CN106701990B - Detect the application of the reagent of long-chain non-coding RNA LINC00472 expression quantity in the tumor tissues of oral cavity - Google Patents

Abstract

The invention discloses a kind of applications of the reagent of long-chain non-coding RNA LINC00472 expression quantity in detection oral cavity tumor tissues, it is used to prepare the prognosis preparation of mouth neoplasm patient, especially prepares the kit of real time fluorescent quantitative detection method prediction mouth neoplasm patient prognosis.Confirm that LncRNA LINC00472 expresses downward in mouth neoplasm tissue by research, mouth neoplasm patient's poor prognosis of the mouth neoplasm patient Bi Gao expression LncRNA LINC00472 of low expression LncRNA LINC00472, therefore, the expression of LncRNA LINC00472 is used for the prognosis prediction of mouth neoplasm patient, strong molecular biology foundation can be provided for the prognosis of mouth neoplasm patient, there is far-reaching clinical meaning and important popularization and application foreground.

Description

Detect the examination of long-chain non-coding RNA LINC00472 expression quantity in the tumor tissues of oral cavity The application of agent

Technical field

The invention belongs to oncomolecularbiology fields, and in particular to detection long-chain non-coding RNA LINC00472 expression Primer, detection reagent and its application of amount.

Background technique

Oral and maxillofacial malignancy is common high-incidence head-neck malignant tumor, is easy to happen metastasic cervical lymph nodes, Prognosis is poor.Studies have shown that the occurrence and development of this tumour are polygenes participation, multi-step, multistage complex process, wherein The inactivation of tumor suppressor gene and the activation of oncogene have played crucial effect.

Long-chain non-coding RNAs (long non-coding RNAs, lncRNAs) are that a kind of length is more than 200nt, lacks The RNAs molecule of special complete open reading frame, nothing or few encoding histone functions.It has recently been demonstrated that lncRNAs By adjusting the expression of gene extensively in epigenetic, transcription and post-transcriptional level, they take part in biology growing, development, The regulation of the important vital movement such as aging and death.More and more researches show that the unconventionality expression or afunction of lncRNAs It is closely related with the occurrence and development of tumour.Some lncRNAs molecules have great importance in terms of the diagnosing and treating of tumour, And it can be used as tumor prognosis and judge new molecular labeling.Currently, the mankind lncRNAs gene cloned is more than 40,000 It is a, but the Unknown Function of overwhelming majority lncRNA.

We have detected expression of the LINC00472 in clinical Ex vivo Tumor sample, and analyze LINC00472 table Up to the horizontal correlation with clinical case data.We are after extract RNA in mouth neoplasm and corresponding normal control tissue By reverse transcription, real-time quantitative PCR has detected the expression of LINC00472, and LINC00472 is in mouth neoplasm as the result is shown It expresses and lowers in tissue.The method that we then utilize in situ hybridization again is further demonstrated in archive paraffin sample LINC00472 expresses significant downward in mouth neoplasm, therefore can be used for mouth neoplasm for the detection preparation of LINC00472 Auxiliary diagnosis carries out survivorship curve analysis in conjunction with clinical return visit data and finds that LINC00472 expresses its life span of low patient The shorter than lncRNA expresses high patient, therefore the prognosis that can be used for mouth neoplasm for the detection preparation of the lncRNA is sentenced It is disconnected.

Summary of the invention

The object of the present invention is to provide the examinations of long-chain non-coding RNA LINC00472 expression quantity in detection oral cavity tumor tissues The application of agent, the reagent are used to prepare mouth neoplasm auxiliary diagnosis or outcome prediction preparation, the long-chain non-coding RNA The sequence of LINC00472 is shown in SEQ NO:1.

The mouth neoplasm auxiliary diagnosis or outcome prediction preparation is existed according to long-chain non-coding RNA LINC00472 Expression in mouth neoplasm carries out auxiliary diagnosis and outcome prediction, and in mouth neoplasm tissue LINC00472 expression is significant It lowers.

The mouth neoplasm auxiliary diagnosis or outcome prediction preparation is real time fluorescent quantitative detection reagent.

Include in the real time fluorescent quantitative detection reagent

Primer sequence for real time fluorescent quantitative detection LINC00472 expression:

Forward primer: 5 '-GCATCTGTCAACGCTCCTCT-3 ',

Reverse primer: 5 '-CCGCGTTCTTTGTGTGTCTC-3 '.

The real time fluorescent quantitative detection preparation includes:

Reference gene GAPDH Specific PCR primers:

Forward primer 5 '-ACCACAGTCCATGCCATCAC-3 '

Reverse primer 5 '-TCCACCACCCTGTTGCTGTA-3 '.

Also contain in reagent:

(1) the extracted total RNA agents useful for same from mouth neoplasm tissue, including RNA stablizing solution, Trizol reagent, trichlorine Methane, isopropanol, without enzyme water；

It (2) is cDNA agents useful for same, including reverse transcription buffering by LncRNA LINC00472 reverse transcription by template of total serum IgE Liquid, triphosphoric acid base deoxynucleotide, RNase inhibitor, MMLV reverse transcriptase and random primer；

(3) by cDNA real-time quantitative PCR agents useful for same, including real time fluorescent quantitative SYBR dyestuff, without enzyme water.

