CN105288659B - The application of TENM1 gene and its expression product in diagnosis and treatment papillary adenocarcinoma - Google Patents
The application of TENM1 gene and its expression product in diagnosis and treatment papillary adenocarcinoma Download PDFInfo
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Abstract
The present invention relates to TENM1 gene and its expression product diagnosis and treatment papillary adenocarcinoma application.Inventor is based on high-flux sequence result and carries out genescreen using bioinformatics method analysis, pick out candidate gene TENM1, further, confirm that TENM1 has good correlation with thyroid papillary carcinoma by molecular cytobiology experiment, it can be used for preparing thyroid papillary carcinoma assisting in diagnosis and treatment preparation, there is important clinical value.
Description
Technical field
The present invention relates to biomedicine fields, and specifically the present invention relates to TENM1 genes and its expression product in diagnosis and treatment cream
The application of head gland cancer.
Background technique
Thyroid cancer is the most common malignant tumour of endocrine system, and disease incidence ranks first in head-neck malignant tumor.First
The reason of pathogenesis and disease incidence of shape gland cancer sharply increase is still not clear.Thyroid papillary carcinoma (papillary
Thyroid carcinoma, PTC) disease incidence account for about the 80% of all thyroid cancer incidence types.PTC accounts for about adult first shape
The whole of 60% and pediatric thyroid carcinomas of gland cancer, grade malignancy are lower.PTC is mainly in young women, is more common in 30-45 years old
Between.About 80% tumour is multicentricity, and about 1/3 involves bilateral thyroid gland.It is relatively early Cervical Lymph Node Metastasis just occur, but prognosis compared with
It is good.PTC early stage non-evident sympton shows as the without pain lump of region thyreoidea or accidentally finds in physical examination, and lump is denumerable
The moon, quality was soft or hard different, and lump more than half is tougher, and the patient less than 1/3 shows as hard mass to many decades；Neck occurs
There is enlarged lymph nodes of neck when lymphatic metastasis, invades recurrent nerve and hoarseness occur.Infringement can intratracheally cause to have difficulty in breathing
Or hemoptysis.Thyroid mini-carcinoma clinical examination is not easy to find, and often using enlarged lymph nodes of neck as onset symptoms, can also oppress larynx
Nervus recurrens first appears as hoarseness.The diagnostic method of thyroid papillary carcinoma mainly includes medical history and the disease of patient at present
Shape, laboratory inspection, imageological examination and fine needle puncture aspirate cytology inspection etc., but still come with some shortcomings.Therefore, it visits
The risk factor for begging for thyroid papillary carcinoma generation studies the pathogenesis of thyroid papillary carcinoma, discovery from molecular level
The molecular marker of specificity, for early prevention, early detection, early treatment thyroid papillary carcinoma has important meaning
Justice.
Inventor carries out high-flux sequence to 6 thyroid papillary carcinoma case samples and 3 normal controls, in conjunction with biology
Informatics Method carries out genescreen, picks out candidate gene TENM1.Further, the present invention has carried out molecular cytobiology
Method confirms relationship of the TENM1 with thyroid papillary carcinoma: having good correlation with thyroid papillary carcinoma, can be used for
Thyroid papillary carcinoma assisting in diagnosis and treatment preparation is prepared, there is important clinical value.
Summary of the invention
The purpose of the present invention is to provide TENM1 genes and/or protein inhibitor to prepare antithyroid papillary carcinoma system
Application in agent.
To achieve the above object, the present invention passes through high-flux sequence combination bioinformatics method first and screens candidate base
Because of TENM1, and then pass through the molecular cytobiology method validation relationship of TENM1 and thyroid papillary carcinoma: TENM1 and first
Shape papillocarcinoma of breast has good correlation, can be used for preparing thyroid papillary carcinoma assisting in diagnosis and treatment preparation, has important
Clinical value.
