CN106868204A - A kind of biomarker for sdenocarcinoma of stomach diagnosis - Google Patents

Abstract

The invention discloses a kind of biomarker for sdenocarcinoma of stomach diagnosis, the biomarker is LINC01234.LINC01234 expressions in patients with gastric adenocarcinoma are significantly raised, this differential expression of TCGA database cross validations；ROC curve analysis is carried out to LINC01234, it is found that LINC01234 has AUC higher, and accuracy and specificity higher, point out LINC01234 that the early diagnosis of sdenocarcinoma of stomach can be applied to as potential label.

Description

A kind of biomarker for sdenocarcinoma of stomach diagnosis

Technical field

The invention belongs to biomedicine field, it is related to a kind of biomarker for sdenocarcinoma of stomach diagnosis, it is specific described Biomarker is LINC01234.

Background technology

Stomach cancer is produced to human health and existence and causes serious prestige as a kind of tumour in the world with higher incidence The side of body, the morbidity and mortality of stomach cancer all come the prostatitis of malignant tumour in the world.Stomach cancer is various pernicious swollen in China Ranked first in knurl, incidence gastric cancer has obvious region gender gap, in the northwest of China and coastal region in east China incidence gastric cancer rate ratio Southern area is evident as height.The age is sent out well more than 50 years old, and the ratio between men and women's incidence of disease is 2：1.The canceration of stomach cancer be more than one because Element, multi-step, Multi stage development process, worldwide statistical data display gastric cancerous area difference also clearly, This may have certain relation with the environment of different geographical and cooking culture difference.

Stomach cancer can be divided into two big types from the angle of histopathology：A kind of is the stomach cancer of general type, that is, I Most common gland cancer, gland cancer has the different Differentiation Types such as high, medium and low, also has some special again according to the different of differentiation degree Gland cancer such as signet ring cell cancer, myxoadenocarcinoma etc. of different Differentiation Types.Another is then the stomach cancer of some more rare types, The adenosquamous carcinoma of such as stomach, the squamous cell carcinoma of stomach, class cancer and undifferentiated carcinoma etc..The stomach cancer of early stage is usually without obvious clinical Symptom, and the discomfort such as sour regurgitation, nausea that some patientss are showed also usually can mutually be obscured with gastritis, gastric ulcer, so for Even if tumor development also occurs to local advanced or late period without specific clinical symptoms for most of patients with gastric cancer.

It is very low in the diagnosis of China's early carcinoma of stomach, clinic conventional diagnostic techniques such as gastroscope, UGI radiography, CT Deng due to inspection fee higher and undertaking certain pain and risk is difficult popularization, tumour in conventional Serological testing Whether unsatisfactory, the searching novel tumor mark that Sensitivity Specificity is all showed such as label such as CEA, CA199, CA724 Thing is always the important topic in tumor research field.

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.As the research on lncRNA is increasingly goed deep into, people are gradually found that lncRNA's is various important Cell adjusting function, and participate in various life processes such as cell metabolism, development, including Genomic Imprinting, chromosome are reinvented, carefully Born of the same parents' cycle regulating, montage regulation and control, mRNA degradeds and antisense modulation etc..In recent years, it is found that some are different successively in tumor tissues The lncRNA for often expressing, it even more increasingly manifests as the potentiality of tumor markers, cancer target.LncRNA is explored in stomach The diagnosis and treatment that act as stomach cancer and Mechanism Study in cancer provide new strategy.

The content of the invention

In order to make up the deficiencies in the prior art, the invention provides a kind of biological marker related to the development of gastric gland carcinogenesis Thing, judges whether patient suffers from sdenocarcinoma of stomach or suffer from the risk of sdenocarcinoma of stomach by the expression of detection molecules mark, from And instruct the clinician to carry out early intervention and treatment, improve 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 It is LINC01234.

Further, lncRNA up-regulateds in patients with gastric adenocarcinoma sample.

Further, the product is detected by the method for sequencing technologies, nucleic acid hybridization technique, nucleic acid amplification technologies The expression of LINC01234 genes.

