CN108949987A - Target of the GPR19 as diagnosis and treatment cervical carcinoma - Google Patents
Target of the GPR19 as diagnosis and treatment cervical carcinoma Download PDFInfo
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- CN108949987A CN108949987A CN201810868578.7A CN201810868578A CN108949987A CN 108949987 A CN108949987 A CN 108949987A CN 201810868578 A CN201810868578 A CN 201810868578A CN 108949987 A CN108949987 A CN 108949987A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57411—Specifically defined cancers of cervix
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
Abstract
The invention belongs to biomedicine fields, disclose purposes of the GPR19 gene as the diagnosis and treatment marker of cervical carcinoma.The experiment proves that there are significant differences for expression of the GPR19 gene in normal tissue and cervical cancer tissues., can be using GPR19 gene as the new molecular marker of clinically diagnosing cervical according to the correlation of GPR19 gene and cervical carcinoma, while it can also be as the new target drone for the treatment of of human cervical cancer drug.
Description
Technical field
The present invention relates to fields of biomedicine, more particularly it relates to which GPR19 gene is in preparation diagnosis and treatment cervical carcinoma
Application in product.
Background technique
Cervical carcinoma is the most common gynecologic malignant tumor, the account for the first in tumors of female reproductive organ.According to world health
Statistical data in 2014 is organized, the year new cases of cervical carcinoma are about 52.8 ten thousand, and year death number of cases is about 26.6 ten thousand, wherein
85% patient occurs in developing country, and rural area is higher than city.China is the big country of cervical cancer pathogenesis, according to country
The newest cancer statistical data that tumor center announces, the cervical carcinoma new cases in China in 2015 are about 9.89 ten thousand, and year is dead
Number of cases is about 3.05 ten thousand, and the data presentation rise year by year and the trend for rejuvenation of falling ill [Chen W, Zheng R,
Baade P D,et al.Cancer statistics in China,2015[J].CA Cancer J Clin,2016,66
(2):115-132].The generation of cervical carcinoma and many factors are closely related, such as early marriage, early childbirth, fecund and sexual life disorder, but
Human papilloma virus (Human papilloma virus, HPV) infection is considered as the necessary condition of cervical cancer pathogenesis,
99.7% cervical cancer patient HPV screening is positive, and the risk of HPV persistent infection patients' cervical carcinoma is HPV negative patient
More than 250 times [Wentzensen N, Arbyn M.HPV-based cervical cancer screening-facts,
fiction,and misperceptions[J].Prev Med,2017,98:33-35l；Torre L A,Islami F,
Siegel R L,et al.Global cancer in women:burden and trends[J].Cancer Epidemiol
Biomarkers Prev,2017].From the seventies and eighties in last century, clear HPV infection is the important origin cause of formation of cervical cancer pathogenesis for the first time
Afterwards, further investigation is unfolded in the mechanism that scholars induce cervical cancer pathogenesis for HPV infection, and therefore has developed for cervical carcinoma
Diagnostic method and therapeutic strategy, immunoprophylaxis measure and treatment including diagnostic method, targeting before sensitive special cancer
Method greatly improves cervical cancer patient existence and quality of life.
With the development of modern biotechnology detection technique and the research that deepens continuously of Tumorigenesis, it is related to tumorigenic
Different kinds of molecules during various biological, as dissociated in nucleic acid, protein, carbohydrate, lipid, small molecule metabolites or even blood
Tumour cell, can be used as important tumor markers, provide specific foundation to clinical prevention, diagnosing and treating.In palace
In neck cancer, oneself is it is found that some tumor markers are used for the clinical preventions of cervical carcinoma at present.
