CN105238782A - Breast tumor prognosis biomarker LncRNA detection method and clinical application thereof - Google Patents

Abstract

The invention discloses a breast tumor prognosis biomarker LncRNA detection method and clinical application thereof, provides a group of new LncRNA in sequence shown as SEQ ID No1, SEQ ID No2, SEQ ID No3, SEQ ID No4 and SEQ ID No5 and relates to a method of taking specific LncRNA in samples (tissues, serum, urine, body fluid and the like) of patients suffering from breast tumors and corresponding juxtacancerous samples or normal samples as detection markers and biomarkers for prognostic auxiliary detection of breast tumors, related kits and a high-throughput sequencing and screening method. In-vitro diagnosis and judgment of canceration and process of breast tumors are realized by identification of differences of LncRNA expression quantity in the breast tumor samples and the corresponding juxtacancerous samples or normal samples. The invention further provides usage of the LncRNA as a breast tumor marker and probes and primers for LncRNA detection.

Description

A kind of method and clinical application detecting breast tumor prognosis biomarker LncRNA

Technical field

The invention belongs to biomedicine field, be specifically related to a kind of method and the clinical application that detect breast tumor prognosis biomarker LncRNA.

Background technology

In human body, the nearly 2-3 of the gene of coded protein ten thousand, only accounts for 2% of human genome, and the genomic dna of all the other 98% not coded proteins is considered to not have function at first, is the rubbish in organism, is commonly called " junk DNA.But current research shows, the most transcribed generation non-coding RNA (non-codingRNA, ncRNA) of these junk DNAs.According to the difference of ripe transcript size, ncRNA can be divided into small molecules ncRNA (as siRNA, miRNA, piRNA etc.), moderate-length ncRNA (70-200nt) and long ncRNA (longncRNA, LncRNA, >200nt).At present, that ncRNA area research is more is small molecules ncRNA, and is also in the starting stage to the research of LncRNA.Because interior sequences exists too much terminator codon, LncRNA can not be translated into protein, they normally exercise its biological function with rna transcription form originally, as the cytodifferentiation in growth course, cell proliferation, apoptosis and steroid metabolism etc., increasing evidence shows that LncRNA plays the vital role of regulate gene expression in cell.Nearest research finds that the canceration of LncRNA and cell has extremely close contact, the LncRNA playing the effect of breast tumor suppressor gene expresses and to decline or disappearance can cause the generation of breast tumor, has been found that the breast tumor diseases such as LncRNA and lung cancer, non-hodgkin lymphoma, cutaneous T cell lymphoma and chronic lymphocytic leukemia are relevant.These results of study all show that LncRNA plays an important role in cell proliferation, differentiation and canceration, and the be correlated with discovery of LncRNA of breast tumor may produce great impact to the diagnosis of breast tumor and gene therapy.By analyzing the IncRNA of breast tumor and healthy tissues on a large scale, relevant LncRNA special to breast tumor can be identified, becoming potential pathological diagnosis mark.Therefore, find the LncRNA that breast tumor is specific expressed, this has important Clinical significance of MG to breast tumor Prognosis and treatment.

Summary of the invention

Object of the present invention just in order to overcome the deficiency of above-mentioned technology, and provides a kind of method and the clinical application that detect breast tumor prognosis biomarker LncRNA.

The object of the invention is to have come by following technical solution.

This LncRNA molecule, described nucleic acid molecule is selected from:

1), the nucleic acid molecule shown in SEQ ID No 1, SEQIDNo2, SEQIDNo3, SEQIDNo4, SEQIDNo5;

2), there is with SEQ ID No 1, the nucleotide sequence shown in SEQIDNo2, SEQIDNo3, SEQIDNo4, SEQIDNo5 the nucleic acid molecule of 90% or more homology.

Described LncRNA molecule is as the application of breast tumor prognostic marker.

A detection method for LncRNA molecule, the method detecting LncRNA molecule in the breast tumor sample be separated adopts hybrid method, TRAP or sequencing.

Described hybrid method is Northern hybridization, gene chip hybridization and hybridization in situ.

Described TRAP is Fluorescent quantitative PCR.

Described sequencing is high-flux sequence.

Described breast tumor sample is breast tumor tissues sample, serum, blood plasma, mouth mucus, urine or body fluid.

A kind of probe, this probe can detect described LncRNA molecule.

Described probe has the nucleotide sequence shown in SEQIDNo6, comprises the probe sequence designed by SEQIDNo1, SEQIDNo2, SEQIDNo3, SEQIDNo4, SEQIDNo5 in claim 1 simultaneously.

