CN105907879B - Carcinoma of endometrium biomarker - Google Patents
Carcinoma of endometrium biomarker Download PDFInfo
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- CN105907879B CN105907879B CN201610491574.2A CN201610491574A CN105907879B CN 105907879 B CN105907879 B CN 105907879B CN 201610491574 A CN201610491574 A CN 201610491574A CN 105907879 B CN105907879 B CN 105907879B
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Abstract
Purposes the invention discloses SPARCL1 as the diagnosis and treatment marker of carcinoma of endometrium.SPARCL1 can be used to develop the product of diagnosis of endometrial carcinoma, the drug of exploitation treatment carcinoma of endometrium accordingly.Research achievement of the invention provides fundamental basis for clinician's formulation personalized therapy program and can provide new drug target for the exploitation of endometrial cancer drug.
Description
Technical field
The present invention relates to cancer diagnosis, treatment, prediction prognosis fields, more particularly it relates to detect SPARCL1
Abnormal is cancer diagnosis, the prediction method of prognosis of means；And the cancer therapeutic agent of activation SPARCL1 gene or protein.
Background technique
Carcinoma of endometrium (endometrial carcinoma or carcinoma of endometrium, EC) is to occur
It is most common with the gland cancer from endometrial gland in one group of epithelial malignancy of endometrium, it is female genital tract
One of three big malignant tumours, account for women whole body malignant tumour 7%, and in worldwide different regions, its disease incidence is variant, is
The most common gynecologic malignant tumor of western industrialization country, North America, Europe disease incidence highest, Asia Japan, India and in
The area such as South America disease incidence is lower.China still lacks the more detailed epidemiological survey data of carcinoma of endometrium, estimation and day
This incidence is similar.North America and countries in Europe are higher than developing country, the former is 10 times of the latter, and disease incidence is located at
It is the 4th common cancer of women whole body after breast cancer, colorectal cancer, lung cancer, occupies female genital tract malignant tumour
One.
Although carcinoma of endometrium symptom occurs, relatively early, diagnosis is opposite is easier to.But about son in actual clinical work
There are still some disputes for the diagnosis and treatment of endometrial carcinoma.The grade malignancy and extent of disease of tumour, including Surgical staging, tissue
It is related with the prognosis of carcinoma of endometrium to learn transfer etc. outside type, tumor grade, Myometrial invasion, Lymph Node Metastasis and uterus.Cause
This, early diagnosis carcinoma of endometrium is extremely important.Currently, the diagnosis of carcinoma of endometrium is mainly according to medical history and clinical manifestation, B
The imageological examinations such as super, CT and MRI, diagnostic curettage, hysteroscope, the auxiliary examinations such as tumor markers CA-125.Wherein CA-
125 are also used as the index of observation of curative effect.But the inorganizable sensibility and specificity of CA-125, it is swollen in ovarian epithelial
CA-125 level can be detected in the diseases such as tumor, carcinoma of endometrium, endometriosis, digestive system tumor to increase.Cause
This, finding the tumor markers high to carcinoma of endometrium specificity becomes research hotspot.
Summary of the invention
One of the objects of the present invention is to provide one kind to diagnose son by detection SPARCL1 gene or protein expression difference
The method of endometrial carcinoma.
The second object of the present invention is to provide one kind by detection SPARCL1 gene or protein expression difference to predict son
The method of endometrial carcinoma prognosis.
The third object of the present invention is to provide one kind by activation SPARCL1 gene or SPARCL1 albumen to treat son
The method of endometrial carcinoma.
The fourth object of the present invention is to provide a kind of method for screening the drug for the treatment of carcinoma of endometrium.
The fifth object of the present invention is to provide a kind of for treating the drug of carcinoma of endometrium.
To achieve the goals above, present invention employs following technical solutions:
The present invention provides the products of detection SPARCL1 gene or SPARCL1 albumen to diagnose work in preparation carcinoma of endometrium
Purposes in tool.
The present invention also provides the products of detection SPARCL1 gene or SPARCL1 albumen to predict carcinoma of endometrium in preparation
Purposes in prognostic tool.
Further, it is described detection SPARCL1 gene or SPARCL1 albumen product include detection SPARCL1 gene or
The product of the expression of SPARCL1 albumen.The product includes that can combine the nucleic acid of SPARCL1 gene or can combine
The substance (such as antibody) of SPARCL1 albumen.The nucleic acid is able to detect the expression of SPARCL1 gene；The substance energy
Enough detect the expression of SPARCL1 albumen.
