CN105288659A

CN105288659A - Application of TENM1 gene and its expression product on diagnosis and treatment of papillary adenocarcinoma

Info

Publication number: CN105288659A
Application number: CN201510767426.4A
Authority: CN
Inventors: 杨承刚
Original assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Current assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Priority date: 2015-06-01
Filing date: 2015-11-11
Publication date: 2016-02-03
Anticipated expiration: 2035-11-11
Also published as: CN105238872B; CN105238872A; CN105288659B

Abstract

The invention relates to an application of TENM1 gene and its expression product on diagnosis and treatment of papillary adenocarcinoma. An inventor employs a bioinformatics method for analysis based on a high-flux sequencing result and performs gene screening, a candidate gene TENM1 can be selected, further, a molecular cell biology experiment confirms that TENM1 and papillary adenocarcinoma have good correlation, TENM1 can be used for preparing an auxiliary diagnosis and treatment preparation for papillary adenocarcinoma, and has an important clinical application value.

Description

TENM1 gene and expression product thereof are in the application of diagnosis and treatment papillary adenocarcinoma

Technical field

The present invention relates to biomedicine field, concrete the present invention relates to the application at diagnosis and treatment papillary adenocarcinoma of TENM1 gene and expression product thereof.

Background technology

Thyroid carcinoma is the most common cancer of hormonal system, and sickness rate ranks first in head-neck malignant tumor.The reason that the pathogenesis of thyroid carcinoma and sickness rate sharply increase is still not clear.The sickness rate of thyroid papillary carcinoma (papillarythyroidcarcinoma, PTC) accounts for 80% of all thyroid carcinoma incidence types.PTC accounts for into 60% of human thyroid carcinomas and the whole of pediatric thyroid carcinomas, and grade malignancy is lower.PTC is mainly in young women, is more common between 30-45 year.About 80% tumor is multicentricity, and about 1/3 involves bilateral thyroid.Comparatively early just occur Cervical Lymph Node Metastasis, but prognosis is better.The early stage non-evident sympton of PTC, shows as accidentally finding without painful lump or when health check-up of region thyreoidea, lump can the several months to many decades, quality soft or hard differs, and lump more than half is more tough, and the patients less than 1/3 is the hard lump of matter; There is deradenoncus when there is Cervical Lymph Node Metastasis, invade recurrent laryngeal nerve and occur hoarseness.Invade tracheal strips and can cause dyspnea or hemoptysis.Thyroid mini-carcinoma clinical examination is not easily found, and is often onset symptoms with deradenoncus, also can oppress recurrent laryngeal nerve and first show as hoarseness.The diagnostic method of current thyroid papillary carcinoma mainly comprises medical history and symptom, lab testing, imaging examination and the inspection of fine needle aspiration aspirate cytology etc. of patient, but still comes with some shortcomings.Therefore, inquire into the risk factor that thyroid papillary carcinoma occurs, study the pathogenesis of thyroid papillary carcinoma from molecular level, find specific molecular marker, for early prevention, early discovery, early treatment's thyroid papillary carcinoma has great importance.

Inventor carries out high-flux sequence to 6 routine thyroid papillary carcinoma case samples and 3 routine normal controls, carries out genescreen, pick out candidate gene TENM1 in conjunction with bioinformatics method.Further, invention has been the relation that molecular cytobiology method confirms TENM1 and thyroid papillary carcinoma: with thyroid papillary carcinoma, there is good dependency, can be used for preparation thyroid papillary carcinoma assisting in diagnosis and treatment preparation, there is important clinical value.

Summary of the invention

TENM1 gene and/or protein inhibitor is the object of the present invention is to provide to prepare the application in antithyroid papillary carcinoma preparation.

For achieving the above object, first the present invention screens candidate gene TENM1 by high-flux sequence in conjunction with bioinformatics method, and then the relation of TENM1 and thyroid papillary carcinoma: TENM1 and thyroid papillary carcinoma have good dependency by molecular cytobiology method validation, can be used for preparation thyroid papillary carcinoma assisting in diagnosis and treatment preparation, there is important clinical value.

