CN105886627A - Marker for diagnosis and treatment of esophagus cancer - Google Patents

Marker for diagnosis and treatment of esophagus cancer Download PDF

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Publication number
CN105886627A
CN105886627A CN201610279226.9A CN201610279226A CN105886627A CN 105886627 A CN105886627 A CN 105886627A CN 201610279226 A CN201610279226 A CN 201610279226A CN 105886627 A CN105886627 A CN 105886627A
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pcp4l1
gene
reagent
product
diagnosis
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CN105886627B (en
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杨承刚
肖枫
宋宏涛
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Qingdao Yangshen Biomedical Co Ltd
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Beijing Medintell Bioinformatic Technology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Abstract

The invention discloses a molecular marker for diagnosis and treatment of esophagus cancer. A PCP4L1 gene can serve as the molecular marker. Whether a subject suffers from the esophagus cancer or has the esophagus cancer risks or not can be judged through detection of content of the PCP4L1 gene in esophagus tissues. Overexpressed PCP4L1 gene expression capable of inhibiting esophagus cancer cell proliferation is discovered through studies on proliferation conditions of esophagus cancer cells cultured in vitro, and study results show that the PCP4L1 gene is a potential drug target for treating the esophagus cancer.

Description

The diagnosis and treatment mark of the esophageal carcinoma
Technical field
The present invention relates to biological technical field, more particularly to PCP4L1 gene in the diagnosis of the esophageal carcinoma, treatment In purposes.
Background technology
The esophageal carcinoma (Esophageal carcinoma, EC) is common malignant tumor of digestive tract, accounts for digestive system The second that each region tumors is dead, the whole world is died from this disease every year and is reached 300,000 people, the wherein every annual of China Death about 150,000 people, is the country that Incidence of esophageal cancer is the highest in the world.Although in recent years in the change of the esophageal carcinoma Treat, the aspect such as radiotherapy and Role of Concurrent Chemoradiotherapy achieves greater advance, but surgical operation is still the esophageal carcinoma the most so far Main treatment means.But therapeutic effect is undesirable, within 5 years, survival rate is only about 30%, and dead is main Reason is postoperative recurrence and transfer, especially the lymphatic metastasis of patient with esophageal carcinoma early postoperation.Report the most both at home and abroad The recurrence of road its early postoperation and the rate of transform (in postoperative 1 year) up to about 20-50%, this to patient and The confidence of surgeon is all heavy blow, and some patients even produces query to surgery operative effect.The esophageal carcinoma Natural history or the title of patient are about the 6-12 month life cycle, occur recurrence in early days and transfer in postoperative 1 year, Mean the failure of operation, take chemicotherapy more useful these patients in other words.
The generation of the esophageal carcinoma, evolution relate to the sudden change of multiple oncogene, antioncogene, produces various enzyme The change learned, shows as polygenes, multifactor, the synergism of multi-step.For esophagus cancer diagnosis and prognosis The research of relevant molecular biology mark has become research emphasis.
The at present research of esophageal carcinoma tumor molecular marker and report more, except the tumor marker cancer of traditional sense Embryonal antigen (CEA), squamous cell cancer related antigen (SCC), cellular keratinization fibroin fragment 19 Beyond (CYFRA2l l), p53 protein antibodies (p53-Ab) etc., focus molecular marked compound in research at present EGFR (EGF-R ELISA), CycIinDl (Cyclin D1 l), COX-2 (Cycloxygenase 2), PCNA (proliferating cell nuclear antigen), VEGF (vascular endothelial cell growth factor) etc. are to the esophageal carcinoma Diagnosis and prognosis have certain clinical meaning, but above-mentioned label lacks specificity and susceptiveness, therefore finds A kind of high sensitivity and strong specific molecular marker are problem demanding prompt solutions.
Summary of the invention
In order to overcome the deficiencies in the prior art, it is an object of the invention to provide one and can be used for the esophageal carcinoma and examine in early days Disconnected molecular marker.Use gene marker to carry out diagnosis of esophageal cancer and there is promptness, specificity and susceptiveness, So that patient just can know disease risks in early days in disease, for risk height, take to prevent accordingly and control Treatment measure.