The product of detection SPARCL1 gene of the invention can play its function based on the known method of nucleic acid molecules is used
Can: such as PCR, such as Southern hybridization, Northern hybridization, dot blot, fluorescence in situ hybridization (FISH), DNA microarray, ASO
Method, high-flux sequence platform etc..It can qualitatively, quantitatively or semi-quantitatively implement analysis using the product.
Include that nucleic acid in the said goods can be obtained by chemical synthesis, or by containing from biomaterial preparation
It is expected that the gene of nucleic acid, then using primer amplification designed for amplification expectation nucleic acid, it is obtained.
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, nesting PCR method etc..Amplification
Nucleic acid can be by using dot blotting hybridization method, Surface Plasmon Resonance (SPR method), PCR-RFLP method, original position 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 for expanding SPARCL1 gene, and the primer for including in product can be by passing through
It is prepared by chemical synthesis, by using those skilled in the art will know that method be suitably designed with reference to Given information, and lead to
Chemical synthesis is crossed to prepare.
In specific embodiments of the present invention, the nucleic acid is amplimer used in QPCR experiment, the primer
Sequence such as SEQ ID NO.1 (positive sequence) and SEQ ID NO.2 (reverse sequence) shown in.
Nucleic acid recited above may also include probe, and the probe can be prepared by chemical synthesis, by using this
The method that field technical staff knows appropriately is designed with reference to Given information, and is prepared by chemical synthesis, or can lead to
The gene for containing desired nucleic acid sequence from biomaterial preparation is crossed, and is expanded using the primer designed for amplification expectation nucleic acid sequence
Increase it to prepare.
The product of detection SPARCL1 albumen of the invention can play its function: example based on the known method of antibody is used
It such as, may include ELISA, radioimmunoassay, immunohistochemical method, western blot etc..
The product of detection SPARCL1 albumen of the invention includes the antibody or its segment for specifically binding SPARCL1 albumen.
The antibody or its segment of any structure and size, immunoglobulin class, origin etc. can be used, as long as it combines target protein
?.The antibody or its segment for including in testing product of the invention can be monoclonal or polyclonal.Antibody fragment refers to
Retain peptide of the antibody to the active antibody of the combination of antigen a part of (Partial Fragment) or containing antibody a part.Antibody fragment can
To include F (ab ')2, Fab ', Fab, scFv (scFv), disulphide bonding Fv (dsFv) or its polymer, dimerization V
Area's (double antibody) or the peptide containing CDR.The product of detection SPARCL1 albumen of the invention may include encoding antibody or coding
The isolated nucleic acid of the amino acid sequence of antibody fragment, the carrier comprising the nucleic acid, and carry the cell of the carrier.
Antibody can be obtained by the way that well known to a person skilled in the art methods.For example, preparation retains target all or in part
The mammalian cell expression vector of their polynucleotides of the polypeptide or integration coding of protein is 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
Tumor culture collects antibody.It finally can be by using SPARCL1 albumen for being used as antigen or part thereof to the antibody of acquisition
Implement antigentic specificity purifying to obtain the monoclonal antibody for SPARCL1 albumen.Polyclonal antibody can be prepared as follows: being used
Antigen-immunized animal same as above collects blood sample from by immune animal, serum is isolated from blood, then
Antigentic specificity purifying is implemented to serum using above-mentioned antigen.It can be by the antibody that is obtained with enzymatic treatment or by using acquisition
The sequence information of antibody obtain antibody fragment.
The combination of marker and antibody or its segment 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. standards
Standby dyestuff, then mixed solution, then at being placed at room temperature for 10 minutes.In addition, the labelling kit of commercialization can be used in label, 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 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-NH2,647 labelling kit-NH2 of HiLyte Fluor (TM) (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- marker protein mark
Remember kit (Funakoshi Corporation).For correct labeling, suitable instrument can be used to detect by label
Antibody or its segment.
As the sample according to testing product of the invention, the tissue sample for example obtained from biopsy subject can be used
Or fluid.Sample is not particularly limited, as long as it is suitable for measurement of the invention；For example, it may include tissue, blood, blood plasma,
Serum, lymph, urine, serous cavity liquid, spinal fluid, synovia, aqueous humor, tear, saliva or its fraction or treated material
Material.
