CN109971854A - For developing the target molecule of the diagnosis product of cervical disease - Google Patents
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Abstract
The invention discloses the target molecules of the diagnosis product for developing cervical disease, the present invention passes through high throughput sequencing technologies combination bioinformatic analysis, have found that FRMD4B expresses downward in cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma for the first time, and the exploitation that FRMD4B is related to the occurrence and development of cervical squamous cell carcinoma, and FRMD4B is prompted to can be used as target molecule applied to cervical squamous cell carcinoma diagnostic products and therapeutic agent is demonstrated by further QPCR and cell experiment.
Description
Technical field
The invention belongs to biomedicine field, it is related to the target molecule of the diagnosis product for developing cervical disease, specifically relates to
And target molecule be FRMD4B.
Background technique
Cervical carcinoma refers to generation in the column of the squamous cell and cervical canal inner membrance of ectocervical or transitional zone
The malignant tumour of chrotoplast intersection.In worldwide, cervical carcinoma is to be only second to the second largest malignant tumour of women of breast cancer,
Nearest global data is shown, in whole world women, annual neopathy number of cases is 460,000, and annual 260000 patients in the whole world are dead
In the disease, 80% case occurs in developing country (Castellsague X.Natural history and
epidemiology of HPV infection and cervical cancer[J].Gyneco Oncol,2008,110
(3): 4-7.), mortality accounts for the 18.4% of whole malignant tumour mortalities, and rural area is higher than city, is that gynaecology three is swollen greatly
One of tumor.The annual new cases 13.15 ten thousand in China, account for about the 28.8% of world's cervical carcinoma new cases, the illness rate of cervical carcinoma
The China Reng Ju women's malignant tumour the 1st, and a kind of rejuvenation trend is just being presented in the morbidity of cervical carcinoma, seriously threatens China female
The quality of life and life and health of property.
Gene therapy is the frontier in the physianthropy molecular genetics to grow up the 1980s, and development is very
Rapidly.Originally the therapeutic studies of single gene inheritance disease are only used for, as the research to malignant tumour obtains on a molecular scale
Breakthrough (JengCJ, KoML, LingQD, et al.Prevalence of cervical human papilloma
Virus in Taiwa-nese women [J] .Clin Invest Med, 2005,28 (5): 261-266.), malignant tumour
Gene therapy has become the hot spot of current research, and laboratory all over the world has conducted extensive research the gene therapy of cervical carcinoma
And exploration.Main treatment method includes tumor suppressor gene treatment, immune-gene therapy and suicide gene therapy etc..Use genetic engineering
The drug of technical research exploitation has achieved many achievements, has interferon (IFN, interleukin 2 using wider at present
(IL-2 and colony-stimulating factor (C-CSF) etc..Currently, the main method for inhibiting oncogene expression has antisense widow's core general
Acid, ribozyme and RNA interfere (RNAi) (Matsukura T, SugaseM.Pitfalls in the epidemiologic
classification of human papillomavirus types associated with cervical cancer
usingpoly merasechain reaction:driver and passenger[J].Int J Gynecol Cancer,
2008,18(5):1042-1050.).Ribozyme is the RNA with catalytic activity, is primarily involved in the processing and maturation of RNA.Catalysis knot
Specific site of the structure domain in target RNA is cut, to inhibit the expression of specific gene.Currently, the gene therapy of cervical carcinoma is still located
In the exploratory stage, especially China's clinical research report is limited, really becomes new clinical treatment means and also needs more to grind
Study carefully and gropes.
Summary of the invention
In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a kind of and cervical diseases: in epithelium of cervix uteri
Tumor-like lesion, the relevant target molecule of cervical squamous cell carcinoma occurrence and development can be used for opening for diagnostic products and treatment product using the molecule
Hair.
To achieve the goals above, the present invention adopts the following technical scheme:
The first aspect of the present invention provides a kind of reagent, and the reagent is able to detect FRMD4B gene or its expression product
Level.
Further, the reagent includes:
The probe of specific recognition FRMD4B gene；Or
The primer of specific amplification FRMD4B gene；Or
Specifically bind the antibody of the albumen of FRMD4B gene coding.
Further, the primer sequence of specific amplification FRMD4B is as shown in NO.1~2 SEQ ID.
The second aspect of the present invention provides a kind of kit, and the kit includes examination described in first aspect present invention
Agent.
The third aspect of the present invention provides a kind of chip, and the chip includes reagent described in first aspect present invention.