Ki-67 and p16INK4a is two kinds and is commonly used in analysis tumor cell proliferation state and malignancy
Molecular marker.Wherein Ki-67 is not expressed in silent GO phase cell, and height is expressed in G1, S, G2 and M phase cell, therefore it can
Be widely used in analysis cell proliferation activity come assess tumor progression [Endl E, Gerdes J.The Ki-67protein:
fascinating forms and an unknown function[J].Exp Cell Res,2000,257(2):231-
237]；Simultaneously as it is expressed, pedigree is extensive, and the tumor markers as specificity still have certain defect, it is clinical it is still necessary to
Other molecular marker auxiliary diagnosis.P16INK4a, can be specific in conjunction with CDK4 and CDK6 as a kind of cycle regulating protein
To inhibit the phosphorylation of its activity and downstream pRb, cell cycle progress and cell differentiation procedure are adjusted；Studies have found that
P16INK4a expression is positively correlated with HR-HPV persistent infection and cervix neoplasms pathological grading, and HPV's postoperative for patient is clear
It removes and persistent infection has certain guiding value [Koh J, Enders G H, Dynlacht B D, et al.Tumour-
derived p16alleles encoding proteins defective in cell-cycle inhibition[J]
.Nature,1995,375(6531):506-510]。
ProExC antibody (BD company) can specificity the karyon albumen MCM2 that is induced by HPV infection of identification and topology
Isomerase TOP2A compound.In cervix gland and squamous cell atypical hyperplasia, the cell S phase gene of E7 oncogene induction
It can promote TOP2A and the MCM2 high expression in nucleus, while TOP2A can form stable structure in conjunction with 6 MCM2 molecules
It is stranded in nucleus [Santin A D, Zhan F, Bignotti E, et al.Gene expression profiles of
primary HPV16-and HPV18-infected early stage cervical cancers and normal
cervical epithelium:identification of novel candidate molecular markers for
cervical cancer diagnosis and therapy[J].Virology,2005,331(2):269-291].Because its
It is not expressed in normal cervical epithelial cell, and the significantly high expression in the active squamous cell of the proliferation of HPV induction, clinically may be used
For distinguishing, atypical hyperplasia and squamous cell ateliosis or atrophy etc. are similar to be changed.
Mainly a stable protection is collectively formed by capsid protein L 1 and L2 albumen in HPV DNA integrity and stability
Shell maintains.Wherein, L1 albumen can also promote virion to infect mucous membrane substrate by the corresponding receptor on identification host cell
Film strips cell or cervical epithelial cells.HPV infect it is slight to moderate atypical hyperplasia during can be continuously detected L1 egg
White expression, but as the expression of uterine neck cancerization degree progress L1 albumen can fade away [Mcmurray H R, Mccance
D J.Human papillomavirus type 16E6activates TERT gene transcription through
induction of c-Myc and release of USF-mediated repression[J].J Virol,2003,77
(18):9852-9861].Therefore HPV-L1 expression disappear prompt viral genome oneself be incorporated into host genome, can be used for uterine neck
The diagnosis of cancer CIN3 phase.
Lanminin-5 is a kind of and the closely related tumor markers of tumor invasion, in a variety of different type malignant tumours
The expression of middle Lanminin-5 gene is often closely related with the progress of tumour.In the generating process of cervical carcinoma, Laminin-5 master
It expresses the early stage in tumour, especially at the skin lesion of microinvasion, therefore it can be used for Cervix Squamous Cell infiltration
The detection of early stage.MIB-I has the expression pattern similar with Ki-67, i.e., the proliferation shape of GINS each phase is expressed in significant height
The active tumour cell of state can be used for periodic state locating for assistant analysis tumour cell in conjunction with Ki-67 detection.Therefore MIB-1
It is an important indicator of cell-proliferation activity during another important detection atypical hyperplasia.
Although oneself has tumour in conclusion diagnosis of the tumor markers for clinical cervical carcinoma there are many oneself at present
Marker is horizontal because of constructive expression under normal circumstances or in non-malignant diseases also to be increased, and tumour-specific is lacked.
It would therefore be highly desirable to find tumour specific antigen as biological marker, early diagnosis and prognosis for clinical cervical carcinoma determine.
Summary of the invention
One of the objects of the present invention is to provide one kind to realize diagnosis of cervical cancer by detection GPR19 gene expression difference
Method.
The second object of the present invention is to provide a kind of method for treating cervical carcinoma by inhibiting GPR19 gene expression.
The third object of the present invention is to provide a kind of method for screening treatment of human cervical cancer drug.
The fourth object of the present invention is to provide a kind of for treating the drug of cervical carcinoma.