Further, the antithyroid papillary carcinoma preparation, which refers to, can inhibit TENM1 in papillary thyroid carcinoma cells
The preparation of the expression of gene.The expression of the known suppressor of those skilled in the art usually can using one of following methods and/
It is or several: to be done by the albumen of the inhibition of gene expression of the suppressor of activating genes of interest, activating genes of interest, using RNA
Disturbing technology inhibits destination gene expression, activation to promote the microRNA of target gene mRNA degradation, importing that target gene is promoted to compile
The molecule of code protein degradation inhibits to promote the factor of destination gene expression and the expression of albumen.Pass through activation TENM1 gene
Suppressor, activation inhibit the albumen, the siRNA for importing inhibition TENM1 gene expression, activation of TENM1 gene expression to promote
TENM1mRNA degradation microRNA, import promote TENM1 protein degradation molecule, inhibit promote TENM1 gene expression because
The expression of son and albumen.
RNA interference (RNAi) refers to that exogenous and endogenous double-stranded RNA induces the mRNA of homologous target gene in vivo
Selective degradation, the phenomenon that leading to posttranscriptional gene silencing, be it is a kind of efficiently, specifically blocked using small double-stranded RNA it is internal
The expression of certain specific gene, promotes mRNA to degrade, and cells show is made to go out the technology of specific gene missing phenotype.SiRNA design
After the completion can be using direct synthesis technique or building SiRNA expression vector, the siRNA prepared can be coprecipitated by calcium phosphate
The Mechanical Methods, cationic-liposome such as shallow lake method, electroporation, DEAE- glucan and polybrene method, microinjection or particle gun
The approach such as reagent method transfect cell.
Further, the siRNA sequence for inhibiting TENM1 gene expression is selected from one of following sequence and/or several:
SEQ ID NO.1,SEQ ID NO.2,SEQ ID NO.3,SEQ ID NO.4,SEQ ID NO.5,SEQ ID NO.6.It is preferred that
SiRNA sequence is SEQ ID NO.3 and SEQ ID NO.4.
The purpose of the present invention is to provide a kind of antithyroid papillary carcinoma preparation, the antithyroid papillary carcinoma preparation
Inhibit the expression of TENM1 gene in papillary thyroid carcinoma cells.Further, contain in the antithyroid papillary carcinoma preparation
There is the siRNA for inhibiting TENM1 gene expression.
The purpose of the present invention is to provide application of the TENM1 gene in preparation thyroid papillary carcinoma diagnosis and treatment preparation.
Further, the diagnosis and treatment preparation of the thyroid papillary carcinoma includes using fluorescence quantifying PCR method, genetic chip side
Method detects the expression of TENM1 gene in Papillary Thyroid Carcinoma.
Fluorescence quantitative PCR method is that PCR product is marked by the probe of the specificity of fluorescent dye or fluorescent marker
Tracking, real-time online monitoring reaction process can analyze product in conjunction with corresponding software, calculate sample to be tested template
Initial concentration.The appearance of quantitative fluorescent PCR greatly simplifies the process of quantitative detection, and is truly realized absolute quantitation.
The appearance of a variety of detection systems keeps the selectivity of experiment stronger.Automatic operation improves work efficiency, rapid reaction, repetition
The good, high sensitivity of property, high specificity, result are clear.
Genetic chip is also known as DNA microarray (DNA microarray), can be divided into three kinds of main Types: 1) be fixed on poly-
The nucleic acid probe or cDNA segment on object substrate (nylon membrane, nitrocellulose membrane etc.) surface are closed, the target of isotope labelling is usually used
Gene is hybrid with it, and is detected by radiography technology.2) DNA probe array on a glass is fixed with point sample method,
Hybridized by the target gene with fluorescent marker and is detected.3) oligonucleotide probe directly synthesized on the hard surfaces such as glass
Array hybridizes with the target gene of fluorescent marker and is detected.Genetic chip is as a kind of advanced, extensive, high-throughput detection
Technology, applied to the diagnosis of disease, advantage has the following aspects: first is that the sensitivity and accuracy of height；Second is that quickly
It is easy；Third is that a variety of diseases can be detected simultaneously.
The product for TENM1 gene in fluorescence quantifying PCR method detection thyroid papillary carcinoma contains a pair
The primer of specific amplification TENM1 gene；The genetic chip includes the probe with the nucleic acid array hybridizing of TENM1 gene.