The fourth aspect of the present invention provides a kind of nucleic acid film item, and the nucleic acid film item includes described in first aspect present invention
Reagent.
The fifth aspect of the present invention provides a kind of composition, and the composition includes the promotor and/or medicine of FRMD4B
Acceptable carrier on.The promotor includes improving FRMD4B gene or its expression product stability, up-regulation FRMD4B base
The expression of cause or its expression product increases FRMD4B gene or the substance of its expression product effective acting time；The medicine
Acceptable carrier and/or auxiliary material on, including but not limited to diluent, adhesive, surfactant, Humectant, absorption
Carrier, lubricant, filler, disintegrating agent；Described pharmaceutical composition can also include stabilizer, fungicide, buffer, isotonic
The additives such as agent, chelating agent, pH controlling agent and surfactant.
Further, the promotor is the carrier for being overexpressed FRMD4B.
The sixth aspect of the present invention provides a kind of method of the drug candidate of screening treatment cervical squamous cell carcinoma, the method packet
It includes:
The cultivating system of expression or the albumen containing FRMD4B gene or its coding is handled with substance to be screened；With
Detect the expression or activity of the albumen of FRMD4B gene or its coding in the system；
Wherein, if the substance to be screened can promote the level or expression activity of FRMD4B gene, show this wait sieve
Selecting substance is the drug candidate for treating cervical squamous cell carcinoma.
In the present invention, the system includes but is not limited to: cell system, subcellular system, solution system, organizer
System, organ systems or animal system.
In the present invention, the step further include: to the drug candidate of acquisition carry out further cell experiment and/or
Animal experiment, with further selection can treat the drug of cervical squamous cell carcinoma from drug candidate.
The seventh aspect of the present invention provides following described in any item applications:
A. reagent described in first aspect present invention is in preparation early diagnosis cervical intraepithelial neoplasia (CIN), cervical squamous cell carcinoma
Application in product；
B. production of the kit described in second aspect of the present invention in preparation diagnosis cervical intraepithelial neoplasia (CIN), cervical squamous cell carcinoma
Application in product；
C. product of the chip described in third aspect present invention in preparation diagnosis cervical intraepithelial neoplasia (CIN), cervical squamous cell carcinoma
In application；
D. nucleic acid film item described in fourth aspect present invention is in preparation diagnosis cervical intraepithelial neoplasia (CIN), cervical squamous cell carcinoma
Application in product；
E. composition described in fifth aspect present invention preparation treatment cervical squamous cell carcinoma and its transfer, invasion drug in
Using；
F. application of the method described in sixth aspect present invention in the drug candidate of screening treatment cervical squamous cell carcinoma；
G.FRMD4B preparation treatment cervical squamous cell carcinoma and its transfer, invasion drug in application；
Application of the h.FRMD4B in the computation model that cervical intraepithelial neoplasia (CIN), cervical squamous cell carcinoma are predicted in building.
Detailed description of the invention
Fig. 1 is the expression using QPCR detection FRMD4B gene in cervical squamous cell carcinoma patient；
Fig. 2 is FRMD4B gene expression dose figure；
Fig. 3 is the CCK-8 method detection active influence diagram of FRMD4B cell proliferation；
Fig. 4 is using the influence diagram of the cell transwell detection FRMD4B gene pairs cell migration invasion, wherein figure A is
To the influence diagram of cell migration；Figure B is the influence diagram to cell invasion.
Specific embodiment
The present invention is by high throughput sequencing technologies and carries out high-flux sequence analysis, detection cervical intraepithelial neoplasia sample disease
Become, the gene expression dose in cervical squamous cell carcinoma patient tissue, discovery wherein expresses the gene with notable difference, inquires into itself and palace
Neck intraepithelial neoplasia, cervical squamous cell carcinoma generation between relationship, thus be cervical squamous cell carcinoma early detection and targeted therapy
Find better approaches and methods.By screening present invention firstly discovers that FRMD4B expresses downward in cervical squamous cell carcinoma patient,
And FRMD4B is further demonstrated by gene overexpression technology and participates in the proliferation of cervical cancer cell, invasive procedure, it prompts
FRMD4B can be used as the independentpredictor of cervical squamous cell carcinoma, can also be with other gene marker use in conjunction.
