CN109825592A - A kind of purposes of biomarker relevant to breast cancer occurrence and development - Google Patents
A kind of purposes of biomarker relevant to breast cancer occurrence and development Download PDFInfo
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Abstract
The invention discloses a kind of purposes of biomarker relevant to breast cancer occurrence and development, and in particular to application of the biomarker LINC01612 in breast cancer diagnosis and treatment.
Description
Technical field
The invention belongs to biomedicine fields, are related to a kind of use of biomarker relevant to breast cancer occurrence and development
On the way, being specifically related to biomarker is LINC01612.
Background technique
The main reason for breast cancer is the cancer being most often diagnosed in global women and cancer mortality, wherein women
Pathogenesis of breast carcinoma is most.According to the latest news, whole world patient with breast cancer about 167.1 ten thousand newly-increased every year, dies of breast cancer every year
Patient about 52.2 ten thousand (Desantis C, Ma J, Bryan L, et al.Breast cancer statistics, 2013 [J]
.Ca ACancer Journal for Clinicians,2014,64(1):52-62.).In China, breast cancer incidence is
42.55/10 ten thousand people account for the 17.10% of women whole Incidence.The major way of breast cancer treatment is improvement root at present
Art is controlled, but therapeutic effect is not good enough, and still have gap to the target for extending the life cycle of patient and improving the quality of living is reached.According to
The expression difference of ER (estrogen receptor), PR (progesterone receptor) and Her-2 (ErbB-2) can be with
Breast cancer is divided into 4 hypotypes (Luminal A type, Luminal Type B, Her-2 overexpression type and Basal-like type), without
Patient with breast cancer with hypotype shows different radiation sensitivities, studied on the basis of existing different subtype and pathological characters it
Between relationship and different subtype and radiotherapy prognosis situation, need further exploratory development.
At present about mammary gland molecular isoform expression and clinical pathologic characteristic relationship and with armpit after modified skinsuture
Influence research of the patient with breast cancer of lymph node positive to radiotherapy side effect is still rare, in tumor size, lymphatic metastasis mammary gland
Situation is expressed in the tissue such as cancer TNM stage, clinical stages and the relationship of lymphatic metastasis and prognosis is still not clear.
With the development of biotechnology, it has been found that intermediary RNA (mRNA) is the sub-fraction of total serum IgE.Non-coding RNA
It is unable to coding protein, but is played a role in terms of structure, function, regulation.Based on expression and function, non-coding RNA can be by
It is indefinite to be divided into the RNA that runs one's home (rRNA, transfer RNA, small nucleolar RNA), the rna regulation of low expression and Some features
RNA.According to size, rna regulation can be further classified as short chain non-coding RNA (< 200bp, such as miRNAs, siRNAs and
PiRNAs) and long-chain non-coding RNA (> 200bp, such as lincRNAs, macroRNAs), i.e. 1ncRNA (A.Pauli,
J.L.Rinn,A.F.Schier.Non-coding RNAs as regulators of embryogenesis[J].Nature
Reviews Genetics,2011,12(2):136-149).Study lncRNA relevant to breast cancer occurrence and development, for into
One step explains the pathogenesis of breast cancer, realizes the finer parting of breast cancer and reaches the personalization of patient with breast cancer
Precisely treatment has great importance.
Summary of the invention
In order to make up for the deficiencies of the prior art, the present invention provides a kind of gene relevant to breast cancer occurrence and development, it is
The early diagnosis of breast cancer provides a kind of product and means and uses marker compared to the diagnostic method of traditional breast cancer
Diagnosis of Breast cancer has timeliness, sensitivity, to make patient that can know disease risks in disease early stage, to take corresponding
Prevention and treatment measure.
To achieve the goals above, the present invention adopts the following technical scheme:
The present invention provides the purposes of LINC01612 a kind of, are used to prepare the product of Diagnosis of Breast cancer.
Further, the product is by passing through the expression of LINC01612 and reference in measurement sample in measurement sample
Level is determined compared to lowering.
Further, the method detection that the product passes through sequencing technologies, nucleic acid hybridization technique, nucleic acid amplification technologies
The expression of LINC01612 gene.
