CN106011286B - Hypopharyngeal cancer diagnosis and treatment marker and its application - Google Patents
Hypopharyngeal cancer diagnosis and treatment marker and its application Download PDFInfo
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Abstract
The invention discloses the molecular markers that SCLY gene can be used as hypopharyngeal cancer diagnosis.The present invention is using genetic chip and QPCR experiment discovery SCLY gene and its encodes expression of the albumen in normal control tissue and hypopharynx cancerous tissue there are significant differences, it can judges whether subject suffers from hypopharyngeal cancer or with the risk for suffering from hypopharyngeal cancer by SCLY expression conditions in detection hypopharynx tissue.According to the correlation of the two, the present invention develops a kind of kit for diagnosing hypopharyngeal cancer, which carries out the diagnosis of hypopharyngeal cancer by the expression of detection SCLY gene.Diagnostic kit of the invention can be used for the early diagnosis of disease, clinically be with a wide range of applications.In addition, providing the foundation the invention also discloses the molecular target that SCLY gene can be used as treatment hypopharyngeal cancer to carry out the development of hypopharyngeal cancer therapeutic agent.
Description
Technical field
The present invention relates to diagnosing tumor, treatment, prediction prognosis fields, more particularly it relates to different to detect SCLY
It is often the diagnosing tumor of means, prediction method of prognosis；And the tumor therapeutic agent of activation SCLY gene or protein.
Background technique
Hypopharyngeal squamous cell carcinoma is one of most common malignant tumour of ENT & HN Surgery Dept., has generation position hidden
It covers, wellability is extremely strong, diffusion, primary affection are in the characteristics of multicenter is grown under easy mucous membrane.Because early stage is without apparent symptom, shortage
Specific sign and Ke Kao Miasma break measure, so, most of patients already belongs to advanced stage, disease in clinical definite and when receiving treatment
Become and often involves the organs such as cavum laryngis, cervical part of esophagus esophagus and the root of the tongue；Simultaneously as laryngopharynx portion lymphatic vessel is abundant, lymphonodi cervicales easily occurs
Transfer and coating external diffusion, and all a side gate of an imperial palace histoorgans, such as the blood vessel that arteria carotis is important are invaded, so, pharynx squamous carcinoma is incidence
Worst one of the malignant tumour of prognosis.Due to the particularity of hypopharyngeal cancer growth site, after operative treatment, it may cause language, exhale
The dysfunction for inhaling and swallowing organ seriously affects the life quality of patient；And radiation and chemotherapy is not thorough, simultaneously in the presence for the treatment of
Send out the problems such as disease is more, patient suffering is larger.Therefore, new diagnostic method and simplicity are proposed on the basis of molecular biology as early as possible
Effectively, the small treatment means of toxic side effect just become the project that need be solved.
Summary of the invention
It is diagnosed and is swallowed by detection SCLY gene or protein expression difference one of the objects of the present invention is to provide one kind
The method of cancer.
The second object of the present invention is to provide one kind by detection SCLY gene or protein expression difference to predict to swallow
The method of cancer prognosis.
The third object of the present invention, which is to provide, a kind of treats hypopharyngeal cancer by activation SCLY gene or SCLY albumen
Method.
The fourth object of the present invention is to provide a kind of method for screening the drug for the treatment of hypopharyngeal cancer.
The fifth object of the present invention is to provide a kind of for treating the drug of hypopharyngeal cancer.
To achieve the goals above, present invention employs following technical solutions:
The present invention provides the products of detection SCLY gene or SCLY albumen to prepare the purposes in hypopharyngeal cancer diagnostic tool.
The present invention also provides the products of detection SCLY gene or SCLY albumen in preparation prediction hypopharyngeal cancer prognostic tool
Purposes.
Further, the product of the detection SCLY gene or SCLY albumen includes the table for detecting SCLY gene or SCLY albumen
Up to horizontal product.The product includes the nucleic acid that can combine SCLY gene or the substance (example that can combine SCLY albumen
Such as antibody).The nucleic acid is able to detect the expression of SCLY gene；The substance is able to detect the expression water of SCLY albumen
It is flat.
The product of detection SCLY gene of the invention can play its function based on the known method of nucleic acid molecules is used: such as
PCR, such as Southern hybridization, Northern hybridization, dot blot, fluorescence in situ hybridization (FISH), DNA microarray, ASO method, height
Flux microarray dataset etc..It can qualitatively, quantitatively or semi-quantitatively implement analysis using the product.
Include that nucleic acid in the said goods can be obtained by chemical synthesis, or by containing from biomaterial preparation
It is expected that the gene of nucleic acid, then using primer amplification designed for amplification expectation nucleic acid, it is obtained.
