CN107385100A - Purposes of the MCM8 as sdenocarcinoma of stomach Metastatic Marker - Google Patents
Purposes of the MCM8 as sdenocarcinoma of stomach Metastatic Marker Download PDFInfo
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- CN107385100A CN107385100A CN201710823753.6A CN201710823753A CN107385100A CN 107385100 A CN107385100 A CN 107385100A CN 201710823753 A CN201710823753 A CN 201710823753A CN 107385100 A CN107385100 A CN 107385100A
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Abstract
The invention discloses diagnosis and treatment targets of the MCM8 as sdenocarcinoma of stomach.MCM8 can be used to develop the product of diagnosis sdenocarcinoma of stomach, the medicine of exploitation treatment sdenocarcinoma of stomach accordingly.The achievement in research of the present invention is provided fundamental basis for clinician's formulation personalized therapy program and can provide new drug target for the exploitation of gastric gland cancer drug.
Description
Technical field
The present invention relates to diagnosing tumor, therapy field, more particularly it relates to abnormal for means to detect MCM8
Diagnosing tumor method；And suppress the tumor therapeutic agent of MCM8 genes or protein.
Background technology
Stomach cancer is derived from the malignant tumour of gastric epithelial cell, predominantly gland cancer.The whole world is newly diagnosed to be stomach cancer within 2008
Nearly 1,000,000, number of dying of illness 740,000, be the whole world incidence of disease the 4th, the cancer of fatal rate second, is a kind of clinically common
Malignant tumour.Although stomach cancer whole world total incidence has declined, the gastric cancer cases for having nearly 2/3rds concentrate on economic owe
Developed countries and regions, China and other East Asian countries are occurred frequently.According to《Life Times》Latest edition " the Cancer in China of issue in 2014
Map ".It is about 3120000 that tumor cases are newly sent out in China every year, average daily 8550, per minute to have 6 people to be diagnosed as cancer
Disease, there are 5 people to die from cancer.And shown according to the investigation of the World Health Organization, China belongs to high incidence area of gastric cancer, Liaoning, Shandong, sweet
It is even more serious that respectful, Jiangsu, Fujian etc. save situation.Clinically, although the treatment of early carcinoma of stomach oneself through achieving major progress, often
Surgically excision plus regional lymph nodes are cleaned and postoperative chemicotherapy is supported, but the long-term survival rate of late gastric cancer is still very
It is low.Many documents and research show that invasion and attack, the transfer of tumour result in the death of most of patients with gastric cancer and in the bad of stomach cancer
Play the part of pivotal player in prognosis.The invasion and attack of stomach cancer, transfer, which are a polygenes regulation and control, multiple-factor participates in, multi-step is carried out answers
Miscellaneous biological process, it is considered that the motion of cancer cell and invasive ability are the essential conditions for producing transfer.But for late period
Stomach cancer, it is attacked and shifts potential molecular mechanism and is still not clear.The mark sex factor of reliable tumor prognosis is thus found, and
The molecular target of metastases and invasion and attack can effectively be suppressed by finding, and improved the prognosis of tumour, be the focus of Recent study,
It is an emphasis and the difficult point in tumor research.Therefore, urgent need differentiates the important molecule of stomach cancer progressive stage, researches and develops new
Drug target, the prognosis for the treatment of and patient for tumour provide valuable help.
The content of the invention
An object of the present invention is that provide one kind realizes gastric gland metastasis of cancer by detecting MCM8 gene expression differences
Diagnosis, sdenocarcinoma of stomach prediction prognosis, the method for sdenocarcinoma of stomach prognosis evaluation.
The second object of the present invention is to provide a kind of treats the side of gastric gland metastasis of cancer by suppressing MCM8 gene expressions
Method.
The third object of the present invention is to provide a kind of method for screening gastric gland metastasis of cancer medicine.
The fourth object of the present invention is to provide a kind of medicine for being used to treat gastric gland metastasis of cancer.
To achieve these goals, present invention employs following technical scheme：
The invention provides detection MCM8 reagent to prepare gastric gland metastasis of cancer diagnosis, sdenocarcinoma of stomach prediction prognosis, sdenocarcinoma of stomach
Application in prognosis evaluation instrument.
Further, the reagent of the detection MCM8 includes the reagent of detection MCM8 gene expression amounts.
Further, the reagent of the detection MCM8 includes the reagent that can quantify MCM8 gene mRNAs, and/or can determine
Measure the reagent of MCM8 albumen.
The reagent of the quantitative MCM8 gene mRNAs of the present invention can be based on playing its work(using the known method of nucleic acid molecules
Energy：Such as PCR, such as Southern hybridization, Northern hybridization, dot blot, FISH (FISH), DNA microarray, ASO
Method, high-flux sequence platform etc..It can qualitatively, quantitatively or semi-quantitatively implement analysis using the reagent.