Further, the breast cancer is Luminal A type breast cancer.
Further, the nucleic acid amplification technologies are selected from polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction
(RT-PCR), the amplification (TMA) of transcriptive intermediate, ligase chain reaction (LCR), strand displacement amplification (SDA) and be based on nucleic acid sequence
The amplification (NASBA) of column.Wherein, PCR needs directly expand RNA reverse transcription at DNA (RT-PCR), TMA and NASBA before amplification
Increase RNA.
The present invention provides a kind of product of LINC01612 expression in vitro detection sample, the product includes system
Agent, chip or kit.Wherein, the chip includes solid phase carrier；And it is fixed on the oligonucleotide probe on solid phase carrier.
The kit includes genetic chip or polymerase chain reaction system.
Further, the product of LINC01612 expression includes: in the vitro detection sample
The probe of specific recognition LINC01612；Or
The primer of specific amplification LINC01612.
Further, the primer sequence of the specific amplification LINC01612 such as SEQ ID NO.1 and SEQ ID NO.2 institute
Show.
The present invention provides the products of LINC01612 expression in vitro detection sample to prepare Diagnosis of Breast cancer tool
In purposes.
The present invention provides a kind of kits of Diagnosis of Breast cancer, comprising:
Detect one or more reagents of LINC01612；With
One or more substances selected from the group below: container, operation instructions, positive control, negative control object, buffering
Agent, auxiliary agent or solvent.
Further, the kit is used to detect LINC01612:qRT-PCR, biochip test by the method for the following group
Method, southern blotting technique method or RNA blotting hybridization in situ.
The advantages of the present invention:
Present invention firstly discovers that the expression of LINC01612 gene is related to breast cancer, LINC01612 is in breast cancer
It expresses and lowers in patient, pass through the expression of LINC01612 in detection subject's sample, it can be determined that whether subject suffers from
Breast cancer, and the risk to suffer from breast cancer are adopted so that clinician be instructed to provide prevention scheme or therapeutic scheme to subject
It is diagnosed with molecular marked compound, there is timeliness, sensitivity, specificity.
Detailed description of the invention
Fig. 1 is the expression figure using QPCR detection LINC01612 gene in breast cancer tissue.
Specific embodiment
The present invention after extensive and in-depth study, passes through the method for high-flux sequence combination bioinformatic analysis, inspection
Transcriptional level of the lncRNA in breast cancer sample and normal sample is surveyed, discovery is wherein with the lncRNA piece of obvious differential expression
Section, inquires into its relationship between the generation of breast cancer, so that the early detection for breast cancer finds better approaches and methods.
By screening, present invention firstly discovers that LINC01612 conspicuousness is lowered in patient with breast cancer, LINC01612 is prompted to can be used as
Testing index is applied to the clinical diagnosis of breast cancer, while according to the relationship of LINC01612 and breast cancer, being overexpressed by design
Carrier etc. can increase the method treatment breast cancer of LINC01612 expression.
LINC01612 gene
LINC01612 gene is located on No. 4 chromosomes of people, gene I/D 101928223, LINC01612 packet in the present invention
Include LINC01612 polynucleotides or its segment, homologue, variant or derivative.A kind of core of representative LINC01612 gene
Nucleotide sequence is as shown in NR_125889.1 in genebank.
It would be recognized by those skilled in the art that practicability of the invention is not limited to appointing to target gene of the invention
The gene expression of what specific variants is quantified.If when nucleic acid or its segment and other nucleic acid (or its complementary strand) optimal comparison
When (have nucleotides inserted appropriate or missing), at least about 60% nucleotide base, usually at least about 70%, more
Usually at least about 80%, it is preferably at least about 90% and there are nucleosides more preferably at least about in 95-98% nucleotide base
The acid sequence phase same sex, then the two sequences are " substantially homologous " (or substantially similar).
The present invention can use any method known in the art measurement gene expression.Those skilled in the art should manage
Solution, the means for measuring gene expression are not importances of the invention.The table of biomarker can be detected on transcriptional level
Up to level.