Further, the nucleic acid amplification technologies are selected from 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 nucleic acid sequence The amplification (NASBA) of row.Wherein, PCR is needed RNA reverse transcriptions into DNA (RT-PCR) before amplification, and TMA and NASBA directly expand Increase RNA.

Further, the LINC01234 include as shown in sequence SEQ ID NO.1 the polynucleotides of nucleotide sequence or its Fragment, homologue, variant or derivative.

The invention provides a kind of product of LINC01234 expressions in vitro detection sample, the product includes system Agent, chip or kit.

Further, the product includes the probe of specific recognition LINC01234；Or

The primer of specific amplification LINC01234.

Product the invention provides LINC01234 expressions in vitro detection sample is preparing diagnosis early stage sdenocarcinoma of stomach Application in instrument.

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, the oligonucleotide probe specific recognition LINC01234.

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 a kind of kit for diagnosing sdenocarcinoma of stomach, the kit includes that the kit includes detection One or more reagent of LINC01234；With

One or more material being selected from the group：Container, operation instructions, positive control, negative control thing, buffering Agent, auxiliary agent or solvent.

Further, the kit includes passing through RT-PCR, real-time quantitative PCR, in situ hybridization, chip, high-flux sequence Method detects the expression reagent of LINC01234.

Present invention also offers LINC01234 inhibitor prepare treatment sdenocarcinoma of stomach pharmaceutical composition in application.

Further, the inhibitor of the LINC01234 includes：With LINC01234 or its transcript as target sequence and can Suppress the disturbing molecule of LINC01234 gene expressions or genetic transcription, including：ShRNA (children purpura nephritis), siRNA (siRNA), dsRNA, Microrna, antisensenucleic acids, or can express or form the shRNA, it is siRNA, dsRNA, small The construction of RNA, antisensenucleic acids.

Brief description of the drawings

Fig. 1 is the expression figure in patients with gastric adenocarcinoma using QPCR detections LINC01234；

Fig. 2 is using differential expression figures of the TCGA database cross validations LINC01234 in patients with gastric adenocarcinoma；

Fig. 3 is ROC curve figures of the LINC01234 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, present invention finds LINC01234, conspicuousness is raised in gastric adenocarcinoma tissue sample, is demonstrate,proved using bioinformatics method Bright, LINC01234 has accuracy higher as the early diagnosis that molecular marker is applied to sdenocarcinoma of stomach.

Biomarker

" biomarker " 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.Used as nonrestrictive example, marker gene LINC01234 genes can have There is the nucleotide sequence shown in SEQ ID NO.1.In some embodiments, it has identical with listed sequence at least 70% Or similar cDNA sequence, all sequences listed as described above at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%th, 95%, 96%, 97%, 98% or at least 99% same or analogous cDNA sequence.

LINC01234 genes

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.LncRNA in the present invention includes such as sequence The polynucleotides or its fragment, homologue, variant or derivative of nucleotide sequence shown in row SEQ ID NO.1.

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.

The nucleic acid of non-amplification or amplification can be detected by any conventional means in the present invention.

Nucleic acid hybridization technique in the present invention include but is not limited in situ hybridization (ISH), microarray and Southern or Northern traces.In situ hybridization (ISH) be it is a kind of use mark complementary DNA or RNA chains as probe with position tissue one Part or section (original position) or if organize it is sufficiently small if for entirely organize (full organization embedding ISH) in specific DNA or The hybridization of RNA sequence.DNA ISH can be used to determine the structure of chromosome.RNA ISH are used to measure and position tissue section or complete MRNA and other transcripts (for example, ncRNA) in organization embedding.Generally processed sample cell and tissue solid with original position Targeting transcript, and increase the entrance of probe.Probe hybridizes with target sequence at high temperature, then washes unnecessary probe off.Point Shi Yong not autoradiograph, fluorescence microscopy or immunohistochemistry, the base to using radiation, fluorescence or antigenic mark in tissue The probe of mark is positioned and quantified.ISH also can be used two or more to pass through radioactivity or other nonradioactive labelings The probe of substance markers, to detect two or more transcripts simultaneously.