Further, the PCR method is known method, for example, ARMS (Amplification Refractory
Abruptly-changing system is not answered in Mutation System, amplification) method, RT-PCR (reverse transcriptase-PCR) method, nesting PCR method etc..Amplification
Nucleic acid can be by using dot blotting hybridization method, Surface Plasmon Resonance (SPR method), PCR-RFLP method, original position RT-PCR
Method, PCR-SSO (sequence specific oligonucleotide) method, PCR-SSP method, AMPFLP (amplifiable fragment length polymorphism) method,
MVR-PCR method and PCR-SSCP (single-strand conformation polymorphism) method detect.
Nucleic acid recited above includes the primer for expanding SCLY gene, and the primer for including in product can be by passing through chemistry
Synthesis to prepare, by using those skilled in the art will know that method be suitably designed with reference to Given information, and passing through
Synthesis is learned to prepare.
In specific embodiments of the present invention, the nucleic acid is amplimer used in QPCR experiment, the primer
Sequence such as SEQ ID NO.1 (positive sequence) and SEQ ID NO.2 (reverse sequence) shown in.
Nucleic acid recited above may also include probe, and the probe can be prepared by chemical synthesis, by using this
The method that field technical staff knows appropriately is designed with reference to Given information, and is prepared by chemical synthesis, or can lead to
The gene for containing desired nucleic acid sequence from biomaterial preparation is crossed, and is expanded using the primer designed for amplification expectation nucleic acid sequence
Increase it to prepare.
The product of detection SCLY albumen of the invention can play its function based on the known method of antibody is used: for example,
It may include ELISA, radioimmunoassay, immunohistochemical method, western blot etc..
The product of detection SCLY albumen of the invention includes the antibody or its segment for specifically binding SCLY albumen.It can make
With the antibody or its segment of any structure and size, immunoglobulin class, origin etc., as long as it combines target protein.This
The antibody or its segment for including in the testing product of invention can be monoclonal or polyclonal.Antibody fragment refers to reservation antibody
Peptide to the active antibody of the combination of antigen a part of (Partial Fragment) or containing antibody a part.Antibody fragment may include F
(ab′)2, Fab ', Fab, scFv (scFv), the Fv (dsFv) of disulphide bonding or its polymer, the area dimerization V it is (dual anti-
Body) or peptide containing CDR.The product of detection SCLY albumen of the invention may include encoding antibody or Encoding Antibody Fragment
The isolated nucleic acid of amino acid sequence, the carrier comprising the nucleic acid, and carry the cell of the carrier.
Antibody can be obtained by the way that well known to a person skilled in the art methods.For example, preparation retains target all or in part
The mammalian cell expression vector of their polynucleotides of the polypeptide or integration coding of protein is as antigen.Exempted from using antigen
After epidemic disease animal, from the immune animal adaptive immune cell of process and myeloma cell is merged to obtain hybridoma.Then from hybridization
Tumor culture collects antibody.It can finally be implemented by using antibody of the SCLY albumen for being used as antigen or part thereof to acquisition
Antigentic specificity purifies to obtain the monoclonal antibody for SCLY albumen.Polyclonal antibody can be prepared as follows: with it is above
Identical antigen-immunized animal collects blood sample from by immune animal, serum is isolated from blood, then using upper
It states antigen and antigentic specificity purifying is implemented to serum.It can be by the antibody that is obtained with enzymatic treatment or by using the antibody of acquisition
Sequence information obtain antibody fragment.
The combination of marker and antibody or its segment can be implemented by method as commonly known in the art.For example, can
With following fluorescent marker protein or peptide: clean protein or peptide with phosphate buffer, addition DMSO, buffer, etc. standards
Standby dyestuff, then mixed solution, then at being placed at room temperature for 10 minutes.In addition, the labelling kit of commercialization can be used in label, it is all
Such as biotin labeling reagent box, such as biotin labeling reagent box-NH2, biotin labeling reagent box-SH
(DojindoLaboratories)；Alkali phosphatase enzyme mark kit such as alkali phosphatase enzyme mark kit-NH2, alkaline phosphorus
Sour enzyme labelling kit-SH (Dojindo Laboratories)；Peroxidase labelling kit such as peroxidase mark
Remember kit-NH2, peroxidase labelling kit-NH2 (Dojindo Laboratories)；Phycobniliprotein labelled reagent
Box such as phycobniliprotein labelling kit-NH2, phycobniliprotein labelling kit-SH, B- phycoerythrin labelling kit-NH2,
B- phycoerythrin labelling kit-SH, R-PE labelling kit-NH2, R-PE labelling kit SH
(DojindoLaboratories)；Fluorescent labeling reagent box such as fluorescein labelling kit-NH2, HiLyte Fluor (TM)
555 labelling kit-NH2,647 labelling kit-NH2 of HiLyte Fluor (TM) (Dojindo Laboratories)；And
DyLight 547 and DyLight647 (Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody
Labelling kit, Qdot (TM) antibody labeling kit (Invitrogen Corporation) and EZ- marker protein mark
Remember kit (Funakoshi Corporation).For correct labeling, suitable instrument can be used to detect by label
Antibody or its segment.
