CN106048031A - Application of SYNM in preparation of tool for diagnosing or treating endometrial cancer - Google Patents
Application of SYNM in preparation of tool for diagnosing or treating endometrial cancer Download PDFInfo
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Abstract
The invention belongs to the field of biological medicines and discloses application of SYNM in preparation of tool for diagnosing or treating endometrial cancer. Experiments prove that the expressions of an SYNM gene in a normal endometrial tissue and an endometrial cancer tissue are significantly different, so that the SYNM can be used for developing products for diagnosing the endometrial cancer. The invention further discloses the SYNM gene which can be taken as a treatment target of the endometrial cancer. According to the research results of the invention, theoretical basis can be provided for clinicians to formulate individual-based treatment scheme, and a new drug target can be provided for the development of medicines for the endometrial cancer.
Description
Technical field
The present invention relates to cancer diagnosis, treat, predict prognosis field, more particularly it relates to different with detection SYNM
It is often the cancer diagnosis of means, prediction method of prognosis；And activate SYNM gene or the cancer therapeutic agent of protein.
Background technology
Carcinoma of endometrium is the modal malignant tumor being diagnosed to be of American Women's reproductive tract.Great majority suffer from endometrium
The women of cancer uses uterectomy surgical operation therapy；But, the women of about 15% proves that the tumor harboring or recurring is for mesh
Front chemotherapy is difficult to cure.The women of late period, progressivity or recurrent disease is suffered from for these, rate variance of surviving, this is because
Do not prove effectively to assist treatment.Mean survival time after recurrence is 10 months, for the patient recurred 5 years
Survival rate is only 13%.
Carcinoma of endometrium mainly determines diagnosis according to medical history, clinical examination, pathologic finding and various auxiliary examination result
And clinical stages, the method that predominantly detects now includes ultrasound diagnosis, diagnostic curettage, hysteroscopy and lymphography, recall rate
The highest, and easily cause suffering to patient.At present, carcinoma of endometrium does not still have effective early diagnosis and prognostic indicator.
Summary of the invention
An object of the present invention is that providing a kind of diagnoses uterus by detection SYNM gene or protein expression difference
The method of endometrial carcinomas.
The two of the purpose of the present invention are that providing a kind of predicts uterus by detection SYNM gene or protein expression difference
The method of endometrial carcinomas prognosis.
The three of the purpose of the present invention are that providing a kind of treats endometrium by activation SYNM gene or SYNM albumen
The method of cancer.
A kind of method that the four of the purpose of the present invention are to provide medicine for screening treatment carcinoma of endometrium.
The five of the purpose of the present invention are to provide a kind of medicine for treating carcinoma of endometrium.
To achieve these goals, present invention employs following technical scheme:
The invention provides the product of detection SYNM gene or SYNM albumen in preparation carcinoma of endometrium diagnostic tool
Purposes.
Present invention also offers the product of detection SYNM gene or SYNM albumen in preparation prediction carcinoma of endometrium prognosis work
Purposes in tool.
Further, the product of described detection SYNM gene or SYNM albumen includes detecting SYNM gene or the table of SYNM albumen
Reach the product of level.Described product includes can be in conjunction with the nucleic acid of SYNM gene or can be in conjunction with the material (example of SYNM albumen
Such as antibody).Described nucleic acid can detect the expression of SYNM gene；Described material can detect the expression water of SYNM albumen
Flat.
The product of the detection SYNM gene of the present invention can play its function based on the known method using nucleic acid molecules: as
PCR, such as Southern hybridization, Northern hybridization, dot blot, fluorescence in situ hybridization (FISH), DNA microarray, ASO method, height
Flux order-checking platform etc..This product is used can qualitatively, quantitatively or semi-quantitatively to implement to analyze.
The nucleic acid being included in the said goods can be obtained by chemosynthesis, or by containing from biomaterial preparation
Expect the gene of nucleic acid, then use be designed for amplification expectation nucleic acid primer amplification it obtain.
Further, described PCR method is known method, such as, and ARMS (Amplification Refractory
Mutation System, abruptly-changing system is not answered in amplification) method, RT-PCR (reverse transcriptase-PCR) method, nested PCR method etc..Amplification
Nucleic acid can by use dot blotting hybridization method, Surface Plasmon Resonance (SPR method), PCR-RFLP method, in situ RT-PCR
Method, PCR-SSO (sequence specific oligonucleotide) method, PCR-SSP method, AMPFLP (amplifiable fragment length polymorphism) method,
MVR-PCR method and PCR-SSCP (single strand conformation polymorphism) method detect.