The application of described probe in preparation breast tumor prognosis detection reagent.

A kind of prognosis detection system of Diagnosis of Breast tumour, this detection system comprises described probe, and this system also comprises reversed transcriptive enzyme and reaction buffer thereof, four kinds of deoxyribonucleotide substrates, archaeal dna polymerase, quantitative fluorescent PCR reaction buffer, the internal reference of synthetic and normal controls product.

One group of nucleic acid oligomer molecule, described nucleic acid oligomer molecule is:

The application of described nucleic acid oligomer molecular combinations in preparation detection reagent, all probe sequences of the design extended by 5 LncRNA molecules.

A method for Diagnosis of Breast tumor prognosis, the method includes the steps of:

1) level of the LncRNA molecule in vitro sample described in paragraph 1 is measured;

2) the LncRNA molecule described in paragraph 1 in the level of the LncRNA molecule in more determined sample described in paragraph 1 and reference sample

Level, if compared with reference sample, in determined sample, the level of this LncRNA molecule has remarkable change, its express more than 2 times,

Illustrate that this LncRNA has significance to raise in prognosis sample.

The technical problem to be solved in the present invention carries out the diagnosis of breast tumor prognosis.The problem to be solved in the present invention also relates to the method and corresponding diagnostic reagent that are provided for Diagnosis of Breast Tumor.

The present invention through extensive and deep research, find and be separated make new advances can as the LncRNA molecule of breast tumor mark: Linc00657, Linc00346, Linc00654, Linc00925, HCG11.This LncRNA molecule do not express in healthy tissues or cancer beside organism or expression amount very low, in breast tumor tissues, have the expression exceeding in healthy tissues or cancer beside organism 2 times.Complete the present invention on this basis.Application of the present invention also comprises the serum, blood plasma, body fluid, saliva of buccal cavity, urine etc. of breast tumor.

In a first aspect of the present invention, provide the LncRNA molecule of one group of novelty, it comprises and has sequence shown in SEQIDNo1, SEQIDNo2, SEQIDNo3, SEQIDNo4, SEQIDNo5; Or with the nucleotide sequence shown in SEQ ID No 1, SEQIDNo2, SEQIDNo3, SEQIDNo4, SEQIDNo5, there is the nucleotide sequence of 90% or more homology.Above-mentioned nucleic acid detects by hybrid method, TRAP or sequencing.Specifically, TRAP comprises Fluorescence quantitative real-time polymerase chain reaction, and hybrid method comprises chip hybridization and Northern hybridization, and sequencing comprises high-flux sequence method.

Second aspect present invention provides the probe or Auele Specific Primer that detect sequence shown in SEQIDNo1, SEQIDNo2, SEQIDNo3, SEQIDNo4, SEQIDNo5.Preferably, described detection probes has 10 sequences (SEQIDNo6).10 sequences (SEQIDNo6) are a kind of probe of SEQIDNo1, SEQIDNo2, SEQIDNo3, SEQIDNo4, SEQIDNo5 kind, comprise in addition by all probe sequences of the design of 5 LncRNA molecule extensions in paragraph 1.Third aspect present invention provides a kind of detection system of Diagnosis of Breast tumor prognosis, and this system comprises above-mentioned detection probes; Or this system comprises above-mentioned detection primer, in preferably embodiment, this system also comprises reversed transcriptive enzyme and reaction buffer thereof, four kinds of deoxyribonucleotide substrates, archaeal dna polymerase, quantitative fluorescent PCR reaction buffer, the internal reference of synthetic and normal controls product.

Fourth aspect present invention provides a kind of method of Diagnosis of Breast tumor prognosis effect assessment, and the method includes the steps of:

1) expression level of SEQIDNo1, SEQIDNo2, SEQIDNo3, SEQIDNo4, SEQIDNo5 in vitro sample is measured;

2) level of SEQIDNo1, SEQIDNo2, SEQIDNo3, SEQIDNo4, SEQIDNo5 in more determined sample in the level of SEQIDNo1, SEQIDNo2, SEQIDNo3, SEQIDNo4, SEQIDNo5 and reference sample, if compared with reference sample, in determined sample, LncRNA molecule has expression, and express more than more than 2 times, illustrate that breast tumor outcome is poor.

