CN106011291B - A kind of molecular marker of diagnosis and treatment hypopharyngeal cancer - Google Patents
A kind of molecular marker of diagnosis and treatment hypopharyngeal cancer Download PDFInfo
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Abstract
The invention discloses the molecular markers that ZACN gene and its expression product can be used as hypopharyngeal cancer diagnosis and treatment.May determine that whether subject suffers from hypopharyngeal cancer or diagnose subject by the content of ZACN gene and its expression product in detection subject's hypopharynx tissue whether there is the risk with hypopharyngeal cancer.The present invention can inhibit cell Proliferation by discovery inhibition ZACN gene expression situations such as the proliferation of the hypopharynx cancer cell of research in vitro culture, migration, invasion, inhibit cell migration and invasion, and the studies above is the result shows that ZACN gene and its expression product are a potential drug targets for treating hypopharyngeal cancer.
Description
Technical field
The present invention relates to diagnosing tumor, treatment, prediction prognosis fields, more particularly it relates to different to detect ZACN
It is often the diagnosing tumor of means, prediction method of prognosis；And inhibit the tumor therapeutic agent of ZACN gene or protein.
Background technique
Hypopharyngeal cancer is also known as hypopharyngeal carcinoma.Foreign countries account for about the 0.3% of 5%, the Zhan Quanshen malignant tumour of head-neck malignant tumor.State
Inside account for about the 0.2% of 1.4%, the Zhan Quanshen malignant tumour of head-neck malignant tumor.Although hypopharynx cancer morbidity is lower, by
Hidden in its position, clinical symptoms occur later, and biological property is again severe, and when 67%~92% patient assessment has been in
III, IV phase, and range of tumor is often underestimated, prognosis is poor.Although the diagnosis and treatment means of current hypopharyngeal cancer are continuously improving height,
It is hypopharyngeal cancer especially advanced cases with hypopharyngeal carcinoma survival rate but without significantly improving, the reason is that hypopharyngeal cancer early diagnosis is more difficult, arrives
Advanced stage is difficult to control the transfer of cancer cell again.The early diagnosis of cancer and the control of cancer metastasis are focuses clinically,
Generation and the metastatic rule for studying tumour find its marker and potentially contribute to the generation of accurate evaluation cancer, development, be conducive to
The formulation of remedy measures improves the prognosis of patient.
Summary of the invention
It is diagnosed and is swallowed by detection ZACN 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 ZACN 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 inhibiting ZACN gene or ZACN 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 ZACN gene or ZACN albumen to prepare the purposes in hypopharyngeal cancer diagnostic tool.
The present invention also provides the products of detection ZACN gene or ZACN albumen in preparation prediction hypopharyngeal cancer prognostic tool
Purposes.
Further, the product of the detection ZACN gene or ZACN albumen includes the table for detecting ZACN gene or ZACN albumen
Up to horizontal product.The product includes the nucleic acid that can combine ZACN gene or the substance (example that can combine ZACN albumen
Such as antibody).The nucleic acid is able to detect the expression of ZACN gene；The substance is able to detect the expression water of ZACN albumen
It is flat.
The product of detection ZACN 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 ZACN 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 ZACN 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 ZACN albumen of the invention includes the antibody or its segment for specifically binding ZACN 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 ZACN 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 ZACN albumen for being used as antigen or part thereof to acquisition
Antigentic specificity purifies to obtain the monoclonal antibody for ZACN 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, ZACN albumen or coding
The gene of ZACN 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 raised patient of the level of ZACN gene or ZACN 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 ZACN gene or ZACN albumen can be detection ZACN gene or ZACN 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 ZACN gene or ZACN albumen
Expression.The tool include can in conjunction with ZACN gene nucleic acid or can in conjunction with ZACN albumen substance (such as
Antibody).The nucleic acid is able to detect the expression of ZACN gene；The substance is able to detect the expression of ZACN 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 ZACN gene is known in high-flux sequence
The purposes for also belonging to ZACN 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 ZACN gene nucleic acid or can in conjunction with ZACN albumen substance (such as antibody).The nucleic acid is able to detect ZACN base
The mRNA level in-site of cause；The substance is able to detect the expression of ZACN 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, ZACN gene is known in high-flux sequence
The abnormal purposes for also belonging to ZACN gene related to hypopharyngeal cancer, equally within protection scope of the present invention.