To achieve the goals above, present invention employs following technical solutions:
The present invention provides application of the reagent of detection GPR19 in the tool for preparing diagnosing cervical.
Further, the reagent of the detection GPR19 includes the reagent for detecting GPR19 gene expression amount.
Further, the reagent of the detection GPR19 includes the reagent that can quantify GPR19 gene mRNA, and/or can
The reagent of quantitative GPR19 albumen.
The reagent of quantitative GPR19 gene mRNA 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 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, 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.
The reagent that GPR19 gene mRNA can be quantified can be the specific primer of GPR19 gene or transcript,
It can be the specific recognition probe of GPR19 gene or transcript, or simultaneously include primer and probe.
The specific primer of GPR19 gene or transcript recited above includes specifically expanding used in real-time quantitative PCR
Increase the primer of GPR19 gene.In specific embodiment of the 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 refer to known letter
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 refer to known letter
Breath appropriately designs, and is prepared by chemical synthesis, or can be by containing desired nucleic acid sequence from biomaterial preparation
Gene, and it is prepared using the primer amplification designed for amplification expectation nucleic acid sequence.
The reagent of quantitative GPR19 albumen of the invention can play its function based on the known method of antibody is used: for example,
It may include ELISA, radioimmunoassay, immunohistochemical method, western blot etc..
The reagent of quantitative GPR19 albumen of the invention includes the antibody or its segment for specifically binding GPR19 albumen.It can be with
Using the antibody or its segment of any structure and size, immunoglobulin class, origin etc., 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 that reservation is anti-
Peptide of the body to the active antibody of the combination of antigen a part of (Partial Fragment) or containing antibody a part.Antibody fragment may include
F(ab′)2, Fab ', Fab, scFv (scFv), the Fv (dsFv) of disulphide bonding or its polymer, the area dimerization V it is (dual anti-
Body) or peptide containing CDR.The reagent of quantitative GPR19 albumen of the invention may include encoding antibody or Encoding Antibody Fragment
The isolated nucleic acid of amino acid sequence, 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 real by using antibody of the GPR19 albumen for being used as antigen or part thereof to acquisition
Antigentic specificity purifying is applied to obtain the monoclonal antibody for GPR19 albumen.Polyclonal antibody can be prepared as follows: with it is upper
The identical antigen-immunized animal of text collects blood sample from by immune animal, serum is isolated from blood, is then used
Above-mentioned antigen implements antigentic specificity purifying to serum.It can the antibody by being obtained with enzymatic treatment or resisting by using acquisition
The sequence information of body obtains 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.
The acquisition of sample for detecting the detection of GPR19 gene expression amount of the invention is the ordinary skill in the art, excellent
It selects and Noninvasive or the method acquisition with minimally-invasive property may be selected.
The sample can be (but are not limited to): tissue, peripheral blood, marrow, lymph node, abdominal cavity cleaning solution, urine, sweat
Liquid.In specific embodiments of the present invention, tissue of the sample from subject.
The present invention also provides a kind of tool for diagnosing cervical, the tool is able to detect GPR19 gene expression
Amount.
Further, the tool includes the reagent that can quantify GPR19 gene mRNA, and/or can quantify GPR19 albumen
Reagent.
In general, the reagent is present in container appropriate.Diluent can be used to draw as described in deionized water by every kind
Object or probe are adjusted to the concentration of at least one requirement, are sub-packed in container.
Further, the reagent that can quantify GPR19 gene mRNA includes specific amplified used in real-time quantitative PCR
The primer of GPR19 gene, the primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.
Further, the tool for diagnosing cervical includes but is not limited to chip, kit, test paper or high throughput
Microarray dataset；High-flux sequence platform is a kind of special tool(s), with the development of high throughput sequencing technologies, to the gene of a people
The building of express spectra will become very easily work.By comparing the gene expression profile of Disease and normal population, it is easy
The exception for analyzing which gene is related to disease.Therefore, the exception and cervical carcinoma of GPR19 gene are known in high-flux sequence
Generation it is related also belong to the new application for having used GPR19 of the invention, equally within protection scope of the present invention.