We detect from reverse transcription after extract RNA in mouth neoplasm and normal oral epithelium sample, real time fluorescent quantitative method The expression of LINC00472, as the result is shown LINC00472 expresses downward in mouth neoplasm tissue.Prompt LINC00472 can make For the molecular marker of mouth neoplasm prognosis prediction.The present invention provides strong for the auxiliary diagnosis and prognosis prediction of mouth neoplasm Biology tool, have far-reaching clinical meaning and important popularization and application foreground.

Detailed description of the invention

Fig. 1 is the expression water that LINC00472 in the in vitro sample of mouth neoplasm is detected using the method for real-time fluorescence quantitative PCR It is flat；

LINC00472 is in the expression in mouth neoplasm (T) than significantly lowering in normal control (N) sample；

Fig. 2 in situ hybridization detects expression of the LINC00472 in mouth neoplasm and normal oral mucosa；

Left figure be normal oral mucosas tissue, right figure be mouth neoplasm tissue, (amplification factor: 200 ×), LINC00472 Expression is lower in mouth neoplasm；

Fig. 3 is the correlation of the expression and patient's prognosis of LINC00472 in mouth neoplasm；

Expresser is good than LINC00472 low expression or not for the highly expressed patient's prognosis of LINC00472, i.e., LINC00472 is low Expression or the patient not expressed are faster dead.

Specific embodiment

The present invention is further illustrated below in conjunction with specific embodiment, is not intended to limit the present invention.

Embodiment 1, the detection of real time fluorescent quantitative method confirm that LINC00472 expresses downward in mouth neoplasm

1. materials and methods:

9 normal oral mucosas and 9 mouth neoplasm tissue extracted total RNAs, 2 μ g RNA after reverse transcription is at cDNA, into Row real-time fluorescence quantitative PCR.

Forward primer: 5 '-GCATCTGTCAACGCTCCTCT-3 ',

Reverse primer: 5 '-CCGCGTTCTTTGTGTGTCTC-3 '.

Internal reference house-keeping gene GAPDH Specific PCR primers for control:

Forward primer 5 '-ACCACAGTCCATGCCATCAC-3 '

Reverse primer 5 '-TCCACCACCCTGTTGCTGTA-3 '.

Real-time fluorescence quantitative PCR reaction system

Real-time fluorescence quantitative PCR reaction step

The amplification curve and melting curve of real-time fluorescence quantitative PCR, the expression intensity root of each gene are confirmed after reaction After CT value (threshold cycle values), reference gene (GAPDH) markization, using group t-test checking computation P value.

2. result

LncRNA LINC00472 expressed in normal control tissue it is lower, and in mouth neoplasm tissue low expression or P < 0.001 (Fig. 1) is not expressed

Embodiment 2, expression of the in situ hybridization detection discovery LINC00472 in mouth neoplasm are related to patient's prognosis

1. MATERIALS METHODS

1.1 are used for the oligonucleotide probe of mouth neoplasm and normal control tissue in situ hybridization

Root Ju LINC00472 gene sequencing is designed 3 best oligonucleotides of the best specificity of Crossbreeding parameters and is visited Needle, in addition, using house-keeping gene GAPDH as positive control.

Oligonucleotide probe in situ hybridization detection LINC00472 expression:

LINC00472 probe 1:5 '-CAAAACTAGGATTCTGTCTTTGAGGCAGAC-3 '

LINC00472 probe 2:5 '-AAGAATGGAAGTGGGAAGAGAGAAAGACTT-3 '；

LINC00472 probe 3:5 '-TAATTCAGGGGTATCCGTCTTAGTTTAGGC-3 '.

Positive control probe (detection house-keeping gene GAPDH):

GAPDH probe 1:5'-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3'

GAPDH probe 2:5'-GTCAGAGGAGACCACCTGGTGCTCAGTGTA-3'

GAPDH probe 3:5 '-CAGTAGAGGCAGGGATGATGTTCTGGAGAG-3 '.

Oligonucleotide probe is synthesized using chemical synthesis process

1.2 oligonucleotide probe labelling kits and in situ hybridization detection reagent

Dig Oligonucleitide Tailing Kit(2^ndGeneration) kit (Roche company), anti-ly High octyl- horseradish peroxidase complex detection kit (Anti-Digoxigenin-POD, Fab fragments, Roche Company).Enhance the TSA signal amplifying system (TSA of detection of expression signal in situ^TMBiotin System, NEL700 kit, PerkinElmer company).DAB staining kit (Beijing Zhong Shan company).20 × SSC, dextran sulfate (Dextran Sulphate), deionized formamide (Deionized Formamide), polyadenylic acid (polyadenylic acid, Poly A), poly deoxyadenylic acid (polydeoxyadenylic acid, Poly dA) is denaturalized the frog essence DNA (denatured of shearing And sheared salmon spermDNA, ssDNA), yeast transfer RNA (yeast t-RNA, tRNA), DTT, 50 × Denhardts ' s solution, PBSbuffer, pepsin K, BSA (bovine serum albumin), triethanolamine (TEA), TNB Buffer (0.1M Tris-HCl, pH7.5,0.15M NaCL, 0.5%Blocking Reagent), TNT Buffer (0.1M Tris-HCl, pH7.5,0.15M NaCL, 0.05%Tween 20) acetic anhydride, block reagent (Blocking reagent Agent, Roche company).