Southern and Northern traces are respectively used to detect specific DNA or RNA sequence.Make to be extracted from sample DNA or RNA fracture, it is separated by electrophoresis on matrix gel, be then transferred on molecular filter.Make filter combine DNA or RNA and and the complementary label probe hybridization of sequence of interest.Detection is attached to the hybridization probe of filter.A kind of change of the program Change form is reverse northern trace, wherein the substrate nucleic acid for being fixed to film is the set of separate DNA fragmentation, and probe is From tissue extraction and the RNA that is marked.

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.

Chip, kit

Chip of the present invention includes：Solid phase carrier；And the oligonucleotides on the solid phase carrier is fixed in order Probe, described oligonucleotide probe specifically corresponds to the part or all of sequence shown in LINC01234.

Specifically, suitable probe can be designed according to lncRNA of the present invention, is fixed on solid phase carrier, shape Into " oligonucleotide arrays ".Described " oligonucleotide arrays " refer to (addressable i.e. with distinctive with addressable point The position that address is characterized) array, a coupled characteristic oligonucleotides is contained in each addressable point.According to Need, oligonucleotide arrays can be divided into multiple Asia battle arrays.

In the present invention, the solid phase carrier is including plastic products, microparticle, membrane carrier etc..The plastic products can lead to Cross non-covalent or physical absorption mechanism to be combined with antibody or proteantigen, the most frequently used plastic products are made for polystyrene Small test tube, globule and micro-reaction plate；The microparticle is the microballoon or particle aggregated into by high polymer monomer, and its diameter is generally Micron, due to the functional group that can be combined with protein, easily forming chemical coupling with antibody (antigen), binding capacity is big；Institute Stating membrane carrier includes nitrocellulose filter, glass fibre element film and miillpore filter etc. nylon membrane.

The present invention can be DNA, RNA, DNA-RNA chimera, PNA for the oligonucleotide probe of LINC01234 genes Or other derivatives.The length of the probe is not limited, as long as completing specific hybrid and purpose nucleotide sequence specificity With reference to any length can.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the spy The length of pin can be grown to 60,80,100,150,300 base-pairs or longer, or even whole gene.Due to different probe lengths There are different influences to hybridization efficiency, signal specificity, the length of the probe is typically at least 14 base-pairs, it is most long general No more than 30 base-pairs, complementary length is optimal with 15-25 base-pair with purpose nucleotide sequence.The probe itself is mutual Complementary series is most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.

Kit of the invention includes the reagent of the detection LINC01234 genes of detection effective dose, the one kind being selected from the group Or many kinds of substance：Container, operation instructions, positive control, negative control thing, buffer, auxiliary agent or solvent.For example for mixing The solution of outstanding or fixed cell, detectable label or tag makes nucleic acid be easy to the solution of hybridization, for the molten of cell lysis Liquid, or for the solution of nucleic acid purification.

Can also have the operation instructions of kit in kit of the invention, wherein describing how to enter using kit Row detection, and how disease development to be judged using testing result.

Using kit of the invention, can be detected by the various methods (including but not limited to) being selected from the group LINC01234：Real Time RT-PCR, biochip test method, southern blotting technique method or RNA blottings or hybridization in situ, High-flux sequence method.Those of ordinary skill in the art can be according to physical condition and needing to be adjusted detection mode and change.

Chip of the invention or kit can be used for detect including including LINC01234 genes multiple genes (for example with The related multiple genes of sdenocarcinoma of stomach) expression.

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 (coy) number variation, one or more DNA mark or region are presence or absence of 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 very high-quality The reference data set of amount.In some cases, the data of the probe groups of matching RefSeq or Ensembl sequences are due to their expections High reliability especially can be included in microarray analysis experiments.Similarly, the reference data of the relatively low reliability of Self Matching are carried out The data of the probe groups of collection 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

The structure of cDNA library is carried out using the Truseq RNA sample Prep Kit using Illumina, specific behaviour Carried out as by specification.

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 expresses significantly rise in gastric adenocarcinoma tissue.

