A kind of molecular target of esophageal squamous cell carcinoma
Technical field
The invention belongs to biomedicine fields, are related to a kind of molecular target of esophageal squamous cell carcinoma, and the molecular target is
AGXT2L1。
Background technique
The cancer of the esophagus is one of common malignant gastrointestinal tumors, and morbidity and mortality occupy pernicious respectively in the world
The 8th and the 6th, tumour.Its main histological type is divided into two major classes, esophageal squamous cell carcinoma and adenocarcinoma of esophagus, esophagus squameous
Cell cancer is also referred to as esophageal squamous cell carcinoma.The morbidity of the cancer of the esophagus has apparent areal variation, and adenocarcinoma of esophagus is mainly distributed on west state
Family, and the trend of disease incidence rising is also shown in the past 20 years.Esophageal squamous cell carcinoma is distributed mainly on developing country, and in
Sub- and Caspian Sea region disease incidence is concentrated very much.Based on esophageal squamous cell carcinoma, the death rate of the domestic cancer of the esophagus is 15.21/10 in China
Ten thousand, the 4th of malignant tumour is occupied, lung cancer, liver cancer and gastric cancer are only second to, male is higher than women, and rural area is higher than city.National ether
Row mountain, Da Bie Mountain area, Fujian Guangdong have a common boundary and Northern Sichuan Province be district occurred frequently, Jiangsu Province with Along North Jiangsu and raise medium ground disease incidence compared with
It is high.
Studies have shown that the cancer of the esophagus is caused by including the multifactor functionings such as heredity, environment, life style.Induce oesophagus
The risk factor of cancer has, genetic predisposition, smokes, drinks, liking to scald the living habits such as food, nitrosamine compound intake, low water
Flat social and economic condition, precancerous lesion etc..The concealment of this disease onset, has stronger invasion transfer ability, when most of patients is made a definite diagnosis
Entered middle and advanced stage, in a manner for the treatment of is to perform the operation, chemotherapy, radiotherapy combine based on, 5 years survival rates are only 15%-25%, prognosis
Situation is dependent on early diagnosis.The occurrence and development of the previous studies have shown that cancer of the esophagus may swash with the oncogenes such as c-myc, H-ras
Living, the tumor suppressor genes such as Rb, P53 inactivation is related, but specific pathogenesis not yet illustrates completely.
With the development of sequencing technologies and relevant molecule biology techniques, people are also deeper and deeper to the understanding of genome
It carves, the occurrence and development of prior art discovery esophageal squamous cell carcinoma and the change of gene or its expression have important relationship.But mesh
It is preceding to be applied to clinic without effective marker.Give farther insight into the pathogenesis of esophageal squamous cell carcinoma, searching esophageal squamous cell carcinoma is examined
There is the drug target and the effective therapeutic scheme of formulation of disconnected molecular marker, determining research and development clinical treatment important clinic to anticipate
Justice.
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 marks for esophageal squamous cell carcinoma diagnosis and treatment
Object AGXT2L1.
To achieve the goals above, the present invention adopts the following technical scheme:
The present invention provides application of the reagent of detection AGXT2L1 gene in the product of preparation diagnosis esophageal squamous cell carcinoma.
Further, the product judges whether patient suffers from by the expression of AGXT2L1 gene in detection sample
Esophageal squamous cell carcinoma.Wherein, AGXT2L1 gene expresses downward in patients with esophageal squamous cell carcinoma.
" sample " includes cell, tissue, internal organs, body fluid (blood, lymph etc.), digestive juice, expectoration, alveole branch gas
Pipe cleaning solution, urine, excrement etc..Preferably, the sample is tissue, blood.In a specific embodiment of the invention, the sample
This is tissue.
Further, the product includes: by RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization, chip or height
Flux microarray dataset detects the expression of AGXT2L1 gene to diagnose esophageal squamous cell carcinoma.
Wherein, the product with RT-PCR diagnosis esophageal squamous cell carcinoma includes at least a pair of of specific amplified AGXT2L1 gene
Primer;The product with real-time quantitative PCR diagnosis esophageal squamous cell carcinoma includes at least drawing for a pair of of specific amplified AGXT2L1 gene
Object;The product with immune detection diagnosis esophageal squamous cell carcinoma includes: the antibody in conjunction with AGXT2L1 protein-specific;The use
The product of in situ hybridization diagnosis esophageal squamous cell carcinoma includes: the probe with the nucleic acid array hybridizing of AGXT2L1 gene;It is described to be examined with chip
The product of disconnected esophageal squamous cell carcinoma includes: protein chip and genetic chip;Wherein protein chip includes and AGXT2L1 protein-specific knot
The antibody of conjunction, genetic chip include the probe with the nucleic acid array hybridizing of AGXT2L1 gene.