1.3 other main agents and material

Dehydrated alcohol, 90% alcohol, 70% alcohol, 50% alcohol, turpentine oil, distilled water, PBS buffer solution (pH7.2~ 7.4, NaCl 137mmol/L, KCl 2.7mmol/L, Na₂HPO₄4.3mmol/L, KH₂PO₄1.4mmol/L)；3% methanol- Hydrogen peroxide solution (80% methanol and the configuration of 30% hydrogen peroxide)；0.01mol/L citrate buffer (citrate buffer, CB, pH6.0 ± 0.1,9ml 0.1M citric acid solution and 41ml 0.1M sodium citrate solution are added interim in 450ml distilled water With postponing correction work liquid pH value again)；0.1% trypsase；Haematoxylin；1% hydrochloride alcohol (1ml concentrated hydrochloric acid+99ml 70% Alcohol configuration)；The dedicated mounting glue of micro-array tissue (PTS Cure Mount II)；Dedicated coverslip (480 × 240mm²) customization In Zhengzhou Glassware Factory.Leica low melting point (58 DEG C) paraffin, domestic beeswax, absolute alcohol, dimethylbenzene, 10% neutral poly Formaldehyde (0.01mol/L, pH7.4, DEPC distilled water and PBS buffer solution are prepared), haematoxylin, Yihong, neutral mounting natural gum, lid glass Piece, glass slide.

The label of 1.4 oligonucleotide probes

Oligonucleotide probe label is carried out using 3-tailing DIG Olignucleutide Kit, reaction system is such as Under.

100pmol oligonucleotide+ddH2O=9 μ l (5 μ of control:control oligonucleutide l+ddH₂O 4μl)

It mixes, is slightly centrifuged.37 DEG C of water-bath 30min add 2ul EDTA (0.2M, pH 8.0) stopped reaction.

It is purified after 1.5 oligonucleotide probes label

In order to increase the purity of label probe, marked probe need to be purified, concrete operations are as follows:

A) cold ethyl alcohol (- 20 DEG C) of+2.5 μ l 4M LiCL+75 μ l of probe reaction mixture (22 μ l) 100%

B) -70 DEG C of precipitatings 60min, or-20 DEG C of 2h.

C) 13.000 × g, 4 DEG C of centrifugation 15min.

D) supernatant is abandoned, with 70% (V/V) ethanol washing that 50 μ l are ice-cold.

E) 4 DEG C of 13.000xg are centrifuged 5min.

F) supernatant, 4 DEG C of dryings of vacuum are abandoned.

G) with the molten probe of aseptic double-distilled water weight.

1.6 histotomies hybridize pre-treatment

A) histotomy of 4 DEG C of preservations is placed in 58 DEG C of roasting piece 30min, melted surface paraffin.

B) dimethylbenzene successively dewaxes 3 × 5min.

C) step ethanol wash, 100% 1 × 5min of the alcohol of 2 × 2min of alcohol → 95% alcohol of 1 × 5min → 70% → 50% 1 × 5min of alcohol → 2 × 3min of DEPC water washing → DEPC-PBS washs 2 × 5min.

D) 300 μ l pepsin K (10 μ g/ml) are added dropwise on slice, 37 DEG C of digestion 20min.

E) it is sliced and washs 1min, stopped reaction into PBS (0.1M PBS+2mg/ml glutamic acid).

F) it is sliced into 0.2N HCL, in 37 DEG C of reaction 20-30min, increases the permeability of tissue.

G) slice fixes 10min, room temperature with 4% paraformaldehyde (0.1M PBS dissolution) afterwards.

H) in order to increase tissue positive intensity for hybridization, acetylation process is carried out to slice.It is sliced into 0.25% acetic anhydride Buffer I (0.1M triethanolamine), room temperature 10min.

I) 1M PBS washs 2 × 5min.

1.7 tissue prehybridizations and hybridization

A) prehybridization: the prehybridization solution of -20 DEG C of preservations is first placed in 37 DEG C of incubation 60min, and the dosage of prehybridization solution is often to put down Square 12.5 μ l of cm chips area, parafilm covering of corresponding size is sliced, prehybridization 2 hours in 37 DEG C of wet box.(prehybridization solution Composition includes: 2XSSC, 10%Dextran sulphate, 1X Denhardt ' s solution, 50mM Phosphate Buffer (PH 7.0), 50mM DTT, 250 μ l, 100 μ g/ml poly A, 5 μ g/ml poly dA, 250 μ g/ml yeast T-RNA, 500 μ g/ml ssDNA, 47%Deionized formamide).

B) parafilm is removed, prehybridization solution is got rid of, slice is placed in 5min in 2 × SSC.

C) hybridization reaction: 37 DEG C of hybridized overnights (18-20h).Each slice is added 250 μ l hybridization solutions and is covered with parafilm Lid.Corresponding probe is added in prehybridization solution just becomes hybridization solution.Hybridization solution is prepared in prehybridization, is placed 37 DEG C of incubations, is made Probe is completely dissolved in hybridization solution, this experiment is mixed with a plurality of oligonucleotide probe, is matched by each probe 500ng/ml concentration Probe hybridization solution is made.Digoxin tailing labelling kit label probe concentration calculation foundation: the concentration of each probe by its with Colour developing is compared when detection reaction when positive quantitative probe and the naked probe of 30 bases of 100pmol marks reaction theory to visit Needle yield is that two kinds of standards of 900ng carry out the concentration that COMPREHENSIVE CALCULATING goes out label probe.