Measuring the method for the expression level of the LncRNA molecule of SEQIDNo1, SEQIDNo2, SEQIDNo3, SEQIDNo4, SEQIDNo5 in vitro sample, can be hybrid method, TRAP or sequencing.Specifically, TRAP comprises Fluorescence quantitative real-time polymerase chain reaction, and hybrid method comprises chip hybridization and Northern is hybridized and in situ hybridization, and sequencing comprises high-flux sequence method.If the expression level of SEQIDNo1LncRNA molecule raises than the expression level of this nucleic acid oligomer molecule in reference sample in the in vitro sample measured by aforesaid method, and exceed healthy tissues or cancer beside organism more than 2 times, then the risk that there is breast tumor poor prognosis is described.

Beneficial effect of the present invention is:

(1), the present invention filters out one group of new long non-coding RNA, and this RNA can be used as the Specific marker of breast tumor outcome diagnosis.

(2), one group has been screened at mammary cancer and corresponding cancer LncRNA that is other or normally sample differential expression by high throughput sequencing technologies.

(3), using the LncRNA that the filters out one group molecular marked compound as novel breast cancer detection particularly prognosis, the test kit detected for the preparation of Prognosis in Breast Cancer and biochip, have and detect the advantages such as pedigree is wide, highly sensitive, testing cost is low.

(4), adopt one group of LncRNA as new breast tumor prognostic markers thing, by improve single marker the low difference brought of the individual difference that is difficult to overcome and low susceptibility, can effectively improve galactophore disease people prognosis effect appraisal thus guiding clinical treatment guide.

(5), the advantage of LncRNA detection technique is, what it detected is a series of mammary gland mark of correlation things, singlely can see prognostic risk size, also can combine LncRNA molecule and see prognostic risk, thus more effectively instruct prognostic clinical to treat.

Accompanying drawing explanation

Fig. 1 is the dependency diagram of LncRNAs (SEQIDNo1, SEQIDNo2, SEQIDNo3, SEQIDNo4, SEQIDNo5) unconventionality expression situation and pathological grading in breast cancer tissue;

Fig. 2 is the dependency diagram of LncRNAs (SEQIDNo1, SEQIDNo2, SEQIDNo3, SEQIDNo4, SEQIDNo5) unconventionality expression situation and ER level in breast cancer tissue.

Fig. 3 is the dependency diagram of LncRNAs (SEQIDNo1, SEQIDNo2, SEQIDNo3, SEQIDNo4, SEQIDNo5) unconventionality expression situation and PR level in breast cancer tissue.

Fig. 4 is the dependency diagram of LncRNAs (SEQIDNo1, SEQIDNo2, SEQIDNo3, SEQIDNo4, SEQIDNo5) unconventionality expression situation and HER2 level in breast cancer tissue.

Fig. 5 is the dependency diagram of LncRNAs (SEQIDNo1, SEQIDNo2, SEQIDNo3, SEQIDNo4, SEQIDNo5) unconventionality expression situation and tumor size in breast cancer tissue.

Fig. 6 is the dependency diagram of LncRNAs (SEQIDNo1, SEQIDNo2, SEQIDNo3, SEQIDNo4, SEQIDNo5) unconventionality expression situation and lymphatic metastasis in breast cancer tissue.

Fig. 7 is the dependency diagram of LncRNAs (SEQIDNo1, SEQIDNo2, SEQIDNo3, SEQIDNo4, SEQIDNo5) unconventionality expression situation and age groups in breast cancer tissue.

Fig. 8 is that the different expression of quantitative fluorescent PCR Linc00657 between breast cancer tissue and healthy tissues analyzes schematic diagram.

Fig. 9 is that the different expression between quantitative fluorescent PCR Linc00657 breast cancer cell and mammary epithelial cell analyzes schematic diagram.

Figure 10 is expression and the survival rate relation schematic diagram of Linc00657, Linc00654, Linc00925, Linc00346, HCG11.

In Figure 10, in 959 the breast tumor samples analyzed, according to LINC00657:EXP>2.0, LINC00654:EXP>2.0, LINC00346:EXP>2.0, LINC00925:EXP>2.0, HCG11:EXP>2.0 analyzes, be divided into two classes, one class is that the survival time is short, as shown in lines a in Figure 10, one class is patient's number for survival, as shown in lines b in Figure 10, the per-cent that the short patient's number of the 5 kinds of LNCRNA single evaluation survival moon numbers detected accounts for total patient's number is Linc0065711% respectively, Linc006547%, Linc009256%, Linc003468%, HCG116%, if the expression amount of these 5 kinds of LncRNA is about more than 2.0, then patient's number that mammary gland patient prognosis survival moon number survival moon number is short accounts for 29% of total patient's number.