Further, the PCR method is known method, for example, ARMS (Amplification Refractory
Abruptly-changing system is not answered in Mutation System, amplification) method, RT-PCR (reverse transcriptase-PCR) method, nested PCR methods etc..Amplification
Nucleic acid can be by using dot blotting hybridization method, Surface Plasmon Resonance (SPR methods), PCR-RFLP methods, RT-PCR in situ
Method, PCR-SSO (sequence specific oligonucleotide) method, PCR-SSP methods, AMPFLP (amplifiable fragment length polymorphism) method,
MVR-PCR methods and PCR-SSCP (single-strand conformation polymorphism) methods detect.
The reagent that MCM8 gene mRNAs can be quantified can be the specific primer of MCM8 genes or transcript, also may be used
To be the specific recognition probe of MCM8 genes or transcript, or include primer and probe simultaneously.
The specific primer of MCM8 genes or transcript recited above includes the specific amplified used in real-time quantitative PCR
The primer of MCM8 genes.In the specific embodiment of the present invention, the primer sequence such as SEQ ID NO.1 and SEQ ID
Shown in NO.2.
Primer can be prepared by chemical synthesis, by using those skilled in the art will know that method reference known believe
Breath is prepared to be suitably designed by chemical synthesis.
Probe can be prepared by chemical synthesis, by using those skilled in the art will know that method reference known believe
Breath appropriately designs, and is prepared by chemical synthesis, or can be by being prepared from biomaterial containing expectation nucleotide sequence
Gene, and using designed for amplification it is expected nucleotide sequence primer expand it to prepare.
The reagent of the quantitative MCM8 albumen of the present invention can be based on playing its function using the known method of antibody：For example,
ELISA, radioimmunoassay, immunohistochemical method, western blot etc. can be included.
The reagent of the quantitative MCM8 albumen of the present invention includes the antibody or its fragment of specific binding MCM8 albumen.It can make
With the antibody or its fragment of any structure and size, immunoglobulin class, origin etc., as long as it combines target protein.This
The antibody or its fragment that the detection product of invention includes can be monoclonals or polyclonal.Antibody fragment refers to reservation antibody
Antibody a part of (Partial Fragment) to the binding 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 areas it is (dual anti-
Body) or peptide containing CDR.The reagent of the quantitative MCM8 albumen of the present invention can include encoding antibody or Encoding Antibody Fragment
The nucleic acid of the separation of amino acid sequence, the carrier comprising the nucleic acid, and carry the cell of the carrier.
Antibody can obtain by the way that well known to a person skilled in the art method.Retain target all or in part for example, preparing
The polypeptide of protein integrates the mammalian cell expression vector for encoding their polynucleotides as antigen.Exempted from using antigen
After epidemic disease animal, from the immune animal adaptive immune cell of process and myeloma cell is merged to obtain hybridoma.Then from hybridization
Knurl culture collects antibody.The antibody of acquisition can finally be implemented by using MCM8 albumen for being used as antigen or part thereof
Antigentic specificity is purified to obtain the monoclonal antibody for MCM8 albumen.Polyclonal antibody can be prepared as follows：With with it is above
Identical antigen-immunized animal, blood sample is collected from by immune animal, serum is isolated from blood, then using upper
State antigen and antigentic specificity purifying is implemented to serum.The antibody or the antibody by using acquisition that can be obtained by using ferment treatment
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.For example, can
With following fluorescent marker protein or peptide：Clean protein or peptide with phosphate buffer, addition DMSO, buffer, etc. standard
Standby dyestuff, then mixed solution, is placed 10 minutes then at room temperature.In addition, the labelling kit of commercialization can be used in mark, it is all
Such as biotin labeling reagent box, such as biotin labeling reagent box-NH2, biotin labeling reagent box-SH
(DojindoLaboratories)；Alkali phosphatase enzyme mark kit such as alkali phosphatase enzyme mark kit-NH2, alkaline phosphorus
Sour enzyme labelling kit-SH (Dojindo Laboratories)；Peroxidase labelling kit such as peroxidase mark
Remember kit-NH2, peroxidase labelling 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 kits-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, the labelling kit-NH2 of HiLyte Fluor (TM) 647 (Dojindo Laboratories)；And
DyLight 547 and DyLight647 (Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody
Labelling kit, Qdot (TM) antibody labeling kit (Invitrogen Corporation) and EZ- label protein marks
Remember kit (Funakoshi Corporation).For correct labeling, can be detected using suitable instrument by mark
Antibody or its fragment.
The acquisition of the sample for being used to detect the detection of MCM8 gene expression amounts of the present invention is the ordinary skill in the art, preferably
Noninvasive may be selected or the method with minimally-invasive property obtains.
Described sample can be (but are not limited to)：Peripheral blood, marrow, lymph node, abdominal cavity cleaning fluid, parietal cell or stomach
Liquid.In specific embodiments of the present invention, tissue of the sample from subject.