The number for the amino acid that anti-ZACN 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 ZACN.When antibody is as therapeutic agent, preferably it can know
Amino acid not as much as possible, as long as it can inhibit ZACN function.The number of antibody or the amino acid of its segment identification is at least
One, more preferably at least three.The immunoglobulin class of antibody is unrestricted, can be IgG, IgM, IgA, IgE, IgD or
IgY。
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 ZACN gene or albumen in Samples subjects is detected；
(3) it associates whether by the expression of the ZACN gene or albumen that measure with the illness of subject.
(4) compared with the control, the expression of ZACN gene or albumen increases, 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 inhibiting ZACN gene or ZACN egg
It is white.
Further, the method includes inhibiting the expression of ZACN gene, or expression or the inhibition ZACN of inhibition ZACN albumen
The activity of albumen.
The present invention also provides a kind of screening techniques of tumour medicine, can be by adding testing drug to tumour cell
Afterwards or in the expression water to some period measurement ZACN gene or ZACN albumen after tumor model animal application testing drug
It puts down to measure tumour medicine and improve the effect of tumor prognosis.More specifically, when ZACN gene or the expression water of ZACN albumen
It puts down when being reduced after addition or application 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 inhibitor containing ZACN gene or ZACN albumen.
The present invention also provides application of the above-mentioned inhibitor in the drug of preparation treatment hypopharyngeal cancer.
The inhibitor of ZACN gene or ZACN albumen of the invention is unrestricted, as long as can inhibit ZACN or be related to
The expression or activity of the substance of the upstream ZACN or downstream pathway, and for treating the effective drug of tumour.
Further, the inhibitor includes antisense nucleic acid, dsRNA, ribozyme, aptamer, ZACN binding protein segment or antibody
Or its segment.
" antisense nucleic acid " refers to the nucleic acid containing the sequence complementary with the coding mRNA of ZACN.Antisense nucleic acid can by DNA,
RNA or both composition.Antisense nucleic acid does not need complementary with the mRNA100% of target ZACN.Antisense nucleic acid can contain Non-complementary bases,
As long as it being capable of specific hybrid under strict conditions.When antisense nucleic acid is introduced cell, it combines target polynucleotide
And inhibit transcription, RNA processing, translation or stability.In addition to antisense polynucleotides, antisense nucleic acid further includes polynucleotides simulation
Object, it contains by the main chain of modification and 3 ' and 5 ' end parts.Such antisense nucleic acid can be according to ZACN sequence information come just
It generates when design and using well known to a person skilled in the art methods.
" dsRNA " refers to containing duplex-RNA constructs, by RNA interference (RNAi) come the RNA of inhibition of gene expression, including
SiRNA (short interfering rna) and shRNA (short hairpin RNA).DsRNA does not need the homology for having 100% with target-gene sequence,
As long as it can inhibit expression of target gene.In order to stabilize or other purposes, a part of dsRNA can be substituted with DNA.
Preferably, siRNA is the double-stranded RNA of 21-23 base.SiRNA can by well known to a person skilled in the art method come
Preparation, such as by chemical synthesis or as the analog of naturally occurring RNA.ShRNA is with hair clip corner (hairpin
Turn) the Short interfering RNA of structure.ShRNA can be prepared by the way that well known to a person skilled in the art methods, such as be closed by chemistry
Cell and DNA is expressed at or by introducing the DNA for encoding shRNA.
" ribozyme " refers to the RNA with catalytic activity, it can cut, paste, insertion and transfer RNA.The structure of ribozyme can
To include tup, hair clip etc..
" aptamer " refers to the nucleic acid in conjunction with something such as protein.Aptamer can be RNA or DNA.The form of nucleic acid can be with
It is double-strand or single-stranded.The infinite in length system of aptamer, as long as it can specifically bind target molecule, can by such as 10 to
200 nucleotide, preferably 10 to 100 nucleotide, more preferable 15 to 80 nucleotide, even more preferably 15 to 50 nucleosides
Acid composition.Aptamer can be used that well known to a person skilled in the art methods to select.For example, (index can be passed through using SELEX
The phyletic evolution for the ligand that formula enrichment carries out).
" the protein-bonded segment of ZACN " refers in conjunction with ZACN and inhibition ZACN implements the segment of the protein of original function.