Embodiment

Below in conjunction with specific embodiment and accompanying drawing, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.DNA nucleic acid oligomer used in the present invention is synthesized by the raw work in Shanghai, and the RNA nucleic acid oligomer used is synthesized by Ji Ma bio tech ltd, Shanghai.

Embodiment 1.solexa sequencing technologies carries out high-flux sequence to the long segment non-coding RNA in patient with breast cancer's cancerous tissue.

1. mammary cancer and corresponding cancer beside organism Total RNAs extraction

Tissue Lysis:

1) the clear area that less RNase disturbs (1 ‰ DEPC water wipings all used by all apparatus), mortar needs 200 DEG C of sterilizings 5 hours and cools 4 hours, take in vitro mammary tissue sample and be about 20mg to having in the mortar of Liquid nitrogen precooler, be ground to Powdered (output of the RNA that grinding thoroughly then can not have a strong impact on) with pestle;

2) add 700 μ lQIAzol lysates in mortar, continue to be ground to the uniform liquid without obvious tissue block, be then transferred in the 1.5mleppendorf pipe without RNA enzyme;

3) refrigerated centrifuge 4 DEG C, 12000 × g, centrifugal 10min are to remove uncracked complete organization and cell debris in solution;

4) careful draw centrifugal after supernatant liquor, move to another new eppendorf and manage, be sure not to draw lower floor's cell debris residue and precipitate;

5) add 140 μ l chloroforms, turn upside down, after thermal agitation, leave standstill 2min;

6) refrigerated centrifuge 4 DEG C is centrifugal, 12000 × g, 15min, and is transferred to by supernatant liquid one new in RNA enzyme eppendorf pipe;

7) add 1.5 times of dehydrated alcohols, vortex mixes.

RNA adsorbs:

8) be all transferred in adsorption column by all liquid, and be placed in the collection tube of 2ml by adsorption column, the centrifugal 15s of 8000g, abandons the filtrate in collection tube;

9) get the RWT solution of 700 μ l in adsorption column, the centrifugal 15s of 8000g, abandons filtrate;

RNA washing and wash-out

10) bufferRPE getting 500 μ l washs to adsorption column, and the centrifugal 15s of 8000g, abandons filtrate;

11) again adsorption column is washed, the centrifugal 2min of 8000g with the bufferRPE of 500 μ l, abandon filtrate and underlying collection sleeve pipe;

12) adsorption column is transferred in a new 2ml collection tube, at full speed centrifugal 1min;

13) abandon underlying collection pipe, adsorption column is moved in new 1.5mleppendorf pipe, add 45 μ lRNase-freewater wash-out adsorption films, leave standstill the centrifugal 1min of 1min, 8000g;

14) at full speed centrifugal 1min again.

2. the quantitative and quality examination of total serum IgE

Nanodrop2000 is utilized to detect the content of mammary tissue RNA sample, through the quantitative assay of OD value, so that the volume of RNA solution required when calculating LincRNA reverse transcription ^[76]; The RNA sample more serious with DNA pollution of having degraded is got rid of, to ensure the specificity of subsequent experimental through qualitative detection ^[76].Concrete detection method is as follows:

1) total serum IgE is quantitative: the OD value measuring 260nm and 280nm on ultraviolet spectrophotometer, and draws A260/A280, when its numerical value illustrates that between 1.8 ~ 2.0 the purity of total serum IgE is better; Remaining salt ion and the pollution of small molecules magazine is had, when being greater than the degraded that 2.0 illustrate possibility total serum IgE when being less than 2.0 explanations:

2) integrity detection of total serum IgE: 1% denaturing formaldehyde agarose gel electrophoresis is separated the ribosome-RNA(rRNA) (rRNA) in total serum IgE, two maximum band 28sRNA and the brightness of 18sRNA are roughly than during for 2:1, illustrate that the integrity of RNA is better, do not occur signs of degradation.

The library construction of 3.LncRNA

(1) ribosomal removal

The hybridization of sample:

1) water-bath temperature is adjusted to 70-75 DEG C.

2), in the centrifuge tube in sterilizing and without the 1.5ml of RNase, following reagent is added successively:

Total serum IgE 5 μ g

Ribominus ^tMprobe 0.15 μm of ol

HybridizationBuffer100μl

3) by having added the centrifuge tube of reagent above at 70-75 DEG C of water-bath 5min, RNA sex change is made

4) after sex change completes, sample is lowered the temperature, namely sample is put into incubation in 37 DEG C of water-baths and is about 30min, the temperature of sample can be made in this process to drop to 37 DEG C gradually, in order to promote sequence specific hybridization (must guard against and sample is placed directly in fast cooling in cold water, the efficiency of impact hybridization).

