 7 days.After to be determined, collect data and handled, draw chart with Excel.

(7) cell clonal formation is tested

1) it transfects: using Lipofectamine^TM2000(Invitrogen) transfect cell, be overexpressed or silenced cell in The expression of SRRP35 gene；

2) cell after transfecting, in 6 orifice plates or 35mm culture dish, with normal culture solution culture, digestion is counted afterwards for 24 hours, by one Fixed number mesh is seeded to 100mm culture dish (different cell strain numbers are different), continues culture for 24 hours, is then added according to cell category G418(600-1000 μ the g/ml of debita spissitudo), to screen the positive cell clone of transfection；

3) cultivate 2-3 week, therebetween every 3-5 days replacement fresh mediums and plus G418 screen, up to have it is macroscopic Cell clonal formation；

4) the training liquid in culture dish is sucked, 1 × PBS is washed twice, and coomassie brilliant blue R_250 dyes 2h, is gently rinsed with water Afterwards, then with coomassie brilliant blue staining destainer decolourize 30-60min；

5) Clone formation coloration result is taken pictures, according to identical standard (cell clone size) to thin on each culture dish Born of the same parents clone counts.

(8) Western blotting (Western Blot)

1) prepared by protein sample: after the cell of culture sucks culture supernatant, washed twice with the 1XPBS of pre-cooling, addition 2 × SDS lysate (100mM Tri s-Cl, pH=6.8,4%SDS, 20% glycerol), sufficiently after cracking, boiling water bath heats 10min, 12000 × g is centrifuged 10min, and supernatant is transferred in new pipe,BCA ProteinAssayKit carries out the albumen of acquisition It is quantitative, -80 DEG C of preservations；

2) protein electrophoresis separates: the sample-loading buffer (loading containing 200mM DTT in right amount is added in protein sample Buffer), boiling water bath heats 10min, is slightly centrifuged, and SDS-PAGE proteins gel electrophoresis separates sample；

3) transferring film: running gel, nitrocellulose membrane, thickness (thin) filter paper backing plate are dipped in transferring film buffer (24mMTris, 192mM glycine, 20% methanol) balance 15-20min.It is thin by positive -1 thickness filter paper backing plate -2 layers of running gel of-nitrocellulose membrane - Filter paper backing plate-cathode sequence is put well, wet to turn instrument (XCellSureLock^TM, invitrogen) and 30 volts of transferring film 30-40min；

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

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 incubation Overnight, 0.1%PBST is washed three times, each 5min；

6) secondary antibody: fluorescence secondary antibody dilutes (1:1000) with confining liquid, is incubated at room temperature 30min, and 0.1%PBST is washed three times, every time 5min；

7) sweep film: ODYSSEY infrared imaging system scans nitrocellulose filter, saves image.

(9) soft-agar cloning forms experiment

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

2) prepare 2 × DMEM culture solution (2.5 × DMEM contains 20%FBS)；

3) 2%Agarose and 2 × DMEM culture solution kept the temperature 37 DEG C is mixed by same volume, is added to every hole 0.5ml It in 24 orifice plates, is placed in 4 DEG C of refrigerators, is used after to be solidified；

4) sufficiently the cell of digestion culture is counted at individual cells, be diluted to same concentrations (3000-5000/ 0.5ml)；

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

6) 0.2ml culture solution is added on the soft agar of solidification, is placed in 37 DEG C, 5%CO₂In incubator, continue to cultivate 2-3 Week；

7) growing state of each hole the inside cell clone of microscopically observation, counts, is analyzed.

(10) tumor formation in nude mice

1) mouse used in is the male BLAB/c nu nude mice of 5-6 weeks size, by Shanghai Slac Experimental Animal Co., Ltd. It provides, raises in southern model animal Culture Center；

2) it is subcutaneous (different cell categories inoculation numbers are different) to be inoculated in mouse with identical quantity for the cell that takes that treated, To avoid error caused by individual difference, allogenic cell different disposal can symmetrically be inoculated in same mouse；

3) after visual tumors to appear, every 3 days monitoring tumor sizes, vernier caliper reads tumour major diameter and minor axis,

Gross tumor volume is calculated as follows: volume=major diameter x minor axis²；

4) after continuing to monitor about 7-8 times, data, statistics are arranged.