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 inhibitor of ZACN 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 detecting ZACN gene in mRNA level in-site using QPCR；
Fig. 2 shows the differential expression for detecting ZACN gene on protein level using immunoblotting；
Fig. 3 shows the jamming effectiveness using QPCR detection siRNA to ZACN gene expression；
Fig. 4 shows the jamming effectiveness using immune-blotting method siRNA to ZACN gene expression；
Fig. 5, which is shown, inhibits influence of the ZACN gene expression to hypopharynx cancer cell multiplication using MTT detection；
Fig. 6 display inhibits influence of the ZACN protein function 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 ZACN 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:
| Reagent | Dosage |
| 5x1st Strand Synthesis Buffer | 4.0μl |
| PrimeScript RTase | 1.0μl |
| RNase Inhibitor | 1.0μl |
| Rnase free dH2O | 4.0μl |
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:
| 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:
ZACN gene:
5'-GCGATGGAGTTAGAGTTC-3'(SEQ ID NO.1)；
5’-TGGGTCTTCAGATCATAAAC-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-△ΔctIt calculates.Experiment is repeated 3 times.
△ ct=ct (A)-ct (β-actin)
△ Δ ct=△ ct (experimental group)-△ ct (control group)
As a result show such as Fig. 1, compared with normal control tissue, in hypopharynx cancerous tissue the mRNA level in-site of ZACN gene it is obvious on
It adjusts, difference has 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 ZACN albumen variation of conventional Western-blot method, each group experiment is repeated 3 times,
Using β-actin as internal reference, ZACN protein band absorbance quantitative analysis is done, expression quantity is with ZACN albumen/β-actin absorbance
Ratio represents.
As a result as Fig. 2 shows that compared with normal control tissue, ZACN protein level is dramatically increased in hypopharynx cancerous tissue, poor
It is different that there is statistical significance (P < 0.05).
Embodiment 3 inhibits ZACN gene expression
1, siRNA design synthesis
For the siRNA sequence of ZACN:
SiRNA-ZACN:
Positive-sense strand is 5 '-AUCAUAAACUACGUAUUCCCU-3 ' (SEQ ID NO.5)
Antisense strand is 5 '-GGAAUACGUAGUUUAUGAUCU-3 ' (SEQ ID NO.6)；
The above siRNA sequence and negative control siRNA sequence (siRNA-NC) are by the limited public affairs of Shanghai Ji Ma pharmaceutical technology
Department provides.
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/ of streptomysin
1640 culture medium of RPMI of ml is placed in 37 DEG C, 5%CO2, cultivate under saturated humidity environment.
2.2 cell transfecting
(1) it transfects first 24 hours, in 500 μ l nonreactive inoculation of medium 0.5-2*105A cell, cell fusion when transfection
Degree is 30-50%.Cell dissociation is mixed completely when bed board, cell accumulation is avoided to grow.
(2) with 50 μ l Opti-MEM dilution siRNA (the final concentration of 33nM of transfection cell, gently pressure-vaccum 3-5 times mixing.
(3) it is gently mixed by inversion transfection reagent, dilutes 1 μ l Lipofectamine2000 gently with 50 μ l Opti-MEM
Pressure-vaccum 3-5 times mixing, stands 5min at room temperature.
(4) transfection reagent and siRNA dilution are mixed, gently pressure-vaccum 3-5 times mixing, stands 20min at room temperature.
(5) transfection composite is added in 24 porocyte plates, 100 holes μ l/, and front and back jog cell plates are uniformly mixed.
(6) cell plates are placed in 37 DEG C, 5%CO2It is cultivated 18-48 hours in incubator.Interchangeable fresh culture after transfection 4-6 hours
Base.
3, the jamming effectiveness of detection siRNA 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.
3.3QPCR
Step is the same as embodiment 2.
3.4 result
As a result as shown in figure 3, compared with siRNA-NC group, siRNA-ZACN can effectively inhibit ZACN gene mRNA
Expression, difference have statistical significance (P < 0.05).
4, the jamming effectiveness of detection siRNA is tested using Western blot
Step is the same as embodiment 2.
As a result as shown in figure 4, compared with siRNA-NC group, siRNA-ZACN can effectively inhibit the table of ZACN albumen
It reaches, difference has statistical significance (P < 0.05).
Measurement of the expression of embodiment 4ZACN gene to hypopharyngeal cancer ability of cell proliferation
1, step
Cell transfecting is carried out according to the step of embodiment 2,48 hours cell dissociations will be transfected, be inoculated in 96 well culture plates
In, 5000, every hole cell, every 200 μ L cell suspension of hole, cultivate respectively for 24 hours, after 48,72, using the person that is not added cell as blank pair
According to group, 20 μ L MTT (0.5mg/mL) are added in every hole, carefully suck liquid in hole after 4h is incubated in incubator, 100 μ are added in every hole
L DMSO vibrates 10~20min, dissolves crystal sufficiently.Enzyme-linked immunosorbent assay instrument measures each hole OD570 value, with the time and
Absorbance value is respectively transverse and longitudinal coordinate, draws cell growth curve.3 multiple holes are arranged in every hole cell.This experiment is repeated 3 times.