Nucleic acid recited above includes the primer expanding SYNM gene, and the primer that product includes can be by by chemistry
Prepared by synthesis, the method that be those skilled in the art will know that by use is suitably designed with reference to Given information, and by changing
Learn synthesis to prepare.
In specific embodiments of the present invention, described nucleic acid is the amplimer used in QPCR experiment, described primer
Sequence such as SEQ ID NO.1 (forward sequence) and SEQ ID NO.2 (reverse sequence) shown in.
Nucleic acid recited above may also include probe, and described probe can be prepared by chemosynthesis, by using this
The method that skilled person knows appropriately designs with reference to Given information, and is prepared by chemosynthesis, or can lead to
Cross the gene containing expectation nucleotide sequence from biomaterial preparation, and use the primer expansion being designed for amplification expectation nucleotide sequence
Increase it to prepare.
The product of the detection SYNM albumen of the present invention can play its function based on the known method using antibody: such as,
ELISA, radioimmunoassay, immunohistochemical method, western blot etc. can be included.
The product of the detection SYNM albumen of the present invention includes antibody or its fragment of specific binding SYNM albumen.Can make
By antibody or its fragment of any structure, size, immunoglobulin class, origin etc., as long as it combines target protein.This
Antibody or its fragment that the detection product of invention includes can be monoclonal or polyclonal.Antibody fragment refers to retain antibody
To the antibody a part of (Partial Fragment) of combination activity of antigen or the peptide containing an antibody part.Antibody fragment can include F
(ab′)2, Fab ', Fab, scFv (scFv), the Fv (dsFv) of disulphide bonding or its polymer, dimerization V district (dual anti-
Body) or containing the peptide of CDR.The product of the detection SYNM albumen of the present invention can include encoding antibody or Encoding Antibody Fragment
The nucleic acid of the separation of aminoacid sequence, comprises the carrier of this nucleic acid, and carries the cell of this carrier.
Antibody can be by well known to a person skilled in the art that method obtains.Such as, preparation retains target all or in part
The polypeptide of protein or the mammalian cell expression vector of integration their polynucleotide of coding are as antigen.Use antigen is exempted from
After epidemic disease animal, from passing through immune animal adaptive immune cell fused bone myeloma cells to obtain hybridoma.Then from hybridization
Antibody collected by tumor culture.SYNM albumen or the antibody enforcement to obtaining of its part of antigen finally can be used as by use
Antigenic specificity purification obtains the monoclonal antibody for SYNM albumen.Polyclonal antibody can be prepared as follows: with above
Identical antigen-immunized animal, collects blood sample from the animal through immunity, isolates serum from blood, then uses
State antigen and serum is implemented antigenic specificity purification.Can be by the antibody obtained with ferment treatment or the antibody obtained by use
Sequence information obtain antibody fragment.
The combination of label and antibody or its fragment can be implemented by method as commonly known in the art.Such as, may be used
With following fluorescent marker protein or peptide: clean protein or peptide with phosphate buffer, add with DMSO, buffer agent, etc. standard
Standby dyestuff, then mixed solution, place 10 minutes then at room temperature.It addition, labelling can the labelling kit of commodity in use, all
Such as biotin labeling reagent box, such as biotin labeling reagent box-NH2, biotin labeling reagent box-SH
(DojindoLaboratories)；Alkali phosphatase enzyme mark test kit such as alkali phosphatase enzyme mark test kit-NH2, alkalescence phosphorus
Acid enzyme labelling test kit-SH (Dojindo Laboratories)；Peroxidase labelling test kit such as peroxidase mark
Note test kit-NH2, peroxidase labelling test kit-NH2 (Dojindo Laboratories)；Phycobniliprotein labelled reagent
Box such as phycobniliprotein labelling kit-NH2, phycobniliprotein labelling kit-SH, B-phycoerythrin labelling kit-NH2,
B-phycoerythrin labelling kit-SH, R-PE labelling kit-NH2, R-PE labelling kit SH
(DojindoLaboratories)；Fluorescent labeling reagent box such as fluorescein labelling kit-NH2, HiLyte Fluor (TM)
555 labelling kit-NH2, HiLyte Fluor (TM) 647 labelling kit-NH2 (Dojindo Laboratories)；And
DyLight 547 and DyLight647 (Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody
Labelling kit, Qdot (TM) antibody labeling test kit (Invitrogen Corporation) and EZ-label protein mark
Note test kit (Funakoshi Corporation).For correct labeling, it is possible to use suitable instrument detects through labelling
Antibody or its fragment.