As well known to those skilled in the art, the cell of tumor tissues can be shed in body fluid, and these cells to come off are referred to as
Circulating tumor cell, the property of circulating tumor cell is identical with the property of tumour cell in tumor tissues, therefore detects in body fluid
The property can of circulating tumor cell represents the property of tumor tissues especially in blood.For the present invention, detection is passed through
MCM8 gene expressions can be used for diagnosing sdenocarcinoma of stomach transfer in tumor tissues, and can easily draw can also be swollen by detecting circulation
MCM8 gene expressions are shifted to diagnose sdenocarcinoma of stomach in oncocyte.
Present invention also offers a kind of for gastric gland metastasis of cancer diagnosis, sdenocarcinoma of stomach prediction prognosis, sdenocarcinoma of stomach prognosis evaluation
Instrument, the instrument can detect MCM8 gene expression amounts.
Further, the instrument includes the reagent that can quantify MCM8 gene mRNAs, and/or can quantify MCM8 albumen
Reagent.
Generally, described reagent is present in appropriate container.Diluent such as deionized water can be used described to draw every kind of
Thing or probe are adjusted to the concentration of at least one requirement, are sub-packed in container.
Further, the reagent that can quantify MCM8 gene mRNAs includes the specific amplified used in real-time quantitative PCR
The primer of MCM8 genes, the primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
Further, it is described to include for gastric gland metastasis of cancer diagnosis, sdenocarcinoma of stomach prediction prognosis, the instrument of sdenocarcinoma of stomach prognosis evaluation
But it is not limited to chip, kit, test paper or high-flux sequence platform；High-flux sequence platform is a kind of special tool(s), with height
The development of flux sequencing technologies, the structure of the gene expression profile of a people will be turned into and very easily worked.By contrasting disease
Patient and the gene expression profile of normal population, the exception for easily analyzing which gene are related to disease.Therefore, in high flux
Know that the exception of MCM8 genes is related to gastric gland metastasis of cancer in sequencing and fall within the new application for having used the MCM8 of the present invention, equally
Within protection scope of the present invention.
The reagent for extracting nucleic acid is may also include in the kit of the present invention, for PCR reagent, for dyeing or showing
Reagent of color etc..For example, these reagents include but is not limited to：Extract, amplification liquid, hybridization solution, nitrite ion, washing lotion etc..
In addition, the specification of method of description detection gastric gland metastasis of cancer is may also include in described kit etc..
Kit of the present invention, which can include, is suitable to a variety of different of practical (being such as directed to different detection methods)
Reagent, however it is not limited to cited reagent at present, as long as judge that sdenocarcinoma of stomach turns based on the detection of MCM8 genes or transcript
The reagent of shifting is all contained in the scope of the invention.
Present invention also offers the side of a kind of gastric gland metastasis of cancer diagnosis, sdenocarcinoma of stomach prediction prognosis or sdenocarcinoma of stomach prognosis evaluation
Method, methods described comprise the following steps：
(1) sample of subject is obtained；
(2) MCM8 gene expression doses in Samples subjects are detected；
(3) the MCM8 gene expression doses measured are associated with the disease associated of subject.
(4) compared with normal control, MCM8 gene expression doses statistically raise, and show that subject is judged gastric gland
Cancer has occurred and that subject's prognosis mala of transfer or prediction gastric gland metastasis of cancer or assesses the subject of gastric gland metastasis of cancer
Recur.
Present invention also offers a kind for the treatment of method of gastric gland metastasis of cancer, methods described includes suppressing MCM8 genes or MCM8
Albumen.
Further, methods described includes suppressing the expression of MCM8 genes, or suppresses the expression of MCM8 genes or suppress MCM8
The activity of albumen.
, can be by after testing drug be added to cancer cell present invention also offers a kind of screening technique of tumour medicine
Or some period after testing drug is applied to tumor model animal measures the expression of MCM8 genes or MCM8 albumen
Improve the effect of tumor prognosis to determine tumour medicine.More specifically, when MCM8 genes or the expression of MCM8 albumen
When being reduced after addition or administration testing drug or recovering normal level, the medicine may be selected as improvement tumor prognosis
Medicine.
Present invention also offers a kind of medicine for treating gastric gland metastasis of cancer, the medicine includes MCM8 inhibitor.
The MCM8 of present invention inhibitor is unrestricted, as long as the inhibitor can suppress MCM8 or be related to MCM8 upstreams
Or expression or the activity of the material of downstream pathway, and for treating the effective medicine of metastases.
Present invention also offers application of the above-mentioned inhibitor in the medicine for preparing treatment gastric gland metastasis of cancer.
Further, the inhibitor includes the RNA interfering for MCM8 gene expressions, or negative regulation miRNA, negative regulation
Transcription regulatory factor or suppressive the targeting micromolecular compound of type.