As the sample according to testing product of the invention, the tissue sample for example obtained from biopsy subject can be used
Or fluid.Sample is not particularly limited, as long as it is suitable for measurement of the invention；For example, it may include tissue, blood, blood plasma,
Serum, lymph, urine, serous cavity liquid, spinal fluid, synovia, aqueous humor, tear, saliva or its fraction or treated material
Material.
In specific embodiments of the present invention, tissue of the sample from subject.
In the present invention, " prognosis " refers to mistake of the tumor patient after inhibiting by surgical procedure etc. or alleviating tumour growth
Journey or result.In the present specification, prognosis can be by surgical procedure inhibit or alleviate tumour growth after 1,2,3,4,5,6,
7,8,9,10,15,20 years or more long when life state.Prognosis can be by checking biomarker, that is, SCLY albumen or coding
The gene of SCLY albumen is predicted.Prognosis prediction can be performed such that according to biomarker with or without or increasing or drop
It is low, determine that the prognosis of patient is good or bad, or determine the probability of good prognosis or poor prognosis.
In the present invention, " prognosis bona " refer to inhibit or alleviate for patient by surgical procedure etc. tumour growth it
Afterwards, patient's long-term (such as 3,5,6,7,8,9,10,15,20 years or longer) does not have critical condition.Alternatively, good prognosis can anticipate
Refer to and survives in such long-time, sent out again without transfer, without recurrence or nothing.For example, prognosis bona can mean at least 3 years or outstanding
It is to survive at least 5 years, preferably without transfer or recurrence.The most preferred state of prognosis bona is survival for a long time without disease.Such as
Used herein, " prognosis bona " can also include any such state, wherein it can be found that disease such as shifts, still
It is pernicious low and do not severely impact survival ability.
In the present invention, " prognosis mala " refers to that patient is short after inhibiting or alleviating tumour growth by surgical procedure etc.
Fatal condition occurs in period (such as 1,2,3,4,5 year or shorter).Alternatively, poor prognosis refers in such short-term extremely
It dies, shift, recur or sends out again.For example, poor prognosis can mean Preventive or dead at least 3 years or especially at least 5 years
It dies.
Prediction prognosis is referred to the process of prediction status of patient or as a result, is not meant to be predicted with 100% accuracy
The process or result of status of patient.Prediction prognosis refers to whether a possibility that determining certain processes or result increases, and simultaneously unexpectedly
Taste by determining a possibility that certain processes or result occurs or not compared with certain processes or result.Such as this
For invention, in the present invention in the horizontal patient reduced of SCLY gene or SCLY albumen, with the patient's phase for not showing this feature
Than more likely observing particular procedure or result.
Further, the product of the detection SCLY gene or SCLY albumen can be detection SCLY gene or SCLY albumen
Reagent is also possible to include kit, chip, test paper of the reagent etc., is also possible to measure using the high pass of the reagent
Sequence platform.
The present invention also provides a kind of tool for diagnosing hypopharyngeal cancer, the tool is able to detect SCLY gene or SCLY albumen
Expression.The tool include can in conjunction with SCLY gene nucleic acid or can in conjunction with SCLY albumen substance (such as
Antibody).The nucleic acid is able to detect the expression of SCLY gene；The substance is able to detect the expression of SCLY albumen.
Further, the property of the nucleic acid and the substance is the same as noted earlier.
Further, the tool of the diagnosis hypopharyngeal cancer includes but is not limited to chip, kit, test paper or high-flux sequence
Platform；High-flux sequence platform is a kind of tool of special diagnosis hypopharyngeal cancer, with the development of high throughput sequencing technologies, to one
The building of personal gene expression profile will become very easily work.By the gene table for comparing Disease and normal population
Up to spectrum, the exception for being easy to analyze which gene is related to disease.Therefore, the exception of SCLY gene is known in high-flux sequence
The purposes for also belonging to SCLY gene related to hypopharyngeal cancer, equally within protection scope of the present invention.
The present invention also provides a kind of tools for predicting hypopharyngeal cancer prognosis, and the prediction hypopharyngeal cancer prognostic tool includes can
In conjunction with SCLY gene nucleic acid or can in conjunction with SCLY albumen substance (such as antibody).The nucleic acid is able to detect SCLY base
The mRNA level in-site of cause；The substance is able to detect the expression of SCLY albumen.
Further, the property of the nucleic acid and the substance is the same as noted earlier.