Sample as the detection product according to the present invention, it is possible to use the tissue sample such as obtained from biopsy experimenter
Or fluid.Sample is not particularly limited, as long as it is suitable to the mensuration of the present invention；Such as, it can include tissue, blood, blood plasma,
Serum, lymph fluid, urine, serous cavity liquid, spinal fluid, synovial fluid, aqueous humor, tear, saliva or its fraction or treated material
Material.
In specific embodiments of the present invention, described sample is from the tissue of experimenter.
In the present invention, " prognosis " refer to that cancer patient is by the suppression such as surgical procedure or the mistake after alleviating tumor growth
Journey or result.In this manual, prognosis can be suppressed or alleviate after tumor growth 1 by surgical procedure, 2,3,4,5,6,
7,8,9,10,15,20 years or more long time life state.Prognosis can be by checking biomarker i.e. SYNM albumen or coding
The gene of SYNM albumen is predicted.Prognosis prediction can be performed such that according to biomarker with or without, or raise or fall
Low, determine that the prognosis of patient is good or bad, or determine the probability of good prognosis or poor prognosis.
In the present invention, " prognosis bona " refer to suppressed for patient by surgical procedure etc. or alleviate tumor growth it
After, patient (such as 3,5,6,7,8,9,10,15,20 years or longer) over a long time does not has critical condition.Or, good prognosis can be anticipated
Refer to survive in the most long-time, without transfer, without recurrence or without sending out again.Such as, prognosis bona can mean at least 3 years or outstanding
It is survival at least 5 years, preferably without transfer or recurrence.The most preferred state of prognosis bona is the long-term survival without disease.As
Used herein, " prognosis bona " can also include any such state, wherein it appeared that disease is as shifted, but
Pernicious low and do not severely impact survival ability.
In the present invention, " prognosis mala " refers to that patient is by the suppression such as surgical procedure or short after alleviating tumor growth
Fatal condition is there is in period (such as 1,2,3,4,5 years or shorter).Or, poor prognosis refers in such short-term dead
Die, shift, recur or send out again.Such as, poor prognosis can mean at least 3 years or Preventive or dead in especially at least 5 years
Die.
Prediction prognosis refers to predict process or the result of status of patient, is not meant to predict with the accuracy of 100%
The process of status of patient or result.Prediction prognosis refers to whether the probability determining some process or result increases, and the most unexpectedly
Taste to be compared by situation about not occurring with some process or result and is determined some process of generation or the probability of result.Such as this
For invention, in the patient that in the present invention, the level of SYNM gene or SYNM albumen reduces, with the patient's phase not showing this feature
Ratio, more likely observes particular procedure or result.
Further, the product of described detection SYNM gene or SYNM albumen can be detection SYNM gene or SYNM albumen
Reagent, can also be to comprise the test kit of described reagent, chip, reagent paper etc., it is also possible to be that the high pass using described reagent measures
Sequence platform.
Present invention also offers the instrument of a kind of diagnosis of endometrial carcinoma, described instrument can detect SYNM gene or SYNM
The expression of albumen.Described instrument includes can be in conjunction with the nucleic acid of SYNM gene or can be in conjunction with the material of SYNM albumen
(such as antibody).Described nucleic acid can detect the expression of SYNM gene；Described material can detect the expression of SYNM albumen
Level.
Further, the character of described nucleic acid and described material is with noted earlier.
Further, the instrument of described diagnosis of endometrial carcinoma includes but not limited to chip, test kit, reagent paper or high flux
Order-checking platform；High-flux sequence platform is the instrument of a kind of special diagnosis of endometrial carcinoma, along with high throughput sequencing technologies
Development, will become the structure of the gene expression profile of a people and work the most easily.By contrast Disease and normal person
The gene expression profile of group, the exception easily analyzing which gene is relevant to disease.Therefore, high-flux sequence is known SYNM
The exception of the gene purposes that fall within SYNM gene relevant to carcinoma of endometrium, equally within protection scope of the present invention.
Present invention also offers a kind of instrument predicting carcinoma of endometrium prognosis, described prediction carcinoma of endometrium prognostic tool
Including can be in conjunction with the nucleic acid of SYNM gene or can be in conjunction with the material (such as antibody) of SYNM albumen.Described nucleic acid can be examined
Survey the mRNA level in-site of SYNM gene；Described material can detect the expression of SYNM albumen.