The inhibitor of the present invention can be used by any of mode compounding pharmaceutical composition in this area.This group
Compound includes active component, plus one or more pharmaceutically acceptable carrier, diluent, filler, bonding agent and other taxes
Shape agent, this depends on administering mode and designed dosage form.The known treatment of this area branch art personnel it is inert inorganic or
Organic carrier includes but is not limited to lactose, cornstarch or derivatives thereof, talcum, vegetable oil, wax, fat, polyhydroxylated
Compound such as polyethylene glycol, water, sucrose, ethanol, glycerine, such, various preservatives, lubricant, dispersant, flavouring.
Moisturizing is cut to pieces, antioxidant, sweetener, colouring agent, stabilizer, salt, buffer solution is such can also be added thereto, these material roots
The stability of formula is used to help according to needs or is favorably improved activity or its biological effectiveness or is produced in the case of oral
Raw acceptable mouthfeel or smell, the preparation that can be used in such a composition can be the form of its original chemical in itself,
Or the form of its pharmaceutically acceptable salt is optionally used, inhibitor of the invention can be administered alone, or with various combinations
Administration, and combining form is administered together with other healing potions.This area may be selected in the composition so prepared as needed
Any appropriate mode is administered inhibitor known to technical staff.It is by safe and effective amount during using pharmaceutical composition
Inhibitor of the invention be applied to people, wherein oral safe and effective amount typically at least about 100 micrograms/kg body weight.Certainly, have
Body dosage is also contemplated that the factors such as method of administration, patient health situation, within the scope of these are all skilled practitioners technical ability.
The medicine of the present invention can be prepared into various formulations as needed.Including but not limited to, percutaneous, mucous membrane, nose, buccal,
Tablet, solution, granule, patch, paste, capsule, aerosol or suppository sublingual or orally 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 i.e.
Can, include but is not limited to intravenously, intraperitoneal, intraocular, intra-arterial, intrapulmonary, orally, in vesicle, intramuscular, tracheal strips, subcutaneously
, local by pleura by skin, suction, intra-ventricle intra-articular by mucous membrane, skin, stomach, rectum, vagina,
In skull, in urethra, in liver, in knurl.In some cases, can systematically be administered.It is to be partly administered in some cases.
The dosage of the medicine of the present invention is unrestricted, can as long as obtaining desired therapeutic effect or preventive effect
To carry out appropriate determination according to symptom, sex, age etc..The medicine of the present invention or the dosage of prophylactic agent can be with use examples
Therapeutic effect or preventive effect such as to disease determine as index.
" MCM8 genes " (NC_000020.11 (5950652..6000941)) of the present invention) sequence can be with ncbi database
In inquired about.
In the context of the present invention, " gastric gland metastasis of cancer diagnosis " includes judging whether subject has occurred and that sdenocarcinoma of stomach turns
Move, judge that whether subject whether there is the risk of gastric gland metastasis of cancer, judges gastric gland metastasis of cancer patient relapse and metastasis.
" treatment " used herein is covered treatment-related in such as mankind of the mammal with relevant disease or illness
Disease or morbid state, and including：
(1) prevention disease or morbid state occur in mammal, especially when the mammal is susceptible in the disease
Diseased state, but when being not yet diagnosed with this morbid state；
(2) suppress disease or morbid state, that is, prevent its generation；Or
(3) disease or morbid state are alleviated, even if disease or morbid state disappear.
Term " treatment " is usually directed to the treatment mankind or animal (for example, being applied by animal doctor), wherein can reach some pre-
The therapeutic effect of phase, for example, suppressing the development (including reduce development speed, stop development) of illness, improving illness and healing
Illness.Also include the treatment as precautionary measures (such as prevention).Pair illness is not developed into also but develop into illness danger
The purposes of the patient of danger, is also included within term " treatment ".
The advantages of the present invention：
Present invention firstly discovers that and confirm the Close relation of MCM8 gene expressions and gastric adenocarcinoma tissue transfer, the sample of checking
This amount is more, as a result accurately.The it is proposed of the correlation provides new approach for the Clinics and Practices of gastric gland metastasis of cancer.
The present invention have developed the reagent or kit for being suitable for carrying out gastric gland metastasis of cancer detection, and detection sensitivity is good.
Brief description of the drawings
Fig. 1 shows the difference table in metastatic gastric adenocarcinoma tissue and normal control tissue using QPCR detection MCM8 genes
Reach；
Fig. 2 is shown using Western blot experiment detection MCM8 albumen in metastatic gastric adenocarcinoma tissue and Normal group
Differential expression in knitting；
Fig. 3, which is shown, utilizes Western blot experiment detection MCM8 gene expression inhibition rates.
Specific embodiment
The present invention is further detailed explanation with reference to the accompanying drawings and examples.Following examples are merely to illustrate this
Invention rather than limitation the scope of the present invention.The experimental method 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.
