CN105506157B - In situ hybridization detects the application of long-chain non-coding RNA LOC284454 reagent in tongue cancer - Google Patents

In situ hybridization detects the application of long-chain non-coding RNA LOC284454 reagent in tongue cancer Download PDF

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CN105506157B
CN105506157B CN201610066689.7A CN201610066689A CN105506157B CN 105506157 B CN105506157 B CN 105506157B CN 201610066689 A CN201610066689 A CN 201610066689A CN 105506157 B CN105506157 B CN 105506157B
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loc284454
tongue cancer
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熊炜
曾朝阳
李小玲
李桂源
喻建军
刘彦
唐艳艳
杨丽婷
石磊
范松青
张文玲
向波
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Central South University
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Abstract

The invention discloses the applications of long-chain non-coding RNA LOC284454 reagent in situ hybridization detection tongue cancer, mainly for the preparation of tongue cancer auxiliary diagnosis or outcome prediction preparation.Confirm that LOC284454 expresses up-regulation in tongue cancer by research, Tongue Cancer Patients poor prognosis of the Tongue Cancer Patients than low expression LOC284454 of height expression LOC284454, therefore, the expression of LOC284454 is used for the prognosis prediction of Tongue Cancer Patients, strong molecular biology foundation can be provided for the prognosis of Tongue Cancer Patients, there is far-reaching clinical meaning and important popularization and application foreground.

Description

In situ hybridization detects long-chain non-coding RNA LOC284454 reagent in tongue cancer Using
Technical field
The invention belongs to oncomolecularbiology fields, and in particular in situ hybridization detects long-chain non-coding in tongue cancer RNA LOC284454 reagent is used to prepare the purposes of tongue cancer auxiliary diagnosis or outcome prediction preparation.
Background technique
The Human Genome Project and its subsequent DNA element encyclopedia plan (The Encyclopedia of DNA Elements Project, ENCODE) research achievement shows that protein coding gene sequence only accounts for the 1- of human genomic sequence 3%, and in human genome the transcribed sequences of the overwhelming majority be long-chain non-coding RNA (Long non-coding RNA, lncRNA).LncRNA is universally present in various biologies, and with the raising of biological complex degree, lncRNA in genome The ratio of sequence also correspondingly increases, and prompts lncRNA significant during biological evolution.With lncRNA constantly quilt It was found that their function is gradually interpreted, scientists discovery lncRNA actively participates in vital movement different layers extensively In the function controlling in face, the field brand-new as one has become international life science field new forward position and hot spot.
LncRNA can be used as signal (signal), induction (guide), bait (decoy) or the bracket of functional protein (scaffold) various ways such as molecule regulate and control base in chromatin reconstruct, genetic transcription, translation and the multiple levels such as protein modified The expression of cause, and irreplaceable role is played during including the basic physiologicals such as development, immune, reproduction, it is prior It is that the expression of lncRNA and functional disturbance are closely connected with a variety of diseases of the mankind including malignant tumour one It rises.Therefore, the function of lncRNA is furtherd investigate, is disclosed by the lncRNA hereditary information transfer mode mediated and expression regulation net Network not only can annotate and illustrate again the structure and function of genome from the angle other than protein coding gene, deeply find The essence and rule of vital movement are expected to recognize from a new visual angle a variety of mankind's common diseases including tumour Pathogenesis, and new molecular marker and therapy target are provided for the Clinics and Practices of these diseases.
Tongue cancer is the most common carcinoma of mouth, and male is more than women.Tongue cancer majority is squamous carcinoma, mostly occurs in lingual margin, is secondly The tip of the tongue, back and root of the tongue etc. are often ulcer type or infiltrative type.General grade malignancy is higher, and growth is fast, and wellability is stronger, often Involve lingualis, cause tongue limitation of movement, it is difficult to make to speak, feed and swallow generation.The occurrence and development of tumour are polygenes ginsengs With, multi-step, multistage complex process, LncRNA may also play important work during the occurrence and development of tongue cancer With.We have found lncRNA LOC284454 (NCBI accession number:NR_036515) in tongue in tongue cancer recently Significant up-regulation is expressed in cancer, LOC284454 expresses high patient and is easier to shift, and life span is shorter than the lncRNA table Up to low patient, therefore the detection preparation for being directed to the lncRNA can be used for the Index for diagnosis of tongue cancer.
Summary of the invention
For the above-mentioned prior art, the object of the present invention is to provide long-chain non-codings in situ hybridization detection tongue cancer RNA LOC284454 reagent is used to prepare the purposes of tongue cancer auxiliary diagnosis or outcome prediction preparation.
In situ hybridization detects the application of long-chain non-coding RNA LOC284454 reagent in tongue cancer, and the reagent is used In preparation tongue cancer auxiliary diagnosis or outcome prediction preparation；The sequence such as SEQ NO:1 of long-chain non-coding RNA LOC284454 It is shown.
Long-chain non-coding RNA LOC284454 reagent includes below for original in the in situ hybridization detection tongue cancer One or more of the oligonucleotide probe of position hybridization check LOC284454 expression:
LOC284454 probe 1:5 '-CCAACATGGCGAAACTCGGTCTCTACTAAAAATAC-3 '；
LOC284454 probe 2:5 '-ACCCCGTCTCTACTAAAAATACAAAGATTAGCCGG-3 '；
LOC284454 probe 3:5 '-TTATTTACTTGAACAACAGACATGACACACAGCAC-3 '.
Long-chain non-coding RNA LOC284454 reagent includes that the following positive is right in the in situ hybridization detection tongue cancer According to one or more of probe:
GAPDH probe 1:5'-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3'
GAPDH probe 2:5'-CAGTAGAGGCAGGGATGATGTTCTGGAGAG-3'
GAPDH probe 3:5'-GTCAGAGGAGACCACCTGGTGCTCAGTGTA-3'.
By in situ hybridization, the epithelium by the tongue cancer and tongue cancer cancer achieves in paraffin organization sample and finds LOC284454 the present invention Significant up-regulation is expressed in tongue cancer, and the expression of LOC284454 and prognosis are closely related, it is raw that LOC284454 expresses high patient It deposits that the time is shorter, LOC284454 is prompted to can be used as the molecular marker of tongue cancer prognosis prediction.The present invention be tongue cancer auxiliary diagnosis and Prognosis prediction provides strong biology tool, has far-reaching clinical meaning and important popularization and application foreground.
Detailed description of the invention
Fig. 1 is that Real-Time Fluorescent Quantitative PCR Technique detects lncRNA LOC284454 in Tongue Cancer Patients (167) and tongue cancer cancer Expression in side tissue (45)；Expression of the LOC284454 in tongue cancer in cancer beside organism than significantly improving, p < 0.001.
Fig. 2 is that in situ hybridization detects expression of the LOC284454 by the tongue cancer and tongue cancer cancer in epithelium；
Expression is lower (30.2%, 13/43) in tongue cancer cancer beside organism, and in 52.7% tongue cancer (148 tongue cancer groups 78 knitted) in detect the expression of LOC284454, P < 0.001.
Fig. 3 is the relationship of expression Yu the tongue patient prognosis of LOC284454 in tongue cancer,
The high of LOC284454 expresses, i.e. LOC284454 highly expressed trouble related to the poor prognosis of Tongue Cancer Patients in tongue cancer The total life span of person (Over-all survival) will be significantly lower than LOC284454 low expression or the patient not expressed.
Specific embodiment
The present invention is further illustrated below in conjunction with specific embodiment, is not intended to limit the present invention.
Embodiment 1, quantitative real-time PCR detection confirm that LOC284454 expresses up-regulation in tongue cancer
1. materials and methods:
Collect by 45 tongue cancer cancers and 167 tongue cancers, extracted total RNA, 1 μ g RNA after reverse transcription is at cDNA, into Row real-time fluorescence quantitative PCR.LOC284454 forward primer is 5 '-AGCCATCCTCCCACCTTAGT-3 ', such as SEQ NO:2 institute Show and reverse primer 5 '-TCCTCAGTCCCACCCAGAAA-3 '.As shown in SEQ NO:3.
GAPDH forward primer for control is 5 '-ACCACAGTCCATGCCATCAC-3 ' as shown in SEQ NO:4, and Reverse primer 5 '-TCCACCACCCTGTTGCTGTA-3 ', as shown in SEQ NO:5.
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
LOC284454 does not express or expresses very low, and the high expression P in tongue cancer by the tongue cancer cancer in control tissue < 0.001 (Fig. 1)
Embodiment 2, expression of the in situ hybridization detection discovery LOC284454 in tongue cancer are related to patient's prognosis
1. MATERIALS METHODS
1.1 design and synthesize hybridization probe
In order to which using the expression of in-situ hybridization method detection LOC284454, we devise examines in situ hybridization Survey the oligonucleotide probe and two groups of in situ hybridization oligonucleotide probes of positive control each 3 that LOC284454 is expressed.
Oligonucleotide probe in situ hybridization detection LOC284454 expression:
LOC284454 probe 1:5 '-CCAACATGGCGAAACTCGGTCTCTACTAAAAATAC-3 ' such as SEQ NO:6 institute Show,
LOC284454 probe 2:5 '-ACCCCGTCTCTACTAAAAATACAAAGATTAGCCGG-3 ' such as SEQ NO:7 institute Show,
LOC284454 probe 3:5 '-TTATTTACTTGAACAACAGACATGACACACAGCAC-3 ' such as SEQ NO:8 institute Show.
Positive control probe (detection house-keeping gene GAPDH):
GAPDH probe 1:5'-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3', as shown in SEQ NO:9,
GAPDH probe 2:5'-CAGTAGAGGCAGGGATGATGTTCTGGAGAG-3', as shown in SEQ NO:10,
GAPDH probe 3:5'-GTCAGAGGAGACCACCTGGTGCTCAGTGTA-3', as shown in SEQ NO:11.
Each gene specific oligonucleotides probe sequence of above-mentioned design is synthesized using chemical synthesis process.
1.2 oligonucleotide probe labelling kits and in situ hybridization detection reagent
Digoxin oligonucleotides tailing reagent (Dig Oligonucleitide Tailing Kit 2ndGeneration, Roche company), anti-digoxin-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 situTM Biotin System, NEL700 kit, PerkinElmer company), DAB staining kit (Beijing Zhong Shan company), 20x sodium citrate Buffer solution (saline sodium citrate, SSC), dextran sulfate (Dextran sulphate), deionized formamide (Deionized Formamide), polyadenylic acid (polyadenylic acid, Poly A), poly deoxyadenylic acid (polydeoxyadenylic acid, Poly dA) is denaturalized frog essence DNA (the denatured and sheared of shearing Salmon sperm DNA, ssDNA), yeast transfer RNA (yeast t-RNA, tRNA), dithiothreitol (DTT) (DTT), 50x Deng Han Family name's buffer (Denhardts ' s solution), phosphate buffer (PBS buffer), pepsin K, bovine serum albumin(BSA) (BSA), 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, resistance Disconnected 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)；Mounting glue (PTS Cure Mount II)；Dedicated coverslip (480 × 240mm2) customize in Zhengzhou glass apparatus Factory.Leica low melting point (58 DEG C) paraffin, domestic beeswax, absolute alcohol, dimethylbenzene, 10% neutrality paraformaldehyde (0.01mol/L, PH7.4, DEPC distilled water and PBS buffer solution are prepared), haematoxylin, Yihong, neutral mounting natural gum, coverslip, glass slide.1.4 mark Remember probe
Oligonucleotide probe label is carried out using 3-tailing DIG Olignucleutide Kit, reaction system is such as Under.
It mixes, is slightly centrifuged.37 DEG C of water-bath 30min add 2 μ l EDTA (0.2M, PH 8.0) stopped reactions.
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:
1) cold ethyl alcohol (- 20 DEG C) of+2.5 μ l 4M LiCL+75 μ l of probe reaction mixture (22 μ l) 100%
2) -70 DEG C of precipitatings 60min, or-20 DEG C of 2h.
3) 13.000 × g, 4 DEG C of centrifugation 15min.
4) supernatant is abandoned, with 70% (V/V) ethanol washing that 50 μ l are ice-cold.
5) 4 DEG C of 13.000xg are centrifuged 5min.
6) supernatant, 4 DEG C of dryings of vacuum are abandoned.
7) with the molten probe of aseptic double-distilled water weight.
1.6 in situ hybridizations detection achieves the expression of LOC284454 in paraffin section
Paraffin section hybridizes pre-treatment
1) paraffin section of 4 DEG C of preservations is placed in 58 DEG C of roasting piece 30min, melted surface paraffin.
2) dimethylbenzene successively dewaxes 3 × 5min.
3) 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.
4) 300 μ l pepsin K (10 μ g/ml) are added dropwise on slice, 37 DEG C of digestion 20min.
5) it is sliced and washs 1min, stopped reaction into PBS (0.1M PBS+2mg/ml glutamic acid).
6) it is sliced into 0.2N HCL, in 37 DEG C of reaction 20-30min, increases the permeability of tissue.
7) slice fixes 10min, room temperature with 4% paraformaldehyde (0.1M PBS dissolution) afterwards.
8) in order to increase tissue positive intensity for hybridization, acetyl processing is carried out to slice.It is sliced into 0.25% acetic anhydride Buffer I (0.1M triethanolamine), room temperature 10min.
9) 1M PBS washs 2 × 5min.
Prehybridization and hybridization
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 50 μ l, Parafilm is carried out lid and 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)。
1) parafilm is removed, prehybridization solution is got rid of, slice is placed in 5min in 2 × SSC.
2) hybridization reaction: 37 DEG C of hybridized overnights (18-20h).Each slice is added 250 μ l hybridization solutions and is carried out 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.
3) 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 hybridization
1) digoxigenin-probe compound in conjunction with mRNA is detected using Anti-Digoxigenin-POD；TSA amplification system Enhance the positive signal of in situ hybridization reaction solution reaction, DAB colour developing.
2) slice is gone in TNT buffer, 3 × 5min.
3) TNB is added dropwise and blocks buffer, 300 μ l/TMAs, room temperature, 30min.
4) extra blocking agent, the diluted Anti-Digoxigenin-POD of 1:100 (TBS+0.1%Triton X- are sucked 100+1% blocking agent), room temperature 4 hours.
5) TNT Buffer (0.1M Tris-CL, pH7.5,0.15M NaCL, 0.05%Tween 20) is washed, 3x5min。
6) 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.
7) TNT is washed, 3 × 5min.
8) SA-HRP (strepto- avidin-horseradish peroxidase) is added dropwise in slice, 300 μ l/TMAs, room temperature 30min.
9) TNT is washed, 3 × 5min.
10) distilled water washing, 1 × 1min.
11) DAB develops the color, and controls chromogenic reaction under microscope.
12) haematoxylin is redyed,
13) alcohol step is dehydrated, chip drying.
14) mounting glue, the coverslip cover plate of dimension, crosslinking slice 1min under ultraviolet lamp is added dropwise.
The judgement of 1.7 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.
1.8 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
Expression of 2.1 LOC284454 in tongue cancer is significantly increased than the expression in tongue cancer cancer beside organism
LOC284454 high expression in 52.7% tongue cancer (78/148), and only by 30.2% tongue cancer cancer (13 in 43) have expression (Fig. 2) in tissue, have apparent statistical difference (P < 0.001) between the two.
The highly expressed Tongue Cancer Patients prognosis of 2.2 LOC284454 is poor
Effect of follow-up visit by telephone has been carried out to 148 Tongue Cancer Patients, inquired in detail they start time, treatment condition, 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 in tongue cancer The survival analysis that the expression of LOC284454 and the life span of patient and state carry out, discovery LOC284454 high expression patient are flat Equal life span is significantly shorter than LOC284454 low expression or the patient not expressed (Fig. 3).Illustrate that LOC284454 is one and tongue The relevant molecular labeling of cancer prognosis, lncRNA expression is high, patient's poor prognosis.

Claims (2)

1. the application that in situ hybridization detects long-chain non-coding RNA LOC284454 reagent in tongue cancer, which is characterized in that described Reagent be used to prepare tongue cancer auxiliary diagnosis or outcome prediction preparation；The sequence of long-chain non-coding RNA LOC284454 is such as Shown in SEQ NO:1；
Long-chain non-coding RNA LOC284454 reagent includes below in situ miscellaneous in the in situ hybridization detection tongue cancer Hand over one or more of the oligonucleotide probe of detection LOC284454 expression:
LOC284454 probe 1:5 '-CCAACATGGCGAAACTCGGTCTCTACTAAAAATAC -3 '；
LOC284454 probe 2:5 '-ACCCCGTCTCTACTAAAAATACAAAGATTAGCCGG -3 '；
LOC284454 probe 3:5 '-TTATTTACTTGAACAACAGACATGACACACAGCAC -3 '.
2. application according to claim 1, which is characterized in that the non-volume of long-chain in the in situ hybridization detection tongue cancer Code RNA LOC284454 reagent includes one or more of following positive control probe:
GAPDH probe 1:5'-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3'
GAPDH probe 2:5'-CAGTAGAGGCAGGGATGATGTTCTGGAGAG-3'
GAPDH probe 3:5'-GTCAGAGGAGACCACCTGGTGCTCAGTGTA-3'.
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