CN106906305A - Applications of the lncRNA in sdenocarcinoma of stomach diagnosis - Google Patents

Abstract

The invention discloses applications of the lncRNA in sdenocarcinoma of stomach diagnosis, the lncRNA is selected from least two in LINC01234, HOTAIR and FEZF1 AS1.The present invention has been experimentally confirmed LINC01234, HOTAIR and FEZF1 AS1 and significantly rise has been expressed in patients with gastric adenocarcinoma；ROC curve analysis display, above-mentioned lncRNA use in conjunction has AUC higher, points out LINC01234, HOTAIR and FEZF1 AS1 to be united and applied in the diagnosis of sdenocarcinoma of stomach.

Description

Applications of the lncRNA in sdenocarcinoma of stomach diagnosis

Technical field

The invention belongs to biomedicine field, it is related to applications of the lncRNA in sdenocarcinoma of stomach diagnosis, is specifically related to The application of LINC01234, HOTAIR and/or FEZF1-AS1 in sdenocarcinoma of stomach diagnosis.

Background technology

Stomach cancer (gastric carcinoma, GC) is the common malignant tumour of the mankind, and more than 90% is stomach in patients with gastric cancer Gland cancer (gastric adenocarcinoma, GAC), meanwhile, stomach cancer is strong because of its invasion and metastatic, and turns into strong to the mankind Health endangers one of larger malignant tumour.Worldwide, the incidence of disease of stomach cancer comes the 5th of mankind's common cancer Position, but because its early symptom is not obvious, is usually mistaken as chronic gastritis (chronic gastritis) and delay diagnosis, Often find that diagnosis is later, treatment results are not satisfactory, and the death rate is high.In the cancer that the mankind suffer from, stomach cancer case fatality rate Come second.Asia is the district occurred frequently of stomach cancer, and China belongs to one of country of the High Risk For Gastric Cancer, East Coastal and west in China Southern area of the incidence of disease and the death rate of northern territory stomach cancer higher than China.Although modus operandi and change now for stomach cancer Treatment scheme suitable specification, but totality survival rate and prognosis it is still not fully up to expectations.The poor prognosis of patients with gastric cancer may Course of disease major part has much relations in progressive stage when being diagnosed with patient.The occurrence and development of stomach cancer be by it is various it is inside and outside because The coefficient result of element, but to its molecular biology behavior, we know little about it, for stomach cancer occurrence and development molecular mechanism The research tool of aspect is of great significance.

So far, early diagnosis, the excision of early stage row surgical radical treatment has important clinical significance for the prognosis of patients with gastric cancer. The early symptom of patients with gastric cancer is not true to type, and clinical manifestation is more hidden, and is difficult to clarify a diagnosis in early days, has been located when most of patients is made a definite diagnosis In the middle and advanced stage of disease, it is impossible to which surgical radical treatment excision reduce further 5 years survival rates of patient.At present, for progressive stage stomach The treatment method of cancer has invaded abdominal viscera or multiple mainly including surgery excision, new adjuvant chemotherapy and chemoradiation therapy etc. The patient of lymphatic metastasis, it tends to be difficult to which row surgical radical treatment cuts off.Therefore early diagnosis the having for patients with gastric cancer of stomach cancer Effect treatment has great importance.

Long-chain non-coding RNA (long non-coding RNA, abbreviation lncRNA) is the mammal for getting most of the attention recently Transcript profile important member.Compared with protein coding gene, it has obvious tissue specificity and stage of development specificity, more may be used From the expression and modification of multiple angular adjustment protein coding genes, such as epigenetics to protein coding gene is adjusted.Cause This, one of the lncRNA features of imbalance as kinds cancer.Being found to be for lncRNA studies the biological behaviour of malignant tumour, takes off Show that malignant tumor chemotherapy resistance essence provides important opportunity.Research sdenocarcinoma of stomach lncRNA express spectra changes, and further investigate this The relation that a little differential expression lncRNA molecules develop with sdenocarcinoma of stomach, for the Mechanism Study of sdenocarcinoma of stomach provides New Century Planned Textbook, be The treatment of sdenocarcinoma of stomach provides novel targets and new strategy.

The content of the invention

In order to make up the deficiencies in the prior art, an object of the present invention is to provide related to the development of gastric gland carcinogenesis Molecular marker.

The second object of the present invention, is to provide a kind of product and means for diagnosing sdenocarcinoma of stomach, by detection molecules mark The expression of thing judges whether patient with sdenocarcinoma of stomach or suffers from the risk of sdenocarcinoma of stomach, so as to instruct clinician to carry out morning Phase intervenes and treats, and improves the survival rate and life quality of patient.

To achieve these goals, the present invention is adopted the following technical scheme that：

The invention provides detection lncRNA reagent prepare diagnosis sdenocarcinoma of stomach product in application, the lncRNA Any two or three in selected from LINC01234, HOTAIR and FEZF1-AS1.

Further, the lncRNA is three kinds in LINC01234, HOTAIR and FEZF1-AS1.Use three kinds of lncRNA Combining diagnosis are carried out, with accuracy higher, and specificity and sensitiveness.

Further, the reagent of the detection lncRNA is selected from：

The probe of specific recognition LINC01234, HOTAIR or FEZF1-AS1；Or

The primer of specific amplification LINC01234, HOTAIR or FEZF1-AS1.

The invention provides it is a kind of diagnose sdenocarcinoma of stomach product, the product include detection LINC01234, HOTAIR and The reagent of at least two lncRNA expressions in FEZF1-AS1.

Further, the product includes flat by RT-PCR, real-time quantitative PCR, in situ hybridization, chip or high-flux sequence The method of platform detects the reagent of the lncRNA expressions.

The invention provides a kind of chip for diagnosing sdenocarcinoma of stomach, the product includes solid phase carrier, and is fixed on solid phase Oligonucleotide probe on carrier, in oligonucleotide probe specific recognition LINC01234, HOTAIR and FEZF1-AS1 At least two.Preferably, tri- kinds of oligonucleotide probe specific recognition LINC01234, HOTAIR and EZF1-AS1 lncRNA。

The solid phase carrier include inorganic carrier and organic carrier, the inorganic carrier included but is not limited to silicon carrier, Glass carrier, ceramic monolith etc.；The organic carrier includes polypropylene film, nylon membrane etc..

The invention provides it is a kind of diagnose sdenocarcinoma of stomach kit, the kit include specific amplification lncRNA and The primer pair of house-keeping gene, the lncRNA is selected from least two in LINC01234, HOTAIR and FEZF1-AS1.

Preferably, the kit includes tri- kinds of lncRNA of specific amplification LINC01234, HOTAIR and FEZF1-AS1 Primer pair.

Further, the kit also includes reverse transcription system, PCR system and operation instructions.Tool Body, the label for labeled RNA sample, and the substrate corresponding with the label are also contained in kit.Additionally, The various reagents required for extracting RNA, PCR, hybridization, colour developing etc. are may also include in described kit, including but not limited to： Extract, amplification liquid, hybridization solution, enzyme, comparison liquid, nitrite ion, washing lotion etc..Additionally, also including using in described kit saying Bright book and/or chip image analysis software.