The MCM8 gene differential expressions of embodiment 1
1st, sample is collected
Metastatic gastric adenocarcinoma tissue and its Carcinoma side normal tissue 50 are collected, collects non-metastatic gastric adenocarcinoma tissue and its cancer
Other normal structure 45.Tissue samples are helped to sample by Pathologis, and it is as follows to collect sample canonical：(1) primary sdenocarcinoma of stomach, nothing
Other diseases, patient are preoperative without carcinosis radiotherapy and chemotherapy；(2) sample is diagnosed as sdenocarcinoma of stomach, and Carcinoma side normal tissue by pathology department
And it is not detected by tumour cell；(3) two sets of equipment are used in order to avoid unnecessary intersection is stained, during sampling, are first taken from cancer group
That knits minimum 5cm visually observes the Carcinoma side normal tissue sample without obvious lesion, after take the gastric adenocarcinoma tissue sample of holostrome, sample
Originally packing is put into the EP pipes added with 1ml RNAlater immediately after collecting, and 4 DEG C overnight, are then placed in -70 DEG C of refrigerator
It is long-term to preserve.And it is numbered and marks on each EP pipes.The remarks of relevant information are carried out in sample record sheet simultaneously.
2nd, the RNA of extraction pairing sdenocarcinoma of stomach and cancer beside organism's sample
Using RNA extracts kits (the Trans Zol purchased from Beijing Quanshijin Biotechnology Co., LtdTM Up Plus
RNA Kit) extraction tissue samples RNA.Comprise the following steps that：
(1) it after the sample weighing of superfreeze, will be transferred quickly in the mortar with Liquid nitrogen precooler, fully ground with pestle
The sample being ground into powder is transferred in centrifuge tube up to being ground into powder, 1ml is added per 50-100mg samples by mill
Trans Zol TMUp carries out homogenized with Syrup-homogenizing instrument, or pressure-vaccum mixes repeatedly with rifle.It is stored at room temperature 5 minutes.
(2) 1ml Trans Zol are often usedTMUp, add 0.2ml chloroforms, acutely vibration 30 seconds, be incubated at room temperature 3 points
Clock.
(3) 4 DEG C of 10000 × g are centrifuged 15 minutes.Now sample is divided into three layers, colourless aqueous phase (upper strata), intermediate layer, powder
Red organic phase (lower floor).RNA draws the μ l liquid of colourless aqueous phase layer 500 in colourless aqueous phase.
(4) aqueous phase colourless 500 μ l of transfer absorption adds 500 μ l absolute ethyl alcohol, gently in new centrifuge tube
It is reverse to mix.(hereafter centrifuging can be carried out at room temperature)
(5) solution for obtaining 700 μ l and precipitation are added in centrifugal column together, and 12000 × g room temperatures centrifuge 30 seconds, discard
Efflux (such as fruit volume is more than centrifugation column capacity, can divide and complete several times).
(6) 500 μ l CB9,12000 × g of room temperature room temperatures are added to centrifuge 30 seconds, discards efflux.
(7) repeat step (6) is once.
(8) add 500 μ l WB9 (before use please first check whether add absolute ethyl alcohol), 12000 × g of room temperature room temperatures from
The heart 30 seconds, discards efflux
(9) repeat step (8) is once.
(10) 12000 × g of room temperature room temperatures centrifuge 2 minutes, thoroughly remove residual ethanol, be stored at room temperature several minutes it is thorough
Dry centrifugal column in bottom.
(11) centrifugal column is put into RNase-free Tube (kit has been matched somebody with somebody), adds 30 μ l RNase-free
Water is stored at room temperature 1 minute in the center of centrifugal column.
(12) 12000 × g of room temperature room temperatures centrifuge 1 minute, eluted rna.
(13) RNA is placed in into -80 DEG C of refrigerators to preserve.
3rd, the RNA concentration and the measure of purity extracted
RNA concentration and purity are detected using the ND-1000 instruments of Nano Drop companies of the U.S..
Using UV detector Nano Drop 1000, the Nano Drop icons on computer screen are double-clicked, into system
System menu；According to system prompt after selection nucleic acid measurement option, 2 μ l distilled waters are added in well；Click on and determine, with first
Beginning system；2 μ l distilled waters are added in well, select RNA items, click on Blank with measuring system background.With rear lens wiping paper
RNA sample to be measured is clicked and entered after cleaning distilled water, clicks on Measure.Reading numerical values simultaneously record, RNA concentration (ng/ μ l), this trends of the times
It is required it is noted that A values 260/280, if numerical value between 1.8-2.0, illustrates that RNA sample quality is preferable.By each sample
RNA concentration markers it is complete after freeze in -70 DEG C of refrigerators.
4th, the RNA integrity mensuration's extracted
Ago-Gel detects RNA sample operations steps：
1) electrophoresis tank, the cleaning of glue apparatus：Detergent wash clean (general soaked overnight), water are used after rinsing, 3%H2O2
Electrophoresis tank is filled, room temperature places 10min, is rinsed, dried standby with 0.1% (V/V) DEPC water.