The differential expression of the QPCR sequence verification related genes of embodiment 2

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.2)

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

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

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

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

Prepare 25 μ l reaction systems

Add SYBR Green PCRs system 12.5 μ l, each 1 μ l of forward and reverse primer (5 μM), template CDNA2.0 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 reactions In triplicate more than ensureing the reliability of 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 as shown in figure 1, compared with cancer beside organism, LINC01234 up-regulateds in gastric adenocarcinoma tissue；Difference has Statistical significance (P<0.01) it is, consistent with RNA-sep results.

Embodiment 3 analyzes expressions of the LINC01234 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 Expressions of the analysis lncRNA LINC01234 in gastric adenocarcinoma tissue and cancer beside organism；Draw box-shaped figure.

2nd, ROC curve analysis

The Receiver Operating Characteristics of lncRNALINC01234 are analyzed using the pROC bags in R language, binomial is calculated and is accurately put Letter space, draws ROC curve.

3rd, result

The expression of LINC01234 is as shown in Fig. 2 compare control group, LLINC01234 (P=6.78e-16) is in gastric gland Expressed in cancerous tissue and significantly raised.

The ROC curve of LINC01234 as shown in figure 3, the AUC of LINC01234 a height of 0.894, and with spy higher The opposite sex and sensitiveness, illustrate that FLINC01234 is applied to the diagnosis of sdenocarcinoma of stomach with accuracy higher.

A kind of diagnostic kit of the sdenocarcinoma of stomach of embodiment 4

According to the correlation of LINC01234 genes and sdenocarcinoma of stomach, can be by detecting the expression of LINC01234 genes To diagnose whether sdenocarcinoma of stomach occurs, gastric gland is diagnosed based on detection LINC01234 gene expressions the invention provides one kind accordingly The kit of cancer, reagent constituents are as follows：SYBR Green PCRs system, amplification LINC01234 genes and β- The primer pair of actin genes, M-MLV reverse transcriptions system, RNA extracts reagents.Expand the forward primer sequence of LINC01234 genes It is 5 '-CGTGAAGAGTAGATGTAGA-3 ', reverse primer sequences are 5 '-TGTATCATAGGTGCTGTAA-3 '；Amplification β- The forward primer sequence of actin is 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 MgCl2 of KCl, 15mM of 375mM, 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>Beijing Yang Shen biology information technologies Co., Ltd

<120>A kind of biomarker for sdenocarcinoma of stomach diagnosis

<160> 5

<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

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cgtgaagagt agatgtaga 19

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tgtatcatag gtgctgtaa 19

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agcgagcatc ccccaaagtt 20

<210> 5

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<212> DNA

<213>Artificial sequence

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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 is LINC01234。

2. application according to claim 1, it is characterised in that the product passes through sequencing technologies, nucleic acid hybridization technique, core The method of sour amplification technique detects the expression of LINC01234 genes.

3. the purposes according to any one of claim 1 or 2, it is characterised in that the LINC01234 includes such as sequence SEQ The polynucleotides of nucleotide sequence shown in ID NO.1 or its fragment, homologue, variant or derivative.

4. in a kind of vitro detection sample LINC01234 expressions product, it is characterised in that the product include preparation, Chip or kit.

5. product according to claim 4, it is characterised in that including：

The probe of specific recognition LINC01234；Or

The primer of specific amplification LINC01234.

6. application of the product described in claim 4 or 5 in diagnosis early stage sdenocarcinoma of stomach instrument is prepared.

7. 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, the oligonucleotide probe specific recognition LINC01234.

8. it is a kind of diagnose sdenocarcinoma of stomach kit, it is characterised in that the kit include detection LINC01234 one kind or many Plant reagent；With

One or more material being selected from the group：Container, operation instructions, positive control, negative control thing, buffer, help Agent or solvent.

9. kit according to claim 8, it is characterised in that the kit includes passing through RT-PCR, real-time quantitative PCR, in situ hybridization, chip, the expression reagent of high-flux sequence method detection LINC01234.

Application of the inhibitor of 10.LINC01234 in the pharmaceutical composition for preparing treatment sdenocarcinoma of stomach.