FRMD4B
The gene I/D of FRMD4B in the present invention be 23150, in the present invention, FRMD4B include wild type, saltant type or
Its segment.One skilled in the art will appreciate that primitive sequencer result can be compared to the reference gene to people when carrying out sequencing analysis
FRMD4B in group, therefore in the selection result may include different saltant type or its segment.In the present invention, a kind of representativeness
FRMD4B gene order as shown in NM_015123.3.
FRMD4B nucleotide full length sequence or its segment of the invention can usually use PCR amplification method, recombination method or artificial
Synthetic method obtains.
The present invention can use any method known in the art measurement gene expression.Those skilled in the art should manage
Solution, the means for measuring gene expression are not importances of the invention.Biology can be detected on transcriptional level or translation skill
The expression of marker.
Gene and albumen of the invention is detected using multiple technologies known to persons of ordinary skill in the art, these skills
Art includes but is not limited to: nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technologies, protein immunization technology.
The nucleic acid amplification technologies be selected from polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR),
Amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA) and the amplification based on nucleic acid sequence of transcriptive intermediate
(NASBA).Wherein, PCR is needed RNA reverse transcription before amplification into DNA (RT-PCR), TMA and the direct cloning RNA of NASBA.
Nucleic acid hybridization technique in the present invention include but is not limited in situ hybridization (ISH), microarray and Southern or
Northern trace.In situ hybridization (ISH) be it is a kind of use label complementary DNA or RNA chain as probe with position tissue one
Part or slice (original position) or if organize it is sufficiently small if for entirely organize (full organization embedding ISH) in specific DNA or
The hybridization of RNA sequence.
Protein immunization technology of the invention includes sandwich immunoassay, such as sandwich ELISA, wherein using identification biology mark
Two kinds of antibody of different epitopes carry out the detection of the biomarker on will object；Radiommunoassay (RIA), directly, indirectly or
Comparison enzyme linked immunosorbent assay (ELISA) (ELISA), fluorescence immunoassay (FIA), immunoblotting, is exempted from enzyme immunoassay (EIA) (EIA)
The epidemic disease precipitation method and immunoassays based on any particle are (as used gold particle, Argent grain or latex particle, magnetic-particle or quantum
Point).Immunization for example can be implemented in the form of microtiter plate or item.
The reagent for detecting FRMD4B albumen is the specific binding agent of FRMD4B albumen.Specific binding agent is such as albumen
The receptor of matter FRMD4B, conjugated protein FRMD4B agglutinin, for protein FRMD4B antibody, be directed to protein
Peptide antibody (peptidebody), the agent of bispecific dual combination or the bispecific antibody form of FRMD4B.
The example of specific binding agent is peptide, peptide mimics, aptamer, spiegelmer, darpin, ankyrin repetition
Albumen, Kunitz type domain, antibody, single domain antibody and monovalent antibody fragments.In a specific embodiment of the present invention, the specificity
Bonding agent is FRMD4B specific antibody.
The present invention provides the product of the expression of detection FRMD4B, the product includes but is not limited to chip, examination
Agent box, nucleic acid film item.Wherein chip includes: solid phase carrier；And the oligonucleotides being orderly fixed on the solid phase carrier is visited
Needle or antibody, the oligonucleotide probe some or all of specifically correspond to shown in FRMD4B sequence, the antibody
The combination FRMD4B albumen of specificity.
The solid phase carrier includes inorganic carrier and organic carrier, the inorganic carrier include but is not limited to have silicon carrier,
Glass carrier, ceramic monolith etc.；The organic carrier includes polypropylene film, nylon membrane etc..
The present invention provides a kind of kit, the kit can be used for detecting the reagent of FRMD4B gene or albumen.Choosing
From one or more substances of the following group: container, operation instructions, positive control, negative control object, buffer, auxiliary agent or molten
Agent.
In kit of the invention can also have kit operation instructions, be described how using kit into
Row detection.
In certain embodiments, it provided herein is the protein levels for detecting one or more biomarkers
Kit.In certain embodiments, the kit includes the coated examination of antibody with identification of protein biomarker
Paper slip, washing solution, the reagent for carrying out the test, Separation of Proteins or tools for purification, detection instrument and the positive and feminine gender are right
According to.In certain embodiments, the kit also includes the specification using the kit.The kit can customize confession
Use at home, clinical use or research use.
The present invention provides a kind of nucleic acid film item, nucleic acid film item includes substrate and the oligonucleotides that is fixed in the substrate
Probe；The substrate can be any substrate suitable for immobilized oligonucleotide probe, such as nylon membrane, nitrocellulose filter, poly-
Propylene film, sheet glass, silica gel chip, miniature magnetic bead etc..