Further, described antithyroid papillary carcinoma preparation refers to the preparation of the expression that can suppress TENM1 gene in papillary thyroid carcinoma cells.The expression that those skilled in the art know suppressor gene can adopt the one in following method and/or several usually: by the albumen of the suppressor gene of activating genes of interest, the inhibition of gene expression of activating genes of interest, adopt RNA perturbation technique to suppress destination gene expression, activate promote genes of interest mRNA degraded microRNA, import promote the degraded of genes of interest encoding proteins molecule, suppress to promote the factor of destination gene expression and the expression of albumen.Namely by activate TENM1 gene suppressor gene, activate suppress TENM1 gene expression albumen, import suppress TENM1 gene expression siRNA, activate promote TENM1mRNA degraded microRNA, import promote TENM1 protein degradation molecule, suppress to promote the factor of TENM1 gene expression and the expression of albumen.

RNA interference (RNAi) refers to that exogenous and endogenous double-stranded RNA induces the mRNA selective degradation of homology target gene in vivo, cause the phenomenon of PTGS, it is a kind of expression using little double-stranded RNA to block body certain specific gene interior efficiently, specifically, impel mRNA to degrade, make cells show go out the technology of specific gene disappearance phenotype.Can adopt direct synthesis technique after siRNA has designed or build SiRNA expression vector, the siRNA prepared can by approach transfectional cells such as Mechanical Method, lipofectamine reagent method such as calcium phosphate precipitation, electroporation, DEAE-glucosan and polybrene method, microinjection or particle guns.

Further, the siRNA sequence of described suppression TENM1 gene expression is selected from one in one sequence and/or several: SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4, SEQIDNO.5, SEQIDNO.6.Preferred siRNA sequence is SEQIDNO.3 and SEQIDNO.4.

The object of the present invention is to provide a kind of antithyroid papillary carcinoma preparation, described antithyroid papillary carcinoma preparation suppresses the expression of TENM1 gene in papillary thyroid carcinoma cells.Further, containing the siRNA suppressing TENM1 gene expression in described antithyroid papillary carcinoma preparation.

The object of the present invention is to provide the application of TENM1 gene in preparation thyroid papillary carcinoma diagnosis and treatment preparation.

Further, the diagnosis and treatment preparation of described thyroid papillary carcinoma comprises the expression detecting TENM1 gene in Papillary Thyroid Carcinoma with fluorescence quantifying PCR method, method for gene chip.

Fluorescence quantitative PCR method is by fluorescent dye or fluorescently-labeled specific probe, carries out labelling tracking, real time and on line monitoring course of reaction, can analyze in conjunction with corresponding software to product PCR primer, calculates the initial concentration of testing sample template.The appearance of quantitative fluorescent PCR, greatly simplifies the process of detection by quantitative, and really achieves absolute quantitation.The appearance of multiple detection system, makes the selectivity of experiment stronger.Automation mechanized operation improves work efficiency, reacts quick, reproducible, highly sensitive, high specificity, result be clear.

Gene chip is also called DNA microarray (DNAmicroarray), three kinds of main Types can be divided into: 1) be fixed on polymer matrix film (nylon membrane, nitrocellulose membrane etc.) nucleic probe on surface or cDNA fragment, usually be hybrid with it with isotope-labeled target gene, detected by radiography technology.2) fixing DNA probe array on a glass by point sample method, detecting by hybridizing with fluorescently-labeled target gene.3) oligonucleotide probe array of directly synthesis on the hard surfaces such as glass, hybridizes with fluorescently-labeled target gene and detects.Gene chip is as a kind of advanced person, extensive, high throughput testing technology, and be applied to the diagnosis of disease, its advantage has the following aspects: one is susceptiveness highly and accuracy; Two is fast and convenient; Three is can detect various diseases simultaneously.

Described contains the primer of a pair specific amplification TENM1 gene for the product of TENM1 gene in fluorescence quantifying PCR method detection thyroid papillary carcinoma; Described gene chip comprises the probe with the nucleic acid array hybridizing of TENM1 gene.

Further, the diagnosis and treatment preparation of described thyroid papillary carcinoma comprises the expression detecting TENM1 albumen in adenocarcinoma of lung with immunization method.In preferred described immunologic detection method detection thyroid papillary carcinoma, TENM1 protein expression is westernblot and/or ELISA and/gold colloidal detection method.

