CN106319044A - Biomarker for auxiliary diagnosis or curative effect prediction of nasopharyngeal carcinoma and application thereof - Google Patents
Biomarker for auxiliary diagnosis or curative effect prediction of nasopharyngeal carcinoma and application thereof Download PDFInfo
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Abstract
The invention discloses a biomarker for auxiliary diagnosis or curative effect prediction of nasopharyngeal carcinoma and application thereof, and the biomarker is mainly used for preparing a real-time fluorescent quantitative detection reagent for the auxiliary diagnosis or the curative effect prediction of the nasopharyngeal carcinoma. A real-time fluorescent quantitative PCR technology verifies the expression situations of a long-chain non-coding RNA LINC01420 in epithelial tissues of the nasopharyngeal carcinoma and chronic inflammation nasopharynx, and the expression of the LINC01420 in the tissues of the nasopharyngeal carcinoma is remarkably up-regulated. The biomarker provides an important reference basis for early discovery and early treatment of the nasopharyngeal carcinoma.
Description
Technical field
The invention belongs to oncomolecularbiology field, be specifically related to a kind of nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction is used
Biomarker long-chain non-coding RNA LINC01420, and the reagent detecting this rna expression amount is auxiliary for preparing nasopharyngeal carcinoma
Help diagnosis or the purposes of outcome prediction preparation.
Background technology
The Human Genome Project and follow-up DNA element encyclopedia plan (The Encyclopedia of DNA thereof
Elements Project, ENCODE) achievement in research shows, protein coding gene sequence only accounts for the 1-of human genomic sequence
3%, and in human genome the overwhelming majority transcribed sequences be long-chain non-coding RNA (Long non-coding RNA,
lncRNA).LncRNA is universally present in various biologies, and along with the rising of biological complex degree, and lncRNA in genome
The ratio of sequence the most correspondingly increases, and lncRNA is significant during biological evolution in prompting.Along with lncRNA constantly quilt
Finding, their function is gradually interpreted, and scientists finds that lncRNA extensively and actively participates in vital movement different layers
In the function controlling in face, as a brand-new field, have become as new forward position, international life science field and focus.
LncRNA can be as the signal (signal) of functional protein, induction (guide), bait (decoy) or support
(scaffold) various ways such as molecule, at chromatin reconstruct, genetic transcription, translation and the multiple levels regulation and control base such as protein modified
The expression of cause, and play irreplaceable role during including the basic physiological such as growth, immunity, reproduction, prior
It is that the expression of lncRNA and functional disorder disease multiple with the mankind including malignant tumor are closely connected one
Rise.Therefore, the function of further investigation lncRNA, discloses the hereditary information transfer mode and expression regulation net mediated by lncRNA
Network, is possible not only to the angle beyond protein coding gene and again annotates and illustrate structure and the function of genome, deeply finds
The essence of vital movement and rule, be expected to from new visual angle understanding multiple mankind's common disease including tumor
Pathogeny, and the Clinics and Practices for these diseases provides new molecular marker and therapy target.
Nasopharyngeal carcinoma is common head-neck malignant tumor occurred frequently, is susceptible to metastasic cervical lymph nodes, and prognosis is poor.Research
Showing, the generation development of this tumor is polygenes participation, multi-step, multistage complex process, and LncRNA is in nasopharyngeal carcinoma
Occur evolution may also play important effect.Recently, we found that lncRNA LINC01420 (NCBI
Accession number:NR_015367) up-regulated in tissues of nasopharyngeal carcinoma, the detection preparation for this lncRNA also may be used
Auxiliary for nasopharyngeal carcinoma diagnoses.We increase sample further, have the nasopharyngeal carcinoma paraffin of Clinical Follow-up data to deposit in 110 examples
In shelves specimen, be have detected the expression of LINC01420 by the method for in situ hybridization (in situ hybridization),
Finding LINC01420 up-regulated in tissues of nasopharyngeal carcinoma, the Nasopharyngeal Carcinoma Patients of high expressed LINC01420 is than low expression
The Nasopharyngeal Carcinoma Patients poor prognosis of LINC01420, therefore the detection preparation for this lncRNA can be used for the prognosis of nasopharyngeal carcinoma
Judge.
We, by designing and synthesizing targeting LINC01420siRNA sequence, are transfected in human nasopharyngeal epithelioma 1, in vitro
Culture systems confirms that the expression of targeting interference LINC01420 can substantially suppress invasion and attack and the transfer ability of nasopharyngeal carcinoma cell.
Summary of the invention
For above-mentioned prior art, it is an object of the invention to provide the auxiliary diagnosis of a kind of nasopharyngeal carcinoma or outcome prediction
In biomarker long-chain non-coding RNA LINC01420, and detection tissues of nasopharyngeal carcinoma, the reagent of this rna expression amount is used for making
Standby nasopharyngeal carcinoma auxiliary diagnosis or the purposes of outcome prediction preparation.
The biomarker of the auxiliary diagnosis of a kind of nasopharyngeal carcinoma or outcome prediction is long-chain non-coding RNA
LINC01420, the sequence of this long-chain non-coding RNA LINC01420 is as shown in SEQ NO:1.
The application of the reagent of long-chain non-coding RNA LINC01420 expression, described reagent in detection tissues of nasopharyngeal carcinoma
For preparing nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction preparation；The sequence such as SEQ of this long-chain non-coding RNA LINC01420
Shown in NO:1.
Described nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction preparation be according to lncRNA LINC01420 in nasopharyngeal carcinoma
Expression assist diagnosis and outcome prediction, the lncRNA LINC01420 of Nasopharyngeal Carcinoma Patients expresses notable rise.
Described nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction preparation combine the sex of patient for nasopharyngeal carcinoma auxiliary diagnosis
Or outcome prediction.
Described nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction preparation are real time fluorescent quantitative detectable.
Described real time fluorescent quantitative detectable comprises for drawing that real time fluorescent quantitative detection LINC01420 expresses
Thing sequence:
Forward primer: 5'-CACTCTACCCTCCGCACC-3',
Reverse primer: 5'-AGGAAGTGAAATCGTGCTGA-3'.
Described real time fluorescent quantitative detectable comprises for comparison GAPDH forward primer:
5 '-ACCACAGTCCATGCCATCAC-3 ' and reverse primer: 5 '-TCCACCACCCTGTTGCTGTA-3 '.
The present invention demonstrates lncRNA LINC01420 in nasopharyngeal carcinoma (26 example) and slow by Real-Time Fluorescent Quantitative PCR Technique
Property inflammation nasopharyngeal epithelium tissue (10 example) in expression, LINC01420 expresses and notable raises (P=0.002).But also send out
Existing LINC01420 expression in nasopharyngeal carcinoma (NPC) is closely related with the sex of patient, and LINC01420 expresses high patient man
Property on the high side, develop the auxiliary diagnosis of a kind of nasopharyngeal carcinoma or outcome prediction real time fluorescent quantitative detectable accordingly, for nasopharynx
The early discovery of cancer, early treatment provide important reference frame.
Accompanying drawing explanation
Fig. 1 is that fluorescence quantitative PCR detection finds that LINC01420 raises situation at nasopharyngeal carcinoma；
Real-Time Fluorescent Quantitative PCR Technique checking lncRNA LINC01420 is in nasopharyngeal carcinoma (26 example) and chronic inflammatory disease nasopharynx
Expression in skin tissue (10 example), LINC01420 expresses notable rise (P=0.002).
Fig. 2 is that in situ hybridization detects LINC01420 expression in nasopharyngeal carcinoma and normal nasopharyngeal epithelium；
LINC01420 expression in normal nasopharyngeal epithelium (NPE) is relatively low, only has 14 examples in 42 example normal nasopharyngeal epitheliums
(33.3%) the low expression of LINC01420 detected in, can't detect in remaining 28 example；And at 110 example tissues of nasopharyngeal carcinomas
In have high expressed LINC01420 being detected in 60 example nasopharyngeal carcinoma (54.5%), in remaining 50 example tissues of nasopharyngeal carcinoma detect
Low expression to LINC01420；P=0.02.LINC01420 expression in nasopharyngeal carcinoma (NPC) with Patients on Recurrence and turns at a distance
Moving closely related, LINC01420 expresses high patient and is easier to recurrence, and LINC01420 expresses the energy of high tumor metastasis
Power is higher.LINC01420 expression in nasopharyngeal carcinoma (NPC) is closely related with the sex of patient, and LINC01420 expresses high trouble
Person male is on the high side.
Fig. 3 is LINC01420 high expressed patient's poor prognosis in tissues of nasopharyngeal carcinoma, is more easy to recurrence and transfer；
In nasopharyngeal carcinoma, the high expressed of LINC01420 and the poor prognosis of Nasopharyngeal Carcinoma Patients are relevant, i.e. LINC01420 high expressed
Total life span (Over-all survival, OS) the low expression of LINC01420 to be significantly lower than of patient or the trouble do not expressed
Person.
Fig. 4 is the interference siRNA sequence importing LINC01420 in nasopharyngeal carcinoma cell, can significantly inhibit LINC01420 and exist
Expression in nasopharyngeal carcinoma cell；
After importing the siRNA mixture of targeting LINC01420 in Nasopharyngeal Carcinoma Cell Line 5-8F, real-time fluorescence quantitative PCR
Method have detected the expression of LINC01420 in nasopharyngeal carcinoma cell, and the expression of LINC01420 is the most significantly suppressed.Cloudy
Property comparison (NC) be Scramble disturb siRNA sequence.
Fig. 5 be import in nasopharyngeal carcinoma cell LINC01420 siRNA suppression LINC01420 express after, cell invasion energy
Power reduces；
Cell-penetrating (transwell) is it is experimentally confirmed that proceed to targeting LINC01420's in Nasopharyngeal Carcinoma Cell Line 5-8F
SiRNA, after the expression of suppression LINC01420, can substantially reduce through the nasopharyngeal carcinoma cell number of substrate glued membrane, show that cell is invaded
Ability of attacking reduces.
Detailed description of the invention
The present invention is further illustrated below in conjunction with detailed description of the invention, and the unrestricted present invention.
Embodiment 1, quantitative real-time PCR detection finds that LINC01420 raises at nasopharyngeal carcinoma
1. materials and methods:
Collecting 10 example chronic nasopharyngeal epithelium tissues and 26 example tissues of nasopharyngeal carcinomas, extracted total RNA, 1 μ g RNA becomes through reverse transcription
After cDNA, carry out real-time fluorescence quantitative PCR.LINC01420 forward primer is 5'-CACTCTACCCTCCGCACC-3', such as SEQ
Shown in NO:2, and reverse primer '-AGGAAGTGAAATCGTGCTGA-3'.As shown in SEQ NO:3.
For comparison GAPDH forward primer be 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 reactions steps
Reaction confirms amplification curve and the melting curve of real-time fluorescence quantitative PCR, the expression intensity root of each gene after terminating
After CT value (threshold cycle values), reference gene (GAPDH) markization, group t-test inspection is used to calculate
P value.2. result
LINC01420 does not expresses in normal control tissue or expresses the lowest, and high expressed P=in tissues of nasopharyngeal carcinoma
0.002 (Fig. 1)
Embodiment 2, in situ hybridization detection finds that LINC01420 expression in nasopharyngeal carcinoma is relevant to patient's prognosis
1. MATERIALS METHODS
1.1 design and synthesize hybridization probe
In order to use the expression of in-situ hybridization method detection LINC01420, we devise and examine in situ hybridization
Survey oligonucleotide probe and each 3 of two groups of in situ hybridization oligonucleotide probes of positive control that LINC01420 expresses.
The oligonucleotide probe expressed in situ hybridization detection LINC01420:
LINC01420 probe 1:5 '-ATTTAAAGAGGGTGGGATTTGGTCAGAAACTCAC-3 ' such as SEQ NO:6 institute
Show,
LINC01420 probe 2:5 '-CAGGACTTGGACCTTCAACACGAAAAATTCAGAAT-3 ' such as SEQ NO:7 institute
Show,
LINC01420 probe 3:5 '-CACTTGAGAAAACCACTGTAGGACAAGAACAACAT-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.
Chemical synthesis process is used to synthesize each gene specific oligonucleotides probe sequence of above-mentioned design.
1.2 oligonucleotide probe labelling kits and in situ hybridization detectable
Digoxin oligonucleotide tailing reagent (Dig Oligonucleitide Tailing Kit 2ndGeneration,
Roche company), anti-digoxin-horseradish peroxidase complex detection kit (Anti-Digoxigenin-POD, Fab
Fragments, Roche company), strengthen the TSA signal amplifying system (TSA of expressed in situ detection signalTM Biotin
System, NEL700 test 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), frog essence DNA (the denatured and sheared that degeneration is sheared
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), 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 materials
Dehydrated alcohol, 90% ethanol, 70% ethanol, 50% ethanol, Oleum Terebinthinae, distilled water, PBS (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 add in 450ml distilled water interim
Correction work liquid pH value again after configuration)；0.1% trypsin；Haematoxylin；1% hydrochloride alcohol (1ml concentrated hydrochloric acid+99ml 70%
Ethanol configures)；Mounting glue (PTS Cure Mount II)；Special cap slide (480 × 240mm2) customize in Zhengzhou glass apparatus
Factory.Leica low melting point (58 DEG C) paraffin, domestic Cera Flava, anhydrous alcohol, dimethylbenzene, 10% neutral paraformaldehyde (0.01mol/L,
PH7.4, DEPC distilled water and PBS preparation), haematoxylin, Yihong, neutral mounting natural gum, coverslip, microscope slide.1.4 mark
Note probe
Utilizing 3-tailing DIG Olignucleutide Kit to carry out oligonucleotide probe labelling, reaction system is such as
Under.
100pmol oligonucleotide+ddH2O=9 μ l (control:control oligonucleutide 5 μ
l+ddH2O 4μl)
Mixing, the most centrifugal.37 DEG C of water-bath 30min, add 2 μ l EDTA (0.2M, PH 8.0) stopped reactions.
Purification after 1.5 oligonucleotide probe labellings
In order to increase the purity of label probe, need to be purified marked probe, concrete operations are as follows:
1) probe reaction mixture (22 μ l)+2.5 μ l 4M cold ethanol of LiCL+75 μ l 100% (-20 DEG C).
2)-70 DEG C of precipitations 60min, or-20 DEG C of 2h.
3) 4 DEG C of centrifugal 15min of 13.000 × g.
4) supernatant is abandoned, by 50 70% (V/V) washing with alcohol ice-cold for μ l.
5) 13.000xg 4 DEG C, centrifugal 5min.
6) abandoning supernatant, 4 DEG C of vacuum is dried.
7) with the aseptic double-distilled water molten probe of weight.
1.6 in situ hybridization detections achieve the expression of LINC01420 in paraffin section
Paraffin section hybridization pre-treatment
1) 4 DEG C of paraffin sections preserved are placed in 58 DEG C of roasting sheet 30min, melted surface paraffin.
2) dimethylbenzene dewaxes 3 × 5min successively.
3) step ethanol wash, 100% ethanol 2 × 2min → 95% ethanol 1 × 5min → 70% ethanol 1 × 5min →
50% ethanol 1 × 5min → DEPC water washing 2 × 3min → DEPC-PBS washs 2 × 5min.
4) drip 300 μ l pepsin K (10 μ g/ml) in section on, 37 DEG C digestion 20min.
5) cut into slices and wash 1min, stopped reaction into PBS (0.1M PBS+2mg/ml glutamic acid).
6) cut into slices into 0.2N HCL, react 20-30min in 37 DEG C, increase the permeability of tissue.
7) section fixes 10min, room temperature afterwards with 4% paraformaldehyde (0.1M PBS dissolving).
8) in order to increase the positive intensity for hybridization of tissue, section is carried out acetyl process.Cut into slices into 0.25% acetic anhydride
Buffer I (0.1M triethanolamine), room temperature 10min.
9) 1M PBS washs 2 × 5min.
Prehybridization and hybridization
Prehybridization :-20 DEG C of prehybridization solutions preserved, is first placed in 37 DEG C and hatches 60min, and the consumption of prehybridization solution is 50 μ l,
Parafilm carries out lid section, prehybridization 2 hours in 37 DEG C of wet boxes.(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) removing Parafilm, get rid of prehybridization solution, section is placed in 5min in 2 × SSC.
2) hybridization: 37 DEG C of hybridized overnight (18-20h).Each section adds 250 μ l hybridization solutions and carries out with Parafilm
Lid.Prehybridization solution adds corresponding probe and just becomes hybridization solution.Hybridization solution is prepared when prehybridization, places 37 DEG C and hatches, makes
Probe is completely dissolved in hybridization solution, and this experiment mixes with a plurality of oligonucleotide probe, joins by each probe 500ng/ml concentration
Make probe hybridization solution.Digoxin tailing labelling kit label probe concentration basis: the concentration of each probe by its with
During positive quantitatively probe, during detection reaction, colour developing is compared and the naked probe labelling reaction theory of 30 bases of 100pmol is visited
Pin yield is that two kinds of standards of 900ng carry out COMPREHENSIVE CALCULATING and go out the concentration of label probe.
3) post-hybridization washing, cut into slices immersion 2 × SSC, 10min, throws off Parafilm.Shake washing on shaking table successively, 2 ×
SSC (0.5%SDS), 2 × 15min → 0.25 × SSC (0.5%SDS), 2 × 15min.
Color developing detection reaction after hybridization
1) Anti-Digoxigenin-POD detection digoxigenin-probe is used to be combined complex with mRNA；TSA amplification system
Strengthening the positive signal of in situ hybridization reaction solution reaction, DAB develops the color.
2) section goes in TNT buffer, 3 × 5min.
3) dropping TNB blocks buffer, 300 μ l/TMAs, room temperature, 30min.
4) unnecessary blocker is sucked, Anti-Digoxigenin-POD (the TBS+0.1%Triton X-of 1:100 dilution
100+1% blocker), room temperature 4 hours.
5) TNT Buffer (0.1M Tris-CL, pH7.5,0.15M NaCL, 0.05%Tween 20) washing,
3x5min。
6) drip signal in section and amplify reagent Biotinyl Tyamid, 300 μ l/TMAs, (Biotinyl Tyramid
Stock solution: Biotinyl Tyramid is dissolved in 0.2ml DMSO, Biotinyl Tyramid working solution: 1 × diluent, 1:50
Dilution Biotinyl Tyramid stock solution), room temperature 10 minutes.
7) TNT washes, 3 × 5min.
8) section dropping SA-HRP (strepto-avidin-horseradish peroxidase), 300 μ l/TMAs, room temperature 30min.
9) TNT washes, 3 × 5min.
10) aquae destillata washing, 1 × 1min.
11) DAB colour developing, controls chromogenic reaction under microscope.
12) haematoxylin is redyed,
13) dehydration of ethanol step, chip drying.
14) dropping mounting glue, the coverslip cover plate of dimension, crosslinking section 1min under uviol lamp.
1.7 results judge and standard
Application Optics microscope is observed respectively under low power and high power lens, looks first at the positive expression of target RNA
Signal is in the intracellular location of object observing: be positioned at nucleus, cytoplasm or cell membrane.
Carry out with the intensity of this detection rna expression position positive signal and two kinds of standards of cell number of positive expression the most respectively
Comprehensive grading, it is judged that standard is: (1) judges according to positive cell dyeing intensity: a. cell dye-free, remembers 0 point；B. cell is dyed
Light brown is the weak positive, remembers 1 point；C. cell dyes brown and without background coloration, or cell is dyed dark-brown and has light brown to carry on the back
Scape is moderate positive, remembers 2 points；D. cell is dyed dark-brown and is strong positive without background coloration, remembers 3 points.(2) according to positive cell
Expression number is scored: the no positive cell of a. is expressed, and 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 factors of appraisal result as far as possible, by two pathology experts respectively by one of above-mentioned standard each
Carrying out judging and marking, then both scorings be multiplied, result is: 1. 0 point of person be finally calculated as 0 point (-), it is believed that negative express；②1
Point and 2 points of persons be finally calculated as 1 point (+), it is believed that weak positive expression；3. 3 points and 4 points of persons are finally calculated as 2 points (++), it is believed that medium sun
Property express；4. 6 assign to 9 points of persons and be finally calculated as 3 points (+++), it is believed that strong positive is expressed.
1.8 analyze and statistical software
Application SPSS13.0 statistical software carries out statistical analysis to experimental result, compares two-by-two and uses χ2Test or Fisher
Exact test, correlation analysis uses Spearmen correlation method；P ＜ 0.05 i.e. difference is statistically significant.
Survival curve analysis uses Kaplan-Meier method and log-rank test；Multivariate analysis uses Cox ' s
proportional hazards model；P ＜ 0.05 i.e. difference is statistically significant.
2 results
Expression in 2.1LINC01420 expression ratio normal control tissue in nasopharyngeal carcinoma significantly raises
LINC01420 is (60/110 example) high expressed in the tissues of nasopharyngeal carcinoma of 54.5%, and only 33.3%, (42 examples are chronic
14 examples in inflammation epithelial tissue sample) normal nasopharyngeal epithelial tissue in have expression (Fig. 2 A), have significantly between the two
Significant difference (P=0.02, Fig. 2 B).LINC01420 expression in nasopharyngeal carcinoma (NPC) is close with Patients on Recurrence and metastasis
Cut is closed, and LINC01420 expresses high patient and is easier to recurrence, and the ability of LINC01420 metastasis is higher (Fig. 2 C).
LINC01420 expression in nasopharyngeal carcinoma (NPC) is closely related with the sex of patient, and LINC01420 expresses high patient male
(Fig. 2 D) on the high side.
The Nasopharyngeal Carcinoma Patients prognosis of 2.2LINC01420 high expressed is poor
We have carried out Effect of follow-up visit by telephone to 110 example Nasopharyngeal Carcinoma Patients, have inquired their start time, treatment feelings in detail
Condition, with or without recurrence, with or without suffering from other diseases, recurrence and death time etc. again, and register life span and state, and to nasopharynx
The survival analysis that in cancerous tissue, the expression of LINC01420 is carried out with the life span of patient and state, finds LINC01420 height table
Reach the total life span of patient and be significantly shorter than the low expression of LINC01420 or the patient (Fig. 3) not expressed.Illustrate that LINC01420 is
One molecular marker relevant to nasopharyngeal carcinoma prognosis, this lncRNA expresses height, patient's poor prognosis.
Embodiment 3, the expression of siRNA interference LINC01420
1. MATERIALS METHODS
1.1 reagent and test kit
TRIZOLTMReagent(Invitrogen)；
Reverse Transcriptase kit (#A3500, Promega)；
Antibiotic G418 (Ameresc).
The design of 1.2shRNA
First by the Block-It RNAi designer software of LINC01420 sequence inputting Invitrogen company, seek
Look for the siRNA best target of this lncRNA, select the corresponding target sequence in optimal 3 as follows:
SiRNA-1:5'-CAUCUCAGGUCUCUUGGCUUUGCCA-3' as shown in SEQ NO:12,
SiRNA-2:5'-GCGUUGGGAUUAUCCGGAAGGAACU-3' as shown in SEQ NO:13,
SiRNA-3:5'-CCUCUGAGAUUUAAGGCCAUGCCCU-3' as shown in SEQ NO:14,
Negative control Scramble sequence: 5 '-GACACGCGACUUGUACCAC-3 ' are as shown in SEQ NO:15.
1.3 cells are cultivated and transfection
Nasopharyngeal Carcinoma Cell Line 5-8F is purchased from Central South University's cell centre, and cell is cultivated RPMI 1640 used and trained base and tire cattle
Serum, and the trypsin used by peptic cell is U.S.'s Gibco Products.
By Nasopharyngeal Carcinoma Cell Line 5-8F good for growth conditions by 2 × 105Individual cells/well is inoculated in 6 orifice plates, by 6 holes
Plate is placed in 37 DEG C, 5%CO2In incubator, cell to be cultivated grows to 50-70% density can start the transfection of siRNA；Transfection
Process is as follows:
The Hipefect adding 3 μ l in aseptic EP pipe mixes standing 5min in 100 μ l serum-free mediums；
Three siRNA mixing are added in 100 μ l serum-free mediums；Then with the above-mentioned 100 μ l comprising Hipefect
Serum-free medium gentleness mixes, and room temperature stands 30 minutes, makes siRNA form complex with liposome；
With D-Hank's liquid washed cell 3 times；
Said mixture will add 800 μ l serum-free medium (antibiotic-free), add in 6 orifice plates after gentle mixing
1 hole；
6 orifice plates are placed in CO2In incubator, cultivate 6 hours for 37 DEG C, then abandon supernatant, add complete medium and continue training
Support 48 hours.
The effect that 1.5 real-time quantitative PCR detection siRNA interference lncRNA express:
Nasopharyngeal carcinoma cell extracted total RNA after being transfected by siRNA, 2 μ g RNA, after reverse transcription becomes cDNA, carry out the most glimmering
Fluorescent Quantitative PCR.LINC01420 primer is: 5'-CACTCTACCCTCCGCACC-3', reverse primer:
5'-AGGAAGTGAAATCGTGCTGA-3'。
For comparison GAPDH primer be 5 '-ACCACAGTCCATGCCATCAC-3 ' and 5 '-
TCCACCACCCTGTTGCTGTA-3’。
Real-time fluorescence quantitative PCR reaction system
Real-time fluorescence quantitative PCR reactions steps
Reaction confirms amplification curve and the melting curve of real-time fluorescence quantitative PCR, the expression intensity root of each gene after terminating
After CT value (threshold cycle values), reference gene (GAPDH) markization, group t-test inspection is used to calculate
P value.
2. result
After siRNA transfection nasopharyngeal carcinoma cell 5-8F, can significantly lower the expression of LINC01420 in nasopharyngeal carcinoma cell
(Fig. 4).
Embodiment 4, in LINC01420siRNA suppression nasopharyngeal carcinoma cell, the expression of LINC01420 and the invasion and attack of nasopharyngeal carcinoma turn
Move
1. MATERIALS METHODS
1.1 cells are cultivated and transfection
By nasopharyngeal carcinoma cell 5-8F good for growth conditions by 2 × 105Individual cells/well is inoculated in 6 orifice plates, by 6 orifice plates
It is placed in 37 DEG C, 5%CO2In incubator, cell to be cultivated grows to 50-70% density can start LINC01420siRNA and turn
Dye.1.2 cell-penetrating experiments
((Transwell) experiment is the experimental technique verifying tumor cell invasion ability to cell-penetrating.Transwell is little
Room (aperture 8 μm) and matrigel (Matrigel) are purchased from U.S. company BD, 4% paraformaldehyde fixative, crystal violet dye liquor
(0.1%g/ml) purchased from Sigma company.Matrigel is pressed 1:8 dilution, is coated on the upper room of Transwell cell bottom film
Face, puts 37 DEG C and makes Matrigel aggregate into gel in 30 minutes.Matrigel film water is carried out by BD company description before using.
Serum-free medium and 1 × 10 is added on each Transwell cell upper strata5Individual transfect LINC01420siRNA
With the nasopharyngeal carcinoma cell of negative control, add the culture medium containing 20% hyclone in Transwell cell lower floor.Cell continues
After cultivating 36 hours, fix with 4% paraformaldehyde fixative, violet staining, dab off the non-migrating cell in upper strata with cotton swab,
3 times are washed with PBS.Examine under a microscope the nasopharyngeal carcinoma cell through substrate glued membrane.
2. result
After 2.1 import LINC01420siRNA suppression LINC01420 in nasopharyngeal carcinoma cell, cell invasion ability reduces
Cell-penetrating (Transwell) is it is experimentally confirmed that proceed to targeting in Nasopharyngeal Carcinoma Cell Line 5-8F
LINC01420siRNA, after the expression of suppression LINC01420, can substantially reduce through the nasopharyngeal carcinoma cell number of substrate glued membrane,
Show that cell invasion ability reduces (Fig. 5).
Claims (10)
1. the biomarker of the diagnosis of nasopharyngeal carcinoma auxiliary or outcome prediction is a long-chain non-coding RNA LINC01420,
The sequence of this long-chain non-coding RNA LINC01420 is as shown in SEQ NO:1.
2. the application of the reagent of long-chain non-coding RNA LINC01420 expression in detection tissues of nasopharyngeal carcinoma, it is characterised in that institute
The reagent stated is for preparing nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction preparation；The sequence of this long-chain non-coding RNA LINC01420
Row are as shown in SEQ NO:1.
Application the most according to claim 2, it is characterised in that described nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction preparation
It is to carry out auxiliary diagnosis and outcome prediction, nose according to long-chain non-coding RNA LINC01420 expression in nasopharyngeal carcinoma
In pharyngeal cancer tissue, LINC01420 expresses notable rise.
4. according to the application described in Claims 2 or 3, it is characterised in that described nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction
Preparation combines the sex of patient and can be used for nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction.
Application the most according to claim 2, it is characterised in that described nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction preparation
For real time fluorescent quantitative detectable.
Application the most according to claim 5, it is characterised in that described real time fluorescent quantitative detectable comprises for
The primer sequence that real time fluorescent quantitative detection LINC01420 expresses:
Forward primer: 5'-CACTCTACCCTCCGCACC-3',
Reverse primer: 5'-AGGAAGTGAAATCGTGCTGA-3'.
7. according to the application described in claim 5 or 6, it is characterised in that described real time fluorescent quantitative detectable comprises
For comparison GAPDH forward primer: 5 '-ACCACAGTCCATGCCATCAC-3 ' and
Reverse primer: 5 '-TCCACCACCCTGTTGCTGTA-3 '.
8. the diagnosis of nasopharyngeal carcinoma auxiliary or an outcome prediction real time fluorescent quantitative detectable, comprise for detecting long-chain non-
The primer that coding RNA LINC01420 expresses.
Nasopharyngeal carcinoma the most according to claim 8 auxiliary diagnosis or outcome prediction real time fluorescent quantitative detectable, bag
Primer sequence containing expressing for real time fluorescent quantitative detection LINC01420:
Forward primer: 5'-CACTCTACCCTCCGCACC-3',
Reverse primer: 5'-AGGAAGTGAAATCGTGCTGA-3'.
Nasopharyngeal carcinoma auxiliary diagnosis the most according to claim 8 or claim 9 or outcome prediction real time fluorescent quantitative detection examination
Agent, also include for comparison GAPDH forward primer: 5 '-ACCACAGTCCATGCCATCAC-3 ' and