Brief description of the drawings

Fig. 1 is that QPCR detects differential expression figures of five lncRNA in gastric adenocarcinoma tissue and cancer beside organism；Wherein, A is schemed It is differential expression figures of the QPCR detections LINC01234 in gastric adenocarcinoma tissue and cancer beside organism；Figure B is that QPCR detections HOTAIR exists Differential expression figure in gastric adenocarcinoma tissue and cancer beside organism；Figure C is QPCR detection FEZF1-AS1 groups by gastric adenocarcinoma tissue and cancer Differential expression figure in knitting；Figure D is differential expression figures of the QPCR detections LINC01105 in gastric adenocarcinoma tissue and cancer beside organism； Figure E is differential expression figures of the QPCR detections LINC00982 in gastric adenocarcinoma tissue and cancer beside organism.

Fig. 2 is using differences of five lncRNA of TCGA databases cross validation in for gastric adenocarcinoma tissue and cancer beside organism Expression figure；Wherein, figure A is that figure A is the differential expression figure for verifying LINC01234 in gastric adenocarcinoma tissue and cancer beside organism；Scheming B is Differential expression figures of the checking HOTAIR in gastric adenocarcinoma tissue and cancer beside organism；Figure C is checking FEZF1-AS1 in gastric adenocarcinoma tissue With the differential expression figure in cancer beside organism；Figure D is the differential expression for verifying LINC01105 in gastric adenocarcinoma tissue and cancer beside organism Figure；Figure E is the differential expression figure for verifying LINC00982 in gastric adenocarcinoma tissue and cancer beside organism.

Specific embodiment

The present invention, by high-flux sequence method, detects lncRNA in sdenocarcinoma of stomach sample by in-depth study extensively In the expression of tumor tissues and cancer beside organism, the wherein lncRNA fragments with obvious differential expression are found, inquire into itself and gastric gland Relation between the generation of cancer, so that for the early detection and targeted therapy of sdenocarcinoma of stomach find more preferable approaches and methods.Pass through Screening, raises present invention finds LINC01234, HOTAIR and FEZF1-AS1 conspicuousness in sdenocarcinoma of stomach.Use bioinformatics Method proves that LINC01234, HOTAIR or FEZF1-AS1 have as the early diagnosis that molecular marker is applied to sdenocarcinoma of stomach Accuracy higher.

Molecular marker

" molecular marker " is the expression of its expression in tissue or cell and normal or healthy cell or tissue Level compares any gene for changing.

" difference expression gene " or " gene differential expression " refers to, compared with normal or control target expression, is suffering from The gene of the activation of higher or lower level is obtained in patient's body of sdenocarcinoma of stomach.Additionally, " difference expression gene " or " differential gene Expression " includes the gene of the by stages interior activation for obtaining higher or lower level of difference of same disease.This species diversity can be by such as The mRNA level in-site of polypeptide, surface show, secrete or other distribution on change and proved.From the purpose of the present invention, " differential gene expression " is considered as from normal or subject with disease, or from the subject with disease it is each by stages In acquirement gene expression between exist 1.5 times or more, about 4 times or more, about 6 times or more, about 10 times or more Difference when the phenomenon that exists.

It would be recognized by those skilled in the art that practicality of the invention is not limited to marker gene of the invention The gene expression of any specific variants is quantified.As nonrestrictive example, marker gene LINC01234, LINC01105, HOTAIR and FEZF1-AS1 gene can have the nucleotide sequence shown in SEQ ID NO.1-3.In some implementations In scheme, its have with the same or analogous cDNA sequence of listed sequence at least 70%, all sequences listed as described above are at least 75%th, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical or phase As cDNA sequence.

LINC01234, HOTAIR and FEZF1-AS1 gene

LINC01234 genes take positioned at No. 12 areas 4 of chromosome long arm 2, and the gene presently, there are 5 transcripts, a kind of The nucleotide sequence of representational people LINC01234 genes is as shown in SEQ ID NO.1.HOTAIR genes are located at No. 12 chromosomes Long-armed 1st area 3 takes, and the gene presently, there are 5 transcripts, and a kind of nucleotide sequence of representational people HOTAIR genes is such as Shown in SEQ ID NO.2.FEZF1-AS1 genes take positioned at No. 7 areas 1 of chromosome long arm 3, and the gene presently, there are 3 transcriptions This, a kind of nucleotide sequence of representational people FEZF1-AS genes is as shown in SEQ ID NO.3.LncRNA bags in the present invention Include wild type, saltant type or its fragment.

It would be recognized by those skilled in the art that practicality of the invention is not limited to appoint target gene of the invention The gene expression of what specific variants is quantified.If when nucleic acid or its fragment and other nucleic acid (or its complementary strand) optimal comparisons When (have appropriate nucleotides inserted or missing), nucleotide base at least about 60%, usually at least about 70%, more Usually at least about 80%, it is preferably at least about 90% and more preferably at least about there is nucleosides in 95-98% nucleotide bases Acid sequence homogeny, then the two sequences are " substantially homologous " (or substantially similar).

Or, when nucleic acid or its fragment with another nucleic acid (or its complementary strand), a chain or its complementary series in selectivity When hybridizing under hybridization conditions, then there is substantially homologous or (homogeny) therebetween.When hybridization has more than specific general loss When selective, there is cross selection.Typically, exist at least about when at least about 14 the one of nucleotides section of sequences 55% homogeny, preferably at least about 65%, more preferably at least about 75% and most preferably at least about 90% homogeny when, hair Raw selective cross.It is as described herein, cognate pair than length can be sequence section more long, lead in certain embodiments Often it is at least about 20 nucleotides, more frequently at least about 24 nucleotides, typically at least about 28 nucleotides, more Typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides.

Therefore, the representative nucleotide sequence of polynucleotides of the invention and lncRNA preferably have at least 75%, it is more excellent At least 85%, more preferably at least 90% homology of choosing.It is highly preferred that in the presence of at least 95%, more preferably at least 98% homology.

The present invention can determine gene expression using any method known in the art.Those skilled in the art should manage Solution, the means for determining gene expression are not importances of the invention.The table of biomarker can be detected on transcriptional level Up to level.

Detection method

LncRNA of the invention detected using multiple nucleic acids technology known to persons of ordinary skill in the art, these skills Art includes but is not limited to nucleic acid sequencing, nucleic acid hybridization and nucleic acid amplification technologies.

The exemplary, non-limitative example of Nucleic acid sequencing techniques includes but is not limited to chain terminator (Sanger) sequencing and contaminates Material terminator sequencing.One of ordinary skill in the art it will be recognized that due to RNA in cell less stable and in an experiment Nuclease attack is more vulnerable to, therefore generally by RNA reverse transcriptions into DNA before sequencing.

The present invention can simultaneously be expanded before detection or with detection to nucleic acid (for example, ncRNA).Nucleic acid amplification technologies Exemplary, non-limitative example include but is not limited to：PCR (PCR), reverse transcriptase polymerase chain reaction (RT- PCR), the amplification (TMA) of transcriptive intermediate, ligase chain reaction (LCR), strand displacement amplification (SDA) and based on nucleotide sequence Amplification (NASBA).One of ordinary skill in the art will be it will be recognized that some amplification techniques (for example, PCR) needs will before amplification RNA reverse transcriptions are into DNA (for example, RT-PCR), and other amplification techniques then direct cloning RNA (for example, TMA and NASBA).