(11) antibody acquisition and immune detection

1) antigen protein obtains

The cDNA sequence that people's SRRP35 gene is obtained from Genebank database obtains encoder block by PCR amplification, inserts Enter in prokaryotes or eukaryotic expression vector, expresses SRRP35 albumen, and press the purification system of gene engineering expression product Purifying protein.

2) Antibody preparation

Following several method can be used and prepare antibody:

A cell fusion method: with the SRRP35 protein immune animal (including rabbit, goat etc.) of above-mentioned preparation, spleen is obtained Cell, then merged with myeloma cell, and routinely monoclonal antibody technology of preparing prepares monoclonal antibody.

B utilizes phage display library, clones the variable region spleen IgG of immune animal and is expressed as genetic engineering Dan Ke Grand antibody.

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

3) it detects

The antibody (more anti-or monoclonal antibodies) of a preparation, the pathological examination of liver cancer is carried out with histochemical method, negative signal is Liver cancer.

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

C is diagnosed using SRRP35 antibody as one of probe of protein-chip for kinds of tumors.

Embodiment 1: real-time quantitative PCR detects expression of the SRRP35 in clinical sample

The total mRNA obtained using universal method 1~2, and pass through expression of the universal method 4SRRP35 in liver cancer tissue. The primer sequence such as SEQIDNO.:5 and SEQ ID NO.:6 institute used for detecting the real-time quantitative PCR that SRRP35 is expressed in experiment Show.The primer sequence of β-actin as internal reference is as shown in SEQ ID NO.:7 and SEQID NO.:8.

As a result such as Figure 1A: in 32 pairs of clinical samples, having 20 pairs of sample liver cancer tissues by real-time quantitative PCR detection discovery The expression of middle SRRP35 is significantly lower than corresponding cancer beside organism, and downward rate reaches 62.5%, and statistical analysis shows p < 0.01, says The reliability of bright result.We also have detected the expression pattern of SRRP35 in 12 kinds of hepatoma cell strains simultaneously, as shown in Figure 1B, Extremely low expression is presented in 10 kinds of cells in SRRP35, also further illustrates that expression of the SRRP35 in cancer is lower.

Embodiment 2: it is overexpressed the proliferation that SRRP35 gene inhibits liver cancer cells

Carrier for expression of eukaryon is constructed respectively with universal method 5,8, and by the cDNA clone of SRRP35 into pcDNA^TM3.1/ Myc-His (-) A, the plasmid for transfecting building enter liver cancer cells and successful expression, as shown in Figure 2 A.It is true for constructing SRRP35 The primer sequence of nuclear expression carrier such as SEQ ID NO.:9(is positive) and SEQ ID NO.:10(it is reversed) it is shown.

Then, functional experiment growth curve and Clone formation test then with universal method 6~7 are carried out, as a result such as Fig. 2 B, C Shown: the overexpression of SRRP35 inhibits the proliferation and clonality of liver cancer cells Sk-hep-1 and WRL-68.

Embodiment 3: the expression of silencing SRRP35 promotes cell growth

SiRNA for interfering SRRP35 to express is synthesized by Shanghai Ji Ma pharmaceutical Co. Ltd, and sequence is respectively such as SEQIDNO.:11(positive-sense strand) and SEQ IDNO.:12(antisense strand) shown in.NC non-specificity nucleotide as interference control Sequence are as follows: SEQIDNO.:13(positive-sense strand) and SEQIDNO.:14(antisense strand)

Through real-time quantitative PCR testing result as shown in Fig. 3 A, 3B: artificial synthesized interference siRNA can be lowered effectively The expression of SRRP35, the downward that growth curve experiment also demonstrates SRRP35 can promote liver cancer cells Huh-7 and liver cancer The growth of cell Hep3B.

It can be seen that SRRP35 is able to suppress liver cancer cell growth.

The pernicious characterization of embodiment 4:SRRP35 inhibition liver cancer cells

The research object being overexpressed using WRL-68 as SRRP35 is had chosen Huh7 cell and expressed as SRRP35 silencing Research object.The number of cell clones being grown in soft agar is analyzed by universal method 9.