Enzyme-linked immunosorbent assay (ELISA) is adsorbed on surface of solid phase carriers by known antigen or antibody, makes the technology that the antigen antibody reaction of enzyme labelling is carried out at solid phase surface.This technology can be used for detecting macromole antigen and specific antibody etc., has quick, sensitive, easy, carrier and is easy to the advantages such as standardization.ELISA detection kit can be divided into indirect method, double-antibody method, competition law, dibit point one-step method, prize law to survey the ELISA of IgM antibody, application Avidin and biotin according to testing goal and operating procedure.In ELISA detection kit, chromogenic substrate can select horseradish peroxidase (HRP) or alkali phosphatase (AP).

Conventional immune colloid gold detection technique: (1) immune colloid gold light microscopic staining cell suspension smear or tissue slice, the antibody of available colloid gold label dyes, also can on the basis of colloid gold label, labelling is strengthened with silver-colored developer solution, make the silver atoms be reduced be deposited on the gold grain surface of labelling, obviously can strengthen the sensitivity of colloid gold label.(2) immune colloid gold staining method for electron microscopy with the antibody of colloid gold label or anti antibody with negative staining Virus Sample or organize ultrathin section to be combined, then can carry out negative staining.Can be used for observation and the Viral diagnosis of morphology of virus.(3) dot immunogold filtration assay application microporous filter membrane makes carrier, first by antigen or antibody point on film, add sample to be checked after closing, wash the corresponding antigen of antibody test or the antibody of rear colloid gold label.(4) specific antigen or antibody are fixed on film with ribbon by colloidal gold immunity chromatography, colloid gold label reagent (antibody or monoclonal antibody) is adsorbed on pad, after in the sample pad that sample to be checked is added to test strips one end, moved forward by capillarity, react to each other after dissolving the colloid gold label reagent on pad, when moving to the region of fixing antigen or antibody, there is specific binding with it again and be trapped in the conjugate of thing to be checked and gold marked reagent, be gathered in detection zone, by being observed visually colour developing result.This method now develops into diagnosis test paper, uses very convenient.

Further, the ELISA method of described detection TENM1 albumen is for using ELISA detection kit.Antibody in described test kit can adopt commercially available TENM1 monoclonal antibody.Further, described test kit comprises: wrap by the solid phase carrier of TENM1 monoclonal antibody, enzyme labelled antibody, the substrate of enzyme, protein standard substance, negative controls, diluent, cleaning mixture, enzyme reaction stop buffer etc.

Further, the colloidal gold method of described detection TENM1 albumen is for using detection kit, and described antibody can adopt commercially available TENM1 monoclonal antibody.Further, described gold-immunochromatographyreagent reagent for assay box adopts colloidal gold immunochromatographimethod technology or gold colloidal percolation.Further, detection zone (T) specking on described gold-immunochromatographyreagent reagent for assay box nitrocellulose filter has anti-TENM1 monoclonal antibody, quality control region (C) specking has immunoglobulin IgG.

Preferred anti-TENM1 monoclonal antibody is selected from following antibody: the article No. that NovusBiologicals company provides is the article No. that the Teneurin-1Antibody of H00010178-M01, Abcam company provides is the Anti-ODZ1antibody of ab56597.

The object of the present invention is to provide a kind of gene detecting kit detecting thyroid papillary carcinoma, it is characterized in that, described test kit detects gene TENM1, adopts special forward primer and downstream primer, forward primer sequence is SEQIDNO.9, and downstream primer sequence is SEQIDNO.10.

Further, this PCR kit is suitable for all types fluorescence quantitative gene extender deposited at present commercially, highly sensitive, quantitatively quick and precisely, good stability, has a good application prospect.

Further, above-mentioned PCR kit for fluorescence quantitative component comprises: Auele Specific Primer, internal reference primer, fluorescence quantitative PCR reaction solution.Wherein said Auele Specific Primer comprises forward primer and downstream primer, and forward primer sequence is SEQIDNO.9, and downstream primer sequence is SEQIDNO.10.Described internal reference primer is β-actin internal reference primer, and forward primer sequence is SEQIDNO.11, and downstream primer sequence is SEQIDNO.12.

Described test kit also comprises RNA extraction agent.Preferably reagent (invitrogen, article No. 15596-018) carries out sample rna extraction.

The present invention also have detected this test kit susceptiveness, and it is 10 that result shows this test kit detection range ⁶-10 ²copies/ μ l, minimum concentrations is 100copies/ μ l.