The PCR of commonly referred to PCR uses annealing and the primer extend of denaturation, primer pair and opposite strand Multiple circulations, exponentially increase the copy numbers of target nucleic acid sequence；The amplification of the transcriptive intermediate of TMA is (in substantial constant Temperature, ionic strength and pH under conditions of autocatalytically synthesize multiple copies of target nucleic acid sequence, wherein target sequence is more Individual RNA copies autocatalytically generate other copy；The ligase chain reaction of LCR uses miscellaneous with the adjacent area of target nucleic acid The two groups of complementary DNA oligonucleotides handed over；Other amplification methods are included for example：The expansion based on nucleotide sequence of commonly referred to NASBA Increase；Use rna replicon enzyme (commonly referred to Q β replicase) amplification probe molecule amplification in itself；Amplification method based on transcription； And the sequence amplification of self―sustaining.

In some embodiments of the present invention, gene expression product mark and optional montage mark can be by micro- Array analysis determination, for example, use Affymetrix arrays, cDNA microarrays, oligonucleotide microarray, point sample microarray or come From other microarray products of Biorad, Agilent or Eppendorf.Microarray provides special advantage, because they can be wrapped Include the lots of genes or optional splicing variants that can be analyzed in single test.In some cases, micro array apparatus can be wrapped Whole human genome or transcript group or their major part are included, so as to allow thoroughly evaluating gene expression pattern, genome sequence Row or optional montage.

Microarray analysis begin with methods known in the art from biological sample (such as biopsy or Fine needle aspiration Thing) extract and purification of nucleic acid.For expression and optional montage analysis, extracted from DNA and/or purifying RNA is probably favourable. Extracted from the RNA such as tRNA and rRNA of other forms and/or purifying mRNA is probably more favourable.

Can also with fluorescence, radionuclide or chemical labeling (such as biotin or digoxin) for example, by reverse transcription, PCR, engagement, chemical reaction or other technologies mark the nucleic acid of purifying.Mark can be direct or indirect, and it may enter one Step needs conjugation stage.Conjugation stage can occur before hybridization, for example, contaminated using aminoallyl-UTP and NHS amino-reactives Material (such as cyanogen indigo plant dyestuff), or after hybridization, for example, use biotin and the streptavidin of mark.The nucleotides of modification with Relatively low ratio (such as with 1aaUTP: 4TTP ratio) enzymatic is added compared with common nucleotides, generally yields every 60 bases 1 Individual result (using spectrophotometer measurement).Then such as post or diafiltration device purifying DNA be can use.Aminoallyl group The amino on the connector long of nucleoside base is attached to, it reacts with reactivity mark (such as fluorescent dye).

Hybridization probe is the fragment of the DNA or RNA of different length, and it is used to detecting in DNA or RNA sample and probe sequence The presence of complementary nucleotide sequence.Therefore, probe and its base sequence allow to make due to the complementarity between probe and target Obtain single-stranded nucleotide (the DNA or RNA) hybridization of probe-target base pairing.

Unless otherwise noted, term " probe " is often referred to be matched by complementary base and (often claimed with another polynucleotides Be " target polynucleotide ") combine polynucleotide probes.According to the preciseness of hybridization conditions, probe energy and lack with the probe The complementary target polynucleotide of complete sequence is combined.Probe can make direct or indirect mark, and its scope includes primer.Hybridization side Formula, includes, but are not limited to：Solution, solid phase, mixed phase or in situ hybridization determination method.

Exemplary probe in the present invention includes PCR primer and gene specific DNA oligonucleotide probe, for example, fix In the micro probe array on microarray substrate, quantitative nucleic acid enzyme protection inspection probe and molecular barcode connection probe and The probe being fixed on pearl.

The probe has the base sequence with the specific base sequence complementary of target gene.Here, so-called " complementation ", As long as hybridization, can not be complete complementary.These polynucleotides are commonly angled relative to the specific base sequence to be had More than 80%, preferably more than 90%, more preferably more than 95%, particularly preferred 100% homology.These probes can be DNA, Can also be RNA, furthermore it is possible to be to pass through PNA (Polyamide nucleic in one part or whole nucleotides Acid, peptide nucleic acid), LNA (registration mark, locked nucleic acid, Bridged Nucleic Acid, Cross-linked core Acid), ENA (registration mark, 2 '-O, 4 '-C-Ethylene-bridged nucleic acids), GNA (Glycerol Nucleic acid, glycerine nucleic acid), the artificial replacement nucleic acid such as TNA (Threose nucleic acid, threose nucleic acid) obtains Polynucleotides.

Biological analysis

Sample compares with normally

The result of the developed by molecule spectrum that can be carried out on the individual sample (estimating sample) for providing is with known or suspection Normal biological sample compares.Normal specimens be without or expected sample without any cancer, disease or situation or dividing It is negative sample to be tested for any Cancerous disease or situation in sub- expression pattern analysis.Normal specimens may be from and tested Body it is different individuality or from same individual.Normal specimens can be analyzed with test sample in same time or different time.

The analysis result of test sample can be compared with the result of the same analysis carried out in normal specimens.At some In the case of, the analysis result in normal specimens comes from database or benchmark.In some cases, dividing in normal specimens Analysis result is known or the value that is generally received by those of ordinary skill in the art.In some cases, compare is qualitatively. In other cases, it is quantitative for comparing.In some cases, qualitatively or quantitatively compare may include but be not limited to it is following a kind of or It is various：Compare fluorescent value, point intensity, absorbance, chemiluminescence signal, block diagram, threshold value crucial, significance,statistical value, Gene product expression level, the change of gene product expression level, may be selected extron using, changing of using of extron may be selected Change, protein level, DNA polymorphism, copy (copy) number variation, one or more DNA mark or region exist or do not deposit Instruction or nucleotide sequence.

Outcome evaluation

In some embodiments, assess developed by molecule using methods known in the art and compose result by gene outcome table , malignant class for example pernicious with particular phenotype (such as filter blocking cancer), benign or normality are used up to level or optional extron (such as Without disease or situation) it is associated.In some cases, it may be determined that the statistics level of confidence specified is to provide diagnosis Confidence level.For example, it may be determined that, the level of confidence more than 90% can be pernicious, malignant class or benign available pre- Measured value.In other embodiments, the level of confidence of higher or lower stringency can be selected.For example, can select about 70%th, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, 99.5% or 99.9% level of confidence is used as useful Phenotypic predictors.In some cases, the level of confidence for being provided can with the quality of sample, the quality of data, analysis Quality, the ad hoc approach for being used are related to the quantity of the gene expression product analyzed.Specified confidence for providing diagnosis Degree level can be based on expected false positive or the number and/or expense of false negative are selected.Select for realizing that specifies puts Confidence level or the method for the parameter of mark of the identification with diagnosis capability are included but is not limited to：Receiver Operating Characteristics Tracing analysis (Receiver Operator Curve analysis, ROC), binormal ROC, principal component analysis, part are minimum Square analysis, singular value decomposition, minimum absolute retract and selection operation device (least absolute shrinkage and Selection operator) analysis, minimum angular convolution is returned and threshold gradient is oriented to regularization (threshold gradient Directed regularization) method.