Further, the product with real-time quantitative PCR diagnosis of esophageal cancer includes at least a pair of of specific amplification AGXT2L1
The primer of gene, the primer is as shown in SEQ ID NO.5 and SEQ ID NO.6.
The present invention provides a kind of product for diagnosing esophageal squamous cell carcinoma, the product can pass through AGXT2L1 in detection sample
The expression of gene diagnoses esophageal squamous cell carcinoma.
Further, the product includes chip or kit;Wherein, the chip includes genetic chip, protein core
Piece;The kit includes gene detecting kit, protein immunization detection kit.The genetic chip include solid phase carrier with
And it is fixed on the oligonucleotide probe of solid phase carrier, the oligonucleotide probe includes for detecting AGXT2L1 genetic transcription water
The flat oligonucleotide probe for AGXT2L1 gene;The protein-chip include solid phase carrier and be fixed on solid phase load
The specific antibody of the AGXT2L1 albumen of body;The gene detecting kit includes for detecting AGXT2L1 gene transcription level
Reagent;The protein immunization detection kit includes the specific antibody of AGXT2L1 albumen.
Gene detecting kit or genetic chip can be used for detecting multiple bases including AGXT2L1 gene in the present invention
Because of the expression of (for example, multiple genes relevant to esophageal squamous cell carcinoma).The protein immunization detection kit or protein core
Piece can be used for detecting multiple protein (such as multiple protein relevant to esophageal squamous cell carcinoma) including AGXT2L1 albumen
Expression.Multiple markers of esophageal squamous cell carcinoma are detected simultaneously, are greatly improved the accuracy rate of oesophagus squama cancer diagnosis.
The present invention provides application of the AGXT2L1 gene in the pharmaceutical composition of preparation treatment esophageal squamous cell carcinoma.
Further, described pharmaceutical composition include increase AGXT2L1 gene expression, enhancing AGXT2L1 expressive function and/
Or the enhancing active reagent of AGXT2L1 expression product.
Further, the reagent includes: reagent, the AGXT2L1 egg of the nucleic acid containing energy encoding function AGXT2L1 albumen
White activator, the reagent containing AGXT2L1 protein.
Wherein, the reagent of the nucleic acid containing energy encoding function AGXT2L1 albumen can be turns under advantage
The single-chain nucleic acid (such as mRNA) or double-strandednucleic acid (such as DNA), the nucleic acid for being translated into the AGXT2L1 albumen of active form can connect
It connects on expression vector or in recombination to host cell, as long as Viability AGXT2L1 albumen, any AGXT2L1 can be encoded
The carrying mode of gene.The AGXT2L1 protein activator refers to stimulation AGXT2L1 protein active, increases AGXT2L1
Protein active, promote AGXT2L1 protein active, enhancing AGXT2L1 protein activation, make the sensitization of AGXT2L1 protein active or on
The reagent for adjusting AGXT2L1 protein active, as the transcription of demethylation reagent, AGXT2L1 promoter and/or enhancer specificity swashs
Living agent, AGXT2L1 albumen agonist (such as activating antibody).
The present invention provides a kind of pharmaceutical composition for treating esophageal squamous cell carcinoma, described pharmaceutical composition includes increasing
AGXT2L1 gene expression, enhancing AGXT2L1 expressive function, and/or the enhancing active reagent of AGXT2L1 expression product.Wherein,
The reagent includes but is not limited to the activation of the reagent, AGXT2L1 albumen of the nucleic acid containing energy encoding function AGXT2L1 albumen
Agent, the reagent containing AGXT2L1 protein.
Further, described pharmaceutical composition further includes pharmaceutically acceptable carrier, as buffer, emulsifier, suspending agent,
Stabilizer, preservative, physiological saline etc..As buffer, it is able to use phosphate, glycine, sorbic acid, sorbic acid clock is satisfied
With the partial glyceride mixtures of vegetable fatty acid, water, salt or electrolyte such as potassium sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, chlorine
Change sodium, zinc salt, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, the substance based on cellulose, polyethylene glycol, carboxylic first
Base sodium cellulosate, polyacrylate, wax, polyethylene-polyoxypropylene block copolymer, polyethylene glycol and lanolin etc..As cream
Agent is able to use gum arabic, sodium alginate, tragacanth etc..As suspending agent, be able to use glycerol monostearate,
Aluminum monostearate, methylcellulose, carboxymethyl cellulose, hydroxymethyl cellulose, NaLS etc..As stabilizer, energy
Enough using propylene glycol, diethylidene sulphite, ascorbic acid etc..As preservative, it is able to use sodium azide, benzene pricks chlorine
Ammonium, p-hydroxybenzoic acid, methaform etc..Pharmaceutical composition of the invention can also include ion-exchanger such as alumina, aluminum stearate,
Lecithin, semi-emulsifying drug delivery system (SEDDS) such as mono- tocopherol cetomacrogol 1000 succinate of d α, in pharmaceutical dosage form
Used in surfactant such as tween or other similar polymeric delivery matrices, haemocyanin such as human serum albumins,
It is, for example, hydroxyl that cyclodextrin such as alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin or the derivative of chemical modification, which can be used,
Alkyl cyclodextrins promote passing for the compounds of this invention including 2- and 3- hydroxypropyl-beta-cyclodextrin or other solubilized derivatives
It send.The carrier of the present invention for carrying gene is various carriers known in the art, such as commercially available carrier including plasmid, clay,
Bacteriophage, virus etc..