To achieve these goals, the present invention adopts the following technical scheme that
The invention provides the product of detection PCP4L1 gene expression in the instrument preparing diagnosis of esophageal cancer Application.
Further, the product of described detection PCP4L1 gene expression includes detecting PCP4L1 gene mRNA The product of level and/or the product of detection PCP4L1 protein level.
Further, the product of described detection PCP4L1 gene expression includes: by RT-PCR, real-time quantitative The expression of PCR, immune detection, in situ hybridization or chip detection PCP4L1 gene and expression product thereof with The product of diagnosis of esophageal cancer.
Further, the product of described RT-PCR diagnosis of esophageal cancer at least includes a pair specific amplified PCP4L1 The primer of gene；The product of described real-time quantitative PCR diagnosis of esophageal cancer at least includes a pair specific amplified The primer of PCP4L1 gene；The product of described immune detection diagnosis of esophageal cancer includes: special with PCP4L1 albumen Anisogamy antibody；The product of described in situ hybridization diagnosis of esophageal cancer includes: with the core of PCP4L1 gene The probe of acid sequence hybridization；The product of described chip diagnosis of esophageal cancer includes: protein chip and gene chip； Wherein, protein chip includes the antibody being combined with PCP4L1 protein-specific, and gene chip includes and PCP4L1 The probe of the nucleic acid array hybridizing of gene.
A pair specific amplified PCP4L1 base that the product of described real-time quantitative PCR diagnosis of esophageal cancer at least includes The primer of cause is as shown in SEQ ID NO.3 and SEQ ID NO.4.
The product of described detection PCP4L1 gene expression can be the reagent, also of detection PCP4L1 gene expression Can be to comprise the test kit of described reagent, chip, reagent paper etc., it is also possible to be the high flux using described reagent Order-checking platform.
The instrument of described diagnosis of esophageal cancer includes but not limited to that chip, test kit, reagent paper or high-flux sequence are flat Platform；High-flux sequence platform is the instrument of a kind of special diagnosis of esophageal cancer, along with sending out of high throughput sequencing technologies Exhibition, will become the structure of the gene expression profile of a people and work the most easily.By contrast Disease and The gene expression profile of normal population, the exception easily analyzing which gene is relevant to disease.Therefore, in high pass Measure the exception purposes that fall within PCP4L1 gene relevant to the esophageal carcinoma knowing PCP4L1 gene in sequence, with Sample is within protection scope of the present invention.
Present invention also offers the instrument of a kind of diagnosis of esophageal cancer, described instrument includes detecting PCP4L1 gene table The reagent reached；Described reagent includes primer and/or probe, the detection PCP4L1 detecting PCP4L1 gene mRNA The antibody of albumen.
Described instrument includes but not limited to chip, test kit, reagent paper or high-flux sequence platform.
Wherein, described chip includes gene chip, protein chip；Described gene chip include solid phase carrier with And it being fixed on the oligonucleotide probe of solid phase carrier, described oligonucleotide probe includes for detecting PCP4L1 base The oligonucleotide probe for PCP4L1 gene because of transcriptional level；Described protein chip includes solid phase carrier And it is fixed on the specific antibody of the PCP4L1 albumen of solid phase carrier；Described gene chip can be used for detection bag Include the PCP4L1 gene expression at interior multiple genes (such as, relevant to the esophageal carcinoma multiple genes). Multiple protein that described protein chip can be used for detecting including PCP4L1 albumen are (such as with the esophageal carcinoma Relevant multiple protein) expression.By multiple marks with the esophageal carcinoma are detected simultaneously, can be big The big accuracy rate improving esophagus cancer diagnosis.