Data analysis

In some cases, original gene expression and optional montage data can be designed for normalization by application And/or improve the algorithm of the confidence level of data and improve.In some embodiments of the present invention, due to the individual data for processing The enormous quantity of point, data analysis requires computer or other devices, machine or equipment to apply various differences as herein described Algorithm." machine learning algorithm " refers to the computer based Forecasting Methodology for characterizing gene expression profile, the common skill in this area Art personnel are also referred to as " grader ".Corresponding to the signal of particular expression level, (it is analyzed for example, by based on microarray hybridization Obtain) algorithm process is generally gone through with express spectra of classifying.The study of supervision generally includes " training " grader to recognize between class Difference, then " test " accuracy of the grader on independent test collection.For new unknown sample, grader can be used Predict the class belonging to the sample.

In some cases, stablize average (RMA) method of many arrays to can be used to normalize initial data.RMA methods pass through The background correction intensity for calculating each matching cell on many microarrays starts.The value of background correction is limited to positive value.In background school After just, the algorithm of basis -2 of the matching cell intensity of each background correction is then obtained.Then quantile method for normalizing is used Make background correction, logarithmic transformed, matching intensity normalization on each microarray, wherein for each input array and each probe Expression value, array hundredths probe value is replaced by the average value of all array terciles.After quantile normalization, then can be by Normalized data are fitted in linear model, to obtain the expression measured value of each probe on each microarray.Then can make The logarithmic scale expression of normalized probe groups data is determined with Tukey medians smoothing algorithm.

Also further can cross filter data and may be considered that suspicious data to remove.In certain embodiments, from having The data obtained less than about the micro probe arrays of 4,5,6,7 or 8 guanosine+cytidylic acids can be considered as it is insecure, Because their abnormal hybridization tendency or secondary structure problem.Similarly, from greater than about 12,13,14,15,16,17,18, 19th, the data that the micro probe array of 20,21 or 22 guanosine+cytidylic acids is obtained be considered it is insecure, because It is their abnormal hybridization tendency or secondary structure problem.

In certain embodiments, selection can be classified to the reliability of probe groups with reference to data set by for a series of Insecure probe groups are excluded with from data analysis.For example, RefSeq or Ensembl (EMBL) are considered as unusual high-quality Reference data set.In some cases, the data of the probe groups of matching RefSeq or Ensembl sequences are expected from them High reliability especially can be included in microarray analysis experiments.Similarly, the reference data set of the relatively low reliability of Self Matching is carried out The data of probe groups can be excluded from further analysis, or consider to be included on the basis of each case.In some cases, Ensembl high fluxs cDNA (HTC) and/or mRNA can be used to determine separately or together the reliability of probe groups with reference to data set Property.In other cases, the reliability of probe groups can be graded.It is appreciated that to be present in and of the prior art any can be used to comment Estimate the combination between the given probe of developed by molecule spectrum and/or the method and distinct methods of the reliability of probe groups, all include In the present invention.

In a particular embodiment of the present invention, experiment is all completed according to being repeated 3 times, and result data is all with average The mode of value ± standard deviation represents, carries out using SPSS18.0 statistical softwares statistical analysis, gene experimental group with it is right Checked using t according to the difference between group, it is believed that work as P<There is statistical significance when 0.05.

Sample

In the present invention, term " sample " including cell, tissue, internal organs, body fluid (blood, lymph etc.), digestive juice, cough Phlegm, stomach born of the same parents' bronchus cleaning fluid, urine, excrement etc..Preferably, the sample is tissue, blood.In specific embodiment of the invention In, the sample is tissue.

The present invention is further detailed explanation with reference to the accompanying drawings and examples.Following examples are merely to illustrate this Invention rather than limitation the scope of the present invention.The experimental technique of unreceipted actual conditions in embodiment, generally according to conventional strip Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) described in condition, or according to the condition proposed by manufacturer.

Embodiment 1 screens the gene marker related to sdenocarcinoma of stomach

1st, sample collection

Collect 3 cancer beside organisms for having carried out the patients with gastric adenocarcinoma that radical excision is performed the operation and cancerous tissue sample, Huan Zhe Do not receive any chemotherapy or radiotherapy before carrying out gastrectomy, the information of the patient for including include sex, the age, lauren types, Histological classification and neoplasm staging, the acquirement of above-mentioned all samples are signed by the agreement of the committee of organizational ethics, and patient Informed Consent Form is ordered.

2nd, the preparation of RNA sample

The extraction of tissue RNA is carried out using the tissue RNA extracts kits of QIAGEN, the specific steps of by specification are carried out Operation.

The RNA concentration and purity extracted are detected using Nanodrop2000, agarose gel electrophoresis detection RNA Integrality, Agilent2100 determines RIN values.Concentration >=200ng/ μ L, OD260/280 is between 1.8~2.2.

3rd, rRNA is removed

The rRNA in total serum IgE is removed using Ribo-Zero kits.

4th, construction cDNA library (being operated using the Truseq RNA sample Prep Kit of Illumina)

(1) fragmentation RNA

Using metal ion, by RNA random fractures into 200bp or so small fragment.

(2) synthesis of cDNA

In the presence of reverse transcriptase, using random primer, cDNA the first chain cDNA are synthesized by template reverse transcription of mRNA, Replace dTTP with dUTP, synthesize second chain of cDNA.

(3) aptamer is connected

Add End Repair Mix that double-strand cDNA is mended into flat end, then add an A base in 3 ' ends, be used for Connect the joint of Y-shaped.

(4) chains of UNG enzymic digestions cDNA bis- are used

5th, high-flux sequence

(1) library enrichment, PCR expands 15 circulations；

(2) purpose band is reclaimed using 2% Ago-Gel；

(3) TBS380 (Picogreen) is quantitative, by the upper machine of ratio data mixing；

(4) bridge-type PCR amplifications are carried out on cBot, data set is generated；

(5) Hiseq4000 microarray datasets, carry out 2*150bp sequencings.

6th, high flux transcript profile sequencing data analysis

1) RNA-seq reads positioning

First by low-quality read removal obtain clean read, then using TopHat v1.3.1 will clean fragment and UCSC H.sapiens reference genes group (hg19) is matched, the index of the advance structure of H.sapiens UCSC hg19 editions Downloaded from TopHat homepages, and as reference gene group, when being matched with genome using TopHat, it is allowed to each read (acquiescence Sites, most 2 mispairing are matched to 20) there is multiple.TopHat sets up possible according to exon region and GT-AG shear signals Shearing site storehouse, navigates on genome the read for not navigating to genome according to these shearing site storehouses.We use The system default parameter of TopHat methods.