The preparation of Beads:

1) the Plastic Bottle Homogeneous phase mixing Ribominus of magnetic bead will be housed ^tMmagneticBeads (magnetic bead).

2) getting 750 μ l magnetic bead mixed solutions joins aseptic and in EP pipe without the 1.5ml of RNase.

3) the EP pipe that magnetic bead mixed solution is housed is put into standing 1min. magnetic bead on magnetic frame and is adsorbed to the side tube wall be connected with magnetic frame, then draw liquid in pipe and discard.

4) in pipe, add the aseptic DEPC water of 750 μ l, low-speed oscillation makes magnetic bead again dissolve;

5) be separated after EP pipe being put into what upper standing 1min of magnetic frame and discard waste liquid; Repeat this two step once.

6) again dissolve magnetic bead with the HybridizationBuffer (hybridization solution) of 750 μ l and draw 250 μ l mixed solutions to put into another new EP pipe for subsequent use.

7) pipe that 500 μ l mixed solutions are housed is placed on 1min on magnetic frame, discards liquid in pipe.

8) again dissolve magnetic bead with 200 μ l hybridization solutions and be placed in 37 DEG C.

RRNA removes:

1) when after hybridizing, sample has cooled to 37 DEG C, centrifugal fast, bottom sample collection to EP pipe;

2) sample of 320 μ l is transferred to ready Ribominus ^tMmagneticBead; Utilize pipettor gun head to blow and beat up and down to mix;

3) 37 DEG C of water-bath incubation 15min are placed in, between incubation period, interval slight oscillatory sample.Then low-speed centrifugal is bottom sample collection to pipe;

4) be placed on by EP pipe on magnetic frame and leave standstill 1min, the magnetic bead in conjunction with rRNA can be adsorbed on tube wall, Ribominus ^tMrNA is Already in solution;

5) the magnetic bead mixed solution of off-the-shelf 250 μ l (in the preparation of Beads the 7th step) is placed on standing 1min on magnetic frame, draws liquid in pipe and also discard;

6) what in EP pipe, add about 320 μ l contains Ribominus ^tMrNA (the 4th step), blows and beats up and down with rifle and mixes;

7) 37 DEG C of incubation 15min, period is slight oscillatory once in a while, then centrifugal bottom sample collection to EP pipe;

8) EP pipe is placed on magnetic frame leaves standstill 1min, containing Ribominus ^tMthe solution of RNA is transferred in new EP pipe.

Alcohol precipitation concentrates RiboMinus ^tMrNA

1) RiboMinus ^tMrNA sample is transferred in the centrifuge tube of the 2ml of RNase-free,

2) to RiboMinus ^tMfollowing reagent is added in RNA:

Glycogen(20ng/μl)1μl

Sodium-acetate (3M) 53 μ l

The alcohol 1325 μ l of 100%

3) mix ,-80 DEG C of standing 30min.

4) 4 DEG C, the centrifugal 15min of 12000g.Carefully siphon away solution, keep off the precipitation contacting bottom,

5) alcohol of the precooling of 500 μ l70% is added,

6) 4 DEG C, the centrifugal 5min of 12000g; Remove supernatant,

7) 5-6 step is repeated.

8) open test tube cap, natural drying at room temperature is about 5min, again dissolves RiboMinus with 30 μ lDEPC water ^tMrNA.

(3) solexa order-checking

Solexa order-checking is carried out to Hua Da gene by with the ncRNA library built friendship.The sequenator adopted is Hi-seq2000, and strategy is pair end sequencings, and it is all 2 × 100bp that the sequence length that namely 100bp draw respectively is surveyed in both sides.Both-end order-checking is the important technology of in sequencing technologies one, can be calculated the length of sequenced fragments accurately, compensate for the defect of single-ended order-checking, therefore can carry out the location of repeat region sequence and the splicing etc. of sequence more accurately by both-end order-checking.

Embodiment 2 is according to gained sequencing result in solexa, and different pathological Epidemiological Analysis is carried out in the screening data of gained being carried out to LncRNA.