Wherein, described test kit includes gene detecting kit and protein immunization detection kit；Described gene is examined Test agent box includes the reagent for detecting PCP4L1 gene transcription level；Described protein immunization detection kit Specific antibody including PCP4L1 albumen.Further, described reagent includes using RT-PCR, real-time quantitative Needed for during PCR, immune detection, in situ hybridization or chip method detection PCP4L1 gene expression dose Reagent.Preference, described reagent includes the primer for PCP4L1 gene and/or probe.According to PCP4L1 The nucleotide sequence information of gene easily design may be used for detect PCP4L1 gene expression dose primer and Probe.
Described reagent paper includes the reagent detecting PCP4L1 gene expression.
Described high-flux sequence platform includes the reagent detecting PCP4L1 gene expression.
Can be that DNA, RNA, DNA-RNA are embedding with the probe of the nucleic acid array hybridizing of PCP4L1 gene Zoarium, PNA or other derivant.The length of described probe does not limit, if complete specific hybrid, Specific binding with purpose nucleotide sequence, any length can.The length of described probe can be as short as 25, 20,15,13 or 10 bases longs.Equally, the length of described probe can grow to 60,80,100,150, 300 base pairs or longer, the most whole gene.Owing to different probe length is special to hybridization efficiency, signal The opposite sex has different impacts, the length of described probe to be typically at least 14 base pairs, the longest is usually no more than 30 base pairs, optimal with 15-25 base pair with the length of purpose nucleotide sequence complementary.Described probe Self-complementary sequences is most preferably less than 4 base pairs, in order to avoid affecting hybridization efficiency.
Further, the specific antibody of described PCP4L1 albumen includes monoclonal antibody, polyclonal antibody.Institute The specific antibody stating PCP4L1 albumen includes complete antibody molecule, any fragment of antibody or modifies (example As, chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc..As long as described fragment can retain and PCP4L1 The binding ability of albumen.Well known to a person skilled in the art when the preparation of the antibody of protein level, And the present invention can use any method to prepare described antibody.
In specific embodiments of the present invention, the primer of described detection PCP4L1 gene mRNA includes SEQ Primer pair shown in ID NO.3 and SEQ ID NO.4.
Present invention also offers the accelerator of PCP4L1 gene and/or its expression product in the preparation treatment esophageal carcinoma Medicine in application.
Described accelerator includes the reagent promoting PCP4L1 gene expression and promotes PCP4L1 gene expression product Reagent；The reagent of described promotion PCP4L1 gene expression includes promoting the reagent of genetic transcription, promoting gene The reagent of translation, the reagent of promotion PCP4L1 protein content；The examination of described promotion PCP4L1 gene expression product Agent includes promoting the reagent of PCP4L1 gene expression product stability, promoting PCP4L1 gene expression product activity Reagent, promote PCP4L1 gene expression product function reagent.
Specifically, the reagent of described promotion PCP4L1 gene expression includes: reagent containing PCP4L1 gene, Carry the carrier of PCP4L1 gene or reagent that host cell is formed, reagent containing PCP4L1 protein.
On the one hand the accelerator of the present invention may be used for supplementing disappearance or the deficiency of endogenic PCP4L1 albumen, By improving the expression of PCP4L1 albumen, thus the esophageal carcinoma that treatment causes because of PCP4L1 hypoproteinosis.Another Aspect may be used for promoting activity or the function of PCP4L1 albumen, thus treats the esophageal carcinoma.
The carrier carrying gene of the present invention is various carrier known in the art, such as commercially available carrier, includes Plasmid, cosmid, phage, virus etc..
In the present invention, term " host cell " includes prokaryotic cell and eukaryotic cell.Conventional prokaryotic hosts is thin The example of born of the same parents includes escherichia coli, bacillus subtilis etc..Conventional eukaryotic host cell includes yeast cells, insecticide Cell and mammalian cell.It is preferred that this host cell is eukaryotic cell, as thin in Chinese hamster ovary celI, COS Born of the same parents etc..
Present invention also offers a kind of pharmaceutical composition for treating the esophageal carcinoma, described pharmaceutical composition includes PCP4L1 gene described in face and/or the accelerator of its expression product.