Further, the character of described nucleic acid and described material is with noted earlier.
Further, the instrument of described prediction carcinoma of endometrium prognosis includes but not limited to chip, test kit, reagent paper or height
Flux order-checking platform；High-flux sequence platform is the instrument of a kind of special diagnosis of endometrial carcinoma, along with high-flux sequence skill
The development of art, will become the structure of the gene expression profile of a people and work the most easily.By contrast Disease and just
The gene expression profile of ordinary person group, the exception easily analyzing which gene is relevant to disease.Therefore, know in high-flux sequence
The exception of the SYNM gene purposes that fall within SYNM gene relevant to carcinoma of endometrium, equally protection scope of the present invention it
In.
The amino acid whose number that the anti-SYNM antibody used in detection product, the diagnostic tool of the present invention or its fragment are identified
Mesh is not particularly limited, as long as antibody can be in conjunction with SYNM.
Present invention also offers a kind of diagnosis of endometrial carcinoma or the method for prediction carcinoma of endometrium prognosis, described method bag
Include following steps:
(1) sample of experimenter is obtained；
(2) SYNM gene or the expression of albumen in detection Samples subjects；
(3) the SYNM gene recorded or the expression of albumen are associated with the whether ill of experimenter.
(4) compared with the control, the expression of SYNM gene or albumen reduces, then this experimenter is diagnosed as endometrium
Cancer, or this experimenter is confirmed as prognosis mala.
Present invention also offers the Therapeutic Method of a kind of carcinoma of endometrium, described method includes activating SYNM gene or SYNM
Albumen.
Further, described method includes the expression promoting SYNM gene, or promotes the expression of SYNM albumen or strengthen SYNM
The activity of albumen.
Present invention also offers the screening technique of a kind of cancer drug, can be by after cancerous cell be added testing drug
Or certain period after cancer model animal is used testing drug measure SYNM gene or the expression of SYNM albumen
Measure cancer drug and improve the effect of cancer prognosis.More specifically, when SYNM gene or the expression of SYNM albumen
When raising after adding or using testing drug or when recovering normal level, this medicine optional is as improving cancer prognosis
Medicine.
Present invention also offers a kind of containing SYNM gene or the medicine of the activator of SYNM albumen.
Present invention also offers the application in the medicine of preparation treatment carcinoma of endometrium of the above-mentioned activator.
The SYNM gene of the present invention or the activator of SYNM albumen are unrestricted, as long as can promote or strengthen SYNM
Or relate to expression or the activity of the material of SYNM upstream or downstream pathway, and for the treatment effective medicine of cancer.
Further, described activator include SYNM gene, SYNM albumen, promoted type miRNA, promoted type transcriptional control because of
Son or promoted type targeting micromolecular compound.
Described activator also includes comprising carrier or the host cell carrying SYNM gene.
On the one hand the activator of the present invention may be used for supplementing disappearance or the deficiency of endogenic SYNM albumen, by improving
The expression of SYNM albumen, thus the carcinoma of endometrium that treatment causes because of SYNM hypoproteinosis.On the other hand may be used for strengthening
The activity of SYNM albumen, thus treat carcinoma of endometrium.
The medicine of the present invention can be administered alone as medicine or use together with other medicines.Can be with the medicine of the present invention
The other medicines that thing is used together are unrestricted, as long as it does not damage the effect of the therapeutic of the present invention or preventive medicine i.e.
Can, it is preferred that the medicine for treatment or prophylaxis of cancer can include such as alkylating agent, such as ifosfamide, ring phosphinylidyne
Amine, dacarbazine, temozolomide, nimustine, busulfan, procarbazine, melphalan and Ranimustine；Antimetabolite, such as
Enocitabine, capecitabine, carmofur, cladribine, gemcitabine, cytosine arabinoside, cytosine arabinoside octadecyl phosphate
(cytarabine ocfosfate), ftorafur, UFT, ftorafur gimeracil oteracil potassium, deoxidation fluorine urine
Glycosides, hydroxyurea, fluorouracil, fludarabine, pemetrexed, pentostatin, mercaptopurine and methotrexate；Plant alkaloid, all
As irinotecan, etoposide, sobuzoxane, docetaxel, nogitecan, Pa Litasai, vinorelbine, vindesine and
Vinblastine；Antitumor antibiotic, such as actinomycin D, aclarubicin, amrubicin, idarubicin, epirubicin, zinostatin
Stimalamer, daunorubicin, doxorubicin, pirarubicin, bleomycin, peplomycin, ametycin and mitoxantrone；
Medicine based on platinum, such as oxaliplatin, carboplatin, cisplatin and nedaplatin；Hormonal medicaments, such as Anastrozole, exemestane,
Estramustine, ethinylestradiol, chlormadinone, goserelin, tamoxifen, dexamethasone, toremifene, bicalutamide, flutamide,
Prednisolone, fostestrol, mitotane, methyltestosterone, medroxyprogesterone, mepitiostane, leuprorelin and letrozole；Biological respinse is modified
Agent, such as interferon-ALPHA, interferon beta, interferon gamma, interleukin, ubenimex, dry BCG and lentinan；With molecular targeted medicine
Thing, such as imatinib (imatinib), gefitinib (gefitinib), gemtuzumab, ozogamicin, Tamibarotene, song
Appropriate monoclonal antibody, tretinoin, bortezomib (bortezomib) and Rituximab etc..