Further, the tool of the prediction hypopharyngeal cancer prognosis includes but is not limited to chip, kit, test paper or high throughput
Microarray dataset；High-flux sequence platform is a kind of tool of special diagnosis hypopharyngeal cancer, with the development of high throughput sequencing technologies,
Very easily work will be become to the building of the gene expression profile of a people.By the base for comparing Disease and normal population
Because of express spectra, the exception for being easy to analyze which gene is related to disease.Therefore, SCLY gene is known in high-flux sequence
The abnormal purposes for also belonging to SCLY gene related to hypopharyngeal cancer, equally within protection scope of the present invention.
The number for the amino acid that anti-SCLY antibody used in testing product of the invention, diagnostic tool or its segment are identified
Mesh is not particularly limited, as long as antibody can combine SCLY.
The present invention also provides a kind of diagnosis hypopharyngeal cancer or the methods for predicting hypopharyngeal cancer prognosis, and the method includes walking as follows
It is rapid:
(1) sample of subject is obtained；
(2) expression of SCLY gene or albumen in Samples subjects is detected；
(3) it associates whether by the expression of the SCLY gene or albumen that measure with the illness of subject.
(4) compared with the control, the expression of SCLY gene or albumen reduces, then the subject is diagnosed as hypopharyngeal cancer,
Or the subject is confirmed as prognosis mala.
The present invention also provides a kind for the treatment of methods of hypopharyngeal cancer, and the method includes activation SCLY gene or SCLY eggs
It is white.
Further, the method includes promoting the expression of SCLY gene, or expression or the Enhanced SC LY of promotion SCLY albumen
The activity of albumen.
The present invention also provides a kind of screening techniques of tumour medicine, can be by after adding testing drug to cancer cell
Or the expression of some period measurement SCLY gene or SCLY albumen after applying testing drug to tumor model animal
Improve the effect of tumor prognosis to measure tumour medicine.More specifically, when SCLY gene or the expression of SCLY albumen
When increasing after adding or applying testing drug or when restoring normal level, the drug may be selected as improvement tumor prognosis
Therapeutic agent.
The present invention also provides a kind of drugs of activator containing SCLY gene or SCLY albumen.
The present invention also provides application of the above-mentioned activator in the drug of preparation treatment hypopharyngeal cancer.
The activator of SCLY gene or SCLY albumen of the invention is unrestricted, as long as can promote or Enhanced SC LY
Or it is related to the expression or activity of the substance of the upstream SCLY or downstream pathway, and for treating the effective drug of tumour.
Further, the activator include SCLY gene, SCLY albumen, promoted type miRNA, promoted type transcriptional control because
Son or promoted type target small molecule compound.
The activator further includes carrier or host cell comprising carrying SCLY gene.
On the one hand activator of the invention can be used for supplementing the missing or deficiency of endogenic SCLY albumen, pass through raising
The expression of SCLY albumen, thus hypopharyngeal cancer caused by treating because of SCLY hypoproteinosis.On the other hand it can be used for Enhanced SC LY egg
White activity, to treat hypopharyngeal cancer.
Drug of the invention can be used as medicine and be administered alone or apply together with other medicines.It can be with medicine of the invention
The other medicines that object is applied together are unrestricted, as long as it does not damage therapeutic or preventive medicine effect of the invention i.e.
It can, it is preferred that the drug for treating or preventing tumour may include such as alkylating agent, such as ifosfamide, ring phosphinylidyne
Amine, Dacarbazine, Temozolomide, Nimustine, busulfan, procarbazine, melphalan and Ranimustine；Antimetabolite, such as
Enocitabine, capecitabine, Carmofur, Cladribine, gemcitabine, cytarabine, cytarabine octadecyl phosphate
(cytarabine ocfosfate), Tegafur, tegafur-Uracil, Tegafur gimeracil oteracil potassium, deoxidation fluorine urine
Glycosides, hydroxycarbamide, fluorouracil, fludarabine, pemetrexed, Pentostatin, mercaptopurine and methotrexate (MTX)；Plant alkaloid, it is all
As Irinotecan, Etoposide, Sobuzoxane, docetaxel, nogitecan, Palmer altruism, vinorelbine, eldisine and
Vincaleukoblastinum；Antitumor antibiotic, such as actinomycin D, Aclarubicin, Amrubicin, idarubicin, epirubicin, Zinostatin
Stimalamer, daunorubicin, Doxorubicin, pirarubicin, bleomycin, Peplomycin, mitomycin C and mitoxantrone；
Drug based on platinum, such as oxaliplatin, carboplatin, cis-platinum and Nedaplatin；Hormonal medicaments, such as Anastrozole, Exemestane,
Estramustine, ethinyloestradiol, chlormadinone, Goserelin, tamoxifen, dexamethasone, Toremifene, Bicalutamide, Flutamide,
Prednisolone, Fosfestrol, mitotane, methyltestosterone, Medroxyprogesterone, Mepitiostane, Leuprorelin and Letrozole；Biological respinse modification
Agent, such as interferon-' alpha ', interferon beta, interferon gamma, interleukin, ubenimex, dry BCG and lentinan；With molecular targeted medicine
Object, such as Imatinib (imatinib), Gefitinib (gefitinib), gemtuzumab, ozogamicin, Tamibarotene, song
Appropriate monoclonal antibody, Tretinoin, bortezomib (bortezomib) and Rituximab etc..