Promotor and pharmaceutical composition
The present invention provides a kind of drug (compositions), it contains the promotor of a effective amount of FRMD4B, and
Pharmaceutically acceptable carrier.The composition can be used for treating cervical squamous cell carcinoma.
As a kind of preferred embodiment of the invention, the promotor of the FRMD4B is the expression vector of FRMD4B a kind of.
The expression vector is usually also containing promoter, replication orgin and/or marker gene etc..
Pharmaceutical composition of the present invention can be the dosage form of suitable drug administration by injection, be suitble to dosage form (such as glue of orally ingestible
Capsule, tablet, caplet, elixir), the ointment, emulsifiable paste or the lotion form that are suitble to local administration, be suitable as the delivery agents of eye drops
Type, the aerosol form (as sucked by nasal inhalation or mouth) for fitting through inhalation, the dosage form for being suitble to parenteral,
I.e. subcutaneous, intramuscular or intravenous injection.
Pharmaceutical composition of the invention can also be with the drug combination of other treatment cervical squamous cell carcinoma, other therapeutic compound
It can be administered simultaneously with main active constituent, or even be administered simultaneously in same composition.It can also be with individual composition
Or the dosage form different from main active constituent individually gives other therapeutic compounds.
Preferably, the means that gene therapy can be used carry out.For example, can be directly by the promotor of FRMD4B by such as infusing
It the methods of penetrates and to deliver medicine to subject；Alternatively, the ceneme that the promotion for carrying FRMD4B is adjusted (can be compared by certain approach
Such as expression vector or virus) it is delivered on target spot, concrete condition need to be depending on the type of the promotor, these are these
Known to the technical staff of field.
The present invention provides FRMD4B in the computation model that cervical intraepithelial neoplasia (CIN), cervical squamous cell carcinoma are predicted in building
Using.As those of skill in the art can understand, can be implemented in various ways and realize by marker levels and certain may
The step of property or risk association get up.Preferably, the mathematically measurement of combined protein matter and one or more other markers
Concentration, and combined value is associated with basic diagnosis problem.It can be incited somebody to action by any suitable prior art mathematical method
The measurement of marker levels is combined.
Preferably, the mathematical algorithm applied in marker combination is a kind of logarithmic function.Preferably, using such mathematics
Algorithm or such logarithmic function the result is that single value.According to basic diagnosis problem, can easily by such value with it is for example a
Body about cervical intraepithelial neoplasia (CIN) or cervical squamous cell carcinoma risk or with help to assess cervical intraepithelial neoplasia (CIN) or palace
Other intentional diagnostic uses of carcinoma of neck patient associate.In an advantageous manner, such logarithmic function is to obtain as follows
: individual segregation a) is entered into group, such as normal person, have cervical intraepithelial neoplasia (CIN) or cervical squamous cell carcinoma risk individual, have
Cervical intraepithelial neoplasia (CIN) or the patient of cervical squamous cell carcinoma etc., b) difference between these groups is identified by univariate analysis
Significant marker, c) logarithmic regressions analysis with assess marker can be used for assessing these difference group independent difference values, and
D) building logarithmic function carrys out composition independency difference value.In such analysis, marker is no longer independent, but is represented
One marker combination.
Logarithmic function for marker combination to be got up with disease association is preferably developed using by applied statistical method
With the algorithm of acquisition.For example, suitable statistical method is discriminant analysis (DA) (i.e. linear, secondary, regular DA), Kernel method
(i.e. SVM), nonparametric technique (i.e. k- nearest neighbor classifiers), PLS (partial least square), method (the i.e. logic based on tree
Recurrence, CART, random forest method, boosting/bagging method), generalized linear model (i.e. logarithm regression), the side based on principal component
Method (i.e. SIMCA), broad sense Additive Model, the method based on fuzzy logic, the method based on artificial neural network and genetic algorithms.Skillfully
Technical staff merges in the suitable statistical method of selection to assess marker group of the invention thus to obtain suitable mathematical algorithm
Aspect will not be problematic.In one embodiment, make for obtaining in assessment cervical intraepithelial neoplasia (CIN) or cervical squamous cell carcinoma
The statistical method of mathematical algorithm is selected from DA (i.e. linear, secondary, rule based judgment analysis), Kernel method (i.e. SVM), non-ginseng
Counting method (i.e. k- nearest neighbor classifiers), PLS (partial least square), based on tree method (i.e. logistic regression, CART, with
Machine forest method, propelled method) or generalized linear model (i.e. logarithm regression).