The medicine of the present invention can be prepared as various dosage form as required.Include but not limited to, percutaneous, mucosa, nose, buccal,
Tablet, solution, granule, patch, unguentum, capsule, aerosol or the suppository that Sublingual or per os use.
The route of administration of the medicine of the present invention is unrestricted, as long as it can play desired therapeutic effect or preventive effect is
Can, include but not limited to intravenous, intraperitoneal, ophthalmic, intra-arterial, in lung, be administered orally, in vesicle, intramuscular, tracheal strips, subcutaneous
, by skin, by pleura, local, suck, by mucosa, skin, the intestines and stomach, intraarticular, in ventricle, rectum, vagina,
In skull, in urethra, in liver, in tumor.In some cases, can systematically be administered.It is to be administered partly in some cases.
The dosage of the medicine of the present invention is unrestricted, as long as obtaining desired therapeutic effect or preventive effect, and can
To carry out appropriate determination according to symptom, sex, age etc..The medicine of the present invention or the dosage of prophylactic agent can make use-case
As the therapeutic effect of disease or preventive effect are determined as index.
In the context of the present invention, " diagnosis of endometrial carcinoma " both includes judging that experimenter has suffered from intrauterine
Film cancer, also include judging whether experimenter exists the risk suffering from carcinoma of endometrium.
" treatment " used herein contains what treatment in the mammal such as mankind suffering from relevant disease or disease was correlated with
Disease or morbid state, and include:
(1) prevention disease or morbid state occur in mammal, especially susceptible in described disease when this mammal
Diseased state, but not yet it is diagnosed when suffering from this morbid state；
(2) suppression disease or morbid state, i.e. stop it to occur；Or
(3) disease or morbid state are alleviated, even if disease or morbid state disappear.
Term " is treated " and is usually directed to treat the mankind or animal (such as, applied by veterinary), wherein can reach some pre-
The therapeutic effect of phase, such as, suppresses the development (including reducing development speed, making development stop) of disease, improves disease and healing
Disease.Also include the treatment as preventive measure (such as prevention).This disease danger is developed into the most not developing into disease
The purposes of patient of danger, is also included within during term " treats ".
Advantages of the present invention and beneficial effect:
The molecular marker being found that a kind of diagnosis of endometrial carcinoma of the present invention, uses this molecular marker can be at son
The early stage that endometrial carcinoma occurs can be used as judging, it is provided that the survival rate of patient.
It addition, by the prognosis predicting patient, the present invention can provide significant information to determine treatment side for patient
Case strategy.
The medicine of the activator including SYNM gene or albumen of the present invention can be used as controlling of new carcinoma of endometrium
Treat medicine.
Accompanying drawing explanation
Fig. 1 shows and utilizes Western blot detection SYNM albumen endometrial tissues and normal endometrial tissue
In expression；
Fig. 2 show utilize Western blot detect SYNM gene overexpression situation.
Specific embodiment
The present invention is further detailed explanation with embodiment below in conjunction with the accompanying drawings.Following example are merely to illustrate this
Invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment, generally according to conventional strip
Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in, or according to the condition proposed by manufacturer.
Embodiment 1 gene chip screening difference expression gene
1, sample collection:
Endometrial sample: collect endometrial carcinoma, the equal underwent operative of all patients is treated, paraffin mark of performing the operation
These 10 examples.All patients all make a definite diagnosis through pathologic finding suffers from carcinoma of endometrium.Other enter set condition: before all patients are admitted to hospital
Do not accepted any treatment: do not merge other malignant tumor；Do not merge other hormone related disorders；Complete clinical data.