2) transcript abundance assessment

By Cufflinks v1.0.3 treatment, Cufflinks v1.0.3 are by RNA-seq pieces for the read file for matching Hop count mesh is standardized the relative abundance for calculating transcript.FPKM values refer to it is every 1,000,000 sequencing fragment in match it is specific The segment number of exon region gene 1kb long.The confidential interval of FPKM estimates is calculated by Bayesian inference method. The GTF comment files of the reference that Cufflinks is used download (Homo_ from Ensembl databases sapiens.GRCh37.63.gtf)。

3) detection of difference expression gene

Cuffdiff is transferred to by the Ensembl GTF files of download and by the original document that TopHat is matched, Cuffdiff re-evaluates the gene expression abundance of the transcript listed in GTF files using original matching files, detects difference table Reach.Work as FDR<0.05 and | log2FC>1 |, test display is considered as successfully more just differential expression.

7th, result

RNA-seq results show, compared with cancer beside organism, 74 genes of significant difference expression are had, wherein 43 LncRNA up-regulateds, 31 expression are lowered；Wherein LINC01234, HOTAIR and FEZF1-AS1 are expressed in gastric adenocarcinoma tissue Significantly raise；LINC01105, LINC00982 express significantly downward in gastric adenocarcinoma tissue.

The differential expression of embodiment 2QPCR sequence verification related genes

1st, large sample QPCR checkings are carried out to LINC00511 gene differential expressions.According to the sample collection side in embodiment 1 Formula collects gastric adenocarcinoma tissue and each 10 of cancer beside organism's sample.

2nd, RNA extraction steps are with embodiment 1.

3rd, reverse transcription：

(1) configuration of reverse transcription system

Using 25 μ l reaction systems, each sample takes 1 μ g total serum IgEs as template ribonucleic acid, is separately added into PCR pipe following Component：DEPC water, 5 × RT Buffer, 10mM dNTP, 0.1mM DTT, 30 μM of Oligo dT, 200U/ μ l M-MLV, Template ribonucleic acid.42 DEG C are incubated 1h, 72 DEG C of 10min, of short duration centrifugation.

(2) QPCR amplifications inspection

Design of primers

The primer sequence of LINC01234 genes is：

Forward primer：5’-CGTGAAGAGTAGATGTAGA-3’(SEQ ID NO.4)

Reverse primer：5’-TGTATCATAGGTGCTGTAA-3’(SEQ ID NO.5)

The primer sequence of LINC01105 genes is：

Forward primer：5’-AAGCCATAAGCAGGTATTCT-3’(SEQ ID NO.6)

Reverse primer：5’-GCGATTCAGACGACATTG-3’(SEQ ID NO.7)

The primer sequence of HOTAIR genes is：

Forward primer：5’-AATAGACATAGGAGAACACTT-3’(SEQ ID NO.8)

Reverse primer：5’-AATCTTAATAGCAGGAGGAA-3’(SEQ ID NO.9)

The primer sequence of FEZF1-AS1 genes is：

Forward primer：5’-ATTGATTGATTAGCGTTCTG-3’(SEQ ID NO.10)

Reverse primer：5’-GTGTCTTGAATATGGTTGTT-3’(SEQ ID NO.11)

The primer sequence of LINC00982 genes is：

Forward primer：5’-TTTCTGGCACCAGAGATTGAG-3’(SEQ ID NO.12)

Reverse primer：5’-CATCCAGGCAACAGCATTCG-3’(SEQ ID NO.13)

The primer sequence of house-keeping gene β-actin is：

Forward primer：5’-AGCGAGCATCCCCCAAAGTT-3’(SEQ ID NO.14)

Reverse primer：5’-GGGCACGAAGGCTCATCATT-3’(SEQ ID NO.15)

Prepare 25 μ l reaction systems

Add SYBR Green PCRs system 12.5 μ l, forward and reverse primer (5 μM) each 1 μ l, template cDNA 2.0 μ l, without the μ l of enzyme water 8.5.Operations are carried out on ice, and each sample sets 3 parallel pipes, all amplified reaction counterpoises Multiple more than the three times reliabilities to ensure result.

Amplification

Setting program is：95 DEG C of 60s, (95 DEG C of 15s, 60 DEG C of 15s, 72 DEG C of 45s) × 35 circulations.

It is anti-in the Light Cycler enterprising performing PCRs of fluorescence real-time quantitative PCR instrument using SYBR Green as fluorescent marker Should, to be analyzed by melt curve analysis and electrophoresis determines purpose band, Δ Δ CT methods carry out relative quantification.

4th, result

Result is as shown in figure 1, compared with cancer beside organism, LINC01234, HOTAIR and FEZF1-AS1 are in gastric adenocarcinoma tissue Middle up-regulated；LINC01105, LINC00982 express downward in gastric adenocarcinoma tissue；Difference has statistical significance (P< 0.01) it is, consistent with RNA-sep results.

Embodiment 3 analyzes expressions of the candidate lncRNAs in TCGA databases

1st, Data Collection

The lncRNA expression modal datas of 285 gastric adenocarcinoma tissues and 30 cancer beside organisms are collected from TCGA databases, point LncRNA LINC01234, LINC01105, HOTAIR, FEZF1-AS1, LINC00982, LINC00261 are in gastric adenocarcinoma tissue for analysis With the expression in cancer beside organism；Draw box-shaped figure.

2nd, ROC curve analysis

Using the pROC bags in R language analyze lncRNA LINC01234, LINC01105, HOTAIR, FEZF1-AS1, The Receiver Operating Characteristics of LINC00982, calculate the accurate confidence space of binomial, draw ROC curve.

3rd, the ROC curve analysis of candidate lncRNA use in conjunction

4th, result

The expression of candidate lncRNA as shown in Fig. 2 compare control group, LINC01105 (P=0.002724), LINC00982 (P=0.001156) expresses significantly downward in gastric adenocarcinoma tissue；FEZF1-AS1 (P=2.2e-16), HOTAIR (P=2.2e-16), LINC01234 (P=6.78e-16) expresses significantly rise in gastric adenocarcinoma tissue.

The ROC curve of candidate lncRNA shows, LINC01105, FEZF1-AS, HOTAIR, LINC00982, LINC01234 AUC be higher than 0.7.Wherein, FEZF1-AS1, HOTAIR, LINC01234 have specificity and sensitiveness higher, and LINC01105 has sensitiveness higher, illustrates that FEZF1-AS1, HOTAIR, LINC01234, LINC01105 are applied to gastric gland The diagnosis of cancer has accuracy higher.

As shown in table 1, FEZF1-AS1, HOTAIR, LINC01234 combine to be used for the AUC of candidate's lncRNA use in conjunction Diagnosis is compared and is used alone with accuracy higher.