In specific embodiments of the present invention, tissue of the sample from subject.
In the present invention, " prognosis " refers to mistake of the cancer patient after inhibiting by surgical procedure etc. or alleviating tumour growth
Journey or result.In the present specification, prognosis can be by surgical procedure inhibit or alleviate tumour growth after 1,2,3,4,5,6,
7,8,9,10,15,20 years or more long when life state.Prognosis can by check biomarker, that is, SPARCL1 albumen or
The gene of SPARCL1 albumen is encoded to predict.Prognosis prediction can be performed such that according to biomarker with or without, or
It is raised and lowered, determines 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 inhibit or alleviate for patient by surgical procedure etc. tumour growth it
Afterwards, patient's long-term (such as 3,5,6,7,8,9,10,15,20 years or longer) does not have critical condition.Alternatively, good prognosis can anticipate
Refer to and survives in such long-time, sent out again without transfer, without recurrence or nothing.For example, prognosis bona can mean at least 3 years or outstanding
It is to survive at least 5 years, preferably without transfer or recurrence.The most preferred state of prognosis bona is survival for a long time without disease.Such as
Used herein, " prognosis bona " can also include any such state, wherein it can be found that disease such as shifts, still
It is pernicious low and do not severely impact survival ability.
In the present invention, " prognosis mala " refers to that patient is short after inhibiting or alleviating tumour growth by surgical procedure etc.
Fatal condition occurs in period (such as 1,2,3,4,5 year or shorter).Alternatively, poor prognosis refers in such short-term extremely
It dies, shift, recur or sends out again.For example, poor prognosis can mean Preventive or dead at least 3 years or especially at least 5 years
It dies.
Prediction prognosis is referred to the process of prediction status of patient or as a result, is not meant to be predicted with 100% accuracy
The process or result of status of patient.Prediction prognosis refers to whether a possibility that determining certain processes or result increases, and simultaneously unexpectedly
Taste by determining a possibility that certain processes or result occurs or not compared with certain processes or result.Such as this
For invention, in the present invention in the horizontal patient reduced of SPARCL1 gene or SPARCL1 albumen, and this feature is not shown
Patient compares, and more likely observes particular procedure or result.
Further, it is described detection SPARCL1 gene or SPARCL1 albumen product can be detection SPARCL1 gene or
The reagent of SPARCL1 albumen is also possible to include kit, chip, test paper of the reagent etc., is also possible to using the examination
The high-flux sequence platform of agent.
The present invention also provides a kind of tool of diagnosis of endometrial carcinoma, the tool be able to detect SPARCL1 gene or
The expression of SPARCL1 albumen.The tool includes that can combine the nucleic acid of SPARCL1 gene or can combine
The substance (such as antibody) of SPARCL1 albumen.The nucleic acid is able to detect the expression of SPARCL1 gene；The substance energy
Enough detect the expression of SPARCL1 albumen.
Further, the property of the nucleic acid and the substance is the same as noted earlier.
Further, the tool of the diagnosis of endometrial carcinoma includes but is not limited to chip, kit, test paper or high throughput
Microarray dataset；High-flux sequence platform is a kind of tool of special diagnosis of endometrial carcinoma, with high throughput sequencing technologies
Development will become very easily work to the building of the gene expression profile of a people.Pass through comparison Disease and normal person
The gene expression profile of group, the exception for being easy to analyze which gene are related to disease.Therefore, know in high-flux sequence
The exception of the SPARCL1 gene purposes for also belonging to SPARCL1 gene related to carcinoma of endometrium, equally in protection model of the invention
Within enclosing.
The present invention also provides a kind of tool for predicting carcinoma of endometrium prognosis, the prediction carcinoma of endometrium prognostic tools
Including that can combine the nucleic acid of SPARCL1 gene or the substance (such as antibody) of SPARCL1 albumen can be combined.The nucleic acid
It is able to detect the mRNA level in-site of SPARCL1 gene；The substance is able to detect the expression of SPARCL1 albumen.
Further, the property of the nucleic acid and the substance is the same as noted earlier.
