CN106244680B - The application of ORC1L gene and its expression product in diagnosis of disease - Google Patents
The application of ORC1L gene and its expression product in diagnosis of disease Download PDFInfo
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Abstract
The invention discloses the application of ORC1L gene and its expression product in diagnosis of disease, detect instruction of the expression of ORC1L genes amplification as esophageal squamous cell carcinoma phenotype.The present invention provides the levels of detection ORC1L gene or its expression product to diagnose esophageal squamous cell carcinoma, while providing the expression by changing ORC1L to improve or treat the illness of patient.
Description
Technical field
The invention belongs to biomedicine fields, are related to the application of ORC1L gene and its expression product in diagnosis of disease, tool
The disease of body is esophageal squamous cell carcinoma.
Background technique
The cancer of the esophagus is one of most common malignant tumour of the mankind.According to WHO, global cancer of the esophagus new cases are within 2012
455,800, dead 400,200, with east Asia, eastern Africa and southern areas disease incidence highest, African west area
Disease incidence is minimum, disease incidence significant difference, and China is Esophageal Cancer area, and the disease incidence of annual China is 21.88/10 ten thousand,
Occupy all kinds of malignant tumours the 5th；The death rate is 15.85/10 ten thousand, occupies the 4th.The cancer of the esophagus is classified as China by the Ministry of Public Health, China
One of big disease of characteristic ten.There are two types of the main histological types of the cancer of the esophagus, and esophageal squamous cell carcinoma and adenocarcinoma of esophagus, esophageal squamous cell carcinoma are also claimed
For esophageal squamous cell carcinoma.The Nosology types of China's oesophagus carcinogenesis are different from western countries, and 90% the above are esophageal squamous cells
Cancer, and apparent Regional Distribution is presented, be concentrated mainly on Taihang Mountain, the Qinling Mountains, Dabie Mountain and Chuan Bei, Fujian Guangdong, northern Suzhou, Xinjiang and
The geographic areas such as Gansu.
Although diagnosis and treatment technology are constantly progressive in recent years, the prognosis of the cancer of the esophagus is not still very ideal, the lifes in 5 years of patient
It deposits rate and is still no more than 14%.And the reason of leading to its poor prognosis, has very much, including the effective generaI investigation means of shortage, when making a definite diagnosis
Stadium is later, misses best operative treatment period, and postoperative or recurrence after radiotherapy lacks specificity and the higher tumour of sensibility
Marker etc..Improving prognosis is critical issue urgently to be resolved in current esophageal carcinoma therapy.And the state of an illness can be early diagnosed by finding
And the new tumor markers of accurate predicted treatment effect are an importances for improving prognosis.
The cancer of the esophagus is a multistage progressive development process, is not only effected by environmental factors, even more
It is related to the reciprocation of multiple genes, multiple accesses.The formation of the cancer of the esophagus is occurred by normal mucous membrane of esophagus epithelial cell
Different degrees of atypical hyperplasia is developing progressively carcinoma in situ, early invasive carcinoma later, canceration finally occurs, in the meantime
Each stepping exhibition is directed to the expression of related gene or function changes.
The lag of cancer of the esophagus early diagnosis at present, 80% is all middle and advanced stage when making a definite diagnosis.Treatment at this time rely primarily on radiotherapy and
Chemicotherapy, 5 years survival rates only have 10% or so after treatment.Therefore, the important molecule for participating in cancer of the esophagus occurrence and development is found, it is right
In improving cancer of the esophagus early diagnosis and finding new therapy target, the prognosis for improving patient with esophageal carcinoma has great importance.
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 molecular marker of esophageal squamous cell carcinoma,
The molecular marker is applied to clinic, and the early discovery early treatment of esophageal squamous cell carcinoma may be implemented.
To achieve the goals above, the present invention adopts the following technical scheme:
The present invention provides application of the reagent of detection ORC1L in the product of preparation diagnosis esophageal squamous cell carcinoma.
Further, the product diagnoses whether patient suffers from esophageal squamous cell carcinoma by detecting the expression of ORC1L gene.
Wherein ORC1L is expressed in patients with esophageal squamous cell carcinoma and is raised.
Further, the product include by RT-PCR, real-time quantitative PCR, immune detection, marking hybridization, in situ hybridization,
The expression of hybridization array, chip or new-generation sequencing detection ORC1L gene and its expression product is to diagnose esophageal squamous cell carcinoma
Product.