It may also include the reagent for extracting nucleic acid in kit of the invention, for the reagent of PCR, for dyeing or showing
The reagent etc. of color.For example, these reagents include but is not limited to: extract, amplification liquid, hybridization solution, developing solution, washing lotion etc..
In addition, may also include the specification etc. of the method for description detection GPR19 gene expression in the kit.
Kit of the present invention may include suitable for 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 the detection based on GPR19 gene or transcript is come diagnosing cervical
Reagent is all contained in the scope of the invention.
The present invention also provides a kind of methods of diagnosing cervical, and described method includes following steps:
(1) sample of subject is obtained；
(2) GPR19 gene expression dose in Samples subjects is detected；
(3) the GPR19 gene expression dose measured is associated with the disease associated of subject.
(4) compared with normal control, GPR19 gene expression dose is statistically increased, and shows that subject is judged trouble
There is cervical carcinoma or judges that risk of the subject with cervical carcinoma is high.
The present invention also provides a kind for the treatment of methods of cervical carcinoma, and the method includes inhibiting GPR19 gene expression or suppression
The activity of GPR19 gene expression product processed.
The present invention also provides a kind of screening techniques of drug candidate for treating cervical carcinoma, can be by model cell
The expression of some period measurement GPR19 gene or GPR19 albumen after addition testing drug changes to measure drug candidate
The effect of kind prognosis.More specifically, when the expression of GPR19 gene or GPR19 albumen is adding or applying test medicine
When reducing after object or when restoring normal level, the drug may be selected as the therapeutic agent for improving cervical carcinoma.
The present invention also provides a kind of drug for treating cervical carcinoma, the drug includes the reagent for inhibiting GPR19.
The reagent of inhibition GPR19 of the invention is unrestricted, as long as the reagent is able to suppress GPR19 or is related to GPR19
The expression or activity of the substance of upstream or downstream pathway, and for treating the effective drug of cervical carcinoma.
The present invention also provides the application of GPR19 gene or its expression product in the drug of preparation treatment cervical carcinoma.
Further, the drug includes the RNA interfering or negative regulation miRNA, negative regulation for GPR19 gene expression
The transcription regulatory factor or suppressive of type target small molecule compound.
Drug of the invention can by any of mode compounding pharmaceutical composition in this field come using.This combination
Object includes active constituent, in addition one or more pharmaceutically acceptable carriers, diluent, filler, bonding agent and other figurations
Agent, this depends on administration mode and designed dosage form.This field known treatment of branch art personnel is inert inorganic or has
The carrier of machine includes but is not limited to lactose, cornstarch or derivatives thereof, talcum, vegetable oil, wax, fat, polyhydroxy chemical combination
Object such as polyethylene glycol, water, sucrose, ethyl alcohol, glycerol, such, various preservatives, lubricant, dispersing agent, corrigent.It protects
It is wet cut to pieces, antioxidant, sweetener, colorant, stabilizer, salt, buffer is such is added thereto, these substances according to
It needs to be used to help the stability of formula or helps to improve activity or its biological effectiveness or generated in the case where oral
Acceptable mouthfeel or smell, the preparation that can be used in such a composition can be the form of its original chemical itself, or
The form of its pharmaceutically acceptable salt is optionally used, drug of the invention can be administered alone, or with various combination medicine-feedings,
And combining form is administered together with other healing potions.Those skilled in the art may be selected in the composition so prepared as needed
Any mode appropriate known to member is administered drug.It is by the present invention of safe and effective amount when using pharmaceutical composition
Medicament administration in people, specific dosage is also contemplated that the factors such as administration route, patient health situation, these are all skilled practitioners skills
Within energy range.
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, as long as obtaining desired therapeutic effect or preventive effect.This
The therapeutic agent of invention or the dosage of prophylactic agent, which can be used for example to be used as the therapeutic effect of disease or preventive effect, to be referred to
Mark is to determine.
" GPR19 gene " of the invention (Chromosome 12, NC_000012.12 (12659691..12717786,
Complement)) sequence can be to be inquired in ncbi database.