The candidate's lncRNA use in conjunction AUCs of table 1

The preparation of the sdenocarcinoma of stomach diagnostic kit of embodiment 4

According to the correlation of LINC01234, HOTAIR, FEZF1-AS1 gene and sdenocarcinoma of stomach, can be by detection The expression of LINC01234, HOTAIR, FEZF1-AS1 gene diagnoses whether sdenocarcinoma of stomach occurs, accordingly the invention provides It is a kind of that the kit of sdenocarcinoma of stomach, reagent constituents are diagnosed based on detection LINC01234, HOTAIR, FEZF1-AS1 gene expression It is as follows：SYBR Green PCRs system, amplification LINC01234, HOTAIR, FEZF1-AS1 gene and β-actin The primer pair of gene, M-MLV reverse transcriptions system, RNA extracts reagents.Expand the forward primer sequence of LINC01234 genes for 5 '- CGTGAAGAGTAGATGTAGA-3 ', reverse primer sequences are 5 '-TGTATCATAGGTGCTGTAA-3 '；Amplification HOTAIR genes Forward primer sequence be 5 '-AATAGACATAGGAGAACACTT-3 ', reverse primer sequences are 5 '- AATCTTAATAGCAGGAGGAA-3’；Expand the forward primer sequence of FEZF1-AS1 genes for 5 '- ATTGATTGATTAGCGTTCTG-3 ', reverse primer sequences are 5 '-GTGTCTTGAATATGGTTGTT-3 '；Amplification β-actin Forward primer sequence be 5 '-AGCGAGCATCCCCCAAAGTT-3 ', reverse primer sequences are 5 '- GGGCACGAAGGCTCATCATT-3’.SYBR Green PCRs system includes PCR buffer solutions, dNTPs, SYBR Green fluorescent dyes.PCR buffer compositions are：25mM KCl、2.5mM MgCl₂、200mM(NH₄)₂SO₄.M-MLV reverse transcriptions System component is：T repeat oligonucleotides Oligo (dT), reverse transcription reaction liquid, M-MLV reverse transcriptases, RNase inhibitor, dNTPs.Reverse transcription reaction liquid component is：The Tris-HCl (PH8.3) of 250mM, the MgCl of KCl, 15mM of 375mM₂, 50mM's DTT.RNase inhibitor is the recombinant protease of the Noncompetition inhibition RNase of Bacillus coli expression.RNA extracts reagents are included Trizol, chloroform, isopropanol, 75% ethanol.

The preparation of the sdenocarcinoma of stomach diagnostic kit of embodiment 5

According to the correlation of LINC01234, FEZF1-AS1 gene and sdenocarcinoma of stomach, can by detect LINC01234, The expression of FEZF1-AS1 genes diagnoses whether sdenocarcinoma of stomach occurs, accordingly the invention provides one kind based on detection LINC01234, FEZF1-AS1 gene expression diagnose the kit of sdenocarcinoma of stomach, and reagent constituents are as follows：SYBR Green are polymerized Primer pair, the M-MLV reverse transcription bodies of PCR system, amplification LINC01234, FEZF1-AS1 gene and β-actin genes System, RNA extracts reagents.The forward primer sequence for expanding LINC01234 genes is 5 '-CGTGAAGAGTAGATGTAGA-3 ', instead It is 5 '-TGTATCATAGGTGCTGTAA-3 ' to primer sequence；Expand the forward primer sequence of FEZF1-AS1 genes for 5 '- ATTGATTGATTAGCGTTCTG-3 ', reverse primer sequences are 5 '-GTGTCTTGAATATGGTTGTT-3 '；Amplification β-actin Forward primer sequence be 5 '-AGCGAGCATCCCCCAAAGTT-3 ', reverse primer sequences are 5 '- GGGCACGAAGGCTCATCATT-3’.SYBR Green PCRs system includes PCR buffer solutions, dNTPs, SYBR Green fluorescent dyes.PCR buffer compositions are：25mM KCl、2.5mM MgCl₂、200mM(NH₄)₂SO₄.M-MLV reverse transcriptions System component is：T repeat oligonucleotides Oligo (dT), reverse transcription reaction liquid, M-MLV reverse transcriptases, RNase inhibitor, dNTPs.Reverse transcription reaction liquid component is：The Tris-HCl (PH8.3) of 250mM, the MgCl of KCl, 15mM of 375mM₂, 50mM's DTT.RNase inhibitor is the recombinant protease of the Noncompetition inhibition RNase of Bacillus coli expression.RNA extracts reagents are included Trizol, chloroform, isopropanol, 75% ethanol.

The explanation of above-described embodiment is only intended to understand the method for the present invention and its core concept.It should be pointed out that for this For the those of ordinary skill in field, under the premise without departing from the principles of the invention, some improvement can also be carried out to the present invention And modification, these are improved and modification will be also fallen into the protection domain of the claims in the present invention.