The present invention provides a kind of product for diagnosing esophageal squamous cell carcinoma, the product can pass through ORC1L base in detection sample
The expression of cause diagnoses esophageal squamous cell carcinoma.
Wherein, described " sample " not only includes organism sample, such as cell, tissue, internal organs, body fluid (blood, lymph
Deng), digestive juice, expectoration, alveole rami tracheales cleaning solution, urine, feces, also include the nucleic acid extractive obtained by these organism samples
(extracting genome DNA object, mRNA extract, the cDNA prepared product prepared by mRNA extract or cRNA prepared product etc.) or albumen
Matter extract.Alternatively, it is also possible to implement formalin fixing process, alcohol fixing process to said sample, freeze processing or paraffin
Embedding treatment.The preferred sample is tissue.
Further, the product includes chip or kit；Wherein, the chip includes genetic chip, protein core
Piece；The genetic chip includes solid phase carrier and the oligonucleotide probe for being fixed on solid phase carrier, the oligonucleotide probe
Including the oligonucleotide probe for ORC1L gene for detecting ORC1L gene transcription level；The protein-chip includes
Solid phase carrier and be fixed on solid phase carrier ORC1L albumen specific antibody；The kit includes gene detection reagent
Box and protein immunization detection kit；The gene detecting kit includes the reagent for detecting ORC1L gene transcription level；
The protein immunization detection kit includes the specific antibody of ORC1L albumen.
Further, the reagent includes the primer and/or probe for ORC1L gene.
Genetic chip or gene detecting kit of the present invention can be used for detecting multiple including ORC1L gene
The expression of gene (for example, multiple genes relevant to esophageal squamous cell carcinoma).The protein chip or protein immunization detection reagent
Box can be used for detecting the table of multiple protein (such as multiple protein relevant to esophageal squamous cell carcinoma) including ORC1L albumen
Up to level.Multiple markers of esophageal squamous cell carcinoma are detected simultaneously, are 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 ORC1L gene and its expression product
With.
Further, described pharmaceutical composition includes ORC1L gene and/or the inhibitor of its expression product.The inhibitor
Including inhibiting the substance of ORC1L gene expression, inhibiting the substance of ORC1L gene expression product stability, and/or inhibiting ORC1L
The active substance of gene expression product.
Further, the inhibitor is the antibody for the siRNA of ORC1L gene and/or for ORC1L albumen.
In a specific embodiment of the invention, the inhibitor is directed to the siRNA of ORC1L gene.In order to further mention
The validity of high siRNA segment, siRNA segment of the comprehensive design for screening.Finally, passing through sequence analysis (NCBI
BLAST siRNA sequence) is filtered, to improve the specificity of siRNA segment and reduce the undershooting-effect of RNAi interference.Preferably,
The sequence of the siRNA is as shown in SEQ ID NO.9 and SEQ ID NO.10.
The present invention also provides a kind of pharmaceutical composition for treating esophageal squamous cell carcinoma, described pharmaceutical composition includes ORC1L base
Cause and/or its expression product inhibitor.The inhibitor includes the substance for inhibiting ORC1L gene expression, inhibits ORC1L gene table
Up to the substance, and/or the inhibition active substance of ORC1L gene expression product of product stability.
Further, above-mentioned pharmaceutical composition further includes pharmaceutically acceptable carrier, and this kind of carrier includes (but and unlimited
In): diluent, excipient such as lactose, sodium chloride, glucose, urea, starch, water etc., filler such as starch, sucrose etc.；Bonding
Agent such as simple syrup, glucose solution, starch solution, cellulose derivative, alginates, gelatin and polyvinylpyrrolidone；It is wet
Agent such as glycerol；Disintegrating agent such as dried starch, sodium alginate, laminarin powder, agar powder, calcium carbonate and sodium bicarbonate；It absorbs
Promotor quaternary ammonium compound, lauryl sodium sulfate etc.；Surfactant such as polyoxyethylene sorbitan aliphatic ester, 12
Sodium alkyl sulfate, 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 talcum powder, calcium stearate and magnesium, polyethylene glycol, boric acid powder etc..
Pharmaceutical composition of the invention can be used different additives and be prepared, such as buffer, stabilizer, antibacterial
Agent, isotonic agent, chelating agent, pH controlling agent and surfactant.