Further, the tool of the prediction carcinoma of endometrium prognosis includes but is not limited to chip, kit, test paper or height
Flux microarray dataset；High-flux sequence platform is a kind of tool of special diagnosis of endometrial carcinoma, with high-flux sequence skill
The development of art will become very easily work to the building of the gene expression profile of a people.By comparison Disease and just
The gene expression profile of ordinary person group, the exception for being easy to analyze which gene are related to disease.Therefore, know in high-flux sequence
The exception of the SPARCL1 gene purposes for also belonging to SPARCL1 gene related to carcinoma of endometrium, equally in protection model of the invention
Within enclosing.
The amino acid that anti-SPARCL1 antibody used in testing product of the invention, diagnostic tool or its segment are identified
Number be not particularly limited, as long as antibody can combine SPARCL1.
The present invention also provides a kind of diagnosis of endometrial carcinoma or the method for predicting carcinoma of endometrium prognosis, the method packets
Include following steps:
(1) sample of subject is obtained；
(2) expression of SPARCL1 gene or albumen in Samples subjects is detected；
(3) it associates whether by the expression of the SPARCL1 gene or albumen that measure with the illness of subject.
(4) compared with the control, the expression of SPARCL1 gene or albumen reduces, then the subject is diagnosed as uterus
Endometrial carcinomas or the subject are confirmed as prognosis mala.
The present invention also provides a kind for the treatment of method of carcinoma of endometrium, the method includes activation SPARCL1 gene or
SPARCL1 albumen.
Further, the method includes promoting the expression of SPARCL1 gene, or the expression or increasing of promotion SPARCL1 albumen
The activity of strong SPARCL1 albumen.
The present invention also provides a kind of screening techniques of cancer drug, can be by after adding testing drug to cancer cell
Or the table of some period measurement SPARCL1 gene or SPARCL1 albumen after applying testing drug to cancer model animal
Cancer drug is measured up to horizontal improves the effect of cancer prognosis.More specifically, when SPARCL1 gene or SPARCL1 egg
The drug may be selected as changing when increasing after adding or applying testing drug or when restoring normal level in white expression
The therapeutic agent of kind cancer prognosis.
The present invention also provides a kind of drugs of activator containing SPARCL1 gene or SPARCL1 albumen.
The present invention also provides application of the above-mentioned activator in the drug of preparation treatment carcinoma of endometrium.
The activator of SPARCL1 gene or SPARCL1 albumen of the invention is unrestricted, as long as can promote or increase
The expression or activity of strong SPARCL1 or the substance for being related to the upstream SPARCL1 or downstream pathway, and medicine effective for treating cancer
Object.
Further, the activator includes SPARCL1 gene, SPARCL1 albumen, promoted type miRNA, promoted type transcription tune
It controls the factor or promoted type targets small molecule compound.
The activator further includes carrier or host cell comprising carrying SPARCL1 gene.
On the one hand activator of the invention can be used for supplementing the missing or deficiency of endogenic SPARCL1 albumen, pass through
The expression of SPARCL1 albumen is improved, thus carcinoma of endometrium caused by treating because of SPARCL1 hypoproteinosis.It on the other hand can be with
For enhancing the activity of SPARCL1 albumen, to treat carcinoma of endometrium.
Drug of the invention can be used as medicine and be administered alone or apply together with other medicines.It can be with medicine of the invention
The other medicines that object is applied together are unrestricted, as long as it does not damage therapeutic or preventive medicine effect of the invention i.e.
It can, it is preferred that the drug for treating or preventing cancer may 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, cytarabine, cytarabine octadecyl phosphate
(cytarabine ocfosfate), Tegafur, tegafur-Uracil, Tegafur gimeracil oteracil potassium, deoxidation fluorine urine
Glycosides, hydroxycarbamide, fluorouracil, fludarabine, pemetrexed, Pentostatin, mercaptopurine and methotrexate (MTX)；Plant alkaloid, it is all
As Irinotecan, Etoposide, Sobuzoxane, docetaxel, nogitecan, Palmer altruism, vinorelbine, eldisine and
Vincaleukoblastinum；Antitumor antibiotic, such as actinomycin D, Aclarubicin, Amrubicin, idarubicin, epirubicin, Zinostatin
Stimalamer, daunorubicin, Doxorubicin, pirarubicin, bleomycin, Peplomycin, mitomycin C and mitoxantrone；
Drug based on platinum, such as oxaliplatin, carboplatin, cis-platinum and Nedaplatin；Hormonal medicaments, such as Anastrozole, Exemestane,
Estramustine, ethinyloestradiol, chlormadinone, Goserelin, tamoxifen, dexamethasone, Toremifene, Bicalutamide, Flutamide,
Prednisolone, Fosfestrol, mitotane, methyltestosterone, Medroxyprogesterone, Mepitiostane, Leuprorelin and Letrozole；Biological respinse modification
Agent, such as interferon-' alpha ', interferon beta, interferon gamma, interleukin, ubenimex, dry BCG and lentinan；With molecular targeted medicine
Object, such as Imatinib (imatinib), Gefitinib (gefitinib), gemtuzumab, ozogamicin, Tamibarotene, song
Appropriate monoclonal antibody, Tretinoin, bortezomib (bortezomib) and Rituximab etc..