Drug of the invention can be prepared into various dosage forms as needed.Including but not limited to, percutaneous, mucous membrane, nose, buccal,
Tablet, solution, granule, patch, paste, capsule, aerosol or suppository sublingual or orally use.
The administration method of drug of the invention is unrestricted, as long as it can play desired therapeutic effect or preventive effect i.e.
Can, including but not limited to intravenously, in peritonaeum, intraocularly, intra-arterial, intrapulmonary is taken orally, in vesicle, intramuscular, intratracheally, subcutaneously
, local by pleura by skin, sucking, by mucous membrane, skin, stomach is intra-articular, intra-ventricle, rectum, vagina,
In skull, in urethra, in liver, in tumor.In some cases, it can systematically be administered.It is locally to be administered in some cases.
The dosage of drug of the invention is unrestricted, can as long as obtaining desired therapeutic effect or preventive effect
To carry out appropriate determination according to symptom, gender, age etc..Example can be used in the dosage of therapeutic agent or prophylactic agent of the invention
Such as the therapeutic effect of disease or preventive effect are determined as index.
In the context of the present invention, " diagnosis hypopharyngeal cancer " both included judge subject whether suffered from hypopharyngeal cancer or
Including judging that subject whether there is the risk with hypopharyngeal cancer.
" treatment " used herein is covered treatment-related in such as mankind of the mammal with related disease or illness
Disease or morbid state, and include:
(1) prevent disease or morbid state occurs in mammals, especially when the mammal is susceptible in the disease
Diseased state, but when being not yet diagnosed with this morbid state；
(2) inhibit disease or morbid state, that is, prevent its generation；Or
(3) alleviate disease or morbid state, even if disease or morbid state subside.
Term " treatment " is usually directed to treatment mankind or animal (for example, being applied by animal doctor), wherein can reach certain pre-
The therapeutic effect of phase, for example, inhibiting the development (including reduce development speed, stop development) of illness, improving illness and healing
Illness.It further include the treatment as precautionary measures (such as prevention).To not yet development be illness but have development be the illness endanger
The purposes of the patient of danger, is also included in term " treatment ".
The advantages of the present invention:
Of the invention has found a kind of molecular marker for diagnosing hypopharyngeal cancer, can be in hypopharyngeal cancer using the molecular marker
The early stage of generation can be used as judging, provide the survival rate of patient.
In addition, the present invention is capable of providing significant information to determine treatment side for patient by the prognosis of prediction patient
Case strategy.
Of the invention includes that the therapeutic agent of the activator of SCLY gene or albumen can be used as the medicine of new hypopharyngeal cancer
Object.
Detailed description of the invention
Fig. 1 shows the differential expression for studying SCLY gene in mRNA level in-site using QPCR；
Fig. 2 shows the differential expression for detecting SCLY gene on protein level using immunoblotting；
Fig. 3 is shown using QPCR the case where detecting SCLY gene overexpression in mRNA level in-site；
Fig. 4 is shown using immunoblotting the case where detecting SCLY gene overexpression on protein level；
Fig. 5 shows influence of the SCLY gene overexpression to hypopharynx cancer cell multiplication.
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.
1 genetic chip of embodiment screens difference expression gene
1, it draws materials:
The primary Hypopharyngeal Cancer Patients operation excision cancerous tissue of 8 customary same period dissections, separately takes 9 non-hypopharyngeal cancers
The hypopharynx normal mucosa tissue of Disease is as control.All equal verified by postoperative pathology of cancerous tissue are hypopharyngeal cancer.It is all primary
The preoperative non-row Radiotherapy chemotherapy of Hypopharyngeal Cancer Patients, all cases complete clinical data.
2, the acquisition of RNA is organized
Total tissue RNA is extracted using Trizol one-step method.
3, the measurement of RNA purity and concentration
1 μ l of RNA solution, Instrument measuring OD260, OD280 are taken, RNA concentration is OD260 value × extension rate × 40/
1000, OD260/OD280 is calculated, ratio represents RNA solution purity is high in 1.7-2.0, -20 DEG C guarantors few containing impurity such as protein
It deposits.
4, RNA integrity detection
(1) 2 μ l RNA sample row, 1.5% agarose gel electrophoresis (80v, 15min) is taken；
(2) after separating zone, genefinder is dyed, and Zone electophoresis band is observed under blue light；
(3) as 28s/18s about 2:1, illustrate that RNA stablizes without degradation.
5, high-throughput transcript profile sequencing
The positioning of 5.1RNA-seq read
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
TopHat method system default parameter.