Detection technique
LncRNA of the invention is 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 present invention simultaneously can expand nucleic acid (for example, ncRNA) before detection or with detection.Nucleic acid amplification technologies
Exemplary, non-limitative example include but is not limited to: polymerase chain reaction (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
It expands (NASBA).Those skilled in the art will be it will be recognized that certain amplification techniques (for example, PCR) needs will before amplification
RNA reverse transcription is at DNA (for example, RT-PCR), and other amplification techniques then direct cloning RNA (for example, TMA and NASBA).
In general, PCR uses the annealing of denaturation, primer pair and opposite strand and multiple circulations of primer extend, with index side
The copy number of formula increase target nucleic acid sequence；Reverse transcriptase (RT) is then used for the DNA (cDNA) complementary from mRNA preparation by RT-PCR,
Then cDNA is generated to multiple copies of DNA by PCR amplification；TMA is in substantially constant temperature, ionic strength and pH
Under the conditions of autocatalytically synthesize multiple copies of target nucleic acid sequence, wherein multiple RNA copy of target sequence is autocatalytically given birth to
At other copy, TMA is optionally included using blocking, part, terminate part and other modified parts, to improve TMA process
Sensitivity and accuracy；LCR uses the two groups of complementary DNA oligonucleotides hybridized with the adjacent area of target nucleic acid.DNA few nucleosides
Acid is covalently attached in thermal denaturation, hybridization and the multiple circulations of the repetition of connection by DNA ligase, to generate detectable double-strand
Connect oligonucleotide product；SDA uses multiple circulations of following steps: primer sequence pair and the opposite strand of target sequence move back
Fire carries out primer extend under there are dNTP α S to generate (hemiphosphorothioated) of half thiophosphorylation of double-strand
Primer extension product, the nicking that the endonuclease that semi-modified restriction enzyme enzyme recognition site carries out mediates, and from cutting
The polymerase-mediated object drawn that the mouth end 3' carries out extends to replace existing chain and generate and set for next round primer annealing, nicking and chain
The chain changed expands so as to cause the geometry of product.
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 trace.In situ hybridization (ISH) be it is a kind of use label complementary DNA or RNA chain as probe with position tissue one
Part or slice (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 for determining the structure of chromosome.RNA ISH is for measuring with position tissue slice or entirely
MRNA and other transcripts (for example, ncRNA) in organization embedding.Usually sample cell and tissue are handled in situ solid
Targeting transcript, and increase the entrance of probe.Probe hybridizes with target sequence at high temperature, then washes off extra probe.Point
Not Shi Yong autoradiograph, fluorescence microscopy or immunohistochemistry, in tissue with radiation, fluorescence or antigenic mark base
The probe of label is positioned and is quantified.ISH can also 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 trace is respectively used to detection specific DNA or RNA sequence.Make to extract 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 with and the complementary label probe of sequence of interest hybridize.Detection is integrated 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 isolated DNA fragmentation, and probe is
From tissue extraction and the RNA that is marked.
Chip, kit
Chip of the present invention includes: solid phase carrier；And orderly it is fixed on the oligonucleotides on the solid phase carrier
Probe, the oligonucleotide probe some or all of specifically correspond to shown in LINC01612 sequence.
Specifically, can lncRNA according to the present invention, design suitable probe, be fixed on solid phase carrier, shape
At " oligonucleotide arrays "." oligonucleotide arrays " refer to (addressable i.e. with distinctive with addressable point
The position that address is characterized) array, each addressable point is containing a coupled characteristic oligonucleotides.According to
It needs, oligonucleotide arrays can be divided into multiple sub- battle arrays.
In the present invention, the solid phase carrier includes plastic products, microparticle, membrane carrier etc..The plastic products can lead to
It crosses non-covalent or physical absorption mechanism to combine with antibody or proteantigen, most common plastic products are made of polystyrene
Small test tube, globule and micro-reaction plate；The microparticle is the microballoon or particle aggregated by high polymer monomer, and diameter is mostly
Micron easily can form chemical coupling with antibody (antigen), binding capacity is big with the functional group in conjunction with protein due to having；Institute
Stating membrane carrier includes the miillpore filters such as nitrocellulose filter, glass fibre element film and nylon membrane.
