CN105886656B - Application of the GIF gene in esophageal squamous cell carcinoma diagnosis and treatment - Google Patents
Application of the GIF gene in esophageal squamous cell carcinoma diagnosis and treatment Download PDFInfo
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- CN105886656B CN105886656B CN201610471615.1A CN201610471615A CN105886656B CN 105886656 B CN105886656 B CN 105886656B CN 201610471615 A CN201610471615 A CN 201610471615A CN 105886656 B CN105886656 B CN 105886656B
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
Abstract
The invention discloses application of the GIF gene in esophageal squamous cell carcinoma diagnosis and treatment, inventors have demonstrated that GIF gene is related to the occurrence and development of esophageal squamous cell carcinoma, the expression for increasing GIF can promote the apoptosis of esophageal squamous cell, the invention discloses the methods of oesophagus squama cancer diagnosis and treatment, have important clinical value.
Description
Technical field
The invention belongs to biomedicine field, it is related to a kind of application of gene in esophageal squamous cell carcinoma diagnosis and treatment, more particularly to
Application of the GIF in esophageal squamous cell carcinoma diagnosis and treatment.
Background technique
The cancer of the esophagus is one of most common malignant tumor of digestive tract in the world, accounts for about the 2% of all malignant tumours, in the whole world
In range, disease incidence ranking the 8th, death rate ranking the 6th.The histological type of the cancer of the esophagus includes esophageal squamous cell carcinoma
And adenocarcinoma of esophagus, while the cancer of the esophagus also has the characteristics that regional allocations, most esophageal squamous cell carcinoma occur mainly in Asia state
Family.Central Asia republic is passed through to north of China in Iranian the north, this region is referred to as " cancer of the esophagus area ", wherein 90% pathology
Type is squamous cell carcinoma.China is one of higher country of esophageal squamous cell carcinoma disease incidence in the world.With science and technology it is continuous
Development, the treatment of the cancer of the esophagus has been developed as based on surgical operation therapy at present, then the complex treatment for assisting Radiotherapy chemotherapy etc.
Mode.In China, surgical operation be still can resection of esophageal cancer first choice treatment method.Recently as esophagus cancer diagnosis technology
Continuous improvement, surgical procedure technical ability continuously improves, the application of Neoadjuvant chemotherapy, along with postoperative regular Radiotherapy chemotherapy
It implements, the quality of life of patient with esophageal carcinoma is gradually improved to a certain extent, but China's cancer of the esophagus is suffered from the whole
5 years postoperative overall survivals of person are only 20%-30%.
The occurrence and development of esophageal squamous cell carcinoma are the pathologic processes of a complexity for being related to multifactor multi-step, in recent years, with
People deepen continuously to molecular biology understanding, the specific variations of a large amount of research verified many genes are in tumour
It plays an important role during occurrence and development.Although the precise mechanism of up to the present cancer of the esophagus occurrence and development is still unknown
Really, but the research about Molecular Biomarkers relevant to esophageal squamous cell carcinoma be all the time the area research heat
Point.The diagnosis marker for clinically lacking effective esophageal squamous cell carcinoma at present, is clinically often used tumor in digestive tract marker such as
SCC, CEA, CA724 and CA199, but above-mentioned molecule is limited as the diagnostic value of esophageal squamous cell carcinoma tumor markers, therefore finds
More valuable esophageal squamous cell carcinoma related molecular marker object, evaluates its diagnostic value and explores its function, to develop new tumour
Marker and more targeted therapy target, to improve the prognosis of patient.
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 points that can be used for diagnosis and treatment esophageal squamous cell carcinoma
Sub- marker-GIF gene.
To achieve the goals above, the present invention adopts the following technical scheme:
The present invention provides the application of GIF gene and its expression product in the product of preparation diagnosis esophageal squamous cell carcinoma.
Further, the product includes: to be examined by RT-PCR, real-time quantitative PCR, immune detection, in situ hybridization or chip
The expression of GIF gene is surveyed to diagnose the product of esophageal squamous cell carcinoma.