The assessment of 5.2 transcript abundances
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)。
The detection of 5.3 difference expression genes
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.
6, result
RNA-Sep the results show that filter out 211 differential expression bases altogether between hypopharynx cancerous tissue and normal control tissue
Cause, wherein expression up-regulation gene 54, expression lower gene 157.
2 large sample of embodiment verifies the difference expression gene filtered out
Based on high throughput transcript profile deep sequencing early period as a result, according to the size of P value, we select SCLY gene
It is verified.
1, sample collection
Hypopharynx cancerous tissue 45, normal control tissue 50 are collected according to the method for embodiment 1.
2, it is verified in mRNA level in-site
2.1 extract tissue RNA
Step is the same as embodiment 1.
2.2 reverse transcription
Reverse transcription uses Primescript 1stStrand cDNA synthesis kit kit, operating procedure are as follows
It carries out:
(1) following reaction liquid is added in microcentrifugal tube, as shown in table 1:
1 reaction liquid of table
| Reagent | Dosage |
| RNA | 2.0μg |
| dNTP | 1.0μl |
| Oligo(dT) | 2.0μl |
| Rnase free dH2O | Add to 10.0 μ l |
(2) 70 DEG C of incubation 5min, are rapidly cooled to 4 DEG C；
Following reaction reagent is added in microcentrifugal tube, reaction system is made:
The preparation of 2 reaction system of table
It gently shakes, after rapid centrifugation, 42 DEG C of reactions 1h, 70 DEG C of 10min terminate reaction, 4 DEG C of coolings, -20 DEG C of preservations.
Using SYBP Premix Ex TapTMII kit is carried out in Eppendorf Real-time PCR analyzer,
Concrete operations are as follows:
(1) following PCR reaction solution is prepared on ice:
The preparation of 3 PCR reaction solution of table
| Reagent | Dosage |
| SYBR | 10.0μl |
| Forward primer | 1.0μl |
| Reverse primer | 1.0μl |
| cDNA | 2.0μl |
| ddH2O | 6.0μl |
| Total amount | 20.0μl |
Primer sequence design is as follows:
SCLY gene:
5'-GTGGACTTCCTTACAATC-3'(SEQ ID NO.1)；
5’-AAATAGCATAGGGTAGAGA-3’(SEQ ID NO.2)
β-actin:
5'-GTGGGGCGCCCCAGGCACCA-3'(SEQ ID NO.3)；
5’-CTCCTTAATGTCACGCACGATTT-3’(SEQ ID NO.4)
(2) machine on executes following programs: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 15s.59 DEG C of annealing 20s, 72 DEG C of extensions
20s, totally 40 recycle.
As a result relative quantification method, formula 2 are used-△△ctIt calculates.Experiment is repeated 3 times.
△ ct=ct (A)-ct (β-actin)
△ △ ct=△ ct (experimental group)-△ ct (control group)
As a result as shown in Figure 1, compared with normal control tissue, under the mRNA level in-site of SCLY gene is obvious in hypopharynx cancerous tissue
Drop, difference have statistical significance (P < 0.05).
3, it is verified on protein level
Each histone is extracted according to RIPA protein lysate kit specification, uses BCA determination of protein concentration kit
Protein concentration in test sample.With the detection SCLY albumen variation of conventional Western-blot method, each group experiment is repeated 3 times,
Using β-actin as internal reference, SCLY protein band absorbance quantitative analysis is done, expression quantity is with SCLY albumen/β-actin absorbance
Ratio represents.
As a result as shown in Fig. 2, compared with normal control tissue, SCLY protein level is significantly reduced in hypopharynx cancerous tissue, poor
It is different that there is statistical significance (P < 0.05).
3 SCLY gene overexpression of embodiment
1, plasmid construction
Amplimer is designed according to the coded sequence of SCLY gene, the design of primer is well known to those skilled in the art.
From at Human fetal spleen cDNA library (clontech company, article No.: 638831) in amplification overall length SCLY gene coded sequence,
Above-mentioned cDNA sequence is inserted into eukaryotic expression vector pcDNA3.1, connects the recombinant vector pcDNA3.1-SCLY of acquisition
For subsequent experimental.
2, the culture and transfection of hypopharynx cancer cell
2.1 cell culture
Hypopharyngeal cancer FADU cell, which uses, contains 10% fetal calf serum (FBS), penicillin 100U/ml, 100 μ g/ml of streptomysin
1640 culture medium of RPMI be placed in 37 DEG C, 5%CO2, cultivate under saturated humidity environment.
2.2 cell transfecting
(1) day before transfection is by 0.5-2*105A tumour cell is suspended in the not antibiotic culture medium of 500 μ l, inoculation
To 24 well culture plates.