Further, the medicine of the present invention also includes pharmaceutically acceptable carrier, carrier, this kind of carrier include (but It is not limited to): diluent, excipient such as water etc., filler such as starch, sucrose etc.；Binding agent such as cellulose Derivant, alginate, gelatin and polyvinylpyrrolidone；Wetting agent such as glycerol；Disintegrating agent such as agar, carbonic acid Calcium and sodium bicarbonate；Absorption enhancer quaternary ammonium compound；Surfactant such as hexadecanol；Absorption carrier is as high Ridge soil and soap clay；Lubricant such as Pulvis Talci, calcium stearate and magnesium, Polyethylene Glycol etc..
The medicine of the present invention import tissue or cell mode can by be divided into external or internal in the way of.External Mode includes importing in cell by the medicine containing PCP4L1 gene or the medicine containing PCP4L1 protein, Again by cell transplantation or feed back to internal.Internal mode include directly by the medicine containing PCP4L1 gene or In infusion of medicine in-vivo tissue containing PCP4L1 protein.
The medicine of the present invention also can be with the drug combination of the other treatment esophageal carcinoma, and multi-medicament is used in combination can be big Mention greatly the success rate for the treatment of.
In the context of the present invention, " PCP4L1 gene " includes PCP4L1 gene and PCP4L1 gene The polynucleotide of any function equivalent.PCP4L1 gene includes and the most international common core sequence databank In GeneBank, PCP4L1 gene (NC_000001.11) DNA sequence has more than 70% homology, and coding Identical function protein DNA sequence.
Preferably, the coded sequence of PCP4L1 gene includes any DNA molecular following:
(1) DNA sequence shown in SEQ ID NO.1 in sequence table；
(2) under strict conditions with 1) the DNA sequence hybridization that limits and coding identical function protein DNA sequence；
(3) DNA sequence limited with (1) or (2) has 70%, preferably, more than 90% with Source property, and coding identical function protein DNA molecule.
In specific embodiments of the present invention, the coded sequence of described PCP4L1 gene is SEQ ID NO.1 Shown DNA sequence.
In the context of the present invention, PCP4L1 gene expression product includes PCP4L1 albumen and PCP4L1 The partial peptide of albumen.The partial peptide of described PCP4L1 albumen contains the functional domain relevant to the esophageal carcinoma.
" PCP4L1 albumen " includes any function equivalent of PCP4L1 albumen and PCP4L1 albumen. Described function equivalent includes PCP4L1 albumen conservative variation's protein or its active fragment, or its activity Derivant, allelic variant, natural mutation, induced mutants, can be with under high or low stringent condition Protein coded by the DNA of the DNA hybridization of PCP4L1.
Preferably, PCP4L1 albumen is the protein with following amino acid sequences:
(1) protein being made up of the aminoacid sequence shown in SEQ ID NO.2 in sequence table；
(2) aminoacid sequence shown in SEQ ID NO.2 is passed through the replacement of one or several amino acid residue And/or disappearance and/or add and with the aminoacid sequence shown in SEQ ID NO.2 have identical function by The protein that aminoacid sequence shown in SEQ ID NO.2 is derivative.Replace, lack or add is amino acid whose Number is usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10.
(3) with the aminoacid sequence shown in SEQ ID NO.2, there is at least 80% homology and (be also called sequence Homogeneity), it is highly preferred that same with the aminoacid sequence at least about 90% to 95% shown in SEQ ID NO.2 Source property, is often the polypeptide of the aminoacid sequence composition of 96%, 97%, 98%, 99% homology.
In specific embodiments of the present invention, described PCP4L1 albumen has shown in SEQ ID NO.2 The protein of aminoacid sequence.
It is known that, conventionally, in a protein, one or more amino acid whose modifications do not interfere with protein Function.Those skilled in the art can approve change single amino acids or the aminoacid of little percentage ratio or to aminoacid sequence Adding individually, lacking, insert, replace of row is conservative modification, and wherein the change generation of protein has similar The protein of function.It is well known in the art for providing intimate amino acid whose Conservative substitution tables.
It is PCP4L1 egg by adding the example of the protein of an aminoacid or multiple Modification of amino acid residues White fusion protein.Peptide or protein with PCP4L1 protein fusion is not limited, as long as gained Fusion protein retains the biologic activity of PCP4L1 albumen.