Pharmaceutical composition of the invention further includes pharmaceutically acceptable excipient, filler, coagulating agent and blender, such as
Lactose hydrous or Lactis Anhydrous, starch, glucose, sucrose, mannitol, sorbierite, silicic acid, microcrystalline cellulose, hydroxylmethyl cellulose
Plain sodium, sodium starch glycol and its derivative etc..
Pharmaceutical composition of the invention also includes interfacial agent, emulsifier, diffusant, defoaming agent etc..It is any pharmaceutically
Or medically acceptable interfacial agent, emulsifier, diffusant, defoaming agent etc. can all be used.
Pharmaceutical composition of the invention further includes that pharmaceutically acceptable coating material includes but is not limited to fast decoupled
Coating material, coloring agent, enteric polymer, plasticizer, water-soluble polymer, insoluble polymer, dyestuff, pigment, its
His disintegrating agent.Common fast decoupled coating material includes OPADRY;Enteric polymer includes methylacrylic acid polymer, phosphorus hydroxyl
Third methylcellulose phthalic acid ester, hypromellose acetic acid esters, hypromellose succinate, the first and second base of hydroxyl are fine
Dimension element, cellulose acetophthalate;Plasticizer includes polyethylene glycol (PEG), propylene glycol etc..
In the present invention, " probe ", which refers to, to divide in conjunction with the particular sequence of another molecule or subsequence or other parts
Son.Unless otherwise indicated, term " probe " is often referred to match and another polynucleotides (often referred to as " target by complementary base
Polynucleotides ") combine polynucleotide probes.According to the preciseness of hybridization conditions, probe energy and lack complete sequence with the probe
The complementary target polynucleotide of column combines.Probe can make direct or indirect label, and range includes primer.Crossing system, packet
It includes, but is not limited to: solution phase, solid phase, mixed phase or in situ hybridization measuring method.
The probe has the base sequence with the specific base sequence complementary of target gene.Here, so-called " complementation ",
As long as hybridization, can not be complete complementary.These polynucleotides have usually relative to the specific base sequence
80% or more, preferably 90% or more, more preferable 95% or more, particularly preferred 100% homology.These probes can be DNA,
It is also possible to RNA, furthermore it is possible to pass through PNA (Polyamide nucleic in part of it or whole nucleotides
Acid, peptide nucleic acid), LNA (registered trademark, locked nucleic acid, Bridged Nucleic Acid, Cross-linked core
Acid), ENA (registered trademark, 2 '-O, 4 '-C-Ethylene-bridged nucleic acids), GNA (Glycerol
Nucleic acid, glycerol nucleic acid), the artificial replacement nucleic acid such as TNA (Threose nucleic acid, threose nucleic acid) obtains
Polynucleotides.
The specific antibody of heretofore described AGXT2L1 albumen includes monoclonal antibody, polyclonal antibody, polyspecific
Antibody (such as bispecific antibody) and antibody fragment, combinatorial antibody etc., as long as they show desired biological activity.
" monoclonal antibody " refers to the antibody that the antibody from a group substantially homogeneity obtains in the present invention, i.e., composition group is each
A antibody is identical and/or combines same epitope, during producing monoclonal antibody other than issuable possibility variant, this
Class variant is generally with indivisible presence.Such monoclonal antibody is typically include the antibody comprising the polypeptide sequence in conjunction with target,
Wherein target combination polypeptide sequence is by including selecting single target combination polypeptide sequence in the more peptide sequence of comforming
What process obtained.
Clonal antibody clearly includes " chimeric " antibody in the present invention, wherein a part and derivative of heavy chain and/or light chain
From particular species or the corresponding sequence that belongs in the antibody of specific antibodies classification or subclass is identical or homologous, and the remainder of chain
With derived from another species or to belong to corresponding sequence in the antibody of another antibody isotype or subclass identical or homologous and such
The segment of antibody, as long as they show desired biological activity.
" complete antibody " refers to the antibody comprising two antigen binding domains and the area Fc in the present invention.Preferably, complete anti-
Body has the functionality area Fc.