The quantitative analysis that LncRNA expresses and the expression correlation in different pathological index

Breast cancer tissue and the other tissue sample info of mammary cancer, according to database website http://www.cbioportal.org/, press " Mutations in mammary cancer putativecopy-numberalterationsfromGISTIC mRNAExpressionz-Scores (RNASeqV2RSEM) " screen, from 959 routine mammary gland samples to HUGOgenenomenclaturecommittee ( http:// www.genenames.org/) in 2,731lncRNAs the analyzing of accreditation, filter out 959 routine mammary gland patient's sample and have complete pathological information, 959 case informations are as follows:

The clinical pathology information of table 1 mammary cancer and cancer beside organism patient

(1) expression of LncRNA in breast cancer tissue sequencing result analysis (Fig. 1) on pathological grading

Mammary cancer sample is pressed pathological information and arrange mammary cancer pathology stage I (160 example), II (536 example) and III (211 example), find linc0657, linc00346, linc00654, it is higher that linc00925 and HCG11 expresses ratio in mammary cancer pathological staging II phase, and expressing ratio in mammary cancer pathological staging I and III phase, to compare for II phase significantly low.Therefore target LncRNA can be used as mammary cancer pathological staging II phase diagnosis index clinically.

(2) the sequencing result analysis (Fig. 2) of the expression of LncRNA in breast cancer tissue in estrogen receptor (ER)

Because the process LAN of estrogen receptor and progesterone receptor is one of principal element of mammary cancer, there is close relationship with the differentiation of cancer cells, transfer and endocrine therapy.Press pathological information according to mammary cancer sample and arrange ER feminine gender (ER-, totally 288 examples), the ER positive (ER+, totally 671 examples), find linc0657 (7.09%), linc00346 (5.11%), linc00654 (5.11%), linc00925 (3.34%) organizes the ratio of expression higher than linc0657 (3.44%) in the ER positive, linc00346 (2.4%), linc00654 (2.4%), linc00925 (2.4%) expresses in ER feminine gender group, and the ratio that HCG (1.56%) expresses in ER positive group is expressed in ER feminine gender group lower than HCG11 (4.8%).Linc0657 is described, linc00346, linc00654 and linc00925 be possibility high expression level in the ER positive, can carry out effective treatment plan, have potential applicability in clinical practice widely according to expression level.

(3) the sequencing result analysis (Fig. 3) of the expression of LncRNA in breast cancer tissue on progesterone receptor (PR)

Press pathological information according to mammary cancer sample and arrange PR feminine gender (PR-, totally 384 examples), the PR positive (PR+, totally 575 examples), find linc0657 (5.84%), linc00346 (4.07%), linc00654 (4.07%) organizes the ratio of expression higher than linc0657 (4.69%) in the PR positive, linc00346 (3.44%), linc00654 (3.44%) expresses in PR feminine gender group, and linc00925 (2.19%) and HCG11 (1.56%) PR the positive group expression ratio lower than linc00925 (3.55%), HCG11 (4.8%) expresses in PR feminine gender group.Linc00925 and HCG11 can initial reaction PR at the expression of mammary cancer.

(4) the sequencing result analysis (Fig. 4) of the expression of LncRNA in breast cancer tissue on HER2

Proto-oncogene ErbB-2 (HER2) gene, be that important Prognosis in Breast Cancer judges the factor, it is process LAN in patients.Press pathological information according to mammary cancer sample and arrange HER2 feminine gender (ER-, totally 774 examples), the HER2 positive (ER+, totally 185 examples), find linc0657 (2.19%), linc00346 (1.04%), linc00654 (1.04%), linc00925 (0.63%) and HCG11 (0.83%) organizes the ratio of expression lower than linc0657 (8.55%) in the HER2 positive, linc00346 (6.47%), linc00654 (6.47%), linc00925 (5.11%) and HCG (5.53%) HER2 feminine gender group are expressed.These 5 kinds of LncRNA possibility high expression level in HER2 feminine gender is described, effective treatment plan can be carried out according to expression level, there is potential application value clinically.

(5) the sequencing result analysis (Fig. 5) of the expression of LncRNA in breast cancer tissue on tumor size

Mammary cancer sample is pressed pathological information arrangement tumor size T1 phase (248 example), T2 (543 example), T3 (111 example) and T4 (35 example) discovery linc0657, linc00346, linc00654, it is lower that linc00925 and HCG11 expresses ratio at mammary gland tumor T3 and T4, and it is higher to express ratio in mammary gland tumor I and II phase.