Buffer may include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid and corresponding salt (their alkali metal or alkali
Property rare earth metal salt, such as sodium salt, sylvite, calcium salt and magnesium salts).Isotonic agent includes potassium chloride, sodium chloride, sugar and glycerol.Chelating
Agent includes sodium ethylene diamine tetracetate and citric acid.Bacteriostatic agent include but is not limited to effective concentration (such as < 1%w/v) benzylalcohol,
Phenol, metacresol, methaform, methyl p-hydroxybenzoate and/or propylparaben.Stabilizer includes human serum egg
White, l-amino acid, sugar and cellulose derivative.L-amino acid can also include appointing in glycine, cysteine and glutamic acid
Meaning one.Carbohydrate includes monosaccharide, such as glucose, mannose, galactolipin, fructose etc.；Sugar alcohol, such as mannitol, inositol, wood
Sugar alcohol etc.；Disaccharides, such as sucrose, maltose, lactose etc.；Polysaccharide, for example, glucan, hydroxypropul starch, vulcanization chondroitin, thoroughly
Bright matter acid etc. and their derivative.Cellulose derivative includes methylcellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxyl
Propyl cellulose, hypromellose and sodium cellulose glycolate.Surfactant includes ion or non-ionic surface active
Agent, such as polyethylene glycol oxide Arrcostab, sorbitan monoacyl ester, fatty glyceride.
Drug of the invention may also include pharmaceutically acceptable coating material, fast decoupled coating
Material, coloring agent, enteric polymer, plasticizer, water-soluble polymer, insoluble polymer, dyestuff, pigment, other collapse
Powder.Common fast decoupled coating material includes OPADRY；Enteric polymer includes methylacrylic acid polymer, phosphorus hydroxypropyl first
Base cellulose phthalate, hypromellose acetic acid esters, hypromellose succinate, hydroxyl first ethyl cellulose
Element, cellulose acetophthalate；Plasticizer includes polyethylene glycol (PEG), propylene glycol etc..
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 ORC1L albumen, by improve ORC1L albumen expression or enhance ORC1L albumen function, thus treat because
Esophageal squamous cell carcinoma caused by ORC1L albumen is reduced.
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.
The carrier that the present invention carries gene is various carriers known in the art, and such as commercially available carrier including plasmid glue
Grain, bacteriophage, virus 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 ORC1L albumen includes that monoclonal antibody, polyclonal antibody, polyspecific are anti-
Body (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.
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, ORC1L gene expression product includes the portion of people ORC1L albumen and ORC1L albumen
Divide peptide.The partial peptide of the ORC1L albumen contains functional domain relevant to esophageal squamous cell carninomatosis.
" ORC1L albumen " includes any functional equivalent of ORC1L albumen and ORC1L albumen.The functional equivalent
Including ORC1L albumen conservative variation protein or its active fragment or its reactive derivative or its mutant.Mutant packet
It includes allelic variant, natural mutation, induced mutants, its amino acid sequence and passes through missing, substitution, increase and/or insertion hair
Change different mutant, can be with the encoded protein of DNA of the DNA hybridization of people ORC1L under high or low stringent condition.
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 protein of ORC1L albumen.
For the peptide or protein with ORC1L protein fusion, there is no limit as long as resulting fusion protein retains the life of ORC1L albumen
Object activity.
In the present invention, " host cell " includes prokaryotic cell and eukaryocyte.The example of common prokaryotic host cell
Including Escherichia coli, hay bacillus etc..Common eukaryotic host cell includes that yeast cells, insect cell and mammal are thin
Born of the same parents.Preferably, the host cell is eukaryocyte, such as Chinese hamster ovary celI, COS cell.
In the present invention, term " treatment ", which refers to, including but not limited to cures, slow down pathological condition that (reduction) targets or
Illness prevents from recurring.Including but not limited to, (1) inhibiting effect, inhibits progression of disease to a certain extent comprising slow down with
And complete inhibition；(2) quantity of seizure of disease and/or symptom is reduced；(3) focal size is reduced；(4) inhibit (reduce, slow down
Or prevent completely) disease cells penetrate into adjacent peripheral organs and/or tissue；(5) inhibit (to reduce, slow down or hinder completely
Only) transmission；(6) alleviate one or more symptoms associated with disease to a certain extent；(7) increase after treating disease-free
The time span of performance；(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:
Present invention firstly discovers that ORC1L gene differential expression in patients with esophageal squamous cell carcinoma, ORC1L is in patients with esophageal squamous cell carcinoma
Middle high expression provides a kind of new molecular marker for the early detection of esophageal squamous cell carcinoma.
Treat accurate medical procedure the present invention provides a kind of, by target ORC1L treat esophageal squamous cell carcinoma, delay or
The illness for curing patient, provides the life quality of patient.