" antibody fragment " includes a part of complete antibody, preferably comprises its antigen binding domain.The example packet of antibody fragment
Include Fab, Fab ', F (ab ')2With Fv segment;Double antibody;Linear antibodies;Single-chain antibody molecules;And it is formed by antibody fragment more
Specific antibody.
The antibody that AGXT2L1 gene expression product is detected in the present invention, can choose well known method appropriate to prepare,
Such as hybridoma, recombinant DNA method, production method of bacteriophage method monoclonal antibody etc..Using being combined with the anti-of mark substance
When body, by detecting the label, target point protein matter can be directly detected.As mark substance, as long as can in conjunction with antibody,
Substance that can be detected, is not particularly limited, for example, can be peroxidase, beta-D-galactosidase, micro- peroxidating
Object enzyme, horseradish peroxidase (HRP), fluorescein isothiocynate (FITC), rhodamine isothiocyanate (RITC), alkaline phosphatase
Enzyme, biotin and radioactive substance.In addition, being combined with the method that the antibody of mark substance directly detects target point protein matter except using
In addition, also secondary antibodies, protein G or a-protein of mark substance etc. can be combined with using use and detects target spot indirectly
The method of protein.
Pharmaceutical composition Orally-administrable of the present invention, parenteral administration are given by sucking spray delivery, part
Medicine, rectally, nasal administration, cheek administration, vagina administration are administered by the storage medicine device of implantation.It is preferred that oral administration or injection
Administration.Pharmaceutical composition of the present invention contains any commonly employed nontoxic pharmaceutical acceptable carrier, auxiliary material or excipient.In certain situations
Under, medicinal acid, alkali or buffer can be used to adjust the pH of preparation to improve the stabilization of prepared compound or its form of administration
Property.Terms used herein parenteral route include subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, in breastbone,
In bringing up, in damage location and intracranial injection or infusion techniques.As long as destination organization can be reached, pharmaceutical composition of the present invention
Receptor can be given by any approach.
Pharmaceutical composition of the present invention can be administered orally in the form of any peroral dosage form, including but not limited to capsule, piece
Agent, emulsion and water slurry, dispersing agent and solution.For oral tablet, common carrier includes lactose and cornstarch.It is general to go back
Lubricant such as magnesium stearate is added.In order to be administered with capsules per os, applicable diluent includes lactose and anhydrous corn
Starch.When water slurry and/or lotion is administered orally, active component can be suspended or dissolved in oily phase, and and emulsifier
And/or suspending agent merges.If necessary, some sweeteners and/or corrigent and/or colorant can be added.Where appropriate, can
The dosage unit preparations packet micro-capsule that will be used to be administered orally.For example, by the way that particulate matter is coated or is wrapped in polymer, wax etc.
It buries, the preparation can also be prepared and extended or maintained release.Pharmaceutical composition of the invention can be used for supplementing endogenic
The missing or deficiency of AGXT2L1 albumen, by improving the expression of AGXT2L1 albumen or enhancing the function of AGXT2L1 albumen, thus
Esophageal squamous cell carcinoma caused by treatment is reduced because of AGXT2L1 albumen.
Drug of the invention can also can be with master with the drug combination of other treatment esophageal squamous cell carcinoma, other therapeutic compound
The active constituent wanted is administered simultaneously, or even is administered simultaneously in same composition.Can also with individual composition or with it is main
The different dosage form of active constituent individually give other therapeutic compounds.The Fractional of main component can with it is other
Therapeutic compound is administered simultaneously, and other dosage can be administered alone.It over the course for the treatment of, can be according to the serious journey of symptom
The physiologic response of degree, the frequency of recurrence and therapeutic scheme adjusts the dosage of pharmaceutical composition of the present invention.
Pharmaceutical composition of the present invention can also be administered in the form of Liposomal delivery systems, such as small monolayer vesicle, big list
Layer vesica and multi-layer vesicles.Liposome can be formed there are many phosphatide, such as cholesterine, stearic amine or phosphatidyl choline.
Pharmaceutical composition of the present invention can be configured to ointment, cream, suspension with the pharmaceutical composition of local administration
Agent, lotion, powder, solution, paste, gelling agent, spray, aerosol or finish.
It would be recognized by those skilled in the art that practicability of the invention is not limited to marker gene of the invention
The gene expression of any specific variants is quantified.As unrestricted example, marker gene can have SEQ ID NO.1
Or coded sequence or amino acid sequence that SEQ ID NO.2 is specified.In some embodiments, have with listed sequence extremely
Few 85% the same or similar cDNA sequence or amino acid sequence, such as above-mentioned listed sequence at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or at least 99% the same or similar cDNA sequence or amino acid sequence.
In the context of the present invention, AGXT2L1 gene expression product includes people AGXT2L1 albumen and AGXT2L1 egg
White partial peptide.The partial peptide of the AGXT2L1 albumen contains functional domain relevant to esophageal squamous cell carninomatosis.