(2) transfection same day cell density should reach 80%-90%, prepare following compound A: 1 μ g Plasmid DNA is diluted in nothing
In blood serum medium, mix gently；Compound B: taking 4 μ l Lipofectamine2000 to be diluted in serum free medium, mixes
It is even.
(3) compound A and B are mixed, is mixed gently, is incubated at room temperature.
(4) 100 μ l liposome compounds are added in tumour cell, mix gently up and down, cell is put into 37 DEG C
Containing 5%CO2Incubator is incubated for 5-7 hours.
(5) it is small to continue culture cell 18-24 for the growth medium for adding 1ml to contain 2 times of normal serums and antibiotic concentration
When.
3, the overexpression situation of detection pcDNA3.1-SCLY is tested using QPCR
3.1 extraction cell total rnas are operated using conventional method.
3.2 reverse transcription
Step is the same as embodiment 2.
3.3 QPCR
Step is the same as embodiment 2.
3.4 result
As a result as shown in figure 3, pcDNA3.1-SCLY can be successfully overexpressed, difference has statistical significance (P < 0.05).
3, Western blot experiment detection pcDNA3.1-SCLY is overexpressed situation
Step is the same as embodiment 2.
As a result as shown in figure 4, transfecting SCLY albumen in the cell of pcDNA3.1-SCLY compared with transfecting pcDNA3.1 group
Content obviously increase, difference have statistical significance (P < 0.05).
Measurement of the expression of 4 SCLY gene of embodiment to hypopharyngeal cancer ability of cell proliferation
1, step:
Hypopharynx cancer cell FADU after transfecting 24 hours is inoculated in 96 porocyte culture plates, every hole 2*103A cell/
Hole/200 μ l, cell are grouped as follows:
Experimental group 1 (control group): hypopharyngeal cancer cell transfecting pcDNA3.1；
Experimental group 2: hypopharyngeal cancer cell transfecting pcDNA3.1-SCLY.
By cell in 37 DEG C, 5%CO2After incubator is incubated for 24 hours again, according to Brd U cell proliferation reagent box
The specification of (Chemicon International) measures cell proliferation rate.
2, 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.
3, result
As a result as shown in figure 5, compared to experimental group 1, the cells proliferation slowed down of experimental group 2, difference has statistical significance
(P<0.05).It is above-mentioned the experimental results showed that, SCLY expression can inhibit hypopharynx cancer cell multiplication.
Embodiment 5 detects influence of the SCLY gene expression to cell migration
1, experimental procedure is migrated
(1) by the cell dissociation after transfection for 24 hours, adjustment cell density is 105/ml；
600 μ L import fetal calf serums (FBS) are added in the every hole of (2) 24 porocyte culture plates, and the cell Transwell is placed in 24
Porocyte culture plates aperture, each 100 μ L RPMI, 1640 culture medium cell suspension of the small indoor addition of Transwell；
(3) 24 porocyte culture plates are incubated for for 24 hours, and the cell Transwell is taken out from 24 porocyte culture plates, discards culture
Base, PBS rinse the cell Transwell film portion attached cell；
(4) new 24 porocyte culture plates are taken, methanol/PBS mixed liquor ((1:1) 600 μ L, by Transwell is added in aperture
Cell is put into aperture, so that methanol/PBS mixed liquor is immersed upper chamber from lower room (24 porocyte culture plates aperture), is stored at room temperature
15min；
(5) methanol/PBS mixed liquor in the cell Transwell upper chamber and 24 porocyte culture plates apertures is discarded, lower room is added
The cell Transwell is placed in aperture, methanol is made to immerse upper chamber, room from lower room (24 porocyte culture plates aperture) by 600 μ L methanol
Temperature stands 15min；
(6) methanol is discarded, dries 15-30min at room temperature；
(7) it takes 0.1% crystal violet dye liquor, 600 μ L to instill 24 porocyte culture plates apertures, the cell Transwell is placed in small
Dye 15min in hole；
(8) 0.1% crystal violet dye liquor is discarded, distilled water flushing Transwell cell 15min is dried, seen under microscope
It examines, the different visuals field is taken to take pictures counting, experiment is repeated 3 times.
2, result
Average mobility cell number is respectively under transfection pcDNA3.1 group and the transfection each visual field of pcDNA3.1-SCLY group
(166.94 ± 13.87) are a and (75.43 soil 7.96) is a, and difference has statistical significance (P < 0.05).Above-mentioned experimental result table
It is bright, the migration of hypopharynx cancer cell of SCLY gene expression inhibition.