10 patients with endometrial cancer, fall ill 58 years old mean age.Patient's main clinical manifestation be abnormal vaginal bleeding,
Lower abdominal pain, menoxenia, neoplasm etc., also have some patients non-evident sympton to find in Physical Examination.Carcinoma of endometrium
Specimen is diagnosed as carcinoma of endometrium through HE stained, tectology.
Normal endometrial tissue sample: 10 example normal endometrium operation paraffin specimens.The patient of samples sources is suffered from
Disease includes: hysteromyoma, uterine prolapse, wing moon bright bulging, proctoptosis.
2, the acquisition of RNA is organized
Use Trizol one-step method to extract total tissue RNA, read at 260nm and 280nm by Nanodrop ND-1000
Absorbance (A) measure RNA solution purity.Through 1% denaturing formaldehyde agarose gel electrophoresis, observe under ultraviolet transmission light,
The integrity of detection RNA.
3, gene chip hybridization and scanning
After the linearized amplification of total serum IgE, cy3-UTP labelling, the cRNAs after fluorescent labeling uses RNEASY Mini Kit
Purification, carries out fragmentation process with the RNA Fragmentation Reagents of Amhion to the cRNAs that labelling is good.Use U.S.
People's full genome chip of expression spectrum (4x 44K gene) of Agilent company of state, 65 DEG C of hybridization 17h in chip hybridization stove, then
Eluting, dyeing, finally by Agilent DNA MicroarrayScanner scanner scanning.
4, chip data processes and analyzes
Chip after hybridization, after chip scanner reads data point, imports data to analyze software, for two groups of ratios
Natural logrithm absolute value more than 2.0 or less than 0.5 gene as difference expression gene.
5, statistical procedures
Use SPSS 13.0 statistical software carry out data analysis, group difference compare employing one factor analysis of variance method, P <
0.05 difference has significant.
6, result
Chip results shows, filters out 654 difference tables between endometrial and normal endometrial tissue altogether
Reach gene, the gene 421 that wherein expression raises, the gene 233 that expression is lowered.
The difference expression gene filtered out verified by embodiment 2 large sample
Consider prior art yet there are no the gene carrying out studying about this gene and carcinoma of endometrium dependency as time
Select gene, consider the result of gene sequencing simultaneously, select SYNM gene (it is expressed in endometrial tissues and lowers) to carry out
Checking.
1, sample collection
Endometrial 50 example, normal endometrial tissue 60 example is collected according to the method for embodiment 1.
2, verify in mRNA level in-site
2.1 extract tissue RNA
Step is with embodiment 1.
2.2 reverse transcription
Use Reverse Transcriptase kit, with RT Buffer, l μ g total serum IgE is carried out converse record and synthesize cDNA.Use 25 μ l
Reaction system, each sample takes 1 μ g total serum IgE as template ribonucleic acid, is separately added into following components in PCR pipe: DEPC water, 5 × inverse
Transcription buffer, 10mmol/l dNTP, 0.1mmol/l DTT, 30 μm mol/l Oligo dT, 200U/ μ l MMLVRT, template
RNA.Hatch 1 hour for 42 DEG C, 72 DEG C 10 minutes, of short duration centrifugal.
2.3PCR
Primer-design software Primer Premier 5.0 is used to design mRNA fluorescent quantitation upstream and downstream PCR primer, synthesis
Primer sequence, uses the operation of SYBR Green PCR Master Mix test kit, and concrete steps by specification operates, and adopts
By 25 μ l reaction systems, each sample arranges 3 parallel pipes, and all amplified reactions are above can with ensure result
By property.Preparing following reaction system (as shown in table 1), operations is all carried out on ice:
The each component of table 1 quantitative fluorescent PCR and respective volume
With GAPDH as internal reference, using SYBR Green I as fluorescent marker, at Light Cycler quantitative fluorescent PCR
The enterprising performing PCR of instrument reacts, and determines that purpose band, Δ Δ CT method carry out relative quantification by melt curve analysis analysis and electrophoresis.
SYNM gene primer sequence is as follows:
Forward primer: 5 '-AAGTAATCCAGAGATAGTGA-3 ' (SEQ ID NO.1)；
Downstream primer: 5 '-GTGAGTCGGTATAGTGAT-3 ' (SEQ ID NO.2).