" probe " refers to can be with the molecule in conjunction with the particular sequence of another molecule or subsequence or other parts.Unless otherwise finger
Out, term " probe " is often referred to match by complementary base and combine with another polynucleotides (often referred to as " target polynucleotide ")
Polynucleotide probes.According to the preciseness of hybridization conditions, probe energy and with the probe lack sufficient sequence complementarity target it is more
Nucleotide combines.Probe can make direct or indirect label, and range includes primer.Crossing system includes, but are not limited to: molten
Liquid phase, solid phase, mixed phase or in situ hybridization measuring method.
" probe " refers to can be with the molecule in conjunction with the particular sequence of another molecule or subsequence or other parts.Unless otherwise finger
Out, term " probe " is often referred to match by complementary base and combine with another polynucleotides (often referred to as " target polynucleotide ")
Polynucleotide probes.According to the preciseness of hybridization conditions, probe energy and with the probe lack sufficient sequence complementarity target it is more
Nucleotide combines.Probe can make direct or indirect label, and range includes primer.Crossing system includes, but are not limited to: molten
Liquid phase, solid phase, mixed phase or in situ hybridization measuring method.
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 have usually relative to the specific base sequence
80% or more, preferably 90% or more, more preferable 95% or more, particularly preferred 100% homology.These probes can be DNA,
It is also possible to RNA, furthermore it is possible to pass through PNA (Polyamide nucleic in part of it or whole nucleotides
Acid, peptide nucleic acid), LNA (registered trademark, locked nucleic acid, Bridged Nucleic Acid, Cross-linked core
Acid), ENA (registered trademark, 2 '-O, 4 '-C-Ethylene-bridged nucleic acids), GNA (Glycerol
Nucleic acid, glycerol nucleic acid), the artificial replacement nucleic acid such as TNA (Threose nucleic acid, threose nucleic acid) obtains
Polynucleotides.
Term " homology " refers to complementary degree.There may be Homoeologies or complete homology (that is, same
Property).The sequence of partial complementarity is at least partly to inhibit the nucleic acid molecules of complete complementary and " substantially homologous " target nucleic acid miscellaneous
The nucleic acid molecules of friendship.Fully-complementary sequence can be by being surveyed under the conditions of property low strict using hybridization with the inhibition of target sequence hybridized
Fixed (Southern or Northern trace, solution hybridization etc.) is checked.Substantially homologous sequence or probe will compete simultaneously
Inhibit complete homologous nucleic acid molecules under the conditions of property low strict and the combination (that is, hybridization) of target.This is not to say, low strict
Property condition is to allow the condition of non-specific binding；Spy is combined between two sequences of property condition requirement low strict
The interaction of anisotropic (that is, selectivity).Being not present for non-specific binding can be by using substantially incomplementarity (for example, low
In about 30% identity) the second target tested；In the case where non-specific binding is not present, probe will not be with second
Incomplementarity target hybridization.
Term " hybridization " in the present invention is used to refer to the pairing of complementary nucleic acid.Hybridization and intensity for hybridization are (that is, between nucleic acid
Association intensity) influenced by factor such as below: the stringency of complementarity, related condition between nucleic acid is formed
Hybrid Tm and nucleic acid in G:C ratio.The individual molecule of pairing in its structure containing complementary nucleic acid is known as " self
Hybridization ".
The present invention can be DNA, RNA, DNA-RNA chimera, PNA for the oligonucleotide probe of LINC01612 gene
Or other derivatives.There is no limit as long as complete specific hybrid and purpose nucleotide sequence specificity for the length of the probe
In conjunction with any length is ok.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, the spy
The length of needle can be grown to 60,80,100,150,300 base-pairs or longer or even whole genes.Due to different probe lengths
There is different influences to hybridization efficiency, signal specificity, the length of the probe is typically at least 14 base-pairs, and longest is general
No more than 30 base-pairs, the length complementary with purpose nucleotide sequence are best with 15-25 base-pair.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 for detecting a effective amount of detection LINC01612 gene, one kind selected from the group below
Or many kinds of substance: container, operation instructions, positive control, negative control object, buffer, auxiliary agent or solvent.Such as mixing
The solution of outstanding or fixed cell, detectable label or tag, the solution for making nucleic acid be easy to hybridize, for the molten of lytic cell
Liquid, or the solution for nucleic acid purification.