The PCP4L1 albumen of the present invention also includes repairing the non-conservative of aminoacid sequence shown in SEQ ID NO.2 Decorations, as long as the protein through modifying remains able to retain the biologic activity of PCP4L1 albumen.At this In class modifying protein, the amino acid number of sudden change is typically 10 or less, such as 6 or less, Such as 3 or less.
In the context of the present invention, " diagnosis of esophageal cancer " both includes judging that experimenter has suffered from esophagus Cancer, also include judging whether experimenter exists the risk suffering from the esophageal carcinoma.
In the context of the present invention, " the treatment esophageal carcinoma " divides from the state change of disease, can include disease The alleviation of disease, the healing completely of disease, also include the evaluation for disease therapeuticing effect.
Advantages of the present invention and beneficial effect:
Present invention firstly discovers that PCP4L1 gene expression is relevant to the esophageal carcinoma, by detection experimenter's esophagus group Knit the expression of middle PCP4L1, it can be determined that whether experimenter suffers from the esophageal carcinoma or judge whether experimenter deposits Suffering from the risk of the esophageal carcinoma, thus instructing clinicist to provide prevention scheme or therapeutic scheme to experimenter.
Present invention finds a kind of new molecular marked compound-PCP4L1 gene, compare traditional detection means, base Because of diagnosis more in time, more special, sensitiveer, it is possible to realize the early diagnosis of the esophageal carcinoma.
Accompanying drawing explanation
Fig. 1 show utilize RNA-seq detect the expression in human esophageal carcinoma and normal esophageal tissue of the PCP4L1 gene Situation；
Fig. 2 show utilize Western blot detect PCP4L1 albumen table in human esophageal carcinoma and normal esophageal tissue Reach situation；
Fig. 3 shows the process LAN situation utilizing QPCR to detect PCP4L1 gene on transcriptional level；
Fig. 4 shows the process LAN situation utilizing Western blot detection PCP4L1 albumen；
Fig. 5 show utilize MTT experiment detect the PCP4L1 gene expression impact on esophagus carcinoma proliferation.
Specific embodiment
The present invention is further detailed explanation with embodiment below in conjunction with the accompanying drawings.Following example are only used for The bright present invention rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment is logical Often according to normal condition, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or according to the bar proposed by manufacturer Part.
The differential expression of PCP4L1 gene in embodiment 1 normal esophageal tissue and human esophageal carcinoma
1, experiment material:
Human esophageal carcinoma is the specimen of hospital's thoracic surgery excision, and normal esophageal is organized as under Dndoscope Laboratory mirror taking out Specimen, draw materials immediately after the esophageal neoplasm tissue of excision is in vitro, when drawing materials, all wear disposable sterilized hands Set, applies autoclaved knife blade to cut.
All cases are all through definitive pathological diagnosis.Including human esophageal carcinoma 40 example, normal esophageal organizes 30 examples.Wherein Esophageal squamous cell carcinoma 34 example, adenocarcinoma of esophagus 6 example.With normal esophageal setup action matched group.Before all operation in patients all Proved by pathology, the treatment such as the most non-preoperative row radiotherapy, chemotherapy, and without the tumor etc. at other positions.Institute is the most equal Within half an hour of performing the operation after Liquid nitrogen storage, move into-80 degree refrigerators frozen.
2, the extraction of RNA is organized
Use RNAprep pure Tissue Kit (the total RNA from animal tissues extraction reagent of QIAGEN company Box (DP431)) extract according to operating instruction.
3, the quality analysis (Agilent Technologies 2100Bioanalyzer) of RNA sample
Agilent Technologies 2100Bioanalyzer detects RNA sample quality, observes 28S rRNA With 18S rRNA master tape substantially, concentration qualified without degraded, RNA Perfection Index reach meeting of requirement The requirement that RNA-seq order-checking cDNA library builds, may be used for library construction and order-checking.