The object of the invention there is provided a kind of thyroid papillary carcinoma protein detection kit, and described detection kit detects TENM1 albumen.Further, described test kit also comprises other detectable.

The object of the invention there is provided a kind of gene chip detecting thyroid papillary carcinoma, and described gene chip comprises the probe with the nucleic acid array hybridizing of TENM1 gene.

Accompanying drawing explanation

Fig. 1 TENM1 gene relative expression's spirogram in cancerous tissue and normal structure

Each group TENM1mRNA expression after Fig. 2 RNA disturbs

Detailed description of the invention

Below in conjunction with specific embodiment, setting forth the present invention further, only for explaining the present invention, and can not limitation of the present invention be interpreted as.Those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.The experimental technique of unreceipted actual conditions in the following example, the usually conveniently conditioned disjunction condition examinations of advising according to manufacturer.

The collection of embodiment 1 case

During getting in January, 2011 in June, 2012 in Beijing Friendship Hospital because of thyroid tumor hospitalisation for surgery, in art, freezing rapid pathology and the slow pathology of postoperative paraffin confirm as the patient of thyroid papillary carcinoma, select through first merit eight and the normal healthy population of thyroid color ultrasound examination as a control group, to choose 6 routine thyroid papillary carcinoma case samples and 3 routine normal control tissues.

Embodiment 2 high-flux sequence and analysis

RNA extraction is carried out to tissue, RNA extract after agarose gel electrophoresis, from electrophoresis result can preliminary judgement extract RNA sample whether up-to-standard, whether may be used for further transcriptome analysis.And then detected the extraction situation of RNA sample by NanoDrop1000 spectrophotometer, the sample requirement of RNA-seq order-checking: OD260/OD280 is 1.8-2.2.

Order-checking platform is the HiSeq2500 high-flux sequence platform of Illumina company, carry out the order-checking of the high flux transcript profile degree of depth, after order-checking, we use Fast-QC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) software to carry out total evaluation to the quality of sequencing data, comprise the mass value distribution of base, the position distribution of mass value, GC content, PCRduplication content, the frequency etc. of kmer.When differential genes expression analysis, according to the FPKM value obtained, internationally recognized algorithm EBSeq is adopted to carry out differential screening.Wherein, during screening, LOG2FC>1 or <-1, FDR<0.05.In order to better understand the function of difference expression gene, we have carried out GeneOnlogy and signal path analysis to difference expression gene, and functional annotation and protein interaction analysis of network are carried out to difference expression gene, in view of the result of above data analysis, in conjunction with document, we have screened difference expression gene TENM1.

Embodiment 3 Papillary Thyroid Carcinoma and normal structure TENM1 expression conditions

One, materials and methods

1, material

Choose 78 routine Papillary Thyroid Carcinomas and 16 routine normal structures, it is divided into groups and numbers.

2, method

The extraction of 2.1 Papillary Thyroid Carcinomas and normal structure total serum IgE

Adopt reagent (invitrogen, article No. 15596-018) carries out sample rna extraction, and experimental implementation is undertaken by product description, and concrete operations are as follows:

Frozen in liquid nitrogen after collecting sample, organizing the mortar putting into pre-cooling to grind, after tissue samples is powdered after taking-up:

1. Trizol is added, room temperature preservation 5 minutes;

2. add chloroform 0.2ml, use forced oscillation centrifuge tube, fully mix, ambient temperatare puts 5 minutes-10 minutes;

3. 12000rpm high speed centrifugation draws upper strata aqueous phase (inhaling 70%) in another new centrifuge tube pipe after 15 minutes, notes the protein substance be not drawn onto between two-layer aqueous phase.Move into new pipe, add isopyknic-20 DEG C of pre-cold isopropanols, fully put upside down mixing, be placed in 10 minutes on ice;

4. 12000rpm carefully discarded supernatant at a high speed after 15 minutes, added 75%DEPC ethanol and washed paint precipitation (4 DEG C of preservations), wash paint precipitate in the ratio of Iml/mlTrizol, vibration mixing, 12000rpm high speed centrifugation 5 minutes at 4 DEG C;

5. discard ethanol liquid, ambient temperatare puts 5 minutes fully to dry precipitation, adds DEPC treated water dissolution precipitation;

6. RNA purity and concentration is measured with Nanodrop2000 ultraviolet spectrophotometer, frozen in-70 DEG C.RNA quality judging standard: the OD260/OD280 value of RNA sample is between 1.7-2.2; Total serum IgE electrophoresis pattern has 28S, 18S band clearly; The electrophoresis pattern of 70 DEG C of water bath heat preservations after 1 hour and the collection of illustrative plates no significant difference before water bath heat preservation.