Further, the diagnosis and treatment preparation of the thyroid papillary carcinoma includes with TENM1 in immunization method detection adenocarcinoma of lung
The expression of albumen.It is preferred that TENM1 protein expression is western in the immunologic detection method detection thyroid papillary carcinoma
Blot and/or ELISA and/colloidal gold detection method.
Known antigen or antibody are adsorbed on surface of solid phase carriers by enzyme-linked immunosorbent assay (ELISA), make enzyme mark
The technology that the antigen-antibody reaction of note is carried out in solid phase surface.The technology can be used for detecting macromolecular antigen and specific antibody
Deng having many advantages, such as that quick, sensitive, easy, carrier is easy to standardize.ELISA detection kit is according to testing goal and operation
Step can be divided into indirect method, double-antibody method, competition law, double site one-step method, prize law survey IgM antibody, using Avidin and
The ELISA of biotin.Horseradish peroxidase (HRP) or alkaline phosphatase may be selected in chromogenic substrate in ELISA detection kit
Enzyme (AP).
Common immune colloid gold detection technique: (1) immune colloid gold light microscopic decoration method cell suspension smear or tissue are cut
Piece can be dyed with the antibody of colloid gold label, can also be enhanced with silver-colored developer solution and be marked on the basis of colloid gold label,
So that the silver atoms being reduced is deposited on marked gold particle surface, the sensibility of colloid gold label can be remarkably reinforced.(2) it is immunized
Colloidal gold staining method for electron microscopy with the antibody of colloid gold label or antiantibody and negative staining Virus Sample or can be organized in conjunction with ultra-thin section,
Then negative staining is carried out.It can be used for observation and the viral diagnosis of morphology of virus.(3) dot immunogold filtration assay application miillpore filter is made
Antigen or antibody point are first added sample to be examined on film by carrier after closing, corresponding with the antibody test of colloid gold label after washing
Antigen or antibody.(4) antigen of specificity or antibody are fixed on film by colloidal gold immunity chromatography with ribbon, colloidal gold
Labelled reagent (antibody or monoclonal antibody) is adsorbed on bonding pad, when sample to be examined is added in the sample pad of test strips one end
Afterwards, it moves forward, reacts to each other after dissolving the colloid gold label reagent on bonding pad through capillary action, it is fixed when being moved to
When the region of antigen or antibody, the conjugate of object and gold marked reagent to be checked occurs specific binding therewith again and is trapped, and assembles
It is taken in detection, colour developing result can be observed by the naked eye.The method has developed into diagnosis test paper, and use is very convenient.
Further, the ELISA method of the detection TENM1 albumen is to use ELISA detection kit.In the kit
Commercially available TENM1 monoclonal antibody can be used in antibody.Further, the kit includes: coating TENM1 monoclonal antibody
Solid phase carrier, enzyme labelled antibody, the substrate of enzyme, protein standard substance, negative controls, dilution, cleaning solution, enzyme reaction terminate liquid
Deng.
Further, the colloidal gold method of the detection TENM1 albumen is using detection kit, and city can be used in the antibody
The TENM1 monoclonal antibody sold.Further, the gold-immunochromatographyreagent reagent for assay box uses colloidal gold immunochromatographimethod technology or colloid
Golden percolation.Further, detection zone (T) specking on the gold-immunochromatographyreagent reagent for assay box nitrocellulose filter has anti-TENM1
Monoclonal antibody, quality control region (C) specking have Immunoglobulin IgG.
Preferred anti-TENM1 monoclonal antibody is selected from following antibody: the article No. that Novus Biologicals company provides is
The article No. that the Teneurin-1Antibody of H00010178-M01, Abcam company provide is the Anti- of ab56597
ODZ1antibody。
The purpose of the present invention is to provide a kind of gene detecting kit for detecting thyroid papillary carcinoma, feature exists
In the kit detects gene TENM1, and using special upstream primer and downstream primer, upstream primer sequence is SEQ ID
NO.9, downstream primer sequence are SEQ ID NO.10.