" AGXT2L1 albumen " includes any functional equivalent of AGXT2L1 albumen and AGXT2L1 albumen.The function
Equivalent includes AGXT2L1 albumen conservative variation protein or its active fragment or its reactive derivative or its mutant.
Mutant include allelic variant, natural mutation, induced mutants, its amino acid sequence by missing, substitution, increase and/
Or insertion morph mutant, can be coded by the DNA with the DNA hybridization of people AGXT2L1 under high or low stringent condition
Protein.
In general, the modification of one or more amino acid will not influence the function of protein in a protein.This field skill
Art personnel can approve the amino acid for changing single amino acids or small percentage or individual additions to amino acid sequence, missing, slotting
Entering, replacing is conservative modification, and wherein the change of protein generates the protein with identity function.Intimate amino is provided
The Conservative substitution tables of acid are well known in the art.
The modification of amino acid sequence is modified after being originated from spontaneous mutation or heredity, can also be produced with artificial induction's natural gene
It is raw.Example by one amino acid of addition or the protein of more amino acid modification is the fusion egg of AGXT2L1 albumen
It is white.For the peptide or protein with AGXT2L1 protein fusion, there is no limit as long as resulting fusion protein retains AGXT2L1
The biological activity of albumen.
Pharmaceutical composition of the invention can be by effectively amount be administered in pharmacy, term " pharmacy of the invention
Upper effective amount " is referred to be applicable to reasonably the receiving benefits of therapeutic treatment or prevention/risk-benefit risks and be enough to treat or prevent
The amount of disease, can be according to including the severity of disease, the activity of drug, the age of patient, weight, health, gender, patient couple
The susceptibility of drug, the administration time of the used present composition, administration route and discharge ratio, treatment time and institute
Well known element is determined in the element of the composition cooperation of the invention or the drug used simultaneously that use and other medical domains
Determine effective dose level.Pharmaceutical composition of the invention can be used as individual therapeutic agent and be administered, or with other therapeutic agents
And land used is administered, and can in turn or be simultaneously administered with previous therapeutic agent.In addition, can single or multiple times into
Row administration.Importantly, needing to take in above-mentioned element, and maximum effect can be obtained with least amount without side-effects
The amount of fruit is administered.
In the present invention, term " chip " or " array ", " microarray " are that hybridised arrays original part is ordered in matrix,
The hybridised arrays original part such as polynucleotide probe (such as oligonucleotides) or bonding agent (such as antibody).The matrix can be with
It is solid matrix, for example, glass or silica slide, pearl, fibre optics binder or semi-solid matrix, such as cellulose nitrate
Plain film.Nucleotide sequence can be DNA, RNA or in which any arrangement.
Term " treatment " refers to not for the purpose of curing, but slows down pathological condition or illness or prevent that (reduction) targets
Recurrence.Including but not limited to, (1) inhibiting effect, inhibits progression of disease to a certain extent comprising slows down and presses down completely
System;(2) quantity of seizure of disease and/or symptom is reduced;(3) focal size is reduced;(4) inhibit (to reduce, slow down or hinder completely
Only) disease cells penetrate into adjacent peripheral organs and/or tissue;(5) (reduce, slow down or prevent completely) disease is inhibited to pass
It broadcasts;(6) alleviate one or more symptoms associated with disease to a certain extent;(7) treat after increase disease-free performance when
Between length;(8) given point in time after the treatment reduces the death rate;And/or it is without side-effects after (9) treatment.
The advantages of the present invention:
The present invention provides a kind of molecular markers of esophageal squamous cell carcinoma, are mechanism study and the clinical application of esophageal squamous cell carcinoma
It provides fundamental basis.
Present invention firstly discovers that AGXT2L1 differential expression in patients with esophageal squamous cell carcinoma tissue, passes through detection AGXT2L1's
Expression may determine that whether patient suffers from the height of esophageal squamous cell carcinoma or risk.
The present invention provides the methods of personalized treatment esophageal squamous cell carcinoma, by improving the expression of AGXT2L1, to oesophagus
Squamous cell carcinoma patients are treated.
Detailed description of the invention
Fig. 1 shows the expression using QPCR detection AGXT2L1 gene in esophageal squamous cell carcinoma tissue;
Fig. 2 shows the expression using QPCR detection AGXT2L1 gene in esophageal cells;
Fig. 3 shows the transfection efficiency using QPCR detection transfection AGXT2L1 gene in esophageal squamous cell;
Fig. 4 display increases esophageal squamous cell using using soft-agar cloning formation experiment detection AGXT2L1 gene expression
Grow the influence of ability;
Fig. 5 shows the influence using the cell transwell detection AGXT2L1 gene pairs esophageal squamous cell invasion.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this
It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.