Below with reference to specific embodiment further illustrate the present invention, the embodiment of the present invention for explaining only the invention,
It is not intended to limit protection scope of the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1 screens gene marker relevant to cervical squamous cell carcinoma
1, sample collection
Collect 32 cervical squamous cell carcinoma (CESC) tissues and 18 normal tissues (N), 24 cervical intraepithelial neoplasia (CIN)s (CIN)
Tissue, all cases are preoperative not to do any immunosuppressant treatment, radiotherapy and chemotherapy, and all research objects being included in are in receipts
Informed consent form is signed before collection sample.The acquirement of above-mentioned all samples passes through the agreement of the committee, organizational ethics.Every group takes 4
Example sample carries out the detection and analysis of gene expression profile, carries out the screening of difference expression gene, and in each group all cases sample
Carry out confirmatory experiment.
2, the preparation of RNA sample
The total serum IgE in each group tissue, specific steps reference are extracted using the tissue RNA extracts kit of QIAGEN
Book.
3, the quality analysis of RNA sample
The RNA of said extracted is subjected to agarose gel electrophoresis, using Nanodrop2000 to the concentration of mentioned RNA and pure
Degree is detected, and agarose gel electrophoresis detects RNA integrality, and Agilent2100 measures RIN value.Single requirement for construction data base RNA is total
5 μ g are measured, concentration >=200ng/ μ L, OD260/280 is between 1.8~2.2.
4, the building of cDNA library
The building of cDNA library, concrete operations are carried out using Illumina TruseqTM RNA sample Prep Kit
By specification carries out.
5, it is sequenced
CDNA library is sequenced using Illumina X-Ten microarray dataset, concrete operations by specification carries out.
6, high-throughput transcript profile sequencing data analysis
Bioinformatic analysis is carried out to sequencing result, is analyzed using metaMA packet, p value merges side used in meta analysis
Method is inverse normal method, and the screening criteria of difference expression gene is FDR < 0.05.
7, result
RNA-seq is the results show that compared with normal control, and FRMD4B gene is in cervical squamous cell cancer and on uterine neck
Expression quantity in intraepithelial neoplasia tissue is significantly lowered, wherein the expression quantity in cervical squamous cell cancer and cervical intraepithelial neoplasia sample
Pathological tissues are compared, and expression quantity is also significantly lowered, and difference has statistical significance, therefore carries out further full-page proof to FRMD4B
This verifying.
The differential expression of 2 QPCR sequence verification FRMD4B gene of embodiment
1, large sample QPCR verifying is carried out to FRMD4B gene differential expression.
2, RNA is extracted
The total serum IgE in each group tissue, specific steps reference are extracted using the tissue RNA extracts kit of QIAGEN
Book.
3、QPCR
1) reverse transcription reaction
LncRNA reverse transcription is carried out using FastQ μ ant the first chain of cDNA synthetic agent box (article No.: KR106), is gone first
Except genomic DNA reacts, 5 × gDNA B μ ffer, 2.0 μ l is added in test tube, 1 μ g of total serum IgE adds RNase Free ddH2O
Make total volume to 10 μ l, 42 DEG C of heating 3min. in water-bath
By 10 × Fast RT B μ, 2.0 μ l, RT Enzyme Mix of ffer, 1.0 μ l, FQ-RT Primer Mix, 2.0 μ
L, RNase Free ddH25.0 μ l of O is added in above-mentioned test tube after mixing and is mixed together totally 20 μ l, 42 DEG C of heating in water-bath
15min, 95 DEG C of heating 3min.
2) design of primers
QPCR amplimer, You Bomai are designed according to the coded sequence of FRMD4B gene and GAPDH gene in Genebank
The synthesis of moral biotech firm.Specific primer sequence is as follows:
FRMD4B gene:
Forward primer is 5 '-TATACAAGACAAGGTGAA-3 ' (SEQ ID NO.1)；
Reverse primer is 5 '-AGAGAAGAGTTAGCATAC-3 ' (SEQ ID NO.2).
GAPDH gene:
Forward primer is 5 '-AATCCCATCACCATCTTCCAG-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-GAGCCCCAGCCTTCTCCAT-3 ' (SEQ ID NO.4).