4, high flux transcript profile order-checking
(1) RNA-seq reads section location
First low-quality reading section is removed and obtain cleaning reading section, then utilize TopHat v1.3.1 by cleansing tablet Section is mated with UCSC H.sapiens reference genome (hg19), H.sapiens UCSC hg19 version pre- The index first built is downloaded from TopHat homepage, and as with reference to genome, utilizes TopHat and genome Timing, it is allowed to each reading section (defaulting to 20) has multiple coupling site, most 2 mispairing.TopHat root Possible shearing site storehouse is set up, according to these shearing site storehouses according to exon region and GT-AG shear signal The reading section not navigating to genome is navigated on genome.We use the system default of TopHat method Parameter.
(2) transcript abundance assessment
The reading segment file matched is by Cufflinks v1.0.3 process, and Cufflinks v1.0.3 is by RNA-seq Segment number is standardized calculating the relative abundance of transcript.FPKM value refers to each million order-checking fragments In match the segment number of exon region of specific gene 1kb length.Calculated by Bayesian inference method The confidence interval of FPKM estimated value.The GTF comment file of the reference that Cufflinks uses is from Ensembl number (Homo_sapiens.GRCh37.63.gtf) is downloaded according to storehouse.
(3) detection of difference expression gene
Ensembl GTF file and the original document mated by TopHat of download are transferred to Cuffdiff, Cuffdiff uses original matching files to re-evaluate the gene expression abundance of the transcript listed in GTF file, inspection Survey differential expression.Q value ＜ 0.01, test display is only had successfully more just to be considered in Cuffidff exports It it is differential expression.
5, result
Result as it is shown in figure 1, compared with normal esophageal tissue, the mRNA of PCP4L1 gene in human esophageal carcinoma Level significantly reduces, and difference has statistical significance (P < 0.05).
6, on protein level, detect the differential expression of PCP4L1 gene
6.1 extract tissue total protein
The operation of protein extraction is carried out according to the description of EpiQuik tissue/cell total protein extraction test kit.
6.2Western blot detects
The protein quantification of extraction is carried out SDS-PAGE electrophoresis, carry out afterwards transferring film, closing, one anti-hatch, Two anti-hatch, develop the color.
6.3 statistical procedures
Image J software is used to be analyzed, with β-actin as internal reference, by purpose the gray value of protein band The gray value of informal voucher band is normalized.Result data is all to represent in the way of mean+SD, Using SPSS13.0 statistical software to carry out statistical analysis, difference between the two uses t inspection, it is believed that When P < has statistical significance when 0.05.
6.4 result
Result is as in figure 2 it is shown, compared with normal esophageal tissue, in human esophageal carcinoma, PCP4L1 protein content drops Low, difference has statistical significance (P < 0.05).
Embodiment 2PCP4L1 gene expression plasmid builds
1, the structure of PCP4L1 expression vector
Coded sequence (as shown in SEQ ID NO.1) design amplimer according to PCP4L1 gene.From one-tenth The PCP4L1 gene of amplification total length in the cDNA library (clontech company, article No.: 638831) of Human fetal spleen Coded sequence, above-mentioned cDNA sequence is inserted in eukaryotic expression vector pcDNA3.1, connects and obtain Recombinant vector pcDNA3.1-PCP4L1 for subsequent experimental.
2, the cultivation of esophageal cancer cell and transfection
2.1 cells are cultivated
Esophageal carcinoma cell line ECA109 is inoculated in the DMEM culture fluid containing 10% hyclone, by it It is positioned over 37 DEG C, 5%CO2Cultivate in incubator, when cell reaches 80% fusion, use 0.25% trypsin Had digestive transfer culture.
2.2 cell transfecting
By esophageal cancer cell by 1 × 104/ hole is inoculated in 24 porocyte culture plates, at 37 DEG C, 5%CO2Training Supporting cell in case and cultivate 24h, transfection is according to lipofectamine 2000 (purchased from Invitrogen company) Description transfection, making an excessive case more excessive is tested and is divided into matched group (transfection pcDNA3.1) and experimental group (to transfect PcDNA3.1-PCP4L1), the working concentration of transfected plasmids is 0.5 μ g/ml.