2.2 reverse transcription synthesis cDNA

Adopt iIIReverseTranscriptase (invitrogen, article No. 18080-044) carries out cDNA reverse transcription, and experimental implementation is undertaken by product description, and concrete operations are as follows:

Use Reverse Transcriptase kit, with RT Buffer, converse record synthesis cDNA is carried out to l μ g total serum IgE.Adopt 25 μ l reaction systems, each sample gets 1 μ g total serum IgE as template ribonucleic acid, in PCR pipe, add following component respectively:

5 × RT Buffer 5 μ l, 10mmol/ldNTP1.25 μ l, 0.1mmol/lDTT2.5 μ l, 30 μm of mol/lOligodT2 μ l, 200U/ μ lMMLV1.25 μ l, template ribonucleic acid 1 μ g, add aquesterilisa to total system 25 μ l.Hatch 1 hour for 42 DEG C, 72 DEG C 10 minutes, of short duration centrifugal.It is for subsequent use that-20 DEG C of refrigerators are put in cDNA preservation.

2.3Real-TimePCR

2.3.1 instrument and analytical method

With ABI7500 type quantitative real time PCR Instrument, 2-△ △ CT method is adopted to carry out the relative quantitative assay of data.

2.3.2 design of primers

Adopt online primer-design software, gene order, with reference to NCBI:XM_011531237.1, is synthesized by invitrogen company after design of primers.Concrete primer sequence is as follows:

Table 1 primer sequence

Operating process is as follows:

(1) reaction system: use Power greenPCRMasterMix (invitrogen, article No. 4367659) increases, and experimental implementation is undertaken by product description.Amplification program is: 95 ° of 5min, (95 DEG C of 15sec, 60 DEG C of 45sec) × 40 circulations.

Table 2RealTime reaction system

Component	Addition
		2×mix	10μl
Forward primer (10uM)	0.5μl
		Downstream primer (10uM)	0.5μl
Template	2μl
		Add sterile purified water	To 25 μ l

(2) primer screening

After each sample cDNA is mixed, 5 times of gradient dilutions are carried out as template, after dilution, sample is respectively got 2 μ l and is made template, increase with genes of interest primer and reference gene primer respectively, carry out melt curve analysis analysis at 60-95 DEG C simultaneously, carry out primer screening according to amplification efficiency height and the unimodal principle of solubility curve.

(3) sample RealTimePCR detects

Get 2 μ l after doubly being diluted by each sample cDNA10 and make template, increase with genes of interest primer and reference gene primer respectively.Carry out solubility curve analysis at 60-95 DEG C simultaneously.

Two, experimental result

Real-time quantitative PCR amplification curve flex point is clear, and the overall collimation of amplification curve is good, shows that the amplification efficiency of each reaction tube is close, and the limit is flat and without raising up now, exponent phase slope is comparatively large, illustrates that amplification efficiency is higher; Sample amplified production solubility curve is all unimodal, illustrates that amplified production only has one, is specific amplification; Relative quantification formula according to qRT-PCR: 2-Δ Ct × 100%, compares the expression of TENM1 gene in Papillary Thyroid Carcinoma and normal structure.Result display (specifically seeing Fig. 1): qRT-PCR stable amplification result, wherein the expression of TENM1 in cancerous tissue is higher than nearly 4 times of normal structure, the result of confluence analysis TENM1 high expressed in Papillary Thyroid Carcinoma of above result verification high flux transcript profile expression data.

Embodiment 4RNAi suppresses TENM1 gene expression and the impact on human thyroid papillary carcinoma BCPAP cell thereof

One, material

(1) Specimen origin

Human thyroid papillary carcinoma cell system BCPAP is purchased from intelligent clever biological cell storehouse, Shanghai.

(2) main agents

Lipofectamine ^tM2000TransfectionReagent (Invitrogen), MTT (Solarbio), Transwell cell (Corning), Matrigel glue (BD).