3) QPCR amplification is examined
It with SuperReal PreMix Plus (SYBR Green) (article No.: FP205), is expanded, experimental implementation is by production
Product specification carries out.
Using 20 μ l reaction systems: 2 × SuperReal PreMix Plus 10 μ l, each 0.6 μ of forward and reverse primer (10 μM)
L, 5 × ROX Reference Dye△2 μ l, 2 μ l of DNA profiling, 4.8 μ l of sterile purified water.3 parallel pipes are arranged in each sample,
All amplified reactions are repeated three times the above reliability to guarantee result.
Amplification program are as follows: 95 ° of 15min, (95 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 32s) × 40 circulations, 95 DEG C of 15s, 60 DEG C
60s, 95 DEG C of 15s).
4) screening of cDNA template concentrations
After each sample cDNA is mixed, 10 times of gradients (10,100,1000,10000,100000 times) are carried out as template
Dilution, sample respectively takes 2 μ l to make template after dilution, is expanded respectively with target gene primer and reference gene primer, while
60-95 DEG C of progress melt curve analysis analysis carries out the screening of template concentrations according to amplification efficiency height and the unimodal principle of solubility curve.
According to solubility curve, it can be seen that when 10 times of dilutions of carry out of cDNA, the amplification efficiency of PCR is higher, and dissolution is bent
Line is unimodal relatively good.
5) sample RealTime PCR is detected
2 μ l will be taken to make template after 10 times of each sample cDNA dilutions, respectively with target gene primer and reference gene primer into
Row amplification.Simultaneously in 60-95 DEG C of progresss solubility curve analysis, purpose band is determined by melt curve analysis analysis and electrophoresis, 2-ΔΔCT
Method carries out relative quantification.
4, result
As a result as shown in Figure 1, compared with normal tissue, in cervical squamous cell cancer and cervical intraepithelial neoplasia (CIN) tissue
FRMD4B expression quantity is significantly lowered, and wherein cervical intraepithelial neoplasia (CIN) tissue is presented conspicuousness and lowers compared with normal tissue,
Cervical squamous cell cancer is presented conspicuousness and lowers, difference all has statistical significance compared with cervical intraepithelial neoplasia (CIN) tissue
(P<0.05)。
The overexpression of 3 FRMD4B gene of embodiment
Specific primer is designed according to FRMD4B gene sequence information in NCBI, amplification is carried out and obtains corresponding gene, by base
Because being transformed into DH5 α competent cell after being attached respectively with expression vector pEGFP-C1 after the recycling of segment glue, picking is positive
Clone obtains pEGFP-C1-FRMD4B recombinant plasmid after PCR and sequencing identification.
1, cell culture
Cervical squamous cell carcinoma cell (Hela) is with the RPIM-1640 culture medium containing 10% fetal calf serum and 1%P/S in 37 DEG C, 5%
CO2Incubator in cultivate, when cell it is long to 80%~90% when, trypsase conventional digestion of the use 0.25% containing EDTA passes
Generation.
2, it transfects
Experiment is divided into 3 groups, respectively control group (Hela), blank control group (transfection pEGFP-C1), experimental group (transfection
PEGFP-C1-FRMD4B), turned according to 2000 transfection reagent specification of the lipofectamine of invitrogen company
Dye.Steps are as follows:
1) cell dissociation of logarithmic growth phase gently blows and beats into single cell suspension, the not antibiotic culture medium inoculated of use in
6 orifice plates, the cell densities of 6 orifice plates is up to 2 × 105A/hole, when cell confluency carries out transfection assay up to 40%-60%；
2) next day transfects, and takes 10 μ l Lipofectamine 2000 to be diluted in Opti-MEM I of the 200 μ l without serum and trains
Base is supported, 5min is stored at room temperature；
3) 4 μ g plasmids is taken to be dissolved in the Opti-MEM I culture medium of 200 μ l serum-frees；
4) above two dilution is uniformly mixed, is incubated at room temperature 20 minutes；The ratio of plasmid and transfection reagent is every hole 4
μg:10μl；
5) cell in 6 orifice plates is replaced into the fresh RPIM-1640 culture medium of 1.6ml.Above-mentioned suspension is added separately to 6 holes
In plate, every hole culture solution final volume is 2ml；
6) the replacement fresh complete culture solution of 2ml continues to cultivate after transfecting 6 hours；
7) 72h collects cell, extracts RNA, carries out QPCR detection.