In kit of the invention can also have kit operation instructions, be described how using kit into
Row detection, and how disease development to be judged using testing result.
Using kit of the invention, can be detected by various methods (including but not limited to) selected from the group below
LINC01612: Real Time RT-PCR, biochip test method, southern blotting technique method or RNA blotting or hybridization in situ.
Those of ordinary skill in the art can be according to physical condition and needing to be adjusted detection mode and change.
Chip or kit of the invention can be used for detect including LINC01612 gene multiple genes (such as with
The relevant multiple genes of breast cancer) expression.
Sample
In the present invention, " sample " means any wherein can detecte interior cell, tissue or body fluid in gene expression
Sampling.The example of this sample includes, but are not limited to biopsy and smear.Body fluid for use in the present invention includes blood
Liquid, lymph, urine, saliva, nipple aspirate fluid, gynecological fluid or any other body secretion fluid or derivatives thereof.Blood can
To include whole blood, blood plasma, serum or any blood derivatives.In some embodiments, biological sample includes mammary glandular cell, special
It is not the breast tissue from biopsy, such as breast tumor tissue samples.It can be obtained by multiple technologies from main body
Biological sample, including, such as by scraping or one region of erasing, by using needle suction of cells or body fluid or passing through shifting
Except tissue sample (i.e. biopsy).The method for collecting various biological samples is well known in the art.In some embodiments
In, by, for example, fine needle aspiration biopsy, aspiration biopsy or Biopsy obtain sample of breast tissue.It can be to cell or group
It knits using fixative and dyeing liquor to save sample and convenient for checking.Biological sample, especially sample of breast tissue can shift
To glass slide to amplify observation.In one embodiment, biological sample be formalin fix, the mammary gland of paraffin embedding
Tissue sample, especially primary Breast Tumor Samples.In different implementation scenarios, the tissue core sample instructed from virologist
Product obtain tissue sample.
Term " diagnosis " in the present invention refers to through the S&S of disease or genetic analysis, pathological analysis, tissue
The identification disease such as credit analysis.
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this
It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.
Embodiment 1 screens gene marker relevant to breast cancer
1, sample collection
The cancerous tissue and corresponding normal tissue sample for collecting 4 Luminal A type breast cancer respectively are (apart from tumour side
5 centimeters of edge), high-flux sequence is carried out, all patients are preoperative not to carry out chemotherapy, radiotherapy and endocrine therapy, and all patients are equal
Informed consent, the acquirement of above-mentioned all samples pass through the agreement of the committee, organizational ethics, and patient information is as shown in table 1.
1 sample information of table
2, the preparation and quality analysis of RNA sample
The RNA in tissue is extracted using Takara RNA extracts kit (Code NO.9767), steps are as follows:
1) fresh or Cryopreservation animal tissue sample is transferred quickly in the mortar of Liquid nitrogen precooler, uses pestle
Tissue abrasion is continuously added liquid nitrogen therebetween, until being ground into powder.The sample being ground into powder is added to containing cracking
In the 1.5ml sterile centrifugation tube of Buffer RL, blown and beaten repeatedly with pipettor until without obvious sediment in lysate.
2) by lysate in 12,000rpm, 4 DEG C of centrifugation 5min.
3) careful Aspirate supernatant is into new 1.5ml RNase Free Tube.
4) 70% ethyl alcohol isometric with liquid is added, is uniformly mixed solution using liquid-transfering gun.
5) mixed liquor is all transferred to immediately in RNA Spin Column.
6) 12,000rpm, it is centrifuged 1min, abandons filtrate.RNA Spin Column is put back into 2ml collecting pipe.