As a result as shown in Figure 4: being overexpressed SRRP35 can effectively inhibit WRL-68 cell and silencing expression SRRP35 can The grade malignancy for effectively facilitating Huh-7 cell shows that SRRP35 influences the pernicious of liver cancer cells.

Embodiment 5:SRRP35 influences liver cancer cells in the one-tenth knurl ability of nude mice by subcutaneous

By universal method 10, by 1 × 10⁶A WRL-68 cell for being overexpressed SRRP35,3 × 10⁶A silencing expression The Huh-7 cell and control cell of SRRP35 is respectively symmetrically injected into that mice belly is subcutaneous, observes the growing state of knurl.

As a result as shown in Fig. 5 A, 5B: the overexpression of SRRP35 is obviously reduced gross tumor volume compared with control group, illustrates: The overexpression of SRRP35 can effectively inhibit WRL-68 cell in the one-tenth knurl ability of nude mice by subcutaneous；And the silencing of SRRP35 makes tumour body Product significantly increases, and illustrates the ability that the downward of SRRP35 has effectively promoted Huh-7 cell nude mice by subcutaneous tumor formation.

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 make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Claims

1. the purposes of a kind of SRRP35 gene or SRRP35 protein assay reagent, which is characterized in that be used to prepare detection liver cancer Reagent or kit.

2. purposes as described in claim 1, which is characterized in that the kit include: to SRRP35 albumen or mRNA into The reagent of row quantitative detection and corresponding label or specification.

3. purposes as described in claim 1, which is characterized in that the reagent includes SRRP35 specific primer, specificity Antibody, probe and/or chip.

4. purposes as described in claim 1, which is characterized in that the reagent includes detection chip, including nucleic acid chip And protein-chip.

5. purposes as claimed in claim 4, which is characterized in that the nucleic acid chip includes substrate and point sample on substrate The specific oligonucleotide probe of liver cancer related gene, the specific oligonucleotide probe of the liver cancer related gene include with SRRP35 gene or the probe of mRNA specific binding.

6. purposes as claimed in claim 4, which is characterized in that the protein-chip includes substrate and point sample on substrate Hepatoma associated protein specific antibody, the specific antibody of the hepatoma associated protein includes the spy of anti-SRRP35 albumen Heterogenetic antibody.

7. purposes as described in claim 1, which is characterized in that the SRRP35 albumen includes fusion protein and non-fused egg It is white.

8. the purposes of a kind of SRRP35 albumen, SRRP35 gene, which is characterized in that be used to prepare inhibit liver cancer cell growth or The drug of proliferation, or it is used to prepare the drug for the treatment of liver cancer.

9. a kind of method for inhibiting liver cancer cell growth or proliferation of external non-therapeutic, which is characterized in that comprising steps of In the presence of SRRP35 albumen, liver cancer cells are cultivated, to inhibit liver cancer cell growth or proliferation.

10. a kind of method of the candidate compound of screening treatment liver cancer, which is characterized in that the method includes the steps:

Wherein, if the expression quantity of the SRRP35 of cell and/or activity are greater than control group in test group, the test chemical combination is indicated that Object is the candidate compound of the expression and/or the active treatment liver cancer for having facilitation to SRRP35.

11. method as claimed in claim 10, which is characterized in that the cell includes: liver cancer cells or normal cell.

12. method as claimed in claim 10, which is characterized in that the method also includes steps:

(b) for the candidate compound obtained in step (a), its inhibition to liver cancer cell growth or proliferation is further tested Effect.

13. method as claimed in claim 12, which is characterized in that in the step (b) comprising steps of in test group, liver cancer Addition test compound in the cultivating system of cell, and observe the quantity and/or growing state of liver cancer cells；In control group, It does not add test compound in the cultivating system of liver cancer cells, and observes the quantity and/or growing state of liver cancer cells；Its In, if the quantity of liver cancer cells or the speed of growth are less than control group in test group, indicate that the test compound is to liver cancer The growth of cell or proliferation have the candidate compound of the treatment liver cancer of inhibiting effect.