3, QPCR detects the transcriptional level of FRMD4B gene
1) extraction of cell total rna
The extraction of cell total rna is carried out using the cell RNA extracts kit of QIAGEN, specific steps, which are detailed in kit, to be said
Bright book
3.2 reverse transcription steps are the same as embodiment 2.
3.3 QPCR amplification steps are the same as embodiment 2.
4, statistical method
Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD,
Using SPSS18.0 statistical software come difference for statistical analysis, being overexpressed between FRMD4B gene expression panel and control group
It is examined using t, it is believed that there is statistical significance as P < 0.05.
5, result
As a result such as Fig. 2 is shown, compared to the control group Hela and transfection zero load pEGFP-C1, experimental group pEGFP-C1-FRMD4B
The expression of FRMD4B gene can be dramatically increased, difference has statistical significance.
4 CCK-8 method of embodiment detects cervical squamous cell carcinoma cell-proliferation activity
1, with 0.25% trypsin digestion and cell after transfecting 6 hours, list is made into the culture solution containing 10% fetal calf serum
A cell suspension (1 × 104/ hole), (100 hole μ l/) is inoculated in 96 well culture plates, and every group sets 6 multiple holes, while setting up nothing
The culture solution blank control of cell.
2, it is separately added into cell Proliferation detection reagent CCK-8 in detection time point (0h, for 24 hours, 48h, 72h, 96h, 120h),
Working concentration is 1:10, i.e. 10 μ l CCK-8 are added in 100 μ l culture solutions；
3, at 37 DEG C, 5%CO2It after being incubated for 1h in incubator, is detected, microplate reader reads OD450 nm.
4, result judges: 0h after transfection, for 24 hours, 48h, 72h, 96h, 120h observe the proliferation of Hela cell using microplate reader
Activity, absorption photometric value (OD value) of the cell at wavelength 450nm indicates the cell quantity for being in vegetative state, with cell-free
Culture solution group OD value is control.
5, result
As a result it as shown in figure 3, the cell Proliferation of the experimental group of transfection pEGFP-C1-FRMD4B substantially reduces, prompts to change
The expression of FRMD4B can change the proliferative capacity of cervical squamous cell carcinoma cell, illustrate the proliferation of FRMD4B Yu cervical squamous cell carcinoma cell
It is related.
5 Transwell method of embodiment detects cervical squamous cell carcinoma cell migration and invasion
1, cell migration ability detects
1) migration the previous day, complete medium was added in orifice plate, is put into cell and is placed in incubator overnight；
2) cell after transfection 48h is subjected to Nature enemy, cell count is collected, with the serum free medium of 0.2%BSA
It is made 1 × 105Suspension.
3) take 200 μ l cell suspension inoculations in the small indoor culture of Transwell.
4) cell is taken out, remaining liq is blotted and is placed on the fixed 30min of 70% methanol, wipe the thin of upper chamber face with cotton swab
Born of the same parents.
5) cell immerses 0.4% violet staining 10min, PBS twice of cleaning, randomly selects the visual field under microscope and counts film
The cell number of bottom surface.
2, cell invasion ability detects
1) matrigel, each 24 hole suspension type cell bottom are diluted with 1640 1:8 of RPIM on ice on the day before Matrigel
Portion film upper chamber face adds the 60 μ l dried for standby of matrigel after dilution.
2) aquation basilar memebrane: 50 serum-free mediums of the μ l containing 10g/LBSA are added in every hole, and 37 DEG C, 30min, culture is sucked out
Residual liquid in plate.
3) cell after transfection 48h is subjected to Nature enemy, cell count is collected, with the serum free medium of 0.2%BSA
It is made 1 × 105Suspension.
4) cell is taken out, remaining liq is blotted and is placed on the fixed 30min of 70% methanol, wipe the thin of upper chamber face with cotton swab
Born of the same parents.
5) cell immerses 0.4% violet staining 10min, PBS twice of cleaning, randomly selects the visual field under microscope and counts film
The cell number of bottom surface.
3, data processing
Statistical analysis is carried out to data with SPSS18.0 software.Measurement data is indicated with mean ± standard deviation.Multiple samples
This mean compares using one-way analysis of variance, and P < 0.05 is that difference is statistically significant.
4, result
As a result as shown in figure 4, the cell migration of experimental group and invasion number relatively transfect lacking for empty plasmid respectively, illustrated table
Migration and the invasive ability that cervical squamous cell carcinoma cell is reduced up to FRMD4B gene prompt FRMD4B to can be used as target molecule applied to palace
The exploitation of carcinoma of neck therapeutic agent.