In the context of the present invention, " diagnosis of cervical cancer " includes judging whether subject has suffered from cervical carcinoma, judgement
Subject whether there is the risk with cervical carcinoma or judge that cervical cancer patient recurs.
" 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:
Present invention firstly discovers that and confirming the Close relation of GPR19 gene expression Yu uterine neck carcinogenesis, the sample of verifying
Amount is more, as a result accurately.The it is proposed of the correlation provides new approach for the Clinics and Practices of cervical carcinoma.
Detailed description of the invention
Fig. 1 shows the differential expression using QPCR detection GPR19 gene in cervical cancer tissues and normal control tissue
Statistical chart；
Fig. 2 is shown using Western blot experiment detection GPR19 albumen in cervical cancer tissues and normal control tissue
Differential expression statistical chart；
Fig. 3 shows the statistical chart using Western blot experiment detection GPR19 gene expression inhibition rate.
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:ColdSpring HarborLaboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.
Embodiment 1 screens unconventionality expression gene in the cancerous tissue of cervical cancer patient
1, tissue collecting
Cervical cancer tissues 4 that obstetrics and gynecology hospital provides are collected, from postoperative through uterectomy (cone is cut or cut entirely)
Pathological diagnosis is the patient of cervical carcinoma, and wherein surrounding normal tissue is as a control group.
2, tissue RNA is extracted
- 80 DEG C of cervical cancer patient tissues frozen and periphery normal cervical tissues about 50mg are taken, is put into liquid nitrogen and is ground
Mill is added 1m L Trizol and is used for Total RNAs extraction and real-time quantitative until being transferred in 1.5mL EP pipe when without bulky grain tissue
PCR analysis, detailed process are as follows:
1) 200 μ L chloroforms are added in the above-mentioned tissue suspension containing 1mL Trizol, mixing rear chamber fullys shake manually
Temperature stands 10min；
2) 12000rpm, 4 DEG C of centrifugation 15min；
3) after wait be centrifuged, gentle aspiration EP pipe upper strata aqueous phase is secondary to new 1.5mL EP pipe, and 600 μ L isopropyls are added
Alcohol turns upside down and mixes well, and is placed at room temperature for 20min (or -20 DEG C of placement 2h, can increase the precipitation of RNA)；
4) 12000rpm, 4 DEG C of centrifugation 15min；
5) it discards supernatant, water-reducible 75% ethyl alcohol of DEPC is added, gently pressure-vaccum suspension precipitates with pipette tips；
6) 12000rpm, 4 DEG C of centrifugation 15min；
7) it discards supernatant, remaining liquid is drawn with pipette tips, hangs 5min at room temperature；
8) take 20 μ L RNAase-free water dissolution precipitating, it is quantitative after using or to freeze -80 DEG C of refrigerators stand-by.
3, quantitative RNA
It takes the 2 μ L of total serum IgE of said extracted to be diluted in 98 μ L DEPC water, measures wavelength using ultraviolet specrophotometer
Absorbance value at 260nm and 280nm calculates sample according to A260 value according to the purity of this RNA of A260/A280 ratio in judgement
The concentration of RNA.
4, high-throughput transcript profile sequencing
(1) RNA-seq read positions
Low-quality read is removed to obtain cleaning read first, then using TopHat v1.3.1 will clean segment and
UCSC H.sapiens is matched with reference to genome (hg19), the H.sapiens UCSC hg19 editions indexes constructed in advance
It is downloaded from TopHat homepage, and as reference genome, when matching using TopHat with genome, allows each read (default
To 20) having multiple matching sites, most 2 mispairing.TopHat establishes possible according to exon region and GT-AG shear signal
Shearing site library navigates to the read for not navigating to genome on genome according to these shearing site libraries.We use
The system default parameter of TopHat method.
(2) transcript abundance is assessed
The read file matched is handled by Cufflinks v1.0.3, and Cufflinks v1.0.3 is by RNA-seq piece
Number of segment mesh is standardized the relative abundance for calculating transcript.FPKM value refers to being matched in every 1,000,000 sequencing fragment specific
The segment number of the exon region of gene 1kb long.The confidence interval of FPKM estimated value is calculated by Bayesian inference method.