(3) siRNA builds and synthesis

According to Photographing On-line software siDirectversion2.0 (http://design.rnai.jp/), according to TENM1 gene corresponding siRNA of sequential design in GenBank (NCBIReferenceSequence:XM_011531237.1).Synesis Company's synthesis is sent to after design.

Two, experimental technique

(1) RNA perturbation technique specificity suppresses the expression of human thyroid papillary carcinoma cell TENM1 gene

1, the cultivation of human thyroid papillary carcinoma cell BCPAP

The concrete method provided with reference to intelligent clever biological cell storehouse, Shanghai.

2, the Design and synthesis of siRNA

SiRNA expression vector pSIREN-DNR, can the transfection efficiency of Real-Time Monitoring carrier in cell containing neomycin resistance gene and GFP green fluorescent label.According to object mRNA sequence, design 3 RNA and disturb target sequence and negative control (table 3).For the siRNA target sequence that every bar is selected, design siRNA positive-sense strand and antisense strand, be connected with loop (9nt), be called shRNA (shorthairpinRNA).Synthesize two strands of the DNA profiling of every bar coding shRNA, annealed dna strand obtains the DNA double chain template of shRNA.Be connected to RNAPoIyIII polymerase transcription after template strand and stop site, simultaneously BamHI and HindIII restriction enzyme site is designed at two ends respectively, between BamHI and the HindIII restriction enzyme site that can be cloned into siRNA carrier multiple clone site.After siRNA empty carrier BamHI and HindIII double digestion, 1% agarose gel electrophoresis, reclaims linear carrier.The DNA profiling double-strand of annealing is connected in linear carrier.Adopt T4 ligase, the mol ratio inserting segment and carrier is about 3:1.Connect product conversion DH5 α escherichia coli, coated plate in LBAmp culture medium, 37 DEG C of overnight incubation.PCR identifies; Order-checking qualification.Post extracting positive colony carrier is also quantitative.

Table 3siRNA transcription templates sequence

3, cell grouping and transfection

(1) cell grouping

C group: blank group; C1 group: transfection liposome group; C2 group: the nonspecific siRNA group of transfection; S1, S2, S3 group: the specific siRNA group of transfection.

(2) transfection

According to Lipofectamine ^tMthe step that 2000TransfectionReagent provides is carried out.

1. 24h before transfection, the cell trypsinization of trophophase of taking the logarithm also counts, and adjustment cell concentration is 1 × 10 ⁵/ ml, gets 2m1 and is inoculated in six orifice plates, be positioned over 37 DEG C, 5%CO ₂cultivate in incubator, when cell reaches 80% fusion for transfection.With the DMEM culture medium culturing 3-4h not containing serum before transfection.

2. transfection liquid is prepared:

A liquid: 250u1 serum-free medium dilution 4.0ugDNA, gentle mixing;

B liquid: 250u1 serum-free medium dilution 10u1Lipofectamine, gentle mixing, room temperature places 5min;

3. transfection: A liquid and B liquid mix, incubation at room temperature 20min, directly joined by complex in every hole, wave and culture plate, mixes gently.At CO ₂change liquid after 37 DEG C of insulations 24-48h, 6h in incubator, add the culture medium containing serum.

4, the checking of transfection efficiency

(1) observation of cell form and transfected condition under fluorescence inverted microscope

After transfection 24h, culture plate is placed in observation of cell form and growth conditions under fluorescence inverted microscope, under green fluorescence, observes transfected condition.

(2) change that Real-timePCR method detects TENM1 gene expression before and after transfection is applied

1. the structure of standard curve: be chosen at normal 1 bottle, the thyroid papillary carcinoma BCPAP cell cultivated in 50mI culture bottle, extract RNA, measure RNA concentration and purity, carry out reverse transcription reaction, by the DNA profiling ten times dilution that reaction generates, obtain being equivalent to 10 ⁴-10 ⁰the DNA profiling of copies/ul, adds TENM1 primer and internal reference actin primer respectively, and preparation 25u1 reaction system, uses Real-timePCR amplification instrument, carry out pcr amplification reaction.Obtain the standard curve of TENM1 and actin.

2. Real-timePCR method detects the change of TENM1 gene expression before and after transfection: the RNA extracting each group of cell, measure RNA concentration and purity, carry out reverse transcription reaction, often organize the Real-timePCR reaction that DNA profiling carries out TENM1 and actin simultaneously, experiment in triplicate.