2, result
As a result as shown in figure 5, compared with transfecting siRNA-NC group, transfection siRNA-ZACN group cell Proliferation is slow, difference
With statistical significance (P < 0.05).It is above-mentioned the experimental results showed that, ZACN gene expression promotes the proliferation of hypopharynx cancer cell.
In 5 hypopharyngeal cancer cell antibody of embodiment and test
1, step:
Hypopharynx cancer cell FADU is inoculated in 96 porocyte culture plates, every hole 2*103A cells/well/200 μ l, cell
It is handled as follows after adherent:
Experimental group 1 (control group): unrelated monoclonal antibody (1:50) is added in hypopharynx cancer cell；
Experimental group 2: anti-human ZACN monoclonal antibody (1:50) is added in hypopharynx cancer cell.
By cell in 37 DEG C, 5%CO2After incubator is incubated for 24 hours, 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 fig. 6, compared to control group, the group cells proliferation slowed down of anti-human ZACN monoclonal antibody is added.Above-mentioned experiment
The result shows that hypopharynx cancer cell multiplication can be inhibited by inhibiting the function of ZACN albumen.
Embodiment 6, which detects ZACN gene expression, influences 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 siRNA-NC group and the transfection each visual field of siRNA-ZACN group
(167.45 ± 14.98) are a and (79.86 soil 8.34) is a, and difference has statistical significance (P < 0.05).Above-mentioned experimental result table
Bright, ZACN gene expression promotes the migration of hypopharynx cancer cell.
Embodiment 7 detects influence of the ZACN 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 siRNA-NC group and the transfection each visual field of siRNA-ZACN group
(54.14 ± 6.46) are a and (26.25 soil 3.69) is a, and difference has statistical significance (P < 0.05).It is above-mentioned the experimental results showed that,
ZACN gene expression promotes the invasion of hypopharynx cancer cell.
The detection of 8 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 siRNA-NC group average colony and forming number is respectively that (175.30 ± 16.45) are a, transfects siRNA-ZACN
It is that (78.00 soil 5.56) is a that group cell average colony, which forms number,.It is above-mentioned the experimental results showed that, ZACN gene expression promotes down
The one-tenth knurl ability of pharynx 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 ZACN gene or ZACN albumen is preparing the application in the tool for diagnosing hypopharyngeal cancer.
2. application according to claim 1, which is characterized in that the detection ZACN gene or the product of ZACN albumen include
Detect the product of the expression of ZACN gene or ZACN 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 ZACN gene
Acid can be in conjunction with the substance of ZACN albumen；The nucleic acid is able to detect the expression of ZACN gene；The substance can
Detect the expression of ZACN albumen.
4. application according to claim 3, which is characterized in that the nucleic acid is specifically expanded used in real-time quantitative PCR
The primer for increasing ZACN 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 ZACN gene or ZACN egg
The tool of white expression.
6. application according to claim 5, which is characterized in that the tool include can in conjunction with ZACN gene nucleic acid or
Person can be in conjunction with the substance of ZACN albumen；The nucleic acid is able to detect the expression of ZACN gene；The substance is able to detect
The expression of ZACN albumen.
7. application according to claim 6, which is characterized in that the nucleic acid is specifically expanded used in real-time quantitative PCR
The primer for increasing ZACN gene, as shown in SEQ ID NO.1 and SEQ ID NO.2.
Application of the inhibitor of 8.ZACN gene or ZACN albumen in the drug of preparation treatment hypopharyngeal cancer.
9. application according to claim 8, which is characterized in that the inhibitor is able to suppress ZACN or is related to the upstream ZACN
Or the expression or activity of the substance of downstream pathway.
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| Title |
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| Aberrantly expressed genes and miRNAs in human hypopharyngeal squamous cell carcinoma based on RNA sequencing analysis;Hu Li等;《Oncology reports》;20180630;第647-658页 |
| Loss of expression of ZAC/LOT1 in squamous cell carcinonas of head and neck;Koy S等;《Head Neck》;20040430;第26卷(第4期);第338-344页 |
| The candidate tumor suppressor gene ZAC is involved in keratinocyte differentiation and its expression is lost in basal cell carcinomas;Basyuk E等;《Mol Cancer Res》;20051231;第3卷(第9期);第483-492页 |
| 非小细胞肺癌ZAC基因的表达及临床意义;李科宇等;《临床肺科杂志》;20131231;第18卷(第12期);第2139-2141页 |
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