Embodiment 6 detects influence of the SCLY gene expression to cell invasion
1, Matrigel step
Matrigel is taken out from -20 DEG C of refrigerators before experimentTMMatrigel melts on ice chest, takes MatrigelTMMatrigel with
1640 culture medium of RPMI is mixed in 1:6 ratio, mixed liquor is made, the cell Transwell upper chamber is added, every 45 μ L of hole will
The cell Transwell is placed in 24 porocyte culture plates, is transferred to 37 DEG C of 5%CO2Incubator is incubated for 30min, subsequent cell inoculation
And culture operation is the same as above-mentioned Cell migration assay.After culture for 24 hours, Pei Ji is discarded, is gently wiped away on the cell Transwell with cotton swab
Indoor MatrigelTMMatrigel does not damage cell counterdie, the remaining same cell transfer experiments of operation.This experiment is repeated 3 times.
2, result
Average invasion cell number is respectively under transfection pcDNA3.1 group and the transfection each visual field of pcDNA3.1-SCLY group
(53.89 ± 6.02) are a and (26.11 soil 5.74) is a, and difference has statistical significance (P < 0.05).It is above-mentioned the experimental results showed that,
The invasion of hypopharynx cancer cell of SCLY gene expression inhibition.
The detection of 7 body outer clone Forming ability of embodiment
1, step
(1) cell suspension will be made after the cell dissociation after transfection for 24 hours, cell counting board counts；
1640 culture medium of 2ml 10%FBS-RPMI is added in the every hole of (2) six porocyte culture plates, close by 500/hole cell
Inoculating cell suspension is spent, is mixed gently；
(3) six porocyte culture plates move to 37 DEG C of 5%CO2Incubator is incubated for 10 days, replacement in every 3 days culture medium 1 time, every time
The hole 2ml/；
(4) after cultivating, culture medium is discarded, PBS buffer solution is carefully cleaned 3 times, each 5min, drying at room temperature, methanol
Fixed 15min discards methanol, the dry 20min of air at room temperature；
(5) take 0.1% crystal violet dye liquor that tissue culture plate is added, 1ml is added in every hole, dyes 15min；
(6) 0.1% crystal violet dye liquor is recycled, tissue culture plate distilled water cleans 15min；
(7) observation of taking pictures counts, and repeats test 3 times.
2, result
Transfecting pcDNA3.1 group average colony and forming number is respectively that (173.87 ± 15.32) are a, transfects pcDNA3.1-
It is that (75.74 soil 4.82) is a that SCLY group cell average colony, which forms number,.It is above-mentioned the experimental results showed that, SCLY gene expression inhibition
The one-tenth knurl ability of hypopharynx cancer 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 (9)
1. the product for detecting SCLY gene or SCLY albumen diagnoses in hypopharyngeal cancer or the tool for predicting hypopharyngeal cancer prognosis in preparation
Using.
2. application according to claim 1, which is characterized in that the detection SCLY gene or the product of SCLY albumen include
Detect the product of the expression of SCLY gene or SCLY albumen.
3. application according to claim 1 or 2, which is characterized in that the product includes can be in conjunction with the core of SCLY gene
Acid can be in conjunction with the substance of SCLY albumen；The nucleic acid is able to detect the expression of SCLY gene；The substance can
Detect the expression of SCLY albumen.
4. application according to claim 3, which is characterized in that the nucleic acid is specifically expanded used in real-time quantitative PCR
Increase the primer of SCLY gene as shown in SEQ ID NO.1 and SEQ ID NO.2.
5. application according to claim 1, which is characterized in that the tool includes being able to detect SCLY gene or SCLY egg
The tool of white expression.
6. application according to claim 5, which is characterized in that the tool include can in conjunction with SCLY gene nucleic acid or
Person can be in conjunction with the substance of SCLY albumen；The nucleic acid is able to detect the expression of SCLY gene；The substance is able to detect
The expression of SCLY albumen.
7. application according to claim 6, which is characterized in that the nucleic acid is specifically expanded used in real-time quantitative PCR
Increase the primer of SCLY gene as shown in SEQ ID NO.1 and SEQ ID NO.2.
Application of the activator of 8.SCLY gene or SCLY albumen in the drug of preparation treatment hypopharyngeal cancer.
9. application according to claim 8, which is characterized in that the activator can promote or Enhanced SC LY or be related to
The expression or activity of the substance of the upstream SCLY or downstream pathway.
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| CN1852974A (en) * | 2003-06-09 | 2006-10-25 | 密歇根大学董事会 | Compositions and methods for treating and diagnosing cancer |
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Non-Patent Citations (3)
| Title |
|---|
| Differential display of messenger RNA and identification of selenocysteine lyase gene in hepatocellular carcinoma cells transiently expressing hepatitis C virus core protein;Yepes JO 等;《Biomedica》;20061231;194-205 |
| 下咽癌相关血清蛋白质分子标志物的初步研究;程磊等;《中华医学杂志》;20061231;第86卷(第21期);全文 |
| 硒代半胱氨酸裂解酶（SCLY）ELISA定量分析试剂盒;中国化工仪器网;《http://www.chem17.com/st167908/product_22200741.html》;20150914 |
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