3, the effect of QPCR experiment detection plasmid transfection is utilized.
3.1 extract cell total rna utilizes conventional method to operate.
3.2 reverse transcription
The Reverse Transcriptase kit utilizing TAKARA company carries out the reverse transcription of RNA.
3.3QPCR
(1) design of primers
Coded sequence design QPCR amplification according to PCP4L1 gene in Genbank and β-actin gene is drawn Thing, is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Concrete primer sequence is as follows:
PCP4L1 gene:
Forward primer is 5 '-GAGGAGGAGGAGATTGAC-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-GAACTTGCCCTGAATAGC-3 ' (SEQ ID NO.4), β-actin gene:
Forward primer is 5 '-GCAGGTCATCACCATCGG-3 ' (SEQ ID NO.5)；
Reverse primer is 5 '-GCTGTCACCTTCACCGTTC-3 ' (SEQ ID NO.6).
(2) PCR reaction system is prepared according to table 1:
Wherein, SYBR Green polymerase chain reaction system is purchased from Invitrogen company.
Table 1 PCR reaction system
Reagent Volume
Forward primer 1μl
Reverse primer 1μl
SYBR Green polymerase chain reaction 12.5μl
Template 2μl
Deionized water Supply 25 μ l
(3) PCR reaction condition: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 40s) * 45 circulations.With SYBR Green, as fluorescent marker, reacts at the Light enterprising performing PCR of Cycler quantitative real time PCR Instrument, Determine that purpose band, Δ Δ CT method carry out relative quantification by melt curve analysis analysis and electrophoresis.
3.4 statistical method
Experiment is all according to being repeated 3 times, and result data is all to come in the way of mean+SD Representing, using SPSS13.0 statistical software to carry out statistical analysis, the difference between two groups uses t inspection, Think when P < has statistical significance when 0.05.
4, Western detection
Concrete steps are with embodiment 1.
5, result
As it is shown on figure 3, compared with the cell of transfection pcDNA3.1 empty carrier, transfect pcDNA3.1-PCP4L1 Cell in the mRNA level in-site of PCP4L1 significantly raise, difference has statistical significance (P < 0.05)； As shown in Figure 4, compared with the cell of transfection pcDNA3.1 empty carrier, transfection pcDNA3.1-PCP4L1's In cell, the protein level of PCP4L1 significantly raises, and difference has statistical significance (P < 0.05).
The impact of embodiment 3PCP4L1 gene pairs esophagus carcinoma proliferation
1, cell transfecting: esophageal cancer cell is carried out pcDNA3.1-PCP4L1 according to the method for embodiment 2 Transfection with pcDNA3.1.
2, transfection added 3H-TdR (1 μ Ci/ hole) after 24 hours, is further cultured for 24 hours, collects cell, liquid feeding Body scintillation solution, β calculating instrument detection cpm value.
3, statistical method
Experiment is all according to being repeated 3 times, and result data is all to come in the way of mean+SD Represent, use SPSS13.0 statistical software to carry out statistical analysis, PCP4L1 gene overexpression group with compare Difference between group uses t inspection, it is believed that when P < has statistical significance when 0.05.
4, result
Result shown in Fig. 5 shows: the vitro growth rates of transfection pcDNA3.1-PCP4L1 group is significantly lower than The vitro growth rates of transfection pcDNA3.1 empty carrier group, it is above-mentioned that difference has statistical significance (P < 0.05) Result shows that PCP4L1 expresses the growth that can suppress esophageal cancer cell.
The impact of embodiment 4PCP4L1 gene pairs Cell Cycle of Esophageal Carcinoma Cells
Using flow cytomery cell cycle, the mono-transfection reagent of PI used in the process is purchased from U.S. BD Products.
1, step: trypsinization after each group cell transfecting 48h, makes single cell suspension, and PBS washs 2 times, The ethanol of 70% is fixing overnight.Adding corresponding reagent by operating instruction, lucifuge places 30min, and flow cytometer is examined Survey each group of cell cycle.