Further, which is suitable for presently, there are all types fluorescence quantitative gene extender in the market, clever
Sensitivity is high, it is quantitative quick and precisely, stability it is good, have a good application prospect.
Further, above-mentioned PCR kit for fluorescence quantitative component includes: specific primer, internal control primer, quantitative fluorescent PCR
Reaction solution.Wherein the specific primer includes upstream primer and downstream primer, and upstream primer sequence is SEQ ID NO.9,
Downstream primer sequence is SEQ ID NO.10.The internal control primer is β-actin internal control primer, and upstream primer sequence is SEQ ID
NO.11, downstream primer sequence are SEQ ID NO.12.
The kit also includes RNA extraction agent.It is preferred thatReagent (invitrogen, article No.
15596-018) carry out sample rna extraction.
The present invention also has detected this kit sensitivity, this kit detection range is 10 as the result is shown6-102copies/μ
L, minimum concentrations are 100copies/ μ l.
It is an object of the present invention to provide a kind of thyroid papillary carcinoma protein detection kit, the detection kit inspection
Survey TENM1 albumen.Further, the kit further includes other detection reagents.
It is an object of the present invention to provide a kind of genetic chip for detecting thyroid papillary carcinoma, the genetic chip includes
With the probe of the nucleic acid array hybridizing of TENM1 gene.
Detailed description of the invention
Fig. 1 TENM1 gene relative expression's spirogram in cancerous tissue and normal tissue
Each group TENM1 mRNA expression after Fig. 2 RNA interference
Specific embodiment
Present invention will be further explained below with reference to specific examples, for explaining only the invention, and should not be understood as to this
The limitation of invention.It will be understood by those skilled in the art that: without departing from the principle and spirit of the present invention may be used
To carry out a variety of change, modification, replacement and modification to these embodiments, the scope of the present invention is limited by claim and its equivalent
It is fixed.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition or according to item proposed by manufacturer
Part examinations.
The collection of 1 case of embodiment
Take in January, 2011 to during in June, 2012 in Beijing Friendship Hospital because of thyroid tumors hospitalisation for surgery, Jing Shuzhong ice
Freeze rapid pathology and the slow pathology of postoperative paraffin is confirmed as the patient of thyroid papillary carcinoma, selects through first function eight and thyroid gland
The normal healthy population of color ultrasound examination as a control group, chooses 6 thyroid papillary carcinoma case samples and 3 Normal groups
It knits.
2 high-flux sequence of embodiment and analysis
RNA extraction is carried out to tissue, agarose gel electrophoresis after RNA is extracted can be extracted from electrophoresis result with preliminary judgement
RNA sample it is up-to-standard whether, if can be used for further transcriptome analysis.And then it is divided by NanoDrop1000
Photometer detects the extraction situation of RNA sample, the sample requirement of RNA-seq sequencing: OD260/OD280 1.8-2.2.
Microarray dataset is the 2500 high-flux sequence platform of HiSeq of Illumina company, carries out high-throughput transcript profile depth
Sequencing, we use Fast-QC (http://www.bioinformatics.babraham.ac.uk/projects/ after sequencing
Fastqc/) software carries out total evaluation, the quality Distribution value including base, the position point of mass value to the quality of sequencing data
Cloth, G/C content, PCR duplication content, the frequency etc. of kmer.In differential genes expression analysis, according to obtaining
FPKM value, using internationally recognized algorithm EBSeq carry out differential screening.Wherein, when screening, LOG2FC>1 or<-1, FDR<
0.05.In order to better understand the function of difference expression gene, we have carried out Gene Onlogy and letter to difference expression gene
Number path analysis, and functional annotation and protein interaction network analysis are carried out to difference expression gene, in view of above data
Analysis as a result, we have screened difference expression gene TENM1 in conjunction with document.