(6) the sequencing result analysis (Fig. 6) of the expression of LncRNA in breast cancer tissue in lymphadenectasis

The cancer cells of mammary cancer, along lymphatic vessel diffustivity lymphatic metastasis, can determine whether to adopt Breast-consering surgery according to the position of its transfer.Press pathological information according to mammary cancer sample and arrange lymphadenectasis feminine gender (totally 446 examples), positive (totally 493 examples), find linc0657, linc00346, it is suitable that linc00654 and linc00925 expresses the expression of ratio in lymphadenectasis feminine gender and Positive Level, not difference, and HCG11 in the expression ratio of lymphadenectasis feminine gender lower than at Positive Level.HCG11 can the situation of initial reaction lymphadenectasis.

(7) the sequencing result analysis (Fig. 7) of the expression of LncRNA in breast cancer tissue in sicken age

Show that median is 52 years old according to mammary cancer sample by the sicken age in pathological information, with 52 years old for boundary, be divided into two age groups, 327 routine samples of low age group and 612 examples of high age group are analyzed, find linc0657, the level that linc00346, linc00654, linc00925 and HCG11 express in high age group is suitable with low age group expression level.Illustrate that the expression change of these 5 kinds of LncRNA does not have dependency with the age.

The different expression analysis (Fig. 8) of embodiment 3Linc00657 between breast cancer tissue and healthy tissues

This experiment utilizes OriGenebreastcancercDNAarrays to carry out RT-PCR experiment, the experimental data of RT-PCR gained is analyzed, draw breast cancer tissue and the relative change multiple in healthy tissues sample, independent sample T in recycling SPSS statistical analysis software checks, analyze the expression in Linc00657 41 routine breast cancer tissue samples in an experiment and 7 routine healthy tissues samples, Linc00657 is high 1.95 times in expression in breast amount is than healthy tissues, and has statistical significance (P=0.01).This experiment demonstrates the order-checking experimental result of 959 mammary gland patients of TCGAdatabaseanalysis further.

(1) combination of primers is detected.Entrust the following primer for detecting of Invitrogen Beijing Company synthesis.

Combination of primers:

Reverse transcriptase primer: the random reverse transcriptase primer of 6 base;

(2) acquisition of breast tumor and control tissue sample and Total RNAs extraction

Obtain 41, the mammary cancer breast tumor tissues sample that underwent operative is separated, 7, Carcinoma side normal tissue sample, extracts total RNA with animal tissues's RNA purification kit of NORGEN company, carries out quantitatively the total serum IgE extracted.

3. real-time fluorescence quantitative RT-PCR detects the expression of Linc00657 in breast tumor sample

Utilize the expression of Linc00657 in 41 the breast tumor samples and 7 cancer beside organism's samples obtained in real-time fluorescence quantitative RT-PCR detecting step 2, concrete steps are as follows:

1) RNA reverse transcription: the Reverse Transcriptase kit (TIANScriptM-MLV using Tian Gen biochemical technology company, cat:ER104-03) reverse transcription reaction of RNA sample is carried out, specifically carry out according to the method for test kit specification sheets, step is as follows: get the total serum IgE sample that 1 microgram is extracted, add the random reverse transcriptase primer of 2 μ l (10 μMs) 6 base, 2 μ ldNTP (10mM), mix rearmounted 70 DEG C of water-bath sex change 5 minutes; Place on ice after 3 minutes and take out, add 4 μ l5 × First-StrandBuffer, 0.5 μ lRNaseinhibitor (40U/ μ l), 1 μ lM-MLV (200U/ μ l), cumulative volume is 20 μ l; Of short duration centrifugal after mixing, be placed in EppendorfPCR instrument and carry out reverse transcription reaction, reaction parameter is 25 DEG C, 10 minutes; 42 DEG C, 50 minutes; 95 DEG C, 5 minutes; With being placed on 4 DEG C of preservations.Wherein, RNaseinhibitor (cat#N211) product that is Promega company.

2) real-time fluorescence quantitative PCR: use Tian Gen biochemical technology company HotMasterTaqDNApolymerase (cat#:ET106-01-01) detection by quantitative is carried out to the expression of Linc00657 in sample, concrete grammar is undertaken by product description, step is as follows: get 2 μ l reverse transcription product, add 2.5 μ l10 × HotMasterTaqBuffer, 0.5 μ l (10 μMs) upstream primer, 0.5 μ l (10 μMs) downstream primer, 1 μ ldNTP mixture (each 2.5 μMs), 1 μ lSYBRGreen Ι (5 ×), 0.2 μ lHotMasterTaqDNApolymerase (2.5u/ μ l), finally add 17.3 μ lddH2O, reaction cumulative volume is 25 μ l.Of short duration centrifugal after mixing, be placed in EppendorfPCR instrument and carry out pcr amplification reaction, reaction parameter is 95 DEG C of denaturations 2 minutes, 95 DEG C of sex change 15 seconds, 58 DEG C of annealing 15 seconds, and 72 DEG C extend 30 seconds; Cycle index is 40 circulations.Each reaction arranges 3 repetitions.