2) glue：0.5g agarose powders are weighed, addition is placed with the 45ml conical flask of DEPC water, and heating makes agarose
It is completely dissolved.5ml 10 × TAE electrophoretic buffers and final concentration of 0.5 μ g/ml Ethidum Eremide are added after slightly cooling down.Then
The Casting of gels in glue groove, it is plugged comb, horizontal positioned rear use to be solidified.
3) it is loaded：After sample mixes with 6 × electrophoretic buffer, it is loaded onto in gel loading wells.
4) electrophoresis：Open electrophoresis apparatus, voltage stabilizing 80V electrophoresis.
5) observe after electrophoresis terminates (about 30 minutes), under uviol lamp, and taken pictures preservation with gel imaging system.
Integrity mensuration' is detected by agarose gel electrophoresis, if 28s rRNA, 18s r RNA, 5s can be clearly apparent
RRNA three bands, and 28s rRNA brightness should be twice of 18s rRNA.Illustrate that the total serum IgE integrality of extraction is preferable, RNA
Satisfactory quality.
5th, the design and preparation of primer
The primer sequence of qRT-PCR detection MCM8 gene expressions and qRT-PCR amplification internal references GAPDH primer sequence are equal
Designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd., and through the retrieval of UCSC databases, by its sequence information
Typing NCBI software Design primers, and the blast in gene pool confirm correctly.
MCM8 gene primers：
Sense primer：5’-ATACCAGATATAGCAACT-3’(SEQ ID NO.1)；
- the CATCATTAGACAATCCTT-3 ' of anti-sense primer 5 ' (SEQ ID NO.2),
GAPDH gene primers：
Sense primer：5’-GGGAGCCAAAAGGGTCA-3’(SEQ ID NO.3)；
Anti-sense primer：5’-GAGTCCTTCCACGATACCAA-3’(SEQ ID NO.4).
6th, real time fluorescent quantitative cDNA reverse transcriptions detecting step
Reverse transcription synthesis cDNA is carried out to l μ g total serum IgEs with RT Buffer.Using 25 μ l reaction systems, each sample
1 μ g total serum IgEs are taken to be separately added into following components in PCR pipe as template ribonucleic acid：DEPC water, 5 × RT Buffer,
10mmol/L dNTP, 0.1mmol/l DTT, 30 μm of mol/l Oligo dT, 200U/ μ l M-MLV, template ribonucleic acid.42 DEG C of incubations
1h, 72 DEG C of 10min, of short duration centrifugation.
7、PCR
(1) Bio-RAD real-time fluorescence quantitative PCR instruments are applied, reaction system is prepared by table 1.
The PCR reaction systems of table 1
(2) carried out using following qPCR response parameters：95 DEG C of pre-degeneration 10min, then, 95 DEG C of denaturation 15s, 57 DEG C are moved back
Fire, extension 1min, totally 40 circulation；Then, the collection of fluorescence signal and the making of product solubility curve, each 3, sample are carried out
Repeat, take its average value.Using 2-△△CtRelative quantification method analyzes MCM8 expression, and Ct is that thermal cycler detects reaction
The intensity level of fluorescence signal in system.Computational methods are：Δ Δ Ct=(Ct target gene-Ct reference genes) tumor tissues are tested
Group-(Ct target gene-Ct reference genes) normal structure control group, 2-△△CtWhat is represented is the expression phase of experimental group target gene
Change multiple for control group, analysis of experimental data are completed by Bio-RAD analysis softwares.
5th, statistical analysis carries out data analysis using statistics software SPSS19.0, judges gastric gland using paired-samples T-test
MCM8 expression is all bilateral with the presence or absence of all statistical checks of difference of statistical significance in cancerous tissue and cancer beside organism's sample
Examine, P ＜ 0.05 are that difference is statistically significant.
6th, result
As a result as shown in figure 1, in transfer group, gastric adenocarcinoma tissue is compared with cancer beside organism, MCM8 mrna expressions
Increase, difference has statistical significance；And at non-diverting group, gastric adenocarcinoma tissue is compared with cancer beside organism, MCM8 gene mRNAs
Expression does not have the change occurred on statistical significance, is detected simultaneously by group by transfer group cancer beside organism and non-diverting group of cancer
It is identical to knit middle MCM8 mrna expressions.The above results show that MCM8 genes take part in the transfer of gastric adenocarcinoma tissue.
The differential expression of the MCM8 albumen of embodiment 2
1st, the total protein of tissue samples in embodiment 1 is extracted
The operation of protein extraction is carried out according to the specification of EpiQuik tissue/cell total protein extraction kits.