Detailed description of the invention
Fig. 1 shows the expression using QPCR detection ORC1L gene in esophageal squamous cell carcinoma tissue；
Fig. 2 shows the expression using QPCR detection ORC1L gene in esophageal cells；
Fig. 3 shows the influence using QPCR detection siRNA to ORC1L gene expression；
Fig. 4 shows that soft-agar cloning forms the influence of experiment detection ORC1L expression cell proliferation；
Fig. 5 shows the influence using the cell transwell detection ORC1L 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 ORC1L gene in esophageal squamous cell carcinoma tissue is significantly raised.
The differential expression of 2 QPCR sequence verification ORC1L gene of embodiment
1, large sample QPCR verifying is carried out to ORC1L gene differential expression.It is selected according to the sample collection mode in embodiment 1
Select normal esophageal tissue and esophageal squamous cell carcinoma tissue each 50.
2, RNA extraction step is as described in Example 1.
3, reverse transcription:
1) reaction system:
| Reagent | Volume |
| MgCl2 | 2μl |
| 10×RT Buffer | 1μl |
| Without Rnase water | 3.75μl |
| DNTP mixed liquor | 1μl |
| Rnase inhibitor | 0.25μl |
| AMV reverse transcriptase | 0.5μl |
| Oligomerization dT aptamer primer | 0.5μl |
| Laboratory sample | 1μl |
2) reverse transcription reaction condition
It is carried out according to reverse transcription reaction condition in RNAPCR Kit (AMV) Ver.3.0.
42 DEG C~55 DEG C 60min, 99 DEG C of 2min, 5 DEG C of 5min.
3) polymerase chain reaction
1) design of primers
QPCR amplimer is designed according to the coded sequence of ORC1L gene and GAPDH gene in Genebank, by Bo Maide
Biotech firm's synthesis.Specific primer sequence is as follows:
ORC1L gene:
Forward primer is 5 '-TGCCATCCGTGATTCTGA-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-GGAGTAGAGGTCGCTTCAT-3 ' (SEQ ID NO.4).
The primer sequence of house-keeping gene GAPDH are as follows:
Forward primer: 5 '-CTCTGGTAAAGTGGATATTGT-3 ' (SEQ ID NO.5)
Reverse primer: 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.6)
2) PCR reaction system is prepared according to table 1:
1 PCR reaction system of table
| Reagent | Volume |
| Forward primer | 1μl |
| Reverse primer | 1μl |
| Takara Ex Taq HS | 12.5μl |
| Template | 2μl |
| Deionized water | Supply 25 μ l |
3) PCR reaction condition: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 45s) × 30 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 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.
6, result
As a result as shown in Figure 1, compared with surrounding normal mucous membrane of esophagus tissue, ORC1L gene table in esophageal squamous cell carcinoma tissue
Up to up-regulation, difference has statistical significance (P < 0.05), consistent with RNA-sep result.
Differential expression of the 3 ORC1L gene of embodiment in esophageal carcinoma cell line
1, cell culture
Human esophageal squamous cell cancer cell strain KYSE150, 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 cell total 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, ORC1L gene esophageal squamous cell KYSE150,
It expresses in KYSE450 and raises, difference has statistical significance (P < 0.05), consistent with RNA-sep result.
The silencing of 4 ORC1L 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, siRNA is designed
For the siRNA sequence of ORC1L gene:
Negative control siRNA sequence (siRNA-NC):
Positive-sense strand is 5 '-UUCUCCGAACGUGUCACGU-3 ' (SEQ ID NO.7),
Antisense strand is 5 '-ACGUGACACGUUCGGAGAA-3 ' (SEQ ID NO.8)；
SiRNA1-ORC1L:
Positive-sense strand is 5 '-AAGCAAUUUAGCAACAUACGG-3 ' (SEQ ID NO.9),
Antisense strand is 5 '-GUAUGUUGCUAAAUUGCUUGA-3 ' (SEQ ID NO.10)；
SiRNA2-ORC1L:
Positive-sense strand is 5 '-UUUCGAAGCUGCAAUUCGGGU-3 ' (SEQ ID NO.11),
Antisense strand is 5 '-CCGAAUUGCAGCUUCGAAAAC-3 ' (SEQ ID NO.12)；
Cell is pressed 5 × 105/ hole is inoculated into six porocyte culture plates, in 37 DEG C, 5%CO2Cell culture in incubator
For 24 hours, in DMEM culture medium without double antibody, containing 10%FBS, transfection (is purchased from according to lipofectamine 2000
Invitrogen company) specification transfection, experiment is divided into control group (KYSE 150) negative control group (siRNA-NC) and real
Test group (20nM) (siRNA1-ORC1L, siRNA2-ORC1L), wherein the sequence of negative control group siRNA and ORC1L gene without
Homology, concentration is the hole 20nM/, while being transfected respectively.