D) post-hybridization washing, slice immerse 2 × SSC, 10min, throw off parafilm.Successively washed in shake on shaking table, 2 × SSC (0.5%SDS), 2 × 15min → 0.25 × SSC (0.5%SDS), 2 × 15min.

Color developing detection is reacted after 1.8 hybridization

A) digoxigenin-probe compound in conjunction with purpose RNA is detected using Anti-Digoxigenin-POD；TSA amplification system The positive signal of system enhancing in situ hybridization reaction solution reaction, DAB colour developing.

B) slice is gone in TNT buffer, 3 × 5min.

C) TNB is added dropwise and blocks buffer, 300 μ l/TMAs, room temperature, 30min.

D) it does not wash, sucks extra blocking agent, the diluted Anti-Digoxigenin-POD (TBS+0.1% of 1:100 TritonX-100+1% blocking agent), room temperature 4 hours.

E) TNT Buffer (0.1M Tris-HCl, PH7.5,0.15M NaCL, 0.05%Tween 20) washes 3 × 5min.

F) signal is added dropwise on slice and amplifies reagent Biotinyl Tyamid, 300 μ l/TMAs, (Biotinyl Tyramid Store liquid: Biotinyl Tyramid is dissolved in 0.2ml DMSO, Biotinyl Tyramid working solution: 1 × dilution, 1:50 Dilute Biotinyl Tyramid and store liquid), room temperature 10 minutes.

G) TNT is washed, 3 × 5min.

H) SA-HRP (strepto- avidin-horseradish peroxidase) is added dropwise in slice, 300 μ l/TMAs, room temperature 30min.

I) TNT is washed, 3 × 5min.

J) distilled water washing, 1 × 1min.

K) DAB develops the color, and controls chromogenic reaction under microscope.

L) haematoxylin is redyed,

M) alcohol step is dehydrated, chip drying.

N) the dropwise addition dedicated mounting glue of histotomy, the coverslip cover plate of dimension, what dicing-tape transfer system was equipped with Crosslinking slice 1min under ultraviolet lamp.

The judgement of 1.9 results and standard

Application Optics microscope is observed under low power and high power lens respectively, looks first at the positive expression of target RNA The signal positioning intracellular in object observing: it is located at nucleus, cytoplasm or cell membrane.

It is carried out respectively with two kinds of standards of the intensity of the detection rna expression position positive signal and the cell number of positive expression again Comprehensive score, judgment criteria are as follows: (1) judge according to positive cell dyeing intensity: a. cell dye-free remembers 0 point；B. cell is dyed Light brown is weakly positive, remembers 1 point；C. cell dyes brown and dyes dark-brown without background coloration or cell and have light brown back Scape is moderate positive, remembers 2 points；D. cell dyes dark-brown and is strong positive without background coloration, remembers 3 points.(2) according to positive cell Express number score: the no positive cell expression of a. remembers 0 point；B. positive expression cell number≤25% remembers 1 point；C.25% < is positive thin Born of the same parents' number < 50% remembers 2 points；D. positive expression cell number >=50% remembers 3 points.

In order to reduce the subjective factor of appraisal result as far as possible, it is respective that You Liangwei pathology expert presses one of above-mentioned standard respectively Judged and scored, then the two is scored and is multiplied, as a result are as follows: 1. 0 point of person is finally calculated as 0 point, it is believed that feminine gender expression；2. 1 point 1 point is finally calculated as with 2 points of persons, it is believed that weakly positive expression；3. 3 points and 4 points of persons are finally calculated as 2 points, it is believed that moderate positive expression；④ 6, which assign to 9 points of persons, is finally calculated as 3 points, it is believed that strong positive expression.

1.10 analyses and statistical software

Statistical analysis is carried out to experimental result using SPSS13.0 statistical software, compares use χ two-by-two²Test or Fisher Exact test, correlation analysis use Spearmen correlation method；P < 0.05 is that difference is statistically significant. Survivorship curve analysis uses Kaplan-Meier method and log-rank test；Multi-variables analysis uses Cox ' s proportional hazards model；P < 0.05 is that difference is statistically significant.

2 results

1) expression of the lncRNA LINC00472 in mouth neoplasm and normal control tissue

LINC00472 has expression (Fig. 2 is left) in normal control tissue, and expresses in most of mouth neoplasm tissue It is lower or do not express (Fig. 2 right), there is apparent statistical difference (P < 0.05) between the two.

2) Kaplan-Meier survival analysis

We have carried out Effect of follow-up visit by telephone to all 69 mouth neoplasm patients, have inquired their start time in detail, have controlled Treat situation, whether there is or not recurrence, whether there is or not suffering from other diseases, recurrence and death time etc. again, and register life span and state, and right The survival analysis that the expression of LINC00472 and the life span of patient and state carry out in mouth neoplasm tissue, discovery The LINC00472 highly expressed survival of patients time, which expresses patient that is low or not expressing compared with LINC00472, will grow (Fig. 3).Explanation LINC00472 is a molecular labeling relevant to mouth neoplasm prognosis, which expresses low, patient's poor prognosis.

SEQUENCE LISTING

<110>Central South University

<120>application of the reagent of long-chain non-coding RNA LINC00472 expression quantity in the tumor tissues of oral cavity is detected

<130>nothing

<160> 11

<170> PatentIn version 3.3

<210> 1

<211> 9515

<212> RNA

<213>sequence of long-chain non-coding RNA LINC00472

<400> 1

augcgaggcu ggggccgguu gccuaccggc cgcuucucgc cgaggcaguc cagacuuuuc 60

ccccggcggu gcccgcucca agacagcauc ugucaacgcu ccucuucucc ccuccuccuc 120

cugccgggcc gggcuccgcc ggcugcggcc gagaggacgc gggacccggc gcggugagcc 180

caucagcugu caggcgagcg gcgaagcggc uggagggcgg cgagagacac acaaagaacg 240

cggugggcgg cggcggcgaa aggggacggc aacuccuccc cgcgcccgcc ggugccaccg 300

ccggccgugc uuguuccgag gccgcgcaga caaugcggcc gggcucgucc ccgcgugccc 360

cagagugcgg agcgcccgcg cucccccgac cccaacuuga ccgucucccg gcucgcccag 420

cccccucccg ggguaggggc gcccccuccc uccgguggcc ggcgaaggaa gucgguccgc 480

ggccgcagau cccggcaacu ugcgaaccgg gaaaaguuug cggcgccucc gcggggcggc 540

gcgacgcggc ccgccccucg cguccgcggu caucgcgggu gacuuucucg acucgucguc 600

agccggggcc gcagcgcggc cgguggggac ugcggggcgg gccggagucc guccgagggc 660

ucccgcaccu cgggcugcgg auuucaggua cuccacuggg cauuuucucu ucauggagcu 720

uauagcaaca aaaguguuuu acaaaacacc agacauguug gcaucaucuc uuaguugggc 780

cauguggcca gggaacccuu caugaaacca ccucugcagu ccuggagagu ugcuguagaa 840

gaaggaaaag agaggaagac aacacaacac aggaggggau aaguaccucu ggacacaaug 900

gaaguaguag uuguugagca gagggaaagg gauaggagaa ggauguggac ugcagugcug 960

cagugccagg uaauaacuuu gaucauguac cuccuuggcu caggugcugu cucuucuacu 1020

cuccuccuua cuuguacuac cuuauuuagc acuuaauuau auacuuucuu gacucacuuu 1080

gagacacacu gauuaugugg ucugccucaa agacagaauc cuaguuuugg auuuuagcau 1140

uacugaucua uagccuugca ggggcuuaag gauggcagcu gucucucucc cuugggaucu 1200

agcuuuugca aauauugaag ggaagagauu ucuuugaucu uauaggaaua gauccacagu 1260

uccucuccag auuugcuuuu auagagaagu gugugucacc aaaacaaugg agauacgguc 1320

agagaggccc cuacccccag aauuuuaaua uaauuuuacu uucuggggca uuuauucuug 1380

acaaauacuu ucucuccuau cagcucacuu guuccucaca aaaacccuau aagauauuaa 1440

uauuauuaaa cagaccccua ugaagcaccu caagucuuuc ucucuuccca cuuccauucu 1500

ugcacaccuu uugcuagacu agucucugcg uucaucacau cagguuucua aaauaaaagu 1560

ccccuaauaa cuuucuauuu guucucucau cauauugaac cuuuuuugcc uggcuuauaa 1620

gguggccuua aaaccccauu uuccucuaaa cauugucucu guaguucagg aauauggcag 1680

gcucaacacu augcuugagc ccauauugcu ucccucauua gugaugccuc cucucagacc 1740

acccagauuc uacucauucu ucaauuucca gcuucuccuu ggagccuucc cagaucauua 1800

gcuucuuaaa aaucccagcu uucucugaau ucugauggua gcccccguua uuuggacaau 1860

uauguccugg ucuuaaaguc cuaaugggaa aaaaaaguac augauggacu ucauguagag 1920

aagaaagcac uggacugagu uuuaagacag cugcuaagga gcugugugag ccaaaccagg 1980

uuacuuuacc ucucuggauc uauauuugcu gaucuguuaa guggagacaa uugggucaga 2040

uaaucuuggu ggucuuugcu uauucugagg uucuuggaca acuuggccua guucacgugg 2100

gaugggauug aagaucuuac ugaguucuug gauggaaaug ucaguaaugg gcacuuagug 2160

ccuucaggac uuguucuaua cuaucccaga aauuucugau gcuaaacuug gcaauuuuua 2220

aaugucauau uccuuuucuc caaacucuuu aaccacaaga auucagggaa gguuaugggg 2280

ggugacuuag uuaucuggau aauacauauu guggacaaua uagcauuauc cuugggaaug 2340

auuucauauu guugaauaaa uaggaucaac uucuugauaa accuuagucc caagcauuuc 2400

aucuucauug gauaguguua cauaguaaua uauuuauguu uucuuuuaau cauuucauaa 2460

cuuggaaaau acuaacauag ucaaaacucu aggguaggug auacaugagu uucuguagua 2520

aucugguugg agacauguug uaauucugua uauauaugua cauuuauccc augcauguua 2580

ugccuaaacu aagacggaua ccccugaauu aagaggugcu guuauacauu gaccaggcuu 2640

aagaauaucu cuuuaaagug ugucgacauu uaauugaccu uuggaaguuc auucuguuaa 2700

ucauacucaa agugcuaaag cuaugguuga cugcucuggu guuuuuauau ucauucgugc 2760

uuuagcauau aaauucuuca gcauaauugc uacuuauuua gcaagaguuu ccuuuauuug 2820

aaaaugugag uugugcuugu auuuuugugu cuuucuuucu uucuuucuuu uuuuaaacuu 2880

ugcuucaggc uggguagugg uagagguuug aauuaaaaug uuuuccuguc aguaguugua 2940

ugaggugugg cuucuuaauc uagaaguaau ggaaauuucu ugcuuaaaug aagugcacuu 3000

uucuuccugc agaaguuaga acucuuuuag acaagcuuaa cacuucaaaa cuguauaucu 3060

uuaacagaaa cuuuuggaaa uuuuuaaauu ccauuugugu agauuaaaaa uaucaaugcu 3120

aauacuuucu auuaaauaag gauuuaucua uuuucauaaa accucuaauu auauuccuug 3180

aauugcuaug aguagaauuu uguuguaaac acaauauuuu uaaaaaguca aauauauuuu 3240

aaaaaucuua acauuauauu acuauaacuu agagaacagg uguuuacaug uuuaaaauuu 3300

ucuaggccuu aaauuuguga aaucagucuu ucagaaguua uuuuuaauau aguuaucugu 3360

guuacagguu uauaaguuga cauuucaauu uaaaauuuuu ucaauuuaua uuucaaugac 3420

caggaaaaaa uaguuaaacu uauuccuaaa uuuuaauaga aaaauaaaaa accacauagu 3480

uguuaauuuc ugguaauuua ucuugagacu ccaggcaugu uuuuagaaca uuuuuaccuu 3540

aaaagcaauu uugaaauagg gaaauuaaag uuuaaagcag aguuuucacu uuaaugaggu 3600

aauuuuauac uucccagaua gguaggccca uauucaagaa cacucugauc ucuguuuaua 3660

guaaguuuug augccauuau aucauuuacu uauucacgca uuauaagcau aguagauagc 3720

cccauuguca cgaagcuggc auuauuuucu ugucugucau ucaauauugu ccccuuuuuu 3780

uuuucuguuu ucaucauagc cguccuuuuc auuuuauugu agcccugacu cgaaaugaaa 3840

guaaguuuga acucuuaaaa aagaguuccu caaacucuuu uuuaagaaua uacagugaaa 3900

ugucuuaaau aauauuaucu aaauaauacc uaaagaaugc aaaugacuug caaugcugaa 3960

uagucagaca caggauguuc caugcuaagg guagacucau auuuccacuu uauaaaaugu 4020

gguuucagaa aacaaauaga uguguuucuu ccuuaauaau uaguguuuca ggaccaaagu 4080

ugacuaaaua uggaaguuua agauaaaacc cgguuucuua gggaaucuuu cucaguccuu 4140

aguaauucug uagaauugau gauagagugg caacuucaau acacucagua uucuuuucug 4200

uuugaagucu guuuugccau uggaaaaaac uuauuguugu uugaauuugg guauauguau 4260

ucacagaugg uuuacuuuau uuuuauuuug uuuaaacaaa uacuuucaug ucccaaacac 4320

ugagacaugu uucauguccu aaacacuuua uaaauauuag aauauuuaau ccccuuaacu 4380

cuguaguuau aauuaucuuc auuuuacaau ugagaaaacu aagauacgac guuuuauuua 4440

augguaacgc cuuaaguaag gggagaguga uaaguguaag cagaguggaa aacuuaguag 4500

ugcuaaggag caacagaagu augugcaaga guuugggaag augugucugc uuggaaagga 4560

ucagggacuc agcugggauc agucuuaaag agcaacaugu ccucauuccu caauuuuaag 4620

ccuuugggcu cuagaaguaa agggcaaacu uagaggaggc acuccucagg uuucagcugc 4680

cuguuccggc cuggugaggu ggcuguacug gcucaugugu uucucauagg gcuacaugag 4740

ccuuuauguc uuaccuccuc cucauuucuu ggcugacucc ugcuuuuuug gaggguuuaa 4800

cuucucucuc agcuagaagg uuaggcauug caaauaccag uggauaauuu uuuucuuagc 4860

uuuaacccca gcccauuuca acccccucuu ugcccuuugu auauucuuuu gaaaauauga 4920

uccaguagug uuuaugaaug uguguugugu aaaauuuaga gauugauguu aaacaacaga 4980

auuaaaggac aaagcugucu uuuuuguugg aauuggggau gggagagcag cucaaagugg 5040

gaaauaugga gaaaggagag acauugugga aaagaagaga uaaauugagg ggaaaaaaag 5100

aggaugaaaa aaugaagaga gagaauuacu gaugugcuuc acacccacac cuccucucca 5160

acuucacagu aacagaggca augucacuau cuuuuaccug acaagugcuu uaccagggag 5220

uugugccugg caucguggga guggaaaauu uuucccuuuu gcauuuugag acuugggugu 5280

gcugcuuuaa gaugccacug guaucuaagu aauguuuguu uauacuggau auuuuucucu 5340

guagccuuug uggauuggac uuauaauaua aacaauauug uuagcagaac ugcagguacu 5400

cuauugcuau guuuauuuua uuaguuaaaa aacuaccaga gcccuaggac uucugagcac 5460

auuuagaaaa uaccagaggc aauuuuaaga cuagcauuag aaagaaagaa aagaauaaga 5520

auaaaacuaa gacuugcaua ccuuagaauc cuaaccaaug uggugauuuu uaaaaauuaa 5580

uguuucagag acucuaaauu aggguaguug guuucaucaa uuacauuuuu uaaguuaaua 5640

uaaagugcuu uuguauguaa auucucuuaa uagauaauuu agguaaaaug aaaguguggc 5700

auuuuuuguu ccauagaaug uaaaacuaac gucagaauaa gaauucuuuu gggaauuuag 5760

gauuuuuuug augcuucuca aaucaucugu aaugacuuuu auuuauagaa aagguagauu 5820

auucauuagu uuuuaugaaa aacacucagu uuaaauagcc ggguauggug gcgcauggcu 5880

guagucccag cuacucagga ggcugggcac gagaauugcu ugaaccugug aggcagaggu 5940

ugcagugagc caagaucgca ccacugcacu ccagccuggu ccagagugag auuccgucuc 6000

gaacaaaaca aaacaaaaau auugagcuaa ucuaaauggu aaaaaucuag auuugaagaa 6060

auuuauuuua aaauaguugu ucauuuguca aauagaaaag uaugaucuua gcagguuugg 6120

uuagaucccu aucugaaaca aaguaaacua aagauguuuu ucuugaucaa ugcauagcaa 6180

uaacugcugu ucaaacaugu gaggaaucac caguaaauug ggggauagaa agcaggaaaa 6240

ggagcuuuuu ucucuuauuu uuuuucauuu aucaguuuuu cccauuuguc cauccuuucc 6300

uauaaucaug gaaauuuuua uuuuuuacac uccucucccc auuuuuuuau ccuagauugc 6360

caccacacca agguuuucua gaguuaggca gaaagagaac cugggccuag augcuaaugg 6420

aaacuauccc augcuuucuc agacucucuu uccuccccag agacaagagg agcaaaacag 6480

gaguugaugg cucauugcug uugcuucugu ggcugcccug uuugguugac cugagcuuuu 6540

uuuaaaaacc uccacucuug acgcuuaacu cuaugaauaa aauuagggcc agucacacug 6600

uauuuucauc aucaguuauc ccuagugauu caaauuuagc acaaaccugc ugugaguaca 6660

uauuaauuag gugaauacag uagaaugcaa ucauaacaug agucuuuuaa uaacuauucu 6720

uuguuugauu uuuauuuuag aaguuugaag uuauuuuguu gaaaaucuag aaaaucuuua 6780

ugugagcaac ugagguucgg guagcccaca acaguuuaaa ucaaacauua acuugcagua 6840

aaacacuacu gaaagugucu uugauuuuga ggugcagaga gcuguuucaa guuaaguugc 6900

uauauuaguc uguucucaug cugcuaauaa agacauaccu gcaaccgggc aauuuauuaa 6960

aaaaaaaaag agguuuaauu gacugacagu uccacauggc uggggaggcc ucaccgucau 7020

ggugaaagag caagggaugu cuuacauggu ggcaggcaag aaguuugugc agaggaacuc 7080

cuguuuauaa aaccaccaga ucucaugaga cuuacucacu accaggagaa caggaugcgg 7140

gaaaccaccu cggugauuca guuaucugca ccuggcccca cccuugacac auguugauua 7200

uuacaauuca aggugagauu uggguaggua cacagccaaa ccauaucagc ugcccauucu 7260

ccugugcagu gacauaaagg ucugauaacc ugauuuaaag cuuggacucu aagauuuuuu 7320

uuuuucuuuu uuugagucag guuaucucug ucgcucaagc uggaguucag uggcaugaac 7380

agggcucauu gcagccucaa ccuuccaggu ucaagugauu cucuugccuc aaccuccuga 7440

guaucugggg cuacaggcau gugccaccgu gccaggcuaa uauuuuuauu uuuauuuuug 7500

uaaagauggg gucucacuuc ugggcucaag caaucuuucc ucagccuccc aaagugcugg 7560

gauuauaggu gcgagccacu gcacccugcu guaaagaauu ucauaucccu guuuucggug 7620

ugcuguguca cuguuugcca uggguaaagg uauaugaauc ucuuuuauuu ugcugcuugc 7680

aacccaauuc ccaugccaaa cccaauuugu auccaaacaa acaauauugg agacaaguuc 7740

uucuauagaa agcuucauaa gccaguuacu gacuacugug ucuguaaaua uacucaaacu 7800

auuuuguagu cuugcuauuu uaaaaaaugu gugggugugg gaagugaugu ugaaugaauu 7860

caggguaauu ggugguucuu uguauuagga aaacucacua guuaagggaa gucuguuuuc 7920

augauucaug uauguuuaac uuaaaaaaaa auucuuccau ccuucucucc aguuuccacu 7980

cuaccucaua aauucaagua augaaggaau uuaaauaaua gaauuuuaua acugaaagga 8040

uuuuggagag aaucuauuaa cccuccuacu acaguugagg aaauagaugu ccagugaggu 8100

caaaugagau cauguagcua guuuaaaaac ccaaauaaca uuuuaguagu uuguuuccau 8160

uaguaaacac uguucuguuu gauuugcccu aacauguuca uaaucagagu cauaauaagg 8220

aaaaaucacu aauaugugug ugguacuuuc uugugcauac cucagucacu ucucaaaaga 8280

ggucuguaag gugucgguau ccugguuucc auucagggag agaacagucu augagccuaa 8340

gagaccuguc ugugccaguc acuaucagaa cagucuaaug cuaugugucu uccuaccuug 8400

gcaaaagcau uggcuggucu cuggacaguu cucauagacc acaggauugg gcagggggga 8460

gccacaucuu cauguuaggg cucuaaccug uaccaauuau uaacuguugu cucuauuuca 8520

ucauuucuua aagggaaaaa aaagcacuuu uuaaagagca ugagauuuua aauuuuucaa 8580

aagaaggaug cuggaucauu auaaguaagg cauguuuuaa agaucuucuu aucaacagcu 8640

uaaaaauuag ccaguauaua cugaacaucc acuguauaaa aagcacauua uggggugcuc 8700

caguagguac agcagugauc gagacagaau cauagccuuc agggaagagg uauaugcaau 8760

guucucuccu uccaaagauu accuagauag agaacuguuc auaagaaaua ucuugguacu 8820

gucuacauua cucuugagau auaaaauaua uauuauuucu uacgucugaa augagucagu 8880

uguuucuaaa acaauuugua uuaaaguauu cacauuaaaa auuuguaaaa uacagaaaag 8940

uagagagaaa aaaucaugaa uuuuuaacau gguaaugauu auaucaguuu cguucuuggc 9000

guuucuuuaa cuugauggga uagcuaaugu uuuuugucau ugcauagcuu uguaaacuug 9060

uccuaaggau uauauaauuu cagcaugagu uaguuucuaa guaugucaga aaucuagcuu 9120

uuuuguggua auuguuguuu uacacaaaau gauuaauaaa agacucuaaa cuaaccuugg 9180

cuuuagaaau gcuuuuuaaa uuuuaaaaua gugaaugagu agcagauuac uuucuaguau 9240

uucuuuaagg ucaccaaaau cacuuaacuu gcuuaaaaua uugauggaaa uaucaacuac 9300

guacaguuga ccuaaauuag aaaugggugu uuuuuaaaua gugaaauuua uaaauauaau 9360

auguauauuu uaaaauacag uuuauauuau agaguuaugu uccucuguaa uaauuucucu 9420

uuacacugaa ucauguucag uuuuuauuau gcagauaauu uaaucuuaca gcuuugaucu 9480

uuuuuaaaaa auaaaacagc cauuuggagu cuaaa 9515

<210> 2

<211> 20

<212> DNA

<213>reference gene GAPDH specific PCR forward primer

<400> 2

accacagtcc atgccatcac 20

<210> 3

<211> 20

<212> DNA

<213>reference gene GAPDH specific PCR reverse primer

<400> 3

tccaccaccc tgttgctgta 20

<210> 4

<211> 20

<212> DNA

<213>LINC00472 forward primer

<400> 4

gcatctgtca acgctcctct 20

<210> 5

<211> 20

<212> DNA

<213>LINC00472 reverse primer

<400> 5

ccgcgttctt tgtgtgtctc 20

<210> 6

<211> 30

<212> DNA

<213>in situ hybridization detects LINC00472 probe 1

<400> 6

caaaactagg attctgtctt tgaggcagac 30

<210> 7

<211> 30

<212> DNA

<213>in situ hybridization detects LINC00472 probe 2

<400> 7

aagaatggaa gtgggaagag agaaagactt 30

<210> 8

<211> 30

<212> DNA

<213>in situ hybridization detects LINC00472 probe 3

<400> 8

taattcaggg gtatccgtct tagtttaggc 30

<210> 9

<211> 30

<212> DNA

<213>GAPDH probe 1

<400> 9

ccactttacc agagttaaaa gcagccctgg 30

<210> 10

<211> 30

<212> DNA

<213>GAPDH probe 2

<400> 10

gtcagaggag accacctggt gctcagtgta 30

<210> 11

<211> 30

<212> DNA

<213>GAPDH probe 3

<400> 11

cagtagaggc agggatgatg ttctggagag 30

Claims (5)

1. detecting the application of the reagent of long-chain non-coding RNA LINC00472 expression quantity in the tumor tissues of oral cavity, which is characterized in that The reagent is used to prepare mouth neoplasm auxiliary diagnosis or outcome prediction preparation, long-chain non-coding RNA LINC00472 Sequence see SEQ NO:1.

2. application according to claim 1, which is characterized in that the mouth neoplasm auxiliary diagnosis or outcome prediction system Agent is that the expression according to long-chain non-coding RNA LINC00472 in mouth neoplasm is pre- to carry out auxiliary diagnosis and curative effect It surveys, LINC00472 expression is significant in mouth neoplasm tissue lowers.

3. application according to claim 1, which is characterized in that the mouth neoplasm auxiliary diagnosis or outcome prediction system Agent is real time fluorescent quantitative detection reagent.

4. application according to claim 1, which is characterized in that include in the real time fluorescent quantitative detection reagent

Primer sequence for real time fluorescent quantitative detection LINC00472 expression:

Forward primer: 5 '-GCATCTGTCAACGCTCCTCT-3 ',

Reverse primer: 5 '-CCGCGTTCTTTGTGTGTCTC-3 '.

5. application according to claim 3 or 4, which is characterized in that the real time fluorescent quantitative detection preparation includes:

Reference gene GAPDH Specific PCR primers:

Forward primer 5 '-ACCACAGTCCATGCCATCAC-3 '

Reverse primer 5 '-TCCACCACCCTGTTGCTGTA-3 '.