3 Papillary Thyroid Carcinoma of embodiment and normal tissue TENM1 expression conditions
One, material and method
1, material
78 Papillary Thyroid Carcinomas and 16 normal tissues are chosen, it is grouped and is numbered.
2, method
The extraction of 2.1 Papillary Thyroid Carcinomas and normal tissue total serum IgE
UsingReagent (invitrogen, article No. 15596-018) carries out sample rna extraction, experimental implementation
It is carried out by product description, concrete operations are as follows:
It freezes to be put into the mortar being pre-chilled tissue after liquid nitrogen, taking-up after collection sample and be ground, sample to be organized
This is at powdered rear:
1. Trizol is added, room temperature preservation 5 minutes；
2. chlorination imitates 0.2ml, with forced oscillation centrifuge tube, mix well, places -10 minutes 5 minutes at room temperature；
3. drawing upper strata aqueous phase (inhaling 70%) after 12000rpm high speed centrifugation 15 minutes into another new centrifuge tube pipe, pay attention to
The protein substance that be not drawn between two layers of water phase.New pipe is moved into, the pre- cold isopropanol of isometric -20 DEG C is added, it is sufficiently reverse
It mixes, is placed in 10 minutes on ice；
4. 12000rpm high speed from 15 minutes after carefully discard supernatant, in I ml/ml Trizol ratio be added 75%
DEPC ethyl alcohol washes paint precipitating (4 DEG C of preservations), washes paint sediment, oscillation mixes, 12000rpm high speed centrifugation 5 minutes at 4 DEG C；
5. discarding ethanol liquid, 5 minutes are placed at room temperature sufficiently to dry precipitating, it is heavy that the processed water dissolution of DEPC is added
It forms sediment；
6. measuring RNA purity and concentration with Nanodrop2000 ultraviolet specrophotometer, freeze in -70 DEG C.RNA mass is sentenced
Calibration is quasi-: the OD260/OD280 value of RNA sample is between 1.7-2.2；Total serum IgE electrophorogram has clearly 28S, 18S band；
70 DEG C of water-baths keep the temperature 1 hour after electrophorogram and the map no significant difference before water-bath heat preservation.
2.2 reverse transcriptions synthesize cDNA
UsingIII Reverse Transcriptase (invitrogen, article No. 18080-044) into
Row cDNA reverse transcription, experimental implementation are carried out by product description, and concrete operations are as follows:
Using Reverse Transcriptase kit, converse record is carried out to l μ g total serum IgE with RT Buffer and synthesizes cDNA.Using 25 μ l
Reaction system, each sample take 1 μ g total serum IgE as template ribonucleic acid, following components are separately added into PCR pipe:
5 × RT Buffer, 5 μ l, 10mmol/l dNTP, 1.25 μ l, 0.1mmol/l DTT 2.5 μ l, 30 μm of mol/l
Aqua sterilisa is added to 25 μ l of total system in 2 μ l, 200U/ μ l MMLV of OligodT 1.25 μ l, 1 μ g of template ribonucleic acid.42 DEG C are incubated for 1
Hour, 72 DEG C 10 minutes, of short duration centrifugation.It is spare that -20 DEG C of refrigerators are put in cDNA preservation.
2.3 Real-Time PCR
2.3.1 instrument and analysis method
With 7500 type fluorescence quantitative PCR instrument of ABI, the relative quantitative assay of data is carried out using 2- △ △ CT method.
2.3.2 design of primers
Using online primer-design software, gene order referring to NCBI:XM_011531237.1, after design of primers by
The synthesis of invitrogen company.Specific primer sequence is as follows:
1 primer sequence of table
Operating process is as follows:
(1) reaction system: Power is usedGreen PCR Master Mix (invitrogen, article No.
4367659) it is expanded, experimental implementation is carried out by product description.Amplification program are as follows: 95 ° of 5min, (95 DEG C of 15sec, 60
DEG C 45sec) × 40 circulations.
2 RealTime reaction system of table
| Component | Additional amount |
| 2×mix | 10μl |
| Upstream primer (10uM) | 0.5μl |
| Downstream primer (10uM) | 0.5μl |
| Template | 2μl |
| Sterile purified water is added | To 25 μ l |
(2) primer screening
After each sample cDNA is mixed, 5 times of gradient dilutions are carried out as template, sample respectively takes 2 μ l to make template after dilution,
It is expanded respectively with target gene primer and reference gene primer, while in 60-95 DEG C of progress melt curve analysis analysis, according to expansion
Increasing Efficiency height and the unimodal principle of solubility curve carry out primer screening.
(3) sample RealTimePCR 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 progress solubility curve analysis.
Two, experimental result
Real-time quantitative PCR amplification curve inflection point understands that amplification curve entirety collimation is good, shows the amplification effect of each reaction tube
Rate is close, and the limit is flat and present without raising up, and exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production is molten
Solution curve be all it is unimodal, illustrate that amplified production only has one, be specific amplification；According to the relative quantification formula of qRT-PCR: 2-
Δ Ct × 100% compares expression of the TENM1 gene in Papillary Thyroid Carcinoma and normal tissue.As the result is shown
(being specifically shown in Fig. 1): qRT-PCR stable amplification result, wherein expression of the TENM1 in cancerous tissue is higher than normal tissue nearly 4
Times, the confluence analysis TENM1 that result above demonstrates high-throughput transcript profile expression data is high in Papillary Thyroid Carcinoma
The result of expression.
4 RNAi of embodiment inhibits TENM1 gene expression and its influence to human thyroid papillary carcinoma BCPAP cell
One, material
(1) Specimen origin
Human thyroid papillary carcinoma cell system BCPAP is purchased from the intelligent clever biological cell library in Shanghai.
(2) main agents
LipofectamineTM2000Transfection Reagent (Invitrogen), MTT (Solarbio),
The cell Transwell (Corning), Matrigel glue (BD).
(3) siRNA building and synthesis
Foundation Photographing On-line software siDirect version 2.0 (http://design.rnai.jp/), according to
TENM1 gene sequence design in GenBank (NCBI Reference Sequence:XM_011531237.1) is corresponding
siRNA.Synesis Company's synthesis is sent to after design.
Two, experimental method
(1) RNA perturbation technique specificity inhibits the expression of human thyroid papillary carcinoma cell TENM1 gene
1, the culture of human thyroid papillary carcinoma cell BCPAP
The method provided referring in particular to the intelligent clever biological cell library in Shanghai.
2, the design and synthesis of siRNA
SiRNA expression vector pSIREN-DNR is marked containing neomycin resistance gene and GFP green fluorescence, can be monitored in real time
Transfection efficiency of the carrier in cell.According to purpose mRNA sequence, 3 RNA interference target sequences and negative control (table 3) are designed.
The siRNA target sequence selected for every designs siRNA positive-sense strand and antisense strand, is connected with loop (9nt), referred to as shRNA
(shorthairpin RNA).Two for synthesizing the DNA profiling of every coding shRNA are single-stranded, and annealed dna is single-stranded to obtain shRNA
DNA double chain template.Template strand stops site followed by RNA PoIyIII polymerase transcription, while both ends separately design
BamHI and HindIII restriction enzyme site can be cloned into BamHI the and HindIII restriction enzyme site of siRNA carrier multiple cloning sites
Between.After siRNA empty carrier BamHI and HindIII double digestion, 1% agarose gel electrophoresis recycles linear carrier.Moving back
The DNA profiling double-strand of fire is connected in linear carrier.Using T4 ligase, the molar ratio of insertion and carrier is about 3:1.Even
Object of practicing midwifery converts DH5 α Escherichia coli, the coated plate on LB Amp culture medium, 37 DEG C of overnight incubations.PCR identification；Sequencing identification.Column
Extracting positive colony carrier is simultaneously quantitative.
3 siRNA transcription templates sequence of table
3, cell grouping and transfection
(1) cell is grouped
C group: blank control group；C1 group: transfection liposome group；C2 group: nonspecific siRNA group is transfected；S1, S2, S3
Group: the siRNA group of specificity is transfected.
(2) it transfects
According to LipofectamineTMThe step of 2000Transfection Reagent is provided carries out.
1. before transfection for 24 hours, the cell pancreatin of logarithmic growth phase is digested and is counted, and adjustment cell concentration is 1 × 105/
Ml takes 2m1 to be inoculated in six orifice plates, is placed in 37 DEG C, 5%CO2It is cultivated in incubator, when cell is merged up to 80% for turning
Dye.With the DMEM culture medium culture 3-4h without serum before transfection.
2. preparing transfection liquid:
A liquid: 250u1 serum free medium dilutes 4.0ugDNA, mild to mix；
B liquid: 250u1 serum free medium dilutes 10u1 Lipofectamine, mild to mix, and is placed at room temperature for 5min；
3. transfection: A liquid is mixed with B liquid, and compound is directly added in every hole by incubation at room temperature 20min, is shaken
Culture plate mixes gently.In CO2Liquid is changed in incubator after 37 DEG C of heat preservations 24-48h, 6h, the culture medium containing serum is added.
4, the verifying of transfection efficiency
(1) cellular morphology and transfected condition are observed under fluorescence inverted microscope
After transfection for 24 hours, culture plate is placed under fluorescence inverted microscope and observes cellular morphology and growth conditions, green fluorescence
Lower observation transfected condition.
(2) using the variation of Real-time PCR method detection transfection front and back TENM1 gene expression
1. the building of standard curve: being chosen at the thyroid papillary carcinoma BCPAP cell 1 normally cultivated in 50mI culture bottle
Bottle extracts RNA, measures RNA concentration and purity, carries out reverse transcription reaction, and ten times of the DNA profiling dilutions that reaction is generated obtain
It is equivalent to 104-100The DNA profiling of copies/ul is separately added into TENM1 primer and internal reference actin primer, and it is anti-to prepare 25u1
System is answered, using Real-time PCR amplification instrument, carries out pcr amplification reaction.Obtain the standard curve of TENM1 and actin.
2. the variation of Real-time PCR method detection transfection front and back TENM1 gene expression: the RNA of group of cells is extracted,
RNA concentration and purity are measured, reverse transcription reaction is carried out, every group of DNA profiling carries out the Real-time of TENM1 and actin simultaneously
PCR reaction, experiment is in triplicate.
3. carrying out agarose gel electrophoresis to PCR product.
Three, experimental result
Three interference carriers that the present invention constructs as the result is shown are all to TENM1 base in thyroid papillary carcinoma BCPAP cell
Because certain inhibiting effect is played in expression, wherein TENM1-siRNA2 inhibitory effect is most obvious, and inhibiting rate is specifically shown in Fig. 2 up to 50%.
A kind of thyroid papillary carcinoma gene detecting kit of embodiment 5
RNA extracts reagent: ultrapure RNA extracts 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 × UltraSYBR One Step RT-qPCR Buffer (With ROX)
12.5 μ l, upstream primer (10 μM) 0.5 μ l, downstream primer (10 μM) 0.5 μ l, 0.5 μ l of SuperEnzyme Mix plus RNA mould
Plate (final concentration of 10pg -100ng), RNase-Free Water filling-in to 25 μ l.
Reaction is adjusted: reverse transcription: 45 DEG C of 10min；95 DEG C of 5min initial denaturations, connect 30-40 circulation: 95 DEG C of 15s, and 60
℃ 45s。
The present invention filters out thyroid papillary carcinoma pathogenic related gene TENM1 using high-flux sequence, and binding molecule is thin
Born of the same parents' biological experiment verifying, it was confirmed that TENM1 is the close marker of thyroid papillary carcinoma.The present invention is papillary thyroid
Cancer clinic diagnosis provides new target, has good potential applicability in clinical practice.
Claims (1)
1.TENM1 gene inhibitor is preparing the application in anti-papillary adenocarcinoma preparation, which is characterized in that TENM1 gene inhibits
Agent is the siRNA that anti-papillary adenocarcinoma preparation inhibits TENM1 gene expression, sequence are as follows: SEQ ID NO.3, SEQ ID
NO.4。
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| CN103038635A (en) * | 2010-05-11 | 2013-04-10 | 威拉赛特公司 | Methods and compositions for diagnosing conditions |
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| CN103038635A (en) * | 2010-05-11 | 2013-04-10 | 威拉赛特公司 | Methods and compositions for diagnosing conditions |
| CN104321439A (en) * | 2012-03-15 | 2015-01-28 | 凯杰科技有限公司 | Thyroid cancer biomarker |
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