7) the Buffer RWA of 500 μ l is added into RNA Spin Column, 12,000rpm centrifugation 30s abandon filtrate.
8) the Buffer RWB of 600 μ l is added into RNA Spin Column, 12,000rpm centrifugation 30s abandon filtrate.
9) step 8) is repeated.
10) RNA Spin Column is relocated on 2ml collecting pipe, 12,000rpm are centrifuged 2min.
11) by RNA Spin Column be placed in 1.5ml without on RNA enzyme collecting pipe, in RNA Spin Column film
The RNase Free dH of 50~200 μ l is added in centre2O or 0.1%DEPC handles water, is stored at room temperature 5min.
12) 12,000rpm is centrifuged 2min eluted rna.
13) RNA concentration is detected, identifies the yield and purity of RNA.
3, the building and sequencing of cDNA library
1) total serum IgE DNaseI digests: using DNA fragmentation present in DNase I digestion Total RNA sample, magnetic bead is pure
Change recycling reaction product, is finally dissolved in DEPC water；
2) rRNA: the good Total RNA sample of cancellationization is removed, is removed using the Ribo-Zero kit of Epicentre
RRNA carries out Agilent 2100 after removal and detects, verifies rRNA removal effect；
3) RNA is interrupted: taking previous step sample, addition interrupts Buffer, is placed in progress heat in PCR instrument and interrupts, interrupts
140-160nt；
4) synthesis of one chain of reverse transcription: appropriate primer being added into the sample after interrupting, after mixing well
Thermomixer thermophilic reacts certain time, is allowed to open secondary structure and in conjunction with primer, adds the chain prepared in advance
Synthetic reaction system Mix synthesizes a chain cDNA by corresponding program in PCR instrument；
5) synthesis of two chain of reverse transcription: preparing two chain synthesis reaction systems, one timing of thermophilic reaction on Thermomixer
Between, synthesis has the two chain cDNA of dUTP, and reaction product carries out purification and recovery with magnetic bead；
6) end is repaired: being prepared end and is repaired reaction system, thermophilic reacts certain time in Thermomixer, in enzyme
Under the action of, the cohesive end for the cDNA double-strand that reverse transcription obtains is repaired, end reparation product is purified with magnetic bead
Recycling, is finally dissolved in EB Solution for sample；
7) 3 ' end cDNA adds " A ": it prepares and adds " A " reaction system, thermophilic reacts certain time in Thermomixer,
Under the action of enzyme, 3 ' ends for the product cDNA for repairing end are plus A base；
8) connection of 5 ' adapter of cDNA: preparing connector coupled reaction system, the thermophilic reaction one in Thermomixer
It fixes time, under the action of enzyme, connect connector with A base, product carries out purification and recovery with magnetic bead；
9) UNG digests bis- chain of cDNA: UNG digestion reaction system is prepared, two chains in double-stranded DNA are fallen by UNG enzymic digestion,
And purification and recovery is carried out to product with magnetic bead；
10) PCR reaction and product recycling: PCR reaction system is prepared, PCR response procedures appropriate is selected, upper step is obtained
To product expanded, to PCR product carry out magnetic beads for purifying recycling, recovery product is dissolved in EB solution, labelled.
11) Library Quality detects: using 2100 Bioanalyzer and ABI StepOnePlus Real- of Agilent
Time PCR System detects Library Quality；
12) machine is sequenced on: detecting qualified library, NaOH denaturation is added at single-stranded, it is anticipated that upper machine data volume, dilution
To certain upper machine concentration.Library after denaturation dilution is added in FlowCell, is hybridized with the connector on FlowCell,
Bridge-type PCR amplification is completed on cBot, is finally sequenced using IlluminaHiseq x-ten platform.
4, bioinformatic analysis
1) with cutadapt to the 5 ' of reads and 3 ' Duan Jinhang trim, trim falls the base of quality < 20, and it is big to delete N
In 10% reads；
2) hisat2 is compared onto reference genome.With reference to genome from Ensembl database, genome version
GRCh38, gene annotation information are Ensemble 92；
3) stringtie quantifies the expression quantity and normalization output of lncRNA；
4) edgeR packet compares control group with the differential expression of disease group lncRNA, the screening criteria of variances movement lncRNA
It is | log2FC |>1 and pvalue<0.05.