2、Western blot
Using β-actin as internal reference.50 μ g total proteins are after SDS-PAGE points, electrotransfer to pvdf membrane, with containing 5% defatted milk
1 × TBST room temperatures jog closing 1h of powder；It is separately added into rabbit-anti people MCM8 monoclonal antibodies (1: 800 dilution) and the anti-human β-actin of mouse is more
Anti- (1: 3 000 dilution), 4 DEG C overnight；1 × TBST washes film 4 times, adds goat-anti rabbit and sheep anti mouse Ig G (1: 2 000 dilution),
It is incubated at room temperature 1h；After 1 × TBST washes film 4 times, it is placed in Super Signal chemical illuminating reagents and reacts 2min, make X in darkroom
Mating plate exposes, conventional method developing fixing.
3rd, statistical procedures
The gray value of protein band is analyzed using Image J softwares, using β-actin as internal reference, by MCM8 albumen
The gray value of band is normalized.Result data is represented in a manner of mean+SD, is used
SPSS13.0 statistical softwares carry out statistical analysis, and difference between the two is examined using t, it is believed that works as P<There is system when 0.05
Meter learns meaning.
4th, result
As a result as shown in Fig. 2 in transfer group, compared with cancer beside organism, MCM8 protein levels increase gastric adenocarcinoma tissue, poor
It is different that there is statistical significance；And at non-diverting group, compared with cancer beside organism, MCM8 protein levels do not occur gastric adenocarcinoma tissue
Change on statistical significance, being detected simultaneously by MCM8 protein levels in transfer group cancer beside organism and non-diverting group of cancer beside organism is
Identical.The above results show that MCM8 albumen take part in the transfer of gastric adenocarcinoma tissue.
Embodiment 3 disturbs MCM8 gene expressions
1st, RNA interfering design synthesis
SiRNA is synthesized by Shanghai JiMa pharmacy Technology Co., Ltd's design according to MCM8 gene orders.Shanghai Ji agate pharmacy
Technology Co., Ltd. provides and negative control siRNA (siRNA-NC) of the MCM8 genes without sequence homology simultaneously.
siRNA-MCM8：
Positive-sense strand is 5 '-CACAGTTTTTGCTTTCAACAAAG-3 ' (SEQ ID NO.5)；
Antisense strand is 5 '-TGGATCGATTCATACCATATAAA-3 ' (SEQ ID NO.6),
2nd, the culture of human gastric adenocarcinoma
People's gastric adenocarcinoma cell line SGC 7901 uses the RPMI1640 culture mediums containing 10% hyclone to add penicillin 100units/
Ml, the μ g/ml of streptomysin 100, put 37 DEG C, 5%CO2Incubator in cultivate, change 1 nutrient solution every 24h, 48h is passed on 1 time.
Take the logarithm growth period cell carry out subsequent experimental.
3rd, cell transfecting
Liposome Lipofectamine2000 is used as transfection reagent.2 groups of experiment point：Negative control group (transfection siRNA-
NC)；Experimental group (transfection siRNA-MCM8).Take the logarithm the SGC7901 cells in growth period, be inoculated in 6 porocyte culture plates.
Tissue Culture Plate coverage rate is about 70%-80% after 24h.Rotaring transfecting mode is carried out with reference to Lipofectamine2000 specifications.
4th, Western blot experiments detection siRNA-MCM8 jamming effectiveness
Step is the same as embodiment 2.
5th, result
As shown in figure 3, compared with negative control group (transfection siRNA-NC), in experimental group (transfection siRNA-MCM8) cell
MCM8 expressing quantities significantly reduce, and difference has statistical significance (P<0.05).
The expression of the MCM8 genes of embodiment 4 is to Gastric Adenocarcinoma Cell Line, the influence of transfer ability
1st, the detection of the external transfer ability of gastric adenocarcinoma cells
48h after transfection, the transfection SGC7901 cells of each group is unicellular with being prepared into without dual anti-RPMI1640 nutrient solutions
Suspension, with 106It is individual/400 μ l density add Transwell upper stratas cell in, 600 μ l mouse are added in bottom chamber into fibre
Cell line NIH3T3 conditioned mediums are tieed up, are placed in 37 DEG C, 5%CO2In incubator, after cultivating 24h, upper interior is wiped with wet cotton swab
Cell, fix 15min, HE dyeing, light Microscopic observation through absolute ethyl alcohol, and randomly select upper and lower, left and right and middle each 5
The visual field, theca cell is worn in counting, and is averaged, to wear the external transfer ability that the number of theca cell represents SGC7901 cells.
As a result：Experimental group SGC7901 cells wear theca cell number (174.2 ± 13.6) compared with negative control group (368.5 ±
8.9) it is obvious to reduce, no significant difference (P ＞ 0.05).It is above-mentioned test result indicates that, suppressing MCM8 gene expressions can be with
Suppress gastric adenocarcinoma cells migration.