GAPDH gene primer sequence is as follows:
Forward primer: 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.3)；
Downstream primer: 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.4)
2.4 result
Result shows, compared with normal endometrial tissue, in endometrial, the mRNA level in-site of SYNM gene is bright
Aobvious downward, relative expression quantity is 0.13 ± 0.011, and difference has statistical significance (P < 0.05).
3, verify on protein level
3.1 extract tissue total protein
The operation of protein extraction is carried out according to the description of EpiQuik tissue/cell total protein extraction test kit.
3.2Western blot detects
The protein quantification of extraction is carried out SDS-PAGE electrophoresis, carry out afterwards transferring film, closing, one anti-hatch, two anti-hatch,
Colour developing.
3.3 statistical procedures
Image J software is used to be analyzed, with β-actin as internal reference, by SYNM albumen the gray value of protein band
The gray value of band is normalized.Result data is all to represent in the way of mean+SD, uses
SPSS13.0 statistical software carries out statistical analysis, and difference between the two uses t inspection, it is believed that when P < has system when 0.05
Meaning learned by meter.
3.4 result
Result is as it is shown in figure 1, compared with normal endometrial tissue, in endometrial, SYNM protein level is notable
Reducing, difference has statistical significance (P < 0.05).
Embodiment 3 SYNM gene overexpression
1, plasmid construction
Coded sequence design amplimer according to SYNM gene, being designed as of primer is well known to those skilled in the art.
The coded sequence of the SYNM gene of amplification total length from the cDNA library (clontech company, article No.: 638831) becoming Human fetal spleen,
Above-mentioned cDNA sequence is inserted in eukaryotic expression vector pcDNA3.1, connects the recombinant vector pcDNA3.1-SYNM obtained
For subsequent experimental.
2, the cultivation of endometrial carcinoma cell and transfection
2.1 cells are cultivated
Ishikawa3-H-12 cell line all by the culture bottle adhere-wall culture of 50ml in RPMI 1640 culture medium (containing 10%
Hyclone, 100U/ml penicillin, 100g/ml streptomycin) in, at 37 DEG C, 5%CO2The incubator of saturated humidity is trained continuously
Support.
2.2 cell transfecting
1, day before transfection is by 0.5-2*105Individual tumor cell is suspended in the 500 μ l culture medium without antibiotic, is seeded to
24 well culture plates.
2, transfection cell density on the same day should reach 80%-90%, prepares following complex A: 1 μ g plasmid DNA is diluted in nothing
In blood serum medium, mix gently；Complex B: take 4 μ l Lipofectamine2000 and be diluted in serum-free medium, mixed
Even.
3, complex A and B is mixed, mix gently, incubated at room.
4,100 μ l liposome compounds are added in tumor cell, mix the most gently, cell is put into 37 DEG C and contains
5%CO2Incubator hatches 5-7 hour.
5, add 1ml and contain 2 times of normal serums and the growth medium of antibiotic concentration, continue to cultivate cell 18-24 hour.
3, the process LAN situation of QPCR experiment detection pcDNA3.1-SYNM is utilized
3.1 extract cell total rna utilizes conventional method to operate.
3.2 reverse transcription
Step is with embodiment 2.
3.3QPCR
Step is with embodiment 2.
3.4 result
Result shows, pcDNA3.1-SYNM can success process LAN, relative expression quantity is 7.95 ± 0.68, and difference has
Statistical significance (P < 0.05).
3, Western blot experiment detection pcDNA3.1-SYNM process LAN situation
Step is with embodiment 2.
Result is as in figure 2 it is shown, compared with transfection pcDNA3.1 group, transfect SYNM albumen in the cell of pcDNA3.1-SYNM
Content substantially increase, difference has statistical significance (P < 0.05).
The expression of the embodiment 4 SYNM gene mensuration to endometrial carcinoma cell multiplication capacity
1, step:
Application 5-bromo-2 ' Brdurds (Brd U) labelling and detection kit.Reference reagent box operation instruction, cell
Transfection (transfection procedure is with embodiment 3), after 48 hours, is inhaled and is abandoned culture medium, addition Brd U labelling culture medium, 37 DEG C, 5%CO2Training
Support and case is cultivated 60min.Discard culture medium, after PBS rinses, fix with 70% ethanol, overnight.The anti-Brd for mice is resisted with one
U antibody, two resist the fluorescent antibody of the anti-mouse for band FITC, carry out immunoreation.Then flow cyctometry detection is carried out.