SEQUENCE LISTING

<110>Gu Jianbin

<120>Applications of the lncRNA in sdenocarcinoma of stomach diagnosis

<160> 15

<170> PatentIn version 3.5

<210> 1

<211> 2088

<212> DNA

<213>People source

<400> 1

acattttcct ggagcggctg ggaaagagga gtctctcgaa attcagcaac tgctaacagc 60

gaggaggggg tgctagccag gatcactccc tccgaagtca caccagaggg agggctggag 120

ggagaatcaa atgaaagaga gagggagaga aaggaaggaa gagaaggagg gagaatggaa 180

ggatgtatgg atttggatgt atgggttcca ttccttctac cctggcaaaa gcttactcat 240

ccttcagtgt ccatccaaaa tggcatctct tcttgggcac catctccaga gtctcctgca 300

agcaggtagc tacatcccac aaaacaacca cccatctgac catgcaagtg tgtgggggaa 360

tgaagaccag cccaggaatc tgagatgaat gttttgcttc ttgctctgcc actgactcac 420

agggtacccc aagcaagtcc cttcacctcc tcggtctcag tttctccatt tatactacta 480

aggcagtgga catgatggcc tcaagtctca acatttttat tccctgaaaa gaaactcttg 540

gtgctgagtg gtcttcttct gccctgacat ctcacctttc aaacgcttgt ctcttctcac 600

cccacccacc acttagcatg tgctcttgga gaacacccag gactcagggt ctctgcacac 660

atcatgagtc cagctcagca tttgaggtgg aatcagagga aaggaaggag agtggggaga 720

atagtcagta ttttggcaag ctcatcatac cttccccctt tgtatagaaa cttcaaacca 780

tttccctttg aaggcactac ctccttcccc cagttataaa tgagtgaagg tctcaagcct 840

ggacaaccaa atgcacagtg attggttcaa ctctggacct gtgactcaag ccagaccaag 900

ggagtgacat gcagggcttt gcctggaact attctgaaag gggcactctc tttctgctgg 960

gctactgata atatgtgcat ccgtgataga agagcctgcc tgataataaa gccaataagg 1020

gaagagcaga gccaagagat ggtgggagag cagatgcctg aaaatatcat ttgagcccct 1080

gggtccagct gcacctgaag ccaccacgat ctcctggact ttgcagttac ttgagttcat 1140

aaataccctt tggcattaag ccagattgag tcttaatgca tatagaaata agagaaatga 1200

gaaaagaaat tgaaaagaga gacagcagag aactgattct ctactagagc ctccagaagg 1260

aatcaactct gccaacacct tgattttgga cttgtggcct tcagaacagt gaaatgataa 1320

acatctgtta ttttaaggta cctagtgtgt aacattccgt catgacagcc ctaggaaatg 1380

aatacagcga ggaaaatcct accagcacaa aggcatggag gtgccaggat gtctcctctg 1440

cgtgaagagt agatgtagat gaggctggaa ttatctatcc tagctgccca gacccatgtg 1500

cctttgtttt atgtagttac agcacctatg atacatattt gttaccatgt atgtcactat 1560

gaacctcctc tggaggacgg agaagtcaaa taccttaatt attccaacac aagcttgcgt 1620

gaacagataa tcatcactac gagtatattg tgtgcctgct aagcaccaca cctgagataa 1680

gcatttgctg tggtttgaat gtcccctcca aagctcatgc taaaatttaa ttgccattgc 1740

aacagtgttg caaggtgaga actttaagag atgatcaggt catgagaact ctgccctcgt 1800

gaatggatta atacccttat cgcagtagtg gacccccctt ttctcttgct gtctgtctct 1860

catgttagct tgtgcttcct cctttcacca tgggataaca cagcaagaag cccctcacca 1920

gatgctggca ccttgctatt ggacttccgg cctccagaac tgagaaatac atgtcttttc 1980

cttataaatt acccagtctg tggtattctg ttatagcggc agaaaatgga ctaagacggc 2040

attttgcata cattatctgt cttattaaaa ataatgtttt tgcccagg 2088

<210> 2

<211> 2421

<212> DNA

<213>People source

<400> 2

ccagttctca ggcgagagcc gcggctgaca gggtctggga cagaaggaaa gccctccagc 60

ctccaggccc tgccttctgc ctgcacattc tgccctgatt tccggaacct ggaagcctag 120

gcaggcagtg gggaactctg actcgcctgt gctctggagc ttgatccgaa agcttccaca 180

gtgaggactg ctccgtgggg gtaagagagc accaggcact gaggcctggg agttccacag 240

accaacaccc ctgctcctgg cggctcccac ccgggactta gaccctcagg tccctaatat 300

cccggaggtg ctctcaatca gaaaggtcct gctccgcttc gcagtggaat ggaacggatt 360

tagaagcctg cagtagggga gtggggagtg gagagaggga gcccagagtt acagacggcg 420

gcgagaggaa ggaggggcgt ctttattttt ttaaggcccc aaagagtctg atgtttacaa 480

gaccagaaat gccacggccg cgtcctggca gagaaaaggc tgaaatggag gaccggcgcc 540

ttccttataa gtatgcacat tggcgagaga agtgctgcaa cctaaaccag caattacacc 600

caagctcgtt ggggcctaag ccagtaccga cctggtagaa aaagcaacca cgaagctaga 660

gagagagcca gaggagggaa gagagcgcca gacgaaggtg aaagcgaacc acgcagagaa 720

atgcaggcaa gggagcaagg cggcagttcc cggaacaaac gtggcagagg gcaagacggg 780

cactcacaga cagaggttta tgtattttta ttttttaaaa tctgatttgg tgttccatga 840

ggaaaaggga aaatctaggg aacgggagta cagagagaat aatccgggtc ctagctcgcc 900

acatgaacgc ccagagaacg ctggaaaaac ctgagcgggt gccggggcag cacccggctc 960

gggtcagcca ctgccccaca ccgggcccac caagccccgc ccctcgcggc caccggggct 1020

tccttgctct tcttatcatc tccatcttta tgatgaggct tgttaacaag accagagagc 1080

tggccaagca cctctatctc agccgcgccc gctcagccga gcagcggtcg gtggggggac 1140

tgggaggcgc taattaattg attcctttgg actgtaaaat atggcggcgt ctacacggaa 1200

cccatggact cataaacaat atatctgttg ggcgtgagtg cactgtctct caaataattt 1260

ttccataggc aaatgtcaga gggttctgga tttttagttg ctaaggaaag atccaaatgg 1320

gaccaatttt aggaggccca aacagagtcc gttcagtgtc agaaaatgct tccccaaagg 1380

ggttgggagt gtgttttgtt ggaaaaaagc ttgggttata ggaaagcctt tccctgctac 1440

ttgtgtagac ccagcccaat ttaagaatta caaggaagcg aaggggttgt gtaggccgga 1500

agcctctctg tcccggctgg atgcagggga cttgagctgc tccggaattt gagaggaaca 1560

tagaagcaaa ggtccagcct ttgcttcgtg ctgattccta gacttaagat tcaaaaacaa 1620

atttttaaaa gtgaaaccag ccctagcctt tggaagctct tgaaggttca gcacccaccc 1680

aggaatccac ctgcctgtta cacgcctctc caagacacag tggcaccgct tttctaactg 1740

gcagcacaga gcaactctat aatatgctta tattaggtct agaagaatgc atcttgagac 1800

acatgggtaa cctaattata taatgcttgt tccatacagg agtgattatg cagtgggacc 1860

ctgctgcaaa cgggactttg cactctaaat atagacccca gcttgggaca aaagttgcag 1920

tagaaaaata gacataggag aacacttaaa taagtgatgc atgtagacac agaaggggta 1980

tttaaaagac agaaataata gaagtacaga agaacagaaa aaaaatcagc agatggagat 2040

taccattccc aatgcctgaa cttcctcctg ctattaagat tgctagagaa ttgtgtctta 2100

aacagttcat gaacccagaa gaatgcaatt tcaatgtatt tagtacacac acagtatgta 2160

tataaacaca actcacagaa tatattttcc atacattggg taggtatgca ctttgtgtat 2220

atataataat gtattttcca tgcagtttta aaatgtagat atattaatat ctggatgcat 2280

tttctgtgca ctggttttat atgccttatg gagtatatac tcacatgtag ctaaatagac 2340

tcaggactgc acattccttg tgtaggttgt gtgtgtgtgg tggttttatg cataaataaa 2400

gttttacatg tggtgaatat a 2421

<210> 3

<211> 2653

<212> DNA

<213>People source

<400> 3

gaaaactttg ggcttggcat taggagagcc tcggctgaaa tccgaggttt tgaacgcgat 60

tttttccgac agaagctggg cgctttcttt catgtaatgc tgcagctgag cctgggacag 120

gtctttgaaa gctactccag aagggtattt ctccaccgcc gggaccacca gtttattcct 180

ttcggctaaa tacgtttttg gctgcgggtg caaaggggaa ctgaggaagt aggaggccac 240

cgggtggatg ttcacgccgg ctgccgggtg gcatgggccg tcacctcggt tcaggtagca 300