Drug of the invention can be prepared into various dosage forms 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 administration method of drug of the invention is unrestricted, as long as it can play desired therapeutic effect or preventive effect i.e.
Can, including but not limited to intravenously, in peritonaeum, intraocularly, intra-arterial, intrapulmonary is taken orally, in vesicle, intramuscular, intratracheally, subcutaneously
, local by pleura by skin, sucking, by mucous membrane, skin, stomach is intra-articular, intra-ventricle, rectum, vagina,
In skull, in urethra, in liver, in tumor.In some cases, it can systematically be administered.It is locally to be administered in some cases.
The dosage of drug of the invention is unrestricted, can as long as obtaining desired therapeutic effect or preventive effect
To carry out appropriate determination according to symptom, gender, age etc..Example can be used in the dosage of therapeutic agent or prophylactic agent of the invention
Such 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 whether subject has suffered from intrauterine
Film cancer also includes the risk that judges subject and whether there is with carcinoma of endometrium.
" treatment " used herein is covered treatment-related in such as mankind of the mammal with related disease or illness
Disease or morbid state, and include:
(1) prevent disease or morbid state occurs in mammals, especially when the mammal is susceptible in the disease
Diseased state, but when being not yet diagnosed with this morbid state；
(2) inhibit disease or morbid state, that is, prevent its generation；Or
(3) alleviate disease or morbid state, even if disease or morbid state subside.
Term " treatment " is usually directed to treatment mankind or animal (for example, being applied by animal doctor), wherein can reach certain pre-
The therapeutic effect of phase, for example, inhibiting the development (including reduce development speed, stop development) of illness, improving illness and healing
Illness.It further include the treatment as precautionary measures (such as prevention).To not yet development be illness but have development be the illness endanger
The purposes of the patient of danger, is also included in term " treatment ".
The advantages of the present invention:
The molecular marker for having found a kind of diagnosis of endometrial carcinoma of the invention, can be in son using the molecular marker
Endometrial carcinoma occur early stage can be used as judging, provide the survival rate of patient.
In addition, the present invention is capable of providing significant information to determine treatment side for patient by the prognosis of prediction patient
Case strategy.
The therapeutic agent of activator including SPARCL1 gene or albumen of the invention can be used as new carcinoma of endometrium
Therapeutic agent.
Detailed description of the invention
Fig. 1, which is shown, utilizes Western blot detection SPARCL1 albumen endometrial tissues and normal endometrium
Expression in tissue；
Fig. 2, which is shown, detects SPARCL1 gene overexpression situation using Western blot.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this
It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually 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 normal condition proposed by manufacturer.
1 genetic chip of embodiment screens difference expression gene
1, sample collection:
Endometrial sample: endometrial carcinoma, the equal underwent operative treatment of all patients, paraffin mark of performing the operation are collected
This 10.All patients pass through pathologic finding and make a definite diagnosis with carcinoma of endometrium.Other enter a group condition are as follows: before all patients are admitted to hospital
Do not receive any treatment: not merging other malignant tumours；Other hormone related disorders are not merged；Complete clinical data.
10 patients with endometrial cancer are fallen ill average age 58 years old.Patient's main clinical manifestation be Irregular vagina bleeding,
Hypogastralgia, paramenia, neoplasm etc. also have some patientss non-evident sympton and find in Physical Examination.Carcinoma of endometrium
Sample is diagnosed as carcinoma of endometrium through HE stained slice, tectology.
Normal endometrial tissue sample: 10 normal endometriums operation paraffin specimens.The patient of samples sources is suffered from
Disease includes: fibroid, uterine prolapse, the bulging of wing moon bright, Rectocele.