2nd, the detection of gastric adenocarcinoma cells vitro invasion ability
After ECM matrigels are diluted in 1: 7 ratio with serum-free RP-MI1640 nutrient solutions, 30 μ l are taken equably to be coated in
The upper chamber face of Tran-swell cells bottom film, ultraviolet irradiation overnight, make ECM matrigels aggregate into gel naturally.By each group
Single cell suspension is made with without dual anti-RPMI1640 nutrient solutions in SGC7901 cells, with 105It is individual/400 μ l density add
In the cell of Transwell upper stratas, remaining step represents the external of SGC7901 cells with experiment is migrated to wear the number of theca cell
Invasive ability.
As a result：Experimental group SGC7901 cells wear theca cell number (32.8 ± 4.5) compared with negative control group (91.6 ± 3.8)
It is obvious to reduce.It is above-mentioned test result indicates that, suppress MCM8 gene expressions can suppress Gastric Adenocarcinoma Cell Line.
The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.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, some improvement can also be carried out to the present invention
And modification, these are improved and modification will be also fallen into the protection domain of the claims in the present invention.
Sequence table
<110>Beijing Yang Shen biology information technologies Co., Ltd
<120>Purposes of the MCM8 as sdenocarcinoma of stomach Metastatic Marker
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ataccagata tagcaact 18
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
catcattaga caatcctt 18
<210> 3
<211> 17
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gggagccaaa agggtca 17
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
gagtccttcc acgataccaa 20
<210> 5
<211> 21
<212> RNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ugaaaaauuu uucaaaugcu u 21
<210> 6
<211> 21
<212> RNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
gcauuugaaa aauuuuucac a 21
Claims (10)
1. the reagent for detecting MCM8 is preparing gastric gland metastasis of cancer diagnosis, sdenocarcinoma of stomach prediction prognosis or the assessment of sdenocarcinoma of stomach transfer and relapse
Application in instrument.
2. application according to claim 1, it is characterised in that the reagent of the detection MCM8 includes detection MCM8 gene tables
Up to the reagent of amount.
3. application according to claim 1 or 2, it is characterised in that the reagent of the detection MCM8 includes quantifying
The reagent of MCM8 gene mRNAs, and/or the reagent of MCM8 albumen can be quantified.
4. application according to claim 3, it is characterised in that the reagent that can quantify MCM8 gene mRNAs includes real
When quantitative PCR in the primer of specific amplified MCM8 genes that uses；The reagent that MCM8 albumen can be quantified includes specificity
With reference to the antibody of MCM8 albumen.
5. a kind of instrument assessed for gastric gland metastasis of cancer diagnosis, the prognosis of sdenocarcinoma of stomach branch prediction or sdenocarcinoma of stomach transfer and relapse, its
It is characterised by, the instrument includes the instrument that can detect MCM8 gene expression amounts；Preferably, the instrument includes quantifying
The reagent of MCM8 gene mRNAs, and/or the reagent of MCM8 albumen can be quantified.
6. instrument according to claim 5, it is characterised in that the reagent that can quantify MCM8 gene mRNAs includes real
When quantitative PCR in the primer of specific amplified MCM8 genes that uses, the primer sequence such as SEQ ID NO.1 and SEQ ID
Shown in NO.2；The reagent that MCM8 albumen can be quantified includes the antibody of specific binding MCM8 albumen.
7. the instrument according to claim 5 or 6, it is characterised in that the instrument includes kit, chip, test paper, high pass
Measure microarray dataset.
8. a kind of medicine for being used to treat gastric gland metastasis of cancer, it is characterised in that the medicine includes MCM8 inhibitor.
9. medicine according to claim 8, it is characterised in that the inhibitor includes that MCM8 can be suppressed or is related to MCM8
The expression of the material of upstream or downstream pathway or the inhibitor of activity.
10. application of the inhibitor in the medicine for preparing treatment gastric gland metastasis of cancer described in claim 8 or 9.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114073770A (en) * | 2020-08-21 | 2022-02-22 | 广州市妇女儿童医疗中心 | Application of MCM8-cGAS-STING-I type interferon signal channel as disease target |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103459597A (en) * | 2010-12-13 | 2013-12-18 | 社会福祉法人三星生命公益财团 | Marker for predicting stomach cancer prognosis and method for predicting stomach cancer prognosis |
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2017
- 2017-09-13 CN CN201710823753.6A patent/CN107385100B/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103459597A (en) * | 2010-12-13 | 2013-12-18 | 社会福祉法人三星生命公益财团 | Marker for predicting stomach cancer prognosis and method for predicting stomach cancer prognosis |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114073770A (en) * | 2020-08-21 | 2022-02-22 | 广州市妇女儿童医疗中心 | Application of MCM8-cGAS-STING-I type interferon signal channel as disease target |
| WO2022037679A1 (en) * | 2020-08-21 | 2022-02-24 | 广州市妇女儿童医疗中心 | Use of mcm8-cgas-sting-i-type interferon signal pathway as disease target |
| CN114073770B (en) * | 2020-08-21 | 2023-06-06 | 广州市妇女儿童医疗中心 | Use of MCM8-cGAS-STING-I type interferon signal pathway as disease target |
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