caaggcgccc atggcgtgga atgaagagtg gttgaccaca cgcggcctta ccagcttgta 360

ctgctgcagc ggcagcgcgt cgcgggccag gtcgcccttg agactcagtg cgcagttgag 420

caggtcgctg cagctgaatg cgggagccga gggcaccgcc gcgggcgccg ccggggcctc 480

cagactggcc ttccgcggct cggagcccgt cactcctgcc ttggggctcg tgtcgtaggc 540

cacaggcacg aaggggatca tgcaggggat cgacgagttg agatgcagag agtgcttggg 600

ttcccccttg gcatcaccag ccgaacctgc ctgcccagcc caatggactc ctgccagccc 660

atcgcagagt tcttggcgca ccaatgactc ggggacaatc actacttatt tctatcaata 720

gaaagtgttg tgtcaataac gcagccaaga tctcgccaat cgttaacttc caagaggaga 780

agggctcggg gttgcccgct cccggaatcc cggagtctcc gcggcccgaa ccgagtctgg 840

accatttaga agacgccggc aggtaactgg cctccccaaa cgccccgaaa attaaaagcc 900

ttaggaggct tgttctgtgt ttgtggtttt gtaaaggcaa tgcccgcggc aataggcctg 960

ggaaagtcgc actttcatcc acaaagttga ggagttgcaa ggaataattg ggtgtcacac 1020

cacaagcaac cttgactcga tcggacccca cccaggatac acacagacgc gcgcgcgcgc 1080

acgcttccga gtttccattg agagtctgga aagagcattt ctttcttaag tacctgcgct 1140

cagctagagc tctcccggag cattttaaat actgaatgcc gagcggagaa gggaaacaga 1200

gtgtattaat ccccactccg gttgccggtg tctgcaagcc tcctctaagc agcgcaggcg 1260

gcagcctgga tgtctgtgcg catcaaggag acttgccggc gccagagttg tgtccacagg 1320

tgcacttgtg cctcccaaag agcgcgcgta gagaccattt cccctgcaac gccccccagc 1380

cgaacacccg ccgggtgtgg cacctcggcc gaaactcctg gggcggcgcg gtggagcgtg 1440

tggaggtttc cgggaagacg cctacggctg cggctccgac tgtgcgggct cccgccgccc 1500

aggcaagagc gtccaggagc gcgctggaag ctgcggcgcg caccttcccc ggcgcagtct 1560

cttgccagcc ggcgatcacg cagagctgaa ccccgcagct aggcgcgcag gaagggctgc 1620

acagagtttg aagacacggc agctgtaagc tggcgtgagg aggccaaagg agcgcgcaga 1680

gctgcaagca aagggcggtg gggcctggac cctgaagtga gtggccgacc gcagcagctc 1740

aaggtgccat ctgcctggca agctacccgg gtccctccgc cccactctaa ctttccgcga 1800

aaatcagttc cgcctctgcc ccaagagcca agaggggtgg aaaggaagag agcttgggct 1860

gggaacgcgc ctgatgtcta acagaaaggc agtggcagct ggcgaaaaat ccccctcctg 1920

ctgcactcct ccgccaccgg gctaggttga aaggaaaaag ccaagaagcg cgaagcagcc 1980

cggctctgga aggctttgga gagtccagct gacttgtaca caattcgcgg cctctggagc 2040

cttacctgcc ttcttgactg aatgccacca gttcgtttta cgcttgtggc tttttgttga 2100

agtccctatc ataagcaaat gcagtttctg gctatggtta ctgcaattcg aaataaaagg 2160

cctgtgaggt gtgtccctcc agactgagag gctatgactc agggttggac tttatggaaa 2220

aaaagaagtt tataaaacaa gtaacccagt ttctccaacc tttactgtgg agcaacagcg 2280

tcaagcaaca aaacccagca aagttttgct tgtcacttac aagggtgtta ctgaaatctg 2340

gaaggcgaac aattagaaga tagcccaaac atgctctatt gctctacact gaaatacttg 2400

cctccaataa tagaaaaaac ctgttttcgt gcaattgatt gattagcgtt ctgcagcagt 2460

ccctggacaa ttttaaagac attttgtgtt tgtggccaaa attagaaaca agcagacaca 2520

cacacaaaac aaccatattc aagacacggt tcgactgttt ccttgacact acccacgaag 2580

tttaaagcat tttttatgtt attttcaaac tttcttaagt gatcccatga aataaaagtc 2640

gataatagaa att 2653

<210> 4

<211> 19

<212> DNA

<213>Artificial sequence

<400> 4

cgtgaagagt agatgtaga 19

<210> 5

<211> 19

<212> DNA

<213>Artificial sequence

<400> 5

tgtatcatag gtgctgtaa 19

<210> 6

<211> 20

<212> DNA

<213>Artificial sequence

<400> 6

aagccataag caggtattct 20

<210> 7

<211> 18

<212> DNA

<213>Artificial sequence

<400> 7

gcgattcaga cgacattg 18

<210> 8

<211> 21

<212> DNA

<213>Artificial sequence

<400> 8

aatagacata ggagaacact t 21

<210> 9

<211> 20

<212> DNA

<213>Artificial sequence

<400> 9

aatcttaata gcaggaggaa 20

<210> 10

<211> 20

<212> DNA

<213>Artificial sequence

<400> 10

attgattgat tagcgttctg 20

<210> 11

<211> 20

<212> DNA

<213>Artificial sequence

<400> 11

gtgtcttgaa tatggttgtt 20

<210> 12

<211> 21

<212> DNA

<213>Artificial sequence

<400> 12

tttctggcac cagagattga g 21

<210> 13

<211> 20

<212> DNA

<213>Artificial sequence

<400> 13

catccaggca acagcattcg 20

<210> 14

<211> 20

<212> DNA

<213>Artificial sequence

<400> 14

agcgagcatc ccccaaagtt 20

<210> 15

<211> 20

<212> DNA

<213>Artificial sequence

<400> 15

gggcacgaag gctcatcatt 20

Claims (10)

1. application of the reagent of detection lncRNA in the product for preparing diagnosis sdenocarcinoma of stomach, it is characterised in that the lncRNA choosings Any two or three from LINC01234, HOTAIR and FEZF1-AS1.

2. application according to claim 1, it is characterised in that the lncRNA is LINC01234, HOTAIR and FEZF1- Three kinds in AS1.

3. application according to claim 1, it is characterised in that the reagent of the detection lncRNA is selected from：

The probe of specific recognition LINC01234, HOTAIR or FEZF1-AS1；Or

The primer of specific amplification LINC01234, HOTAIR or FEZF1-AS1.

4. it is a kind of diagnose sdenocarcinoma of stomach product, it is characterised in that the product include detection LINC01234, HOTAIR and The reagent of at least two lncRNA expressions in FEZF1-AS1.

5. product according to claim 4, it is characterised in that the product include by RT-PCR, real-time quantitative PCR, The method of in situ hybridization, chip or high-flux sequence platform detects the reagent of the lncRNA expressions.

6. a kind of chip for diagnosing sdenocarcinoma of stomach, it is characterised in that the product includes solid phase carrier, and is fixed on solid phase carrier On oligonucleotide probe, in oligonucleotide probe specific recognition LINC01234, HOTAIR and FEZF1-AS1 extremely It is few two kinds.

7. chip according to claim 6, it is characterised in that the oligonucleotide probe specific recognition LINC01234, Tri- kinds of lncRNA of HOTAIR and FEZF1-AS1.

8. it is a kind of diagnose sdenocarcinoma of stomach kit, it is characterised in that the kit include specific amplification lncRNA and pipe The primer pair of family's gene, the lncRNA is selected from least two in LINC01234, HOTAIR and FEZF1-AS1.

9. kit according to claim 8, it is characterised in that the kit include specific amplification LINC01234, Tri- kinds of primer pairs of lncRNA of HOTAIR and FEZF1-AS1.

10. kit according to claim 8 or claim 9, it is characterised in that the kit also includes reverse transcription system, poly- Polymerase chain reaction system and operation instructions.