2, the acquisition of RNA is organized
Total tissue RNA is extracted using Trizol one-step method, is read at 260nm and 280nm by Nanodrop ND-1000
Absorbance value (A) measurement RNA solution purity.Through 1% denaturing formaldehyde agarose gel electrophoresis, observed under ultraviolet transmission light,
Detect the integrality of RNA.
3, gene chip hybridization and scanning
After the linearized amplification of total serum IgE, cy3-UTP is marked, and the cRNAs after fluorescent marker uses RNEASY Mini Kit
Purifying, carries out fragmentation processing to the cRNAs marked with the RNA Fragmentation Reagents of Amhion.Using beauty
People's full genome chip of expression spectrum (4x 44K gene) of Agilent company, state, 65 DEG C of hybridization 17h in chip hybridization furnace, then
Elution, dyeing, finally use Agilent DNA MicroarrayScanner scanner scanning.
4, chip data processing and analysis
Chip after hybridization imports data to analysis software, for two groups of ratios after chip scanner reads data point
Natural logrithm absolute value be greater than 2.0 or the gene less than 0.5 as difference expression gene.
5, statistical procedures
Data analysis is carried out using 13.0 statistical software of SPSS, group difference compares using one-way analysis of variance method, P <
0.05 difference has significant.
6, result
Chip results are shown, filter out 654 difference tables between endometrial and normal endometrial tissue altogether
Up to gene, wherein gene 421 of expression up-regulation, lower gene 233 of expression.
2 large sample of embodiment verifies the difference expression gene filtered out
Consider to yet there are no the gene studied about the gene with carcinoma of endometrium correlation in the prior art as time
Select gene, at the same consider gene sequencing as a result, selection SPARCL1 gene (its express endometrial tissues in lowers) into
Row verifying.
1, sample collection
Endometrial 50, normal endometrial tissue 60 are collected according to the method for embodiment 1.
2, it is verified in mRNA level in-site
2.1 extract tissue RNA
Step is the same as embodiment 1.
2.2 reverse transcription
Using Reverse Transcriptase kit, converse record is carried out to l μ g total serum IgE with RT Buffer and synthesizes cDNA.Using 25 μ l
Reaction system, each sample take 1 μ g total serum IgE as template ribonucleic acid, following components are separately added into PCR pipe: DEPC water, 5 × inverse
Transcription buffer, 10mmol/l dNTP, 0.1mmol/l DTT, 30 μm of mol/l Oligo dT, 200U/ μ l MMLVRT, template
RNA.42 DEG C be incubated for 1 hour, 72 DEG C 10 minutes, of short duration centrifugation.
2.3PCR
MRNA fluorescent quantitation upstream and downstream PCR primer, synthesis are designed using primer-design software Primer Premier 5.0
Primer sequence, is operated using SYBR Green PCR Master Mix kit, and specific steps by specification is operated, adopted
With 25 μ l reaction systems, each sample is arranged 3 parallel pipes, all amplified reactions be repeated three times it is above can with guarantee result
By property.It prepares following reaction system (as shown in table 1), operations are carried out on ice:
1 quantitative fluorescent PCR each component of table and respective volume
Using GAPDH as internal reference, using SYBR Green I as fluorescent marker, in Light Cycler quantitative fluorescent PCR
PCR reaction is carried out on instrument, determines that purpose band, Δ Δ CT method carry out relative quantification by melt curve analysis analysis and electrophoresis.
SPARCL1 gene primer sequence is as follows:
Upstream primer: 5 '-TCAATAAGCACATTCAAG-3 ' (SEQ ID NO.1)；
Downstream primer: 5 '-ATTACCATCATCAGTAGG-3 ' (SEQ ID NO.2).
GAPDH gene primer sequence is as follows:
Upstream primer: 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.3)；
Downstream primer: 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.4).
2.4 result
The results show that compared with normal endometrial tissue, the mRNA level in-site of SPARCL1 gene in endometrial
Obvious to lower, relative expression quantity is 0.18 ± 0.012, and difference has statistical significance (P < 0.05).
3, it is verified on protein level
3.1 extract tissue total protein
The operation of protein extraction is carried out according to the specification of EpiQuik tissue/cell total protein extraction kit.