Further, the product with RT-PCR diagnosis esophageal squamous cell carcinoma includes at least drawing for a pair of of specific amplified GIF gene
Object；The product with real-time quantitative PCR diagnosis esophageal squamous cell carcinoma includes at least the primer of a pair of of specific amplified GIF gene；It is described
Product with immune detection diagnosis esophageal squamous cell carcinoma includes: the antibody in conjunction with GIF protein-specific；It is described to be diagnosed in situ hybridization
The product of esophageal squamous cell carcinoma includes: the probe with the nucleic acid array hybridizing of GIF gene；The product with chip diagnosis esophageal squamous cell carcinoma
It include: protein chip and genetic chip；Wherein, protein chip includes the antibody in conjunction with GIF protein-specific, genetic chip packet
Include the probe with the nucleic acid array hybridizing of GIF gene.
Further, the genetic chip can be used for detecting multiple genes including GIF gene (for example, and esophageal squamous cell
The relevant multiple genes of cancer) expression.The protein-chip can be used for detecting multiple albumen including GIF albumen
The expression of matter (such as multiple protein relevant to esophageal squamous cell carcinoma).By by multiple markers with esophageal squamous cell carcinoma simultaneously
Detection, is greatly improved the accuracy rate of oesophagus squama cancer diagnosis.
The present invention provides a kind of product for diagnosing esophageal squamous cell carcinoma, the product can pass through GIF in detection esophageal tissue
The expression of gene diagnoses esophageal squamous cell carcinoma, and GIF gene expresses downward in esophageal squamous cell carcinoma tissue.
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 GIF gene transcription level
For the oligonucleotide probe of GIF gene；The protein-chip includes solid phase carrier and the GIF egg for being fixed on solid phase carrier
White specific antibody；The gene detecting kit includes the reagent for detecting GIF gene transcription level；The albumen is exempted from
Epidemic disease detection kit includes the specific antibody of GIF albumen.
Further, the gene detecting kit can be used for detect including GIF gene multiple genes (for example, with
The relevant multiple genes of esophageal squamous cell carcinoma) expression.It includes GIF albumen that the protein immunization detection kit, which can be used for detecting,
The expression of multiple protein (such as multiple protein relevant to esophageal squamous cell carcinoma) inside.By multiple marks of esophageal squamous cell carcinoma
Will object is detected simultaneously, is greatly improved the accuracy rate of oesophagus squama cancer diagnosis.
The answering in the pharmaceutical composition of preparation treatment esophageal squamous cell carcinoma the present invention provides GIF gene and its expression product
With.
Further, described pharmaceutical composition includes increasing GIF gene expression, enhancing GIF expressive function, and/or enhancing GIF
The active reagent of expression product.
Further, the reagent includes: the activation of the reagent, GIF albumen of the nucleic acid containing energy encoding function GIF albumen
Agent, the reagent containing GIF protein.
Wherein, the reagent of the nucleic acid containing energy encoding function GIF albumen can be translates under advantage
The single-chain nucleic acid (such as mRNA) or double-strandednucleic acid (such as DNA) of the GIF albumen of active form, the nucleic acid, which can connect, is expressing
On carrier or in recombination to host cell, as long as Viability GIF albumen can be encoded, the carrying mode of any GIF gene is equal
It can.The GIF protein activator refers to stimulation GIF protein active, increases GIF protein active, promote GIF protein active, increase
Strong GIF protein activation makes the sensitization of GIF protein active or raises the reagent of GIF protein active, as demethylation reagent, GIF are opened
The agonist (such as activation antibody) of the transcription activation factor of mover and/or enhancer specificity, GIF albumen.