The GTF comment file for the reference that Cufflinks is used downloads (Homo_ from Ensembl database
sapiens.GRCh37.63.gtf)。
(3) detection of difference expression gene
It is transferred to Cuffdiff by the Ensembl GTF file of downloading and by the matched original document of TopHat,
Cuffdiff re-evaluates the gene expression abundance for the transcript listed in GTF file using original matching files, detects difference table
It reaches.The only q value < 0.01 in Cuffidff output, test display is more just considered as successfully differential expression.
4, result
For RNA-seq the results show that compared with normal control tissue, highly expressed gene is 351 in cervical cancer tissues, low
The gene of expression is 294, and difference all has statistical significance (P < 0.05).
The expression of 2 large sample of embodiment verifying difference expression gene
One, it is detected on transcriptional level
1, tissue collecting
According to the standard collection cervical cancer tissues of embodiment 1 and corresponding normal control tissue 40.
2, tissue RNA is extracted and is identified
Step is the same as embodiment 1.
3, the design and preparation of primer
Real-time quantitative PCR primer sequence used herein following (commission Sangon Biotech (Shanghai) Co., Ltd.) limited liability company
Design and synthesize):
GPR19 gene primer:
Upstream primer: 5 '-CTACACTGTCATCCACTTCT-3 ' (SEQ ID NO.1)；
Downstream primer 5 '-GCCATCTGTGCCTATTCT-3 ' (SEQ ID NO.2),
GAPDH gene primer:
Upstream primer: 5 '-GACCTGACCTGCCGTCTA-3 ' (SEQ ID NO.3)；
Downstream primer: 5 '-AGGAGTGGGTGTCGCTGT-3 ' (SEQ ID NO.4).
4, reverse transcription PCR
Reverse transcription is carried out using Takara reverse transcription reagent box (going to dezymotize containing genomic DNA), specific reaction system is as follows:
Remove the reaction system of genomic DNA:
The reaction system of the removal genomic DNA of table 1
| Reagent | Volume |
| Total serum IgE | 1.0μg |
| gDNA Eraser | 1.0μL |
| 5*gDNA Eraser B buffer | 2.0μL |
| RNase Free water | It mends to 10 μ L |
5min is reacted at 42 DEG C.
Reverse transcription PCR reaction system:
2 reverse transcription PCR reaction system of table
| Reagent | Volume |
| RT Primer Mix | 1.0μL |
| PrimeScript RT Enzyme Mix I | 1.0μL |
| 5*Prime Script Buffer | 4.0μL |
| Remove the reaction system of genomic DNA | 10.0μL |
| RNase Free water | 4.0μL |
37 DEG C of reactions 15min, 85 DEG C of reaction 5s.
5, real-time quantitative PCR
Reaction system:
3 reverse transcription PCR reaction system of table
Reaction condition: 95 DEG C of initial denaturation 30s；
95 DEG C of 15s, 55.6 DEG C of 15s, 72 DEG C of 20s, global cycle number are 40 times；
72 DEG C of extension 5min (GAPDH is internal reference).
Using 2-△△CtRelative quantification method analyzes the expression of GPR19, and Ct is that thermal cycler detects glimmering in reaction system
The intensity value of optical signal.Calculation method are as follows: Δ Δ Ct=(Ct target gene-Ct reference gene) cervical cancer tissues experimental group-(Ct
Target gene-Ct reference gene) normal control tissue group, 2-△△CtWhat is indicated is the expression of experimental group target gene relative to right
According to the variation multiple of group, analysis of experimental data is completed by Bio-RAD analysis software.
6, statistical analysis carries out data analysis using statistics software SPSS19.0, judges uterine neck using paired-samples T-test
The expression of GPR19 is with the presence or absence of the difference on statistical significance in cancerous tissue and normal control tissue sample.Statistical check is all
Two-sided test, P < 0.05 are that difference is statistically significant.
7, result
Compared with normal control tissue, 36 GPR19 gene expression is significantly increased in 40 cervical cancer tissues.Statistics knot
Fruit is as shown in Figure 1, compared with normal control tissue, and GPR19 gene significantly increases in cervical cancer tissues, and difference is anticipated with statistics
Adopted (P < 0.05).