3) data analysis: the expression same sample being detected respectively to wherein target RNA and internal reference RNA, detects the expression of Linc00657 with primer sets unification; With the expression amount of internal reference for benchmark, the expression of target RNA is normalized; The normally used deltadeltaCt method in this area is used to carry out quantitatively the expression amount of target RNA subsequently.The internal reference of this experiment is beta-actin.Concrete grammar and step can see documents: LivakKJandSchmittgenTD.Analysisofrelativegeneexpressiond atausingreal-timequantitativePCRandthe2 (-DeltaDeltaC (T)) Method.Methods.2001Dec; 25 (4): 402-408.

Expression change (Fig. 9) of embodiment 4.Linc00657 between breast cancer cell and normal epithelium cell

This experimental verification Linc00657 is whether variant expression in breast cancer cell, have chosen mammary epithelial cell HMLE and breast cancer cell MCF7, MDA-MB-231.As shown in Figure 9, Linc00657 is remarkable high expression level in breast cancer cell, wherein high than mammary epithelial cell 2.81 times in MCF7 cell, high 2.69 times in MDA-MB-231.

Embodiment 5. is for detecting the real-time fluorescence quantitative RT-PCR test kit of Linc00657 expression level

Real-time fluorescence quantitative RT-PCR test kit for detecting Linc00657 expression level comprises following composition:

1. reversed transcriptive enzyme and reaction buffer thereof, buffer components and concentration as follows: 1MTris (pH8.5), 10mM; 1MHCl, 2.94mM; 1MKCl, 50mM; 1MMgCl ₂, 2.5mM; 10mMdNTP, 200 μMs; 50 × ROX, 0.02 ×; DdH2O;

2.Linc00657 reverse transcription and pcr amplification combination of primers:

Reverse transcriptase primer: the random reverse transcriptase primer of 6 base;

Forward primer: 5 '-AGATTCCCATTTTGGCTTTG;

Reverse primer: 5 '-GGTGACATGGTGCTGTTTCA;

3.DNA polysaccharase;

4. reference sample;

5. test kit working instructions.

In addition to the implementation, the present invention can also have other embodiments.All employings are equal to the technical scheme of replacement or equivalent transformation formation, all drop on the protection domain of application claims.

Claims (10)

1. a LncRNA molecule, is characterized in that: described nucleic acid molecule is selected from:

1), the nucleic acid molecule shown in SEQ ID No 1, SEQIDNo2, SEQIDNo3, SEQIDNo4, SEQIDNo5;

2), there is with SEQ ID No 1, the nucleotide sequence shown in SEQIDNo2, SEQIDNo3, SEQIDNo4, SEQIDNo5 the nucleic acid molecule of 90% or more homology.

2. LncRNA molecule according to claim 1 is as the application of breast tumor prognostic marker.

3. detect a method for LncRNA molecule as claimed in claim 1, it is characterized in that: the method detecting LncRNA molecule in the breast tumor sample be separated adopts hybrid method, TRAP or sequencing.

4. the detection method of LncRNA molecule according to claim 3, is characterized in that: described hybrid method is Northern hybridization, gene chip hybridization and hybridization in situ.

5. the detection method of LncRNA molecule according to claim 3, is characterized in that: described TRAP is Fluorescent quantitative PCR.

6. the detection method of LncRNA molecule according to claim 3, is characterized in that: described breast tumor sample is breast tumor tissues sample, serum, blood plasma, mouth mucus, urine or body fluid.

7. a probe, is characterized in that: this probe can detect described LncRNA molecule.

8. probe according to claim 7, is characterized in that: described probe has the nucleotide sequence shown in SEQIDNo6, comprises the probe sequence designed by SEQIDNo1, SEQIDNo2, SEQIDNo3, SEQIDNo4, SEQIDNo5 in claim 1 simultaneously.

9. the probe according to claim 7 or 8, is characterized in that: the application of described probe in preparation breast tumor prognosis detection reagent.

10. a prognosis detection system for Diagnosis of Breast tumour, is characterized in that: this detection system comprises described probe.