2, Brd U incorporation result:
Result shows: transfection pcDNA3.1 groups of cells incorporation efficiency average out to: (27.06 ± 1.21) %；pcDNA3.1-SYNM
Groups of cells incorporation efficiency average out to (13.54 ± 0.68) %, difference has statistical significance (P < 0.05).Above-mentioned experimental result table
Bright, the SYNM gene expression inhibition propagation of endometrial carcinoma cell.
The impact of embodiment 7 Transwell experiment detection SYNM gene expression cell migration
Step:
1, from the refrigerator of-80 DEG C, frozen Matrigel thing is taken out, then under 4 DEG C of temperature conditionss overnight so that it is become
Become liquid.
2, take out 200 μ l serum-free cell culture mediums, add the Matrigel reagent of 50 μ l, at cryogenic conditions, preferably
Ice face operates and is mixed evenly, respectively add 100 μ l, be placed on 37 DEG C, the incubator of carbon dioxide is cultivated 5h, this
Between often observe liquid case, when liquid become slightly white time, illustrate that it has turned into solidification state.
3, cell (according to the embodiment 3 step operation) trypsinization that will transfect, clear by the culture medium without serum
Wash 3 times, then count, then be made into cell suspending liquid.
4, gel is washed 1 time gently, then by 2*10 by the culture medium of serum-free5Cell suspension is in 100 μ l RPMI
In 1640, it is inoculated in room on transwell.
5, lower room adds 600 μ l 10%FBS RPMI 1640.
6, in 37 DEG C of incubators, after cultivating 12h, take out transwell cell, often organize all 4 samples of repetition.
7, wherein 1 cell discards culture medium, washes 3 times with the PBS without calcium, and 4% poly first ferment fixes 10min, and cotton swab is wiped
Falling the cell that upper strata does not migrates, PBS washes 3 times, violet staining or Giemsa dyeing, examines under a microscope.Remain 3 cells
Its lower floor's migrating cell is digested with 0.25% membrane proteolytic enzyme, computation migration cell number.
Result:
Migration experimental result shows, transfects pcDNA3.1 groups of cells cell migration number average out to 238, transfection
PcDNA3.1-SYNM groups of cells cell migration number average out to 126, difference has statistical significance (P < 0.05).
The explanation of above-described embodiment is only intended to understand the method for the present invention and core concept thereof.It should be pointed out that, for this
For the those of ordinary skill in field, under the premise without departing from the principles of the invention, it is also possible to the present invention is carried out some improvement
And modification, these improve and modify also by the protection domain falling into the claims in the present invention.
Claims (10)
1. the product of detection SYNM gene or SYNM albumen is being prepared diagnosis of endometrial carcinoma or is being predicted carcinoma of endometrium prognosis
Application in instrument.
Application the most according to claim 1, it is characterised in that the product of described detection SYNM gene or SYNM albumen includes
The product of the expression of detection SYNM gene or SYNM albumen.
Application the most according to claim 1 and 2, it is characterised in that described product includes can be in conjunction with the core of SYNM gene
Acid or can be in conjunction with the material of SYNM albumen；Described nucleic acid can detect the expression of SYNM gene；Described material can
The expression of detection SYNM albumen.
Application the most according to claim 3, it is characterised in that described nucleic acid is the special expansion used in real-time quantitative PCR
Increase the primer of SYNM gene as shown in SEQ ID NO.1 and SEQ ID NO.2.
5. a diagnosis of endometrial carcinoma or the instrument of prediction carcinoma of endometrium prognosis, it is characterised in that described instrument includes energy
Enough instruments of the expression of detection SYNM gene or SYNM albumen.
Instrument the most according to claim 5, it is characterised in that described instrument include can in conjunction with the nucleic acid of SYNM gene or
Person can be in conjunction with the material of SYNM albumen；Described nucleic acid can detect the expression of SYNM gene；Described material can detect
The expression of SYNM albumen.
Instrument the most according to claim 6, it is characterised in that described nucleic acid is the special expansion used in real-time quantitative PCR
Increase the primer of SYNM gene as shown in SEQ ID NO.1 and SEQ ID NO.2.
8. the medicine treating carcinoma of endometrium, it is characterised in that described pharmaceutical pack swashing containing SYNM gene or SYNM albumen
Agent alive.
Medicine the most according to claim 8, it is characterised in that described activator can promote or strengthen SYNM or relate to
The expression of the material of SYNM upstream or downstream pathway or activity.
10. the application in the medicine of preparation treatment carcinoma of endometrium of the activator described in claim 8 or 9.
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