Embodiment 1 screens gene marker relevant to esophageal squamous cell carcinoma
1, sample collection
Respectively collect 6 surrounding normal mucous membrane of esophagus tissues and esophageal squamous cell carcinoma tissue, the equal informed consent of patient, above-mentioned all marks
This obtains the agreement for passing through the committee, organizational ethics.
2, the preparation (being operated using the tissue RNA extracts kit of QIAGEN) of RNA sample
Taking-up freezes the tissue samples in liquid nitrogen, and tissue samples are put into the mortar being pre-chilled and are ground, according to
Specification in kit extracts separation RNA.It is specific as follows:
1) Trizol is added, is placed at room temperature for 5min;
2) chloroform 0.2ml is added to be mixed well with forced oscillation centrifuge tube, places 5-10min at room temperature;
3) 12000rpm is centrifuged 15min, and upper strata aqueous phase is moved on in another new centrifuge tube and (is careful not to be drawn onto two layers of water
Protein substance between phase), the isopropanol of -20 DEG C of isometric pre-coolings is added, is sufficiently mixed by inversion, is placed in 10min on ice;
4) 12000rpm high speed is added 75% in the ratio of 1ml/ml Trizol from supernatant is carefully discarded after 15min
DEPC ethanol washing precipitating (4 DEG C of preservations), washing precipitate, oscillation mixes, and 4 DEG C, 12000rpm is centrifuged 5min;
5) ethanol liquid is discarded, places 5min at room temperature, DEPC water dissolution precipitating is added;
6) it with Nanodrop2000 ultraviolet specrophotometer measurement RNA purity and concentration, freezes in -70 DEG C of refrigerators.
3, reverse transcription and label
With Low RNA Input Linear Amplification Kit by mRNA reverse transcription at cDNA, while using Cy3
Labelling experiment group and control group respectively.
4, hybridize
Genetic chip uses people's full-length genome chip of expression spectrum of Aglient company, and every chip includes 45015 few cores
Thuja acid, wherein having 43376 people's gene probes and 1639 experiment control probes.It is carried out by the step of chip operation instructions,
Temperature rolls through 17h 10r/min and hybridizes, 37 DEG C are developed a film at 65 DEG C.
5, data processing
Chip Agilent scanner scanning after hybridization, resolution ratio are 5 μm, and scanner is automatically with 100% and 10%PMT
Each scanning 1 time, 2 times result Agilent software merges automatically.Scan image data is using at Feature Extraction
Reason analysis, obtained initial data application Bioconductor program bag carry out follow-up data processing.Last Ratio value is experiment
Group and control group.Differential gene screening criteria: ratio >=4 are up-regulation gene, and ratio≤0.25 is down-regulated gene.
6, result
Compared with normal esophageal mucosal tissue, expression quantity of the AGXT2L1 gene in esophageal squamous cell carcinoma tissue is significantly lowered.
The differential expression of 2 QPCR sequence verification AGXT2L1 gene of embodiment
1, large sample QPCR verifying is carried out to AGXT2L1 gene differential expression.According to the sample collection mode in embodiment 1
Select surrounding normal mucous membrane of esophagus tissue and esophageal squamous cell carcinoma tissue each 50.
2, RNA extraction step is the same as embodiment 1.
3, reverse transcription:
Using 25 μ l reaction systems, each sample takes 1 μ g total serum IgE as template ribonucleic acid, is separately added into PCR pipe following
Component: DEPC water, 5 × RT Buffer, 10mM dNTP, 0.1mM DTT, 30 μM of Oligo dT, 200U/ μ l M-MLV,
Template ribonucleic acid.42 DEG C of incubation 1h, 72 DEG C of 10min, of short duration centrifugation.
(3) QPCR amplification is examined
Design of primers
The primer sequence of house-keeping gene GAPDH are as follows:
Forward primer: 5 '-CTCTGGTAAAGTGGATATTGT-3 ' (SEQ ID NO.3)
Reverse primer: 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.4)
The primer sequence of AGXT2L1 gene are as follows:
Forward primer: 5 '-ACCAACTCCAGATACTTAC-3 ' (SEQ ID NO.5)
Reverse primer: 5 '-CTTCCTTCCACTGTTATGA-3 ' (SEQ ID NO.6)
Using 25 μ l reaction systems, 3 parallel pipes are arranged in each sample, and all amplified reactions are repeated three times above to protect
Demonstrate,prove the reliability of result.
Prepare following reaction system: 12.5 μ l of SYBR Green polymerase chain reaction system, forward and reverse primer (5 μM)
Each 1 μ l, template cDNA 2.0 μ l, no 8.5 μ l of enzyme water.Operations are carried out on ice.
Amplification program are as follows: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 45s) × 30 circulations.
Using SYBR Green as fluorescent marker, it is anti-that PCR is carried out on Light Cycler fluorescence real-time quantitative PCR instrument
It answers, determines that purpose band, Δ Δ CT method carry out relative quantification by melt curve analysis analysis and electrophoresis.