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
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<120>for developing the target molecule of the diagnosis product of cervical disease
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<211> 18
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<213>artificial sequence (Artificial Sequence)
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tatacaagac aaggtgaa 18
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
agagaagagt tagcatac 18
<210> 3
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<213>artificial sequence (Artificial Sequence)
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gagccccagc cttctccat 19
Claims (10)
1. a kind of reagent, which is characterized in that the reagent is able to detect FRMD4B gene or the level of its expression product.
2. reagent according to claim 1 characterized by comprising
The probe of specific recognition FRMD4B gene；Or
The primer of specific amplification FRMD4B gene；Or
Specifically bind the antibody of the albumen of FRMD4B gene coding.
3. reagent according to claim 2, which is characterized in that the primer sequence of specific amplification FRMD4B such as SEQ ID
Shown in NO.1~2.
4. a kind of kit, which is characterized in that the kit includes the described in any item reagents of claim 1-3.
5. a kind of chip, which is characterized in that the chip includes the described in any item reagents of claim 1-3.
6. a kind of nucleic acid film item, which is characterized in that the nucleic acid film item includes the described in any item reagents of claim 1-3.
7. a kind of composition, which is characterized in that the composition includes the promotor of FRMD4B and/or pharmaceutically acceptable
Carrier.
8. composition according to claim 7, which is characterized in that the promotor is the carrier for being overexpressed FRMD4B.
9. a kind of method of the drug candidate of screening treatment cervical squamous cell carcinoma, which is characterized in that the described method includes:
The cultivating system of expression or the albumen containing FRMD4B gene or its coding is handled with substance to be screened；With
Detect the expression or activity of the albumen of FRMD4B gene or its coding in the system；
Wherein, if the substance to be screened can promote the level or expression activity of FRMD4B gene, show the object to be screened
Matter is the drug candidate for treating cervical squamous cell carcinoma.
10. following described in any item applications:
A. claim 1-3 described in any item reagents are in preparation early diagnosis cervical intraepithelial neoplasia (CIN), cervical squamous cell carcinoma
Application in product；
B. kit as claimed in claim 4 answering in the product that preparation diagnoses cervical intraepithelial neoplasia (CIN), cervical squamous cell carcinoma
With；
C. application of the chip described in claim 5 in the product that preparation diagnoses cervical intraepithelial neoplasia (CIN), cervical squamous cell carcinoma；
D. nucleic acid film item as claimed in claim 6 is in the product that preparation diagnoses cervical intraepithelial neoplasia (CIN), cervical squamous cell carcinoma
Using；
E. composition described in claim 7 or 8 preparation treatment cervical squamous cell carcinoma and its transfer, invasion drug in application；
F. application of the method as claimed in claim 9 in the drug candidate of screening treatment cervical squamous cell carcinoma；
G.FRMD4B preparation treatment cervical squamous cell carcinoma and its transfer, invasion drug in application；
Application of the h.FRMD4B in the computation model that cervical intraepithelial neoplasia (CIN), cervical squamous cell carcinoma are predicted in building.
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|---|---|---|---|---|
| US20130065791A1 (en) * | 2010-05-19 | 2013-03-14 | Signature Diagnostics Ag | Methods and kits for diagnosing colorectal cancer |
| US20150361396A1 (en) * | 2013-02-27 | 2015-12-17 | The Broad Institute Inc. | T cell balance gene expression, compositions of matters and methods of use thereof |
| CN105256051A (en) * | 2015-11-11 | 2016-01-20 | 首都儿科研究所 | Probe set and reagent kit used for detecting pathopoiesia/susceptibility genes of congenital megacolon and relative syndromes |
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2019
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|---|---|---|---|---|
| US20130065791A1 (en) * | 2010-05-19 | 2013-03-14 | Signature Diagnostics Ag | Methods and kits for diagnosing colorectal cancer |
| US20150361396A1 (en) * | 2013-02-27 | 2015-12-17 | The Broad Institute Inc. | T cell balance gene expression, compositions of matters and methods of use thereof |
| CN105256051A (en) * | 2015-11-11 | 2016-01-20 | 首都儿科研究所 | Probe set and reagent kit used for detecting pathopoiesia/susceptibility genes of congenital megacolon and relative syndromes |
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