3.2Western blot detection
The protein quantification of extraction is subjected to SDS-PAGE electrophoresis, carry out later transferring film, closing, primary antibody is incubated for, secondary antibody is incubated for,
Colour developing.
3.3 statistical procedures
The gray value of protein band is analyzed using Image J software, using β-actin as internal reference, by SPARCL1 egg
The gray value of informal voucher band is normalized.Result data is indicated in a manner of mean+SD, is used
SPSS13.0 statistical software is next for statistical analysis, and difference between the two is examined using t, it is believed that has system as P < 0.05
Meter learns meaning.
3.4 result
As a result as shown in Figure 1, compared with normal endometrial tissue, SPARCL1 protein level in endometrial
It significantly reduces, difference has statistical significance (P < 0.05).
3 SPARCL1 gene overexpression of embodiment
1, plasmid construction
According to the coded sequence of SPARCL1 gene design amplimer, primer be designed as those skilled in the art institute it is ripe
Know.From cDNA library (clontech company, the article No.: the 638831) volume of the SPARCL1 gene of amplification overall length at Human fetal spleen
Code sequence, above-mentioned cDNA sequence are inserted into eukaryotic expression vector pcDNA3.1, connect the recombinant vector of acquisition
PcDNA3.1-SPARCL1 is used for subsequent experimental.
2, the culture and transfection of endometrial carcinoma cell
2.1 cell culture
Ishikawa3-H-12 cell line uses the culture bottle adhere-wall culture of 50ml in 1640 culture medium of RPMI (containing 10%
Fetal calf serum, 100U/ml penicillin, 100g/ml streptomysin) in, at 37 DEG C, 5%CO2It is continuously trained in the incubator of saturated humidity
It supports.
2.2 cell transfecting
1, the day before transfection is by 0.5-2*105A tumour cell is suspended in the not antibiotic culture medium of 500 μ l, is seeded to
24 well culture plates.
2, transfection same day cell density should reach 80%-90%, prepare following compound A: 1 μ g Plasmid DNA is diluted in nothing
In blood serum medium, mix gently；Compound B: taking 4 μ l Lipofectamine2000 to be diluted in serum free medium, mixes
It is even.
3, compound A and B are mixed, is mixed gently, is incubated at room temperature.
4,100 μ l liposome compounds are added in tumour cell, mix gently up and down, cell is put into 37 DEG C and is contained
5%CO2Incubator is incubated for 5-7 hours.
5, the growth medium for adding 1ml to contain 2 times of normal serums and antibiotic concentration continues culture cell 18-24 hours.
3, the overexpression situation of detection pcDNA3.1-SPARCL1 is tested using QPCR
3.1 extraction cell total rnas are operated using conventional method.
3.2 reverse transcription
Step is the same as embodiment 2.
3.3QPCR
Step is the same as embodiment 2.
3.4 result
The results show that pcDNA3.1-SPARCL1 can be successfully overexpressed, relative expression quantity is 5.94 ± 0.78, difference tool
Statistically significant (P < 0.05).
3, Western blot experiment detection pcDNA3.1-SPARCL1 is overexpressed situation
Step is the same as embodiment 2.
As a result as shown in Fig. 2, being transfected in the cell of pcDNA3.1-SPARCL1 compared with transfecting pcDNA3.1 group
The content of SPARCL1 albumen obviously increases, and difference has statistical significance (P < 0.05).
Measurement of the expression of 4 SPARCL1 gene of embodiment to endometrial carcinoma cell proliferative capacity
1, step:
Using the bromo- 2 ' Brdurd of 5- (Brd U) label and detection kit.Referring to kit operation instruction, cell
(transfection procedure is with embodiment 3) is transfected after 48 hours, inhales and abandons culture medium, addition Brd U label culture medium, 37 DEG C, 5%CO2Training
It supports in case and cultivates 60min.It discards culture medium to fix after PBS is rinsed with 70% ethyl alcohol, overnight.It is the anti-Brd of mouse with primary antibody
U antibody, secondary antibody are the fluorescence antibody of the anti-mouse with FITC, are immunoreacted.Then flow cyctometry detection is carried out.
2, Brd U mixes result:
As the result is shown: transfection pcDNA3.1 groups of cells incorporation efficiency is average are as follows: (28.59 ± 1.37) %；pcDNA3.1-
SPARCL1 groups of cells incorporation efficiency average out to (11.64 ± 0.53) %, difference have statistical significance (P < 0.05).Above-mentioned experiment
The result shows that the SPARCL1 gene expression inhibition proliferation of endometrial carcinoma cell.