5, result
Sequencing data is as shown in table 2, and bioinformatic analysis discovery, LINC01612 is expressed significantly in patient with breast cancer
It lowers, prompts LINC01612 that may be applied to the early diagnosis of breast cancer as detection target.
2 sequencing data of table
The differential expression of embodiment 2QPCR sequence verification LINC01612 gene
1, according to 25 Luminal A type breast cancer patients tissue samples of the collection mode collection of embodiment 1 and just
Normal tissue samples carry out large sample QPCR verifying to LINC01612 gene differential expression.
2, RNA is extracted
Takara RNA extracts kit (Code NO.9767) extracts the RNA in tissue, and specific steps are referring to embodiment 1.
3、QPCR
According to the gene order design primer of LINC01612 and GADPH, primer sequence is as shown in table 3.
3 amplimer of table
Use TaKaRa One Step TB GreenTMPrime ScriptTMRT-PCR kit (Code
No.RR066A PCR reaction) is carried out, reaction system and reaction condition is as shown in table 4.In Thermal CyclerReal
PCR amplification is carried out on Time System amplification instrument, confirms that the amplification curve of Real Time PCR and dissolution are bent after reaction
Line, Δ Δ CT method carry out relative quantification.
4 QPCR reaction system and reaction condition of table
4, result
QPCR result is as shown in Figure 1, compared with normal tissue, and LINC01612 is lowered in expression in breast, difference
It is consistent with high-flux sequence result with statistical significance (P < 0.05), it prompts to judge by the horizontal of detection LINC01612
Whether subject suffers from breast cancer, and when the horizontal significant decrease of LINC01612, subject suffers from breast cancer or exists and suffers from
The risk of breast cancer can design the overexpression for increasing LINC01612 level by the relationship between LINC01612 and breast cancer
Carrier to treat breast cancer, while can the relationship based on LINC01612 and breast cancer, the calculating mould of building prediction breast cancer
Type.
The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.
Sequence table
<110>People's Hospital of Deyang City
<120>a kind of purposes of biomarker relevant to breast cancer occurrence and development
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gttaattgtg acctctatga t 21
<210> 2
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ttctacagcc tcttatctc 19
<210> 3
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
aatcccatca ccatcttcca g 21
<210> 4
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gagccccagc cttctccat 19
Claims (10)
1.LINC01612 purposes, which is characterized in that be used to prepare the product of Diagnosis of Breast cancer.
2. purposes according to claim 1, which is characterized in that the product in measurement sample by passing through in measurement sample
The expression of LINC01612 is lowered compared with reference levels and is determined.
3. purposes according to claim 2, which is characterized in that the product passes through sequencing technologies, nucleic acid hybridization technique, core
The expression of the method detection LINC01612 gene of sour amplification technique.
4. purposes according to claim 1-3, which is characterized in that the breast cancer is Luminal A type mammary gland
Cancer.
5. the product of LINC01612 expression in a kind of vitro detection sample, which is characterized in that the product include preparation,
Chip or kit.
6. product according to claim 5 characterized by comprising
The probe of specific recognition LINC01612；Or
The primer of specific amplification LINC01612.
7. product according to claim 6, which is characterized in that the primer sequence of the specific amplification LINC01612 is such as
Shown in SEQ ID NO.1 and SEQ ID NO.2.
8. the described in any item products of claim 5-7 are preparing the purposes in Diagnosis of Breast cancer tool.
9. a kind of kit of Diagnosis of Breast cancer characterized by comprising
Detect one or more reagents of LINC01612；With
One or more substances selected from the group below: container, positive control, negative control object, buffer, helps operation instructions
Agent or solvent.
10. kit according to claim 9, which is characterized in that the kit is used to detect by the method for the following group
LINC01612:qRT-PCR, biochip test method, southern blotting technique method or RNA blotting hybridization in situ.
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