Two, it is detected on protein level
1, the cervical cancer tissues total protein being collected into is extracted
(1) it is removed from liquid nitrogen the cervical cancer patient tissue and periphery normal cervical tissues of preservation, room temperature is after it thaws
Cutting tissue about 100mg is transferred in tissue homogenizer and is shredded tissue using clean scissors；
(2) the about 300 μ L of RIPA lysate containing 1%PMSF is added, is then homogenized on ice；
(3) tissue homogenate is transferred in 1.5mL EP pipe, places 15min on ice；
(4) 12000rpm, 4 DEG C of centrifugation 15min；
(5) transfer supernatant takes 10 μ L to carry out BCA protein quantification, remaining is set -20 DEG C and freezes into new 1.5mL EP pipe.
2, electrophoresis
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 temperature jog of powder closes 1h；It is separately added into GPR19 monoclonal antibody and β-actin monoclonal antibody, 4 DEG C overnight；1 × TBST washes film 4
It is secondary, secondary antibody is added, is incubated at room temperature 1h；After 1 × TBST washes film 4 times, it is placed in Super Signal chemical illuminating reagent and reacts
2min exposes X-ray in darkroom, conventional method developing fixing, therefrom chooses photo expose and utilizes Image Pro software
Carry out gray analysis.
3, statistical procedures
The gray value of protein band is analyzed using Image J software, using β-actin as internal reference, by GPR19 albumen
The gray value of 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.
4, result
Compared with normal control tissue, 36 GPR19 protein levels are significantly increased in 40 cervical cancer tissues, difference tool
It is statistically significant.Statistical result is as shown in Fig. 2, compared with normal control tissue, and GPR19 protein level is aobvious in cervical cancer tissues
It writes and increases, difference has statistical significance (P < 0.05).
Embodiment 2 interferes GPR19 gene expression
1, RNA interfering design synthesis
SiRNA is synthesized by Shanghai JiMa pharmacy Technology Co., Ltd's design according to GPR19 gene order.Shanghai Ji Ma pharmacy
Technology Co., Ltd. provides and negative control siRNA (siRNA-NC) of the GPR19 gene without sequence homology simultaneously.
SiRNA-GPR19:
Positive-sense strand is 5 '-AAUUCCAUCAGGUAUUGGCUU-3 ' (SEQ ID NO.5)；
Antisense strand is 5 '-GCCAAUACCUGAUGGAAUUAA-3 ' (SEQ ID NO.6),
2, the culture of Hela cell
Cell is placed in 37 DEG C with containing dual anti-and 10% fetal calf serum 1640 culture mediums, 5%CO2Incubator in training
It supports, every changing 1 culture solution for 24 hours, 48h is passed on 1 time.The cell of logarithmic growth phase carries out subsequent experimental.
3, cell transient transfection
In day before transfection vitellophag, and by cell inoculation 6 orifice plates, every hole about 2x105A cell, overnight incubation are seen
It examines cell fusion degree (about 50-70%) and carries out cell siRNA transient transfection, detailed process is as follows:
1) according to the explanation on siRNA synthesis book, siRNA to 20 μM is diluted using RNase Free water；
2) two sterile EP tubes are taken to be respectively labeled as A and B, 125 μ L/ hole serum free mediums and 5 μ L are added in A pipe
125 μ L/ hole serum free mediums and 5 μ L Lipofectamine 3000 are added in siRNA, B pipe；
3) above-mentioned A is mixed well, the solution of B pipe is stored at room temperature 5min；
4) replacing the culture medium in 6 orifice plates is serum free medium, every hole 2mL；
5) A of above-mentioned mixing, B mixed solution is added, cross shake mixes well cell；
6) it is placed in CO2In incubator, 37 DEG C of progress routine cultures；
7) after transfecting 48h, 1ml Trizol or 200 μ L RIPA lytic cells are separately added into, extract RNA and protein
Real-time quantitative PCR and Western blot detection are carried out, carries out subsequent experimental after analyzing interference effect.