3, 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 for statistical analysis, difference between the two is examined using t, it is believed that is had as P < 0.05
It is statistically significant.
4, result
As a result as shown in Figure 1, AGXT2L1 gene is in esophageal squamous cell carcinoma tissue compared with surrounding normal mucous membrane of esophagus tissue
Expression is lowered, and difference has statistical significance (P < 0.05), consistent with RNA-sep result.
Differential expression of the 3 AGXT2L1 gene of embodiment in esophageal carcinoma cell line
1, cell culture
Human esophageal squamous cell cancer cell strain KYSE 150, KYSE450 are purchased from institute of oncology, the Chinese Academy of Sciences, normal esophageal epithelial cell
Strain Het-1a is purchased from Guangzhou Ji Niou company.With the culture medium DMEM containing 10% fetal calf serum and 1%P/S in 37 DEG C, 5%
CO2, relative humidity be 90% incubator in cultivate.It changes within 2-3 days liquid 1 time, trypsase of the use 0.25% containing EDTA is conventional
Had digestive transfer culture.
2, the extraction of RNA
1) pancreatin digests attached cell, the cell of acquisition is blown and beaten after centrifugation, resuspension, cleaning, with 1640 culture mediums (10%
Calf serum) it is resuspended;
2) cell of resuspension is transferred in 6 orifice plates (/ hole), adds culture medium to the hole 2m1/, jog 6 orifice plates keep cell uniform
It is resuspended;
3) cell adherent growth 48h, removes culture medium;
4) with 1ml Trizol reagent lytic cell, 6 orifice plates wall is blown and beaten repeatedly, cracks cell completely as far as possible;
5) metastatic cells lysate is placed on ice into the processed EP pipe of 1.5ml DEPC.0.2m1 chloroform is added, remains
Remaining operating procedure is the same as RNA extraction process in tissue.
3, reverse transcription
Specific 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 for statistical analysis, difference between the two is examined using t, it is believed that is had as P < 0.05
It is statistically significant.
5, result
As a result as shown in Fig. 2, compared with esophageal epithelial cell, AGXT2L1 gene esophageal squamous cell KYSE150,
It expresses in KYSE450 and lowers, difference has statistical significance (P < 0.05), consistent with RNA-sep result.
The overexpression of 4 AGXT2L1 gene of embodiment
1, cell culture
Human esophageal squamous cell cancer cell strain KYSE 150, with the culture medium DMEM containing 10% fetal calf serum and 1%P/S 37 DEG C,
5%CO2, relative humidity be 90% incubator in cultivate.It changes within 2-3 days liquid 1 time, trypsase of the use 0.25% containing EDTA is normal
Advise had digestive transfer culture.
2, the overexpression of AGXT2L1 gene
The building of 2.1AGXT2L1 expression vector
Amplimer is designed according to the coded sequence (as shown in SEQ ID NO.1) of AGXT2L1 gene, primer sequence is such as
Under:
Forward primer: 5 '-CCGAAGCTTGCCACCATGTGCGAGCTGTACAGTA-3 ' (SEQ ID NO.7)
Reverse primer: 5 '-CGGCTCGAGTGTCTTGAGCCTCTTACTGAGCAG-3 ' (SEQ ID NO.8)
From cDNA library (clontech company, the article No.: 638831) the AGXT2L1 gene of amplification overall length at Human fetal spleen
Coded sequence, above-mentioned cDNA sequence is inserted into after restriction enzyme HindIII and XhoI double digestion through restriction enzyme
In the eukaryotic expression vector pcDNA3.1 of enzyme HindIII and XhoI double digestion, the recombinant vector pcDNA3.1- of acquisition is connected
AGXT2L1 is used for subsequent experimental.
2.2 transfection
Esophageal squamous cell is divided into two groups, respectively control group (transfection pcDNA3.1 empty carrier) and AGXT2L1 crosses table
Up to group (transfection pcDNA3.1-AGXT2L1).The transfection of carrier is carried out using liposome 2000, specific transfection method is according to explanation
The instruction of book carries out.The transfection concentrations of pcDNA3.1 empty carrier and pcDNA3.1-AGXT2L1 are 0.5 μ g/ml.
2.3RT-PCR detection
Specific steps are the same as embodiment 2.
3, 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 for statistical analysis, difference between the two is examined using t, it is believed that is had as P < 0.05
It is statistically significant.
4, result
As shown in figure 3, being transfected in the cell of pcDNA3.1-AGXT2L1 compared with the cell of transfection pcDNA3.1 empty carrier
The content of AGXT2L1 significantly raises, and difference has statistical significance (P < 0.05).
The influence of 5 AGXT2L1 gene pairs esophageal squamous cell apoptosis of embodiment
Use the influence of flow cytomery AGXT2L1 gene pairs Apoptosis.
1, cell culture step is the same as embodiment 3.
2, cell transfecting step is the same as embodiment 3.
3, step
After cell transfecting 72h, cell is washed using pre-cooling PBS, then with 0.25% trypsin digestion cell, stops digestion,
The cell being collected by centrifugation is resuspended using PBS, is 1 × 10 by cell quantification6A/ml takes the 200 above-mentioned cell suspensions of μ l to be placed into
In Eppendorf pipe, 10 μ l Annexin-V-FITC are added and mix, room temperature dark place is incubated for dyeing 15min, and 5min adds before upper machine
Enter 10mg/L propidium iodide (PI) and dyes 5 μ l.The cell of untransfected siRNA is used Annexin-V-FITC and PI to dye respectively and is used for
Standard quantitative.Two Colour Fluorescence cell cytometry, observing apoptosis cell percentages are carried out with FACS flow cytometer.
3, 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 t inspection for statistical analysis, that difference between the two uses, it is believed that as P < 0.05
With statistical significance.
4, result:
The apoptosis rate for transfecting pcDNA3.1-AGXT2L1 group is (15.14 ± 0.021) %, and transfection pcDNA3.1 is unloaded
The apoptosis rate of body group is (6.21 ± 0.011) %, and above-mentioned difference has statistical significance (P < 0.05), the above results table
Bright, the overexpression of AGXT2L1 gene promotes the apoptosis of esophageal squamous cell.
6 soft-agar cloning of embodiment forms experiment
1, the cell of logarithmic growth phase is in 0.25% trypsin digestion, gently piping and druming makes unicellular outstanding
Cell precipitation is collected by centrifugation in liquid.
2, it is resuspended with the DMEM complete medium containing 20% fetal calf serum, is counted after appropriate dilution, adjustment cell concentration is 5
×103A/ml.
3, the low melting point agar liquid glucose that two concentration are respectively 1.2% and 0.7% is prepared, after high pressure sterilization, maintains 40
In DEG C water-bath.
4,1.2% agarose and 2 × DMEM culture medium 1:1 mixing, are added the calf serum of 2 × antibiotic and 20%,
It takes in 3ml mixed liquor injection diameter 6cm plate and places 5min cooled and solidified, be placed in as bottom-layer agar spare in CO2 incubator.
5, in sterile test tube 1:1 mixing 0.7% agarose and 2 × DMEM culture medium, then into pipe be added 0.2ml it is dense
Degree is 5 × 103A/ml's stablizes infection cell suspension, mixes well, injects in above-mentioned plate, gradually forms double agar layers, often
A experimental group repeats 4 samples.
6, after top-layer agar solidification, 37 DEG C of 5%CO are placed in2It is cultivated in incubator, every 3 days plus culture medium 1.5ml.
7, culture dish is taken out after cultivating 14 days, dyes 90min with the gentian violet that 1ml concentration is 0.005%.Plate is placed
It is observed under inverted microscope, every group of cell randomly selects 10 low-power fields, the number of cell clones that technology is formed under mirror.
8, result
As a result as shown in figure 4, compared with other groups, the groups of cells single cell clone colony of pcDNA3.1-AGXT2L1 is transfected
Forming number significantly reduces.
7 Transwell cells in vitro Matrigel of embodiment
1, the serum-free medium Matrigel that 100 μ l are added into the upper hole of the cell Transwell impregnates 30min.
2, the group of cells after stablizing infection with pancreatin digestion in logarithmic growth phase, PBS is cleaned cell 3 times, with containing
Cell is resuspended in the culture solution of 10% serum, and adjustment cell concentration is 1 × 105/ml。
3,200 μ l of cell suspension is added into the upper chamber for the cell Transwell for being coated with Matrigel.
4, DMEM culture solution of the 600 μ l containing 20%FBS is added in the lower room of the cell Transwell.
5,37 DEG C, 5%CO2It is cultivated for 24 hours in incubator.
6, the culture solution in upper chamber and lower room is removed, the cell for striking off the Matrigel in upper chamber with cotton swab and retaining is used
PBS is cleaned 2 times.
7,45min is dyed with 1% violet staining liquid, PBS is cleaned 1 time.
8,4 100 times of visuals field are randomly selected, count invasion cell number respectively under the microscope, it is every kind different types of thin
Born of the same parents set 3 multiple holes, are repeated 3 times altogether.
9, 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, and P < 0.01 is significant difference.
10, result
As a result as shown in figure 5, KYSE150, pcDNA3.1 be unloaded, pcDNA3.1-AGXT2L1 group cell is in transwell
After cultivating for 24 hours in cell, the cell number in room face is substantially reduced under pcDNA3.1-AGXT2L1 group polycarbonate membrane.
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.