2, statistical method
Experiment is all according to being repeated 3 times, and result data is all to come in the way of mean+SD Representing, using SPSS13.0 statistical software to carry out statistical analysis, the difference between two groups uses t inspection, Think when P < has statistical significance when 0.05.
3, result
Result as shown in table 2, compared with transfection pcDNA3.1 empty carrier group cell, transfects pcDNA3.1- PCP4L1 group cell is in the cell showed increased of G1 phase, is in the cell of S phase and significantly reduces.Above-mentioned knot Fruit shows, PCP4L1 gene expression can suppress cell cycle.
Cell cycle change (percentage ratio) after table 2 cell transfecting
Group The G1 phase The G2 phase The S phase
Transfection pcDNA3.1 empty carrier group 25.47±0.69 14.31±0.29 60.22±0.58
Transfection pcDNA3.1-PCP4L1 53.12±0.98* 14.02±1.24 32.86±1.03*
Note: compared with transfection pcDNA3.1 empty carrier group, * P < 0.05.
The explanation of above-described embodiment is only intended to understand the method for the present invention and core concept thereof.It is right to it should be pointed out that, For those of ordinary skill in the art, under the premise without departing from the principles of the invention, it is also possible to the present invention Carrying out some improvement and modification, these improve and modify also by the protection domain falling into the claims in the present invention.

Claims (10)

1. the product of detection PCP4L1 gene expression application in the instrument preparing diagnosis of esophageal cancer.
Application the most according to claim 1, it is characterised in that described product includes: by RT-PCR, Real-time quantitative PCR, immune detection, in situ hybridization chip or high-flux sequence detection of platform PCP4L1 gene table Reach the product with diagnosis of esophageal cancer.
Application the most according to claim 2, it is characterised in that described RT-PCR diagnosis of esophageal cancer Product at least include the primer of a pair specific amplified PCP4L1 gene；Described real-time quantitative PCR diagnoses The product of the esophageal carcinoma at least includes the primer of a pair specific amplified PCP4L1 gene；Described immune detection diagnoses The product of the esophageal carcinoma includes: the antibody being combined with PCP4L1 protein-specific；Described in situ hybridization diagnosis food The product of pipe cancer includes: with the probe of the nucleic acid array hybridizing of PCP4L1 gene；Described chip diagnoses esophagus The product of cancer includes: protein chip and gene chip；Wherein, protein chip includes special with PCP4L1 albumen Property combine antibody, gene chip includes the probe of the nucleic acid array hybridizing with PCP4L1 gene.
Application the most according to claim 3, it is characterised in that described real-time quantitative PCR diagnoses esophagus The primer such as SEQ ID NO.3 and SEQ ID of a pair specific amplified PCP4L1 gene that the product of cancer at least includes Shown in NO.4.
5. the instrument of a diagnosis of esophageal cancer, it is characterised in that described instrument includes detecting PCP4L1 gene table The reagent reached；Described reagent includes primer and/or probe, the detection PCP4L1 detecting PCP4L1 gene mRNA The antibody of albumen.
Instrument the most according to claim 5, it is characterised in that described detection PCP4L1 gene mRNA Primer include the primer pair shown in SEQ ID NO.3 and SEQ ID NO.4.
The accelerator of 7.PCP4L1 gene and/or its expression product answering in the medicine of the preparation treatment esophageal carcinoma With.
Application the most according to claim 7, it is characterised in that described promotion PCP4L1 gene expression Reagent, the reagent of promotion PCP4L1 gene expression product stability, promotion PCP4L1 gene expression product activity Reagent, promote PCP4L1 gene expression product function reagent.
Application the most according to claim 8, it is characterised in that promote the reagent of PCP4L1 gene expression Reagent that the carrier including the reagent containing PCP4L1 gene, carrying PCP4L1 gene or host cell are formed, Reagent containing PCP4L1 protein.
10. the pharmaceutical composition being used for treating the esophageal carcinoma, it is characterised in that described pharmaceutical composition includes Accelerator according to any one of 7-9.
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