Further, aforementioned pharmaceutical compositions further include pharmaceutically acceptable carrier, and the carrier can be one kind can also
Be it is a variety of, the carrier includes but is not limited to diluent such as lactose, sodium chloride, glucose, urea, starch, water etc.；Adhesive
As starch, pregelatinized starch, dextrin, maltodextrin, sucrose, Arabic gum, gelatin, methylcellulose, carboxymethyl cellulose,
Ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginic acid and alginate, xanthan gum, hydroxypropyl are fine
Dimension element and hydroxypropyl methyl cellulose etc.；Surfactant such as polyoxyethylene sorbitan aliphatic ester, dodecyl sulphate
Sodium, glyceryl monostearate, hexadecanol etc.；Humectant such as glycerol, starch etc.；Absorption carrier for example starch, lactose, bentonite,
Silica gel, kaolin and soap clay etc.；Lubricant such as zinc stearate, glycerin monostearate, polyethylene glycol, talcum powder, stearic acid
Calcium and magnesium, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyl 40 stearate, Dan Yuegui sugarcane
Furamide, sldium lauryl sulfate, magnesium laurylsulfate, Stepanol MG etc.；Filler such as mannitol (granular or powdery),
Xylitol, sorbierite, maltose, erythrose, microcrystalline cellulose, polymerization sugar, coupling sugar, glucose, lactose, sucrose, dextrin, shallow lake
Powder, sodium alginate, laminarin powder, agar powder, calcium carbonate and sodium bicarbonate etc.；Disintegrating agent such as cross-linked ethylene pyrrolidines
Ketone, sodium carboxymethyl starch, low-substituted hydroxypropyl ylmethyl, croscarmellose sodium, soybean polyoses etc..
The pharmaceutical composition can be used different additives and be prepared, such as stabilizer, fungicide, buffering
Agent, isotonic agent, chelating agent, pH controlling agent and surfactant.
Stabilizer includes Human serum proteins, l-amino acid, sugar and cellulose derivative.L-amino acid can also include sweet
Any one in propylhomoserin, cysteine and glutamic acid.Carbohydrate includes monosaccharide, such as glucose, mannose, galactolipin, fructose
Deng；Sugar alcohol, such as mannitol, inositol, xylitol etc.；Disaccharides, such as sucrose, maltose, lactose etc.；Polysaccharide, such as Portugal
Glycan, hydroxypropul starch, vulcanization chondroitin, hyaluronic acid etc. and their derivative.Cellulose derivative includes Methyl cellulose
Element, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hypromellose and sodium cellulose glycolate.
Surfactant includes ion or nonionic surfactant, such as polyethylene glycol oxide Arrcostab, sorbitan list
Acyl ester, fatty glyceride.
Additive buffer may include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid and corresponding salt (their alkali gold
Category or alkaline rare earth metal salt, such as sodium salt, sylvite, calcium salt and magnesium salts).Isotonic agent includes potassium chloride, sodium chloride, sugar and sweet
Oil.Chelating agent includes sodium ethylene diamine tetracetate and citric acid.
The present invention also provides a kind of pharmaceutical composition for treating esophageal squamous cell carcinoma, described pharmaceutical composition includes increasing GIF
Gene expression, enhancing GIF expressive function, and/or the enhancing active reagent of GIF expression product.
Drug of the invention can be used for supplementing the missing or deficiency of endogenic GIF albumen, by improving GIF albumen
Expression, thus esophageal squamous cell carcinoma caused by treatment is reduced because of GIF albumen.
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..
Further, the nucleic acid of GIF albumen or coding GIF albumen can be given by liposome in the present invention, the lipid
The effect of body is by drug targeting in specific tissue, and increases the half-life period of drug.Liposome includes emulsifier, blistering
Agent, liquid fatty substance, Solid lipid, insoluble monolayer, phospholipid dispersions, surfactant etc..It can be in the liposome
Including can be in conjunction with the acceptor molecule in the cell of targeting or other therapeutic or immunogenic composition.
Pharmaceutical composition of the invention can be formulated into any administration fashion, for example, utilizing syringe or other dresses
The intracutaneous injection set, subcutaneous injection, intravenous injection, intraperitoneal injection, intrapleural injection, Intravesical administration, in coronary artery
Or intra-tumoral injection, oral administration, rectally.
Drug of the invention, which imports tissue or the mode of cell, can be divided into external or intracorporal mode.Vitro formats
Including importing the drug containing GIF gene or the drug containing GIF protein in cell, then by cell transplantation or feed back to
In vivo.Internal mode includes directly by the drug containing GIF gene or the infusion of medicine in-vivo tissue containing GIF protein
In.
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 (for example, nucleic acid of GIF albumen or encoding said proteins) wanted is administered simultaneously, or even in same composition simultaneously
Administration.Other therapeuticization can also be individually given with individual composition or the dosage form different from main active constituent
Close object.The Fractional of main component (such as GIF albumen or the nucleic acid of encoding said proteins) can be with other therapeutic compounds
It is administered simultaneously, and other dosage can be administered alone.