Influence of 7 Transwell of the embodiment experiment detection SPARCL1 gene expression to cell migration
Step:
1, the Matrigel object frozen is taken out from -80 DEG C of refrigerator, then under the conditions of 4 DEG C of temperature overnight, makes its change
At liquid.
2,200 μ l serum-free cell culture mediums are taken out, the Matrigel reagent of 50 μ l are added, in cryogenic conditions, preferably
It operates and is mixed evenly in ice face, 100 μ l are then respectively added, is placed on 37 DEG C, cultivates 5h in the incubator of carbon dioxide, this
Between often observe liquid case, when liquid becomes slightly white, illustrate that it has turned into solidification state.
3, the cell transfected (according to 3 step operation of embodiment) is digested with pancreatin, it is clear with the culture medium without serum
It washes 3 times, then counts, then be made into cell suspending liquid.
4, gel is gently washed 1 time with the culture medium of serum-free, then by 2*105Cell is suspended in 100 μ l RPMI
In 1640, it is inoculated in transwell upper chamber.
5, lower room adds 600 μ l 10%FBS RPMI 1640.
6, in 37 DEG C of incubators, after cultivating 12h, the cell transwell is taken out, every group repeats 4 samples.
7, wherein 1 cell discards culture medium, is washed 3 times with the PBS of no calcium, and the fixed 10min of 4% poly first ferment, cotton swab is wiped
Fall the cell that upper layer does not migrate, PBS is washed 3 times, and violet staining or Giemsa dyeing are observed under the microscope.Remaining 3 cells
Its 0.25% membrane proteolytic enzyme of lower layer's migrating cell is digested, computation migration cell number.
As a result:
Migration experimental result is shown, transfects pcDNA3.1 groups of cells cell migration number average out to 242, transfection
PcDNA3.1-SPARCL1 groups of cells cell migration number average out to 147, difference have statistical significance (P < 0.05).
The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.
Claims (9)
1. the product of detection SPARCL1 gene or SPARCL1 albumen is preparing diagnosis of endometrial carcinoma or prediction carcinoma of endometrium
Application in the tool of prognosis.
2. application according to claim 1, which is characterized in that the production of the detection SPARCL1 gene or SPARCL1 albumen
Product include the product for detecting the expression of SPARCL1 gene or SPARCL1 albumen.
3. application according to claim 1 or 2, which is characterized in that the product includes that can combine SPARCL1 gene
Nucleic acid can be in conjunction with the substance of SPARCL1 albumen；The nucleic acid is able to detect the expression of SPARCL1 gene；It is described
Substance is able to detect the expression of SPARCL1 albumen.
4. application according to claim 3, which is characterized in that the nucleic acid is specifically expanded used in real-time quantitative PCR
Increase the primer of SPARCL1 gene as shown in SEQ ID NO.1 and SEQ ID NO.2.
5. application according to claim 1, which is characterized in that the tool include be able to detect SPARCL1 gene or
The tool of the expression of SPARCL1 albumen.
6. application according to claim 5, which is characterized in that the tool includes can be in conjunction with the core of SPARCL1 gene
Acid can be in conjunction with the substance of SPARCL1 albumen；The nucleic acid is able to detect the expression of SPARCL1 gene；The object
Matter is able to detect the expression of SPARCL1 albumen.
7. application according to claim 6, which is characterized in that the nucleic acid is specifically expanded used in real-time quantitative PCR
Increase the primer of SPARCL1 gene as shown in SEQ ID NO.1 and SEQ ID NO.2.
Application of the activator of 8.SPARCL1 gene or SPARCL1 albumen in the drug of preparation treatment carcinoma of endometrium.
9. application according to claim 8, which is characterized in that the activator can promote or enhance SPARCL1 or relate to
And the expression or activity of the substance of the upstream SPARCL1 or downstream pathway.
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| CN110387420B (en) * | 2019-08-26 | 2020-11-13 | 武汉艾米森生命科技有限公司 | Kit for endometrial cancer diagnosis and application |
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| Title |
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| SPARCL1 在结直肠癌中的表达及临床意义;王向阳 等;《实用医学杂志》;20141231;第30卷(第14期);2242-2245 |
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