3. agarose gel electrophoresis is carried out to PCR primer.

Three, experimental result

Three interference carriers that result shows the present invention's structure all play certain inhibitory action to TENM1 gene expression in thyroid papillary carcinoma BCPAP cell, and wherein TENM1-siRNA2 inhibition is the most obvious, and suppression ratio reaches 50%, specifically sees Fig. 2.

Embodiment 5 one kinds of thyroid papillary carcinoma gene detecting kits

RNA extracts reagent: ultrapure RNA extracts test kit (article No. CW0597)

Fluorescent quantitation reagent: UltraSYBR one-step method PCR kit for fluorescence quantitative (article No. CW0660)

Quantitative fluorescent PCR reaction system and method:

RT-PCR reaction system (25 μ l): 2 × UltraSYBROneStepRT-qPCRBuffer (WithROX) 12.5 μ l, forward primer (10 μMs) 0.5 μ l, downstream primer (10 μMs) 0.5 μ l, SuperEnzymeMix0.5 μ l, add RNA template (final concentration is 10pg – 100ng), RNase-FreeWater fills to 25 μ l.

Reaction regulates: reverse transcription: 45 DEG C of 10min; 95 DEG C of 5min denaturations, connect 30-40 circulation: 95 DEG C of 15s, 60 DEG C of 45s.

The present invention adopts high-flux sequence to filter out thyroid papillary carcinoma pathogenic related gene TENM1, and binding molecule Cell Biology Experiment is verified, confirms that TENM1 is the close mark of thyroid papillary carcinoma.The present invention is that thyroid papillary carcinoma clinic diagnosis provides new target, has good potential applicability in clinical practice.

Claims

1.TENM1 gene and/or the application of TENM1 protein inhibitor in the anti-papillary adenocarcinoma preparation of preparation.

2. application according to claim 1, it is characterized in that, described anti-papillary adenocarcinoma preparation can adopt the expression of TENM1 gene in a kind of and/or several suppression papillary adenocarcinoma cell in following method: suppress the albumen of TENM1 gene expression by the suppressor gene, the activation that activate TENM1 gene, import the siRNA of suppression TENM1 gene expression, activate the microRNA of promotion TENM1mRNA degraded, import the molecule of promotion TENM1 protein degradation, suppress the factor of promotion TENM1 gene expression and the expression of albumen.

3. application according to claim 2, it is characterized in that, the siRNA sequence of described suppression TENM1 gene expression is selected from one in one sequence and/or several: SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4, SEQIDNO.5, SEQIDNO.6.

4. an anti-papillary adenocarcinoma preparation, is characterized in that, anti-papillary adenocarcinoma preparation suppresses the expression of TENM1 gene in papillary adenocarcinoma cell.

5. anti-papillary adenocarcinoma preparation according to claim 4, is characterized in that, containing the siRNA suppressing TENM1 gene expression in described anti-papillary adenocarcinoma preparation; Preferably, the siRNA sequence of TENM1 gene expression is suppressed to be selected from one in one sequence and/or several: SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4, SEQIDNO.5, SEQIDNO.6.

6.TENM1 gene is preparing the application in papillary adenocarcinoma diagnosis and treatment preparation.

7. application according to claim 6, is characterized in that, papillary adenocarcinoma diagnosis and treatment preparation comprises the expression detecting TENM1 gene in papillary adenocarcinoma tissue with fluorescence quantifying PCR method, method for gene chip.

8. application according to claim 7, is characterized in that, contains the primer of a pair specific amplification TENM1 gene for the product of TENM1 gene in fluorescence quantifying PCR method detection papillary adenocarcinoma; Gene chip comprises the probe with the nucleic acid array hybridizing of TENM1 gene.

9. application according to claim 6, it is characterized in that, papillary adenocarcinoma diagnosis and treatment preparation comprises the expression detecting TENM1 albumen in adenocarcinoma of lung with immunization method, and preferred ELISA detection kit and/gold-immunochromatographyreagent reagent for assay box detect TENM1 protein expression in papillary adenocarcinoma.

10. detect a PCR kit for fluorescence quantitative for papillary adenocarcinoma, it is characterized in that, test kit adopts special forward primer and downstream primer to detect the expression of gene TENM1, and forward primer sequence is SEQIDNO.9, and downstream primer sequence is SEQIDNO.10.