4, result
As shown in figure 3, compared with negative control group (transfection siRNA-NC), experimental group (transfection siRNA-GPR19) cell
Middle GPR19 expressing quantity significantly reduces, and difference has statistical significance (P < 0.05).
Influence of the expression of 4 GPR19 gene of embodiment to Hela cell Proliferation
The detection of cell Proliferation uses CCK-8 method
(1) it is transfected according to the method for embodiment 3；
(2) the Hela cell after transfection for 24 hours is inhaled abandoning culture medium and is contaminated with 0.25% trypsin digestion and cell with crystal violet
Liquid counts cell, and it is 2x 10 that cell, which is resuspended, with 1640 culture mediums containing 0.5%FBS and adjusts cell concentration4/ml；
(3) into 96 new well culture plates, 100 μ l of cell suspension, i.e. 2x10 is added in every hole under aseptic condition3A cell, 37
DEG C, 5%CO2It stands for 24 hours；
(4) 10 μ l CCK solution are added into each hole respectively, cultivate 2h；
(5) 450nm wavelength, ultraviolet specrophotometer measure each experimental group light absorption value.
As a result:
The results show that negative control group (transfection siRNA-NC) cell OD value is 0.984 ± 0.138, experimental group (transfection
SiRNA-GPR19) cell OD value is 0.517 ± 0.083.The above results show inhibit GPR19 expression cervical cancer cell proliferation by
To inhibition, difference has 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.
Sequence table
<110>Beijing Yang Shen biology information technology Co., Ltd
<120>target of the GPR19 as diagnosis and treatment cervical carcinoma
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<213>artificial sequence (Artificial Sequence)
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gccatctgtg cctattct 18
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<213>artificial sequence (Artificial Sequence)
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gacctgacct gccgtcta 18
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aggagtgggt gtcgctgt 18
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Claims (10)
1. detecting application of the reagent of GPR19 in the diagnostic tool of preparation cervical carcinoma.
2. application according to claim 1, which is characterized in that the reagent of the detection GPR19 includes detection GPR19 gene
The reagent of expression quantity.
3. application according to claim 1 or 2, which is characterized in that the reagent of the detection GPR19 includes that can quantify
The reagent of GPR19 gene mRNA, and/or the reagent of GPR19 albumen can be quantified.
4. application according to claim 3, which is characterized in that the reagent that can quantify GPR19 gene mRNA includes
The primer of specific amplified GPR19 gene used in real-time quantitative PCR；The reagent that GPR19 albumen can be quantified includes spy
The opposite sex combines the antibody of GPR19 albumen.
5. a kind of tool for diagnosis of cervical cancer, which is characterized in that the tool includes being able to detect GPR19 gene expression amount
Tool；Preferably, the tool includes the reagent that can quantify GPR19 gene mRNA, and/or can quantify GPR19 albumen
Reagent.
6. tool according to claim 5, which is characterized in that the reagent that can quantify GPR19 gene mRNA includes
The primer of specific amplified GPR19 gene used in real-time quantitative PCR, the primer sequence such as SEQ ID NO.1 and SEQ ID
Shown in NO.2；The reagent that GPR19 albumen can be quantified includes the antibody for specifically binding GPR19 albumen.
7. tool according to claim 5 or 6, which is characterized in that the tool includes kit, chip, test paper, high pass
Measure microarray dataset.
8. a kind of for treating the drug of cervical carcinoma, which is characterized in that the drug includes the reagent for inhibiting GPR19.
9. drug according to claim 8, which is characterized in that the reagent is including being able to suppress GPR19 or being related to GPR19
The expression of the substance of upstream or downstream pathway or active reagent.
The application of 10.GPR19 gene or its expression product in the drug of preparation treatment cervical carcinoma.
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| US20120121615A1 (en) * | 2010-11-17 | 2012-05-17 | Flygare John A | Alaninyl maytansinol antibody conjugates |
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| US20120121615A1 (en) * | 2010-11-17 | 2012-05-17 | Flygare John A | Alaninyl maytansinol antibody conjugates |
| CN107106700A (en) * | 2013-12-16 | 2017-08-29 | 基因泰克公司 | Peptide simulated compound and its antibody drug conjugate |
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