3, QPCR detects the transcriptional level of ORC1L gene
3.1 the extraction of cell total rna
Specific steps are the same as embodiment 3.
3.2 reverse transcription steps are the same as embodiment 2.
3.3 QPCR amplification steps are the same as embodiment 2.
4, statistical method
Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD,
Using SPSS18.0 statistical software come for statistical analysis, the difference between ORC1L gene expression panel and control group is interfered to adopt
It is examined with t, it is believed that there is statistical significance as P < 0.05.
5, result
As a result such as Fig. 3 is shown, compared to KYSE 150, transfection zero load siRNA-NC, siRNA2-ORC1L group, siRNA1-
ORC1L group can significantly reduce the expression of ORC1L gene, and difference has statistical significance (P < 0.05).
The influence of 5 ORC1L gene pairs esophageal squamous cell apoptosis of embodiment
Use the influence of flow cytomery ORC1L 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 siRNA1-ORC1L group is (17.35 ± 0.016) %, transfects siRNA-NC empty carrier group
Apoptosis rate be (5.83 ± 0.013) %, above-mentioned difference have statistical significance (P < 0.05), the above results show
The overexpression of ORC1L gene inhibits the apoptosis of esophageal squamous cell.
6 soft-agar cloning of embodiment forms experiment
1, with the cell for being in logarithmic growth phase after the transfection of 0.25% trypsin digestion, gently piping and druming makes slender
Born of the same parents' suspension, is collected by centrifugation cell precipitation.
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 CO as bottom-layer agar2It is spare in 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, siRNA1-ORC1L group single cell clone Colony forming digital display writes drop
It is low.
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.
10, result
As a result as shown in figure 5, KYSE150, siRNA1-ORC1L, siRNA-NC group cell are trained in the cell transwell
After supporting for 24 hours, the cell number in room face is significantly reduced under siRNA1-ORC1L 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.
Claims (8)
1. detecting application of the reagent of ORC1L in the product of preparation diagnosis esophageal squamous cell carcinoma.
2. application according to claim 1, which is characterized in that the expression that the product passes through detection ORC1L gene
To diagnose whether patient suffers from esophageal squamous cell carcinoma.
3. application according to claim 2, which is characterized in that the product include by RT-PCR, real-time quantitative PCR,
Immune detection, marking hybridization, in situ hybridization, hybridization array, chip or new-generation sequencing detection ORC1L gene and its expression product
Expression to diagnose the product of esophageal squamous cell carcinoma.
4. application according to claim 3, which is characterized in that the product includes chip or kit；Wherein, described
Chip includes genetic chip, protein-chip；The kit includes gene detecting kit, protein immunization detection kit.
The application of 5.ORC1L gene and its expression product in the pharmaceutical composition of preparation treatment esophageal squamous cell carcinoma.
6. application according to claim 5, which is characterized in that described pharmaceutical composition includes ORC1L gene and/or its table
Up to the inhibitor of product.
7. application according to claim 6, which is characterized in that the inhibitor includes the object for inhibiting ORC1L gene expression
Matter, the substance for inhibiting ORC1L gene expression product stability, and/or the inhibition active substance of ORC1L gene expression product.
8. application according to claim 6 or 7, which is characterized in that the inhibitor be for ORC1L siRNA and/or
Antibody.
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Non-Patent Citations (3)
| Title |
|---|
| Butyrate Induced Cell Cycle Arrest in Bovine Cells through Targeting Gene Expression Relevant to DNA Replication Apparatus;Cong-jun Li等;《Gene Regulation and Systems Biology》;20081231;第2卷;113–123 |
| NCBI Reference Sequence: NM_004153.3;NCBI;《GenBank》;20151115;核苷酸和氨基酸序列表 |
| Orc1 Binding to Mitotic Chromosomes Precedes Spatial Patterning during G1 Phase and Assembly of the Origin Recognition Complex in Human Cells;Nihan Kara等;《THE JOURNAL OF BIOLOGICAL CHEMISTRY》;20150508;第290卷(第19期);12355–12369 |
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