In the course of disease treatment, it can be answered according to the physiology of the severity of symptom, the frequency of recurrence and therapeutic scheme
It answers, adjusts the dosage of pharmaceutical composition of the present invention.
In the present invention, term " host cell " includes prokaryotic cell and eukaryocyte.Common prokaryotic host cell
Example includes Escherichia coli, hay bacillus etc..Common eukaryotic host cell includes yeast cells, insect cell and mammal
Cell.Preferably, the host cell is eukaryocyte, such as Chinese hamster ovary celI, COS cell.
Probe in the present invention for GIF gene can be DNA, RNA, DNA-RNA chimera, PNA or other derivatives.
There is no limit as long as completing specific hybrid, specifically binding with purpose nucleotide sequence, any length for the length of the probe
It is ok.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the length of the probe can be grown
To 60,80,100,150,300 base-pairs or longer or even whole genes.Due to different probe lengths to hybridization efficiency,
Signal specificity has different influences, and the length of the probe is typically at least 14 base-pairs, and longest is usually no more than 30
Base-pair, the length complementary with purpose nucleotide sequence are best with 15-25 base-pair.The probe self-complementary sequences are best
Less than 4 base-pairs, in order to avoid influence hybridization efficiency.
The specific antibody of heretofore described GIF albumen includes monoclonal antibody, polyclonal antibody.The GIF albumen
Specific antibody include complete antibody molecule, antibody any segment or modification, for example, chimeric antibody, scFv, Fab, F
(ab ') 2, Fv etc..As long as the segment can retain the binding ability with GIF albumen.For detecting protein level
The preparation of antibody is well known to those skilled in the art, and the present invention may use any method to prepare the antibody, such as
The segment can be synthesized by chemical method de novo formation or using recombinant DNA technology.
In the context of the present invention, " GIF gene " includes that any function of hGIF gene and hGIF gene is equivalent
The polynucleotides of object.GIF gene includes and GIF gene (NC_ in the public GenBank GeneBank in the current world
000011.10) DNA sequence dna has 70% or more homology, and encodes the DNA sequence dna of identical function protein；
Preferably, the coded sequence of GIF gene includes following any DNA molecular:
(1) DNA sequence dna shown in SEQ ID NO.1 in sequence table；
(2) hybridize under strict conditions with the DNA sequence dna that (1) limits and encode the DNA sequence dna of identical function protein；
(3) DNA sequence dna limited with (1) or (2) has 70%, preferably, 90% or more homology, and encodes identical function
The DNA molecular of energy protein.
In specific embodiments of the present invention, the coded sequence of the GIF gene is DNA shown in SEQ ID NO.1
Sequence.
In the context of the present invention, GIF gene expression product includes the partial peptide of hGIF albumen and hGIF albumen.
The partial peptide of the GIF albumen contains functional domain relevant to esophageal squamous cell carcinoma.
" GIF albumen " includes any functional equivalent of GIF albumen and GIF albumen.The functional equivalent includes GIF
Albumen conservative variation protein or its active fragment or its reactive derivative, allelic variant, natural mutation, induction are prominent
Variant, can be with the encoded protein of DNA of the DNA hybridization of hGIF under high or low stringent condition.
Preferably, GIF albumen is the protein with following amino acid sequences:
(1) protein that the amino acid sequence shown in SEQ ID NO.2 in sequence table forms；
(2) amino acid sequence shown in SEQ ID NO.2 by the substitution of one or several amino acid residues and/or is lacked
Lose and/or addition and with the ammonia with the same function as shown in SEQ ID NO.2 of amino acid sequence shown in SEQ ID NO.2
Protein derived from base acid sequence.The number for the amino acid for replacing, lacking or adding is usually 1-50, preferably 1-30
It is a, more preferably 1-20, most preferably 1-10.
(3) there is at least 80% homology (also known as sequence identity) with amino acid sequence shown in SEQ ID NO.2,
Preferably, with the homology of amino acid sequence at least about 90% to 95% shown in SEQ ID NO.2, Chang Wei 96%, 97%,
98%, the polypeptide that the amino acid sequence of 99% homology is constituted.
In specific embodiments of the present invention, the GIF albumen is with amino acid sequence shown in SEQ ID NO.2
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.
Example by one amino acid of addition or the protein of more amino acid modification is the fusion egg of GIF albumen
It is white.For the peptide or protein with GIF protein fusion, there is no limit as long as resulting fusion protein retains the life of GIF albumen
Object activity.
GIF albumen of the invention also includes the non-conservative modification to amino acid sequence shown in SEQ ID NO.2, as long as through
The protein for crossing modification still is able to retain the biological activity of GIF albumen.The ammonia being mutated in such modification protein
Base acid number is usually 10 perhaps less such as 6 perhaps less such as 3 or less.
In the context of the present invention, " treatment esophageal squamous cell carcinoma " includes that can eliminate, mitigate, alleviate, reverse or prevent or prolong
The generation and recurrence of any symptom of slow illness, that is, include the therapeutic intervention and Primary preventive intervention to disease.
The advantages of the present invention:
Present invention firstly discovers that the new molecular marked compound-GIF gene of esophageal squamous cell carcinoma a kind of, passes through detection subject's food
The expression of GIF in tubing, it can be determined that whether subject suffers from esophageal squamous cell carcinoma, to instruct clinician to subject
Personalized prevention scheme or therapeutic scheme are provided.
Detailed description of the invention
Fig. 1 shows the expression using QPCR detection GIF gene in esophageal squamous cell carcinoma tissue；
Fig. 2 shows the expression using QPCR detection GIF gene in esophageal squamous cell；
Fig. 3 shows the influence using MTT detection GIF gene expression to esophageal squamous cell proliferative capacity.
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 esophageal squamous cell carcinoma cancer beside organisms and esophageal squamous cell carcinoma tissue sample.The acquirement of above-mentioned all samples passes through
The agreement of the committee, organizational ethics.
2, the preparation of RNA sample (utilizesKit is operated)
The tissue of above-mentioned acquisition shred after put into liquid nitrogen in and be ground to it is powdered, according in kit specification extract
Separate RNA.It is specific as follows:
1) separation of RNA:
A. RNA is added in tissue homogenate or cellII 1ml；
B. it is placed at room temperature for 3min, is aggressively shaken 15s after 0.2ml chloroform is added；
C. be placed in prevents 10min on ice；
D.12000g, 4 DEG C of centrifugation 15min；
E. the water phase of transfer 80% enters in new 2ml EP pipe, and the dehydrated alcohol of 1/2 amount, shaking is added；
F. the aforesaid liquid less than 700 μ l is transferred toMini column, 10000g room temperature after shaking
It is centrifuged 60s.
2) RNA is purified:
A. toMini column is added 500 μ l RWC Wash Buffer, 10000g and is centrifuged 30s；
B. 500 μ l RWB Wash Buffer, 10000g centrifugation 30s are added, take maximum centrifugal complete after being repeated twice
It is dryMini column；
C. the DEPC water that 15 μ l are preheated to 70 DEG C is added to pillar, is centrifuged at full speed after being placed at room temperature for 2min.
3, high-throughput transcript profile sequencing
1) RNA-seq read positions
Low-quality read is removed to obtain cleaning read first, then using TopHat v1.3.1 will clean segment and
UCSC H.sapiens is matched with reference to genome (hg19), the H.sapiens UCSC hg19 editions indexes constructed in advance
It is downloaded from TopHat homepage, and as reference genome, when matching using TopHat with genome, allows each read (default
To 20) having multiple matching sites, most 2 mispairing.TopHat establishes possible according to exon region and GT-AG shear signal
Shearing site library navigates to the read for not navigating to genome on genome according to these shearing site libraries.We use
The system default parameter of TopHat method.
2) transcript abundance is assessed
The read file matched is handled by Cufflinks v1.0.3, and Cufflinks v1.0.3 is by RNA-seq piece
Number of segment mesh is standardized the relative abundance for calculating transcript.FPKM value refers to being matched in every 1,000,000 sequencing fragment specific
The segment number of the exon region of gene 1kb long.The confidence interval of FPKM estimated value is calculated by Bayesian inference method.
The GTF comment file for the reference that Cufflinks is used downloads (Homo_ from Ensembl database
sapiens.GRCh37.63.gtf)。
3) detection of difference expression gene
It is transferred to Cuffdiff by the Ensembl GTF file of downloading and by the matched original document of TopHat,
Cuffdiff re-evaluates the gene expression abundance for the transcript listed in GTF file using original matching files, detects difference table
It reaches.The only q value < 0.01 in Cuffidff output, test display is more just considered as successfully differential expression.
4, result
RNA-seq is the results show that expression quantity of the GIF gene in esophageal squamous cell carcinoma tissue is substantially less than the table in cancer beside organism
Up to amount.
The differential expression of embodiment 2QPCR sequence verification GIF gene
1, large sample QPCR verifying is carried out to GIF gene differential expression.It is selected according to the sample collection mode in embodiment 1
Esophageal squamous cell carcinoma cancer beside organism and esophageal squamous cell carcinoma tissue each 50.
2, RNA extraction step is the same as embodiment 1.
3, it reverse transcription: is operated using the reverse transcription reagent box of TAKARA company.Specific step is as follows:
(1) it takes 1 μ g of total serum IgE to carry out reverse transcription, Oligo (dT) 2 μ l is added, mixes well.After 70 DEG C of water-bath 5min immediately
Ice bath 2min.
(2) 25 μ l reaction systems are constructed, including 5 × RT Buffer 5 μ l, dNTP (2.5mM) 5 μ l, RNasin
40U/ μ l, M-MLV 200U/ μ l mends nuclease-free water to anticipated volume.
After (3) 42 DEG C of water-bath 60min, 95 DEG C of water-bath 5min are to inactivate M-MLV.
(4) -20 DEG C store for future use.
4, QPCR is expanded
(1) design of primers
QPCR amplimer is designed according to the coded sequence of GIF gene in Genebank and β-actin gene, is given birth to by Shanghai
The synthesis of work biotechnology Services Co., Ltd.Specific primer sequence is as follows:
The primer sequence of GIF:
Forward primer: 5 '-GTGTTACTTGTTGTCCTA-3 ' (SEQ ID NO.3),
Reverse primer: 5 '-CGATATTGTTGATAGAAGAG-3 ' (SEQ ID NO.4)
The primer sequence of β-actin:
Forward primer: 5 '-CCTGGGCATGGAGTCCTGTG-3 ' (SEQ ID NO.5)
Reverse primer: 5 '-TCCTTCTGCATCCTGTCG-3 ' (SEQ ID NO.6)
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.
(2) PCR reaction system is prepared according to table 1:
Wherein, SYBR Green polymerase chain reaction system is purchased from Invitrogen company.
Table 1PCR reaction system
| Reagent | Volume |
| Forward primer | 1μl |
| Reverse primer | 1μl |
| SYBR Green polymerase chain reaction system | 12.5μl |
| Template | 2μl |
| Deionized water | Supply 25 μ l |
(3) PCR reaction condition: 95 DEG C of 10min, (95 DEG C of 30s, 60 DEG C of 60s) × 35 circulations.Using SYBR Green as
Fluorescent marker carries out PCR reaction on Light Cycler fluorescence quantitative PCR instrument, true by melt curve analysis analysis and electrophoresis
Determine purpose band, Δ Δ CT method carries out relative quantification.
5, statistical method
Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD,
Using SPSS13.0 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.
6, result
As a result as shown in Figure 1, compared with esophageal squamous cell carcinoma cancer beside organism, GIF gene is lowered in esophageal squamous cell carcinoma tissue, difference
It is consistent with RNA-sep result with statistical significance (P < 0.05).
The overexpression of embodiment 3GIF gene
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 GIF gene
The building of 2.1GIF expression vector
Amplimer is designed according to the coded sequence (as shown in SEQ ID NO.1) of GIF gene, primer sequence is as follows:
Forward primer: 5 '-CCGAAGCTTGCCACCATGGCCTGGTTTGCCCT-3 ' (SEQ ID NO.7)
Reverse primer: 5 '-CGGGCGGCCGCGTACTGTGTGTGAAATTGGCT-3 ' (SEQ ID NO.8)
From cDNA library (clontech company, the article No.: the 638831) volume of the GIF gene of amplification overall length at Human fetal spleen
Code sequence, above-mentioned cDNA sequence are inserted into after restriction enzyme HindIII and NotI double digestion through restriction enzyme
In the eukaryotic expression vector pcDNA3.1 of HindIII and NotI double digestion, the recombinant vector pcDNA3.1- of acquisition is connected
GIF 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 GIF overexpression group
(transfection pcDNA3.1-GIF).The transfection of carrier, the instruction of specific transfection method to specifications are carried out using liposome 2000
It carries out.The working concentration of pcDNA3.1 empty carrier and pcDNA3.1-GIF 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 SPSS13.0 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 Fig. 2, transfecting GIF in the cell of pcDNA3.1-GIF compared with the cell of transfection pcDNA3.1 empty carrier
Content significantly raise, difference have statistical significance (P < 0.05).
The influence of embodiment 4GIF gene pairs esophageal squamous cell proliferation
It is influenced using MTT experiment detection GIF gene pairs esophageal squamous cell proliferative capacity.
1, step: pancreatin digests after group of cells transfects 12h, single cell suspension is made, with 6000, every hole cell inoculation
In 96 well culture plates, at 7 time points of every component, each time point sets 6 multiple holes.After cell is adherent, the 1st detection is carried out:
The 20 μ l of MTT liquid of 5g/L is added in every hole, continues after cultivating 4h, sucks culture medium, and 150 μ l of DMSO is added, and carefulness piping and druming makes purple
Blue precipitate sufficiently dissolves, and surveys absorbance value (A value) in 490nm wavelength with microplate reader.Then every 12h is detected 1 time, continuous to survey
72h, totally 7 times.This experiment is repeated 3 times.
2, statistical method
Experiment is completed according to being repeated 3 times, using SPSS13.0 statistical software come for statistical analysis, two groups
Between difference using t examine, it is believed that as P < 0.05 have statistical significance.
3, result
It is shown in Fig. 3 as the result is shown: transfection pcDNA3.1-GIF group vitro growth rates be obviously butted on transfection
The vitro growth rates of pcDNA3.1 empty carrier group, difference have statistical significance (P < 0.05), and the above results show the table of GIF
Up to the growth for being able to suppress esophageal squamous cell.
The influence of embodiment 5GIF gene pairs esophageal squamous cell apoptosis
Use the influence of flow cytomery GIF 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 plasmid 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 SPSS13.0 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-GIF group is (24.39 ± 0.012) %, transfects pcDNA3.1 empty carrier group
Apoptosis rate be (6.73 ± 0.009) %, above-mentioned difference have statistical significance (P < 0.05), the above results show GIF
The overexpression of gene promotes the apoptosis of esophageal squamous cell.
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.
Claims (8)
- Application of the 1.GIF gene in the product of preparation diagnosis esophageal squamous cell carcinoma, which is characterized in that the gene order of the GIF is such as Shown in SEQ ID NO.1.
- 2. application according to claim 1, which is characterized in that the product include: by RT-PCR, real-time quantitative PCR, In situ hybridization or chip detect the expression of GIF gene to diagnose the product of esophageal squamous cell carcinoma.
- 3. application according to claim 2, which is characterized in that the product with RT-PCR diagnosis esophageal squamous cell carcinoma at least wraps Include the primer of a pair of of specific amplified GIF gene；The product with real-time quantitative PCR diagnosis esophageal squamous cell carcinoma includes at least a pair of special The primer of different amplification GIF gene；The product in situ hybridization diagnosis esophageal squamous cell carcinoma includes: the nucleic acid sequence with GIF gene The probe of hybridization；The product with chip diagnosis esophageal squamous cell carcinoma includes genetic chip, and the genetic chip includes and GIF gene Nucleic acid array hybridizing probe.
- 4. application according to claim 1, which is characterized in that the product includes genetic chip or gene detection reagent Box.
- Application of the 5.GIF gene in the pharmaceutical composition of preparation treatment esophageal squamous cell carcinoma, which is characterized in that the GIF gene Sequence is as shown in SEQ ID NO.1.
- 6. application according to claim 5, which is characterized in that described pharmaceutical composition includes increasing GIF gene expression Reagent.
- 7. application according to claim 6, which is characterized in that the reagent includes: containing energy encoding function GIF albumen Nucleic acid reagent.
- 8. application according to claim 6 or 7, which is characterized in that described pharmaceutical composition further includes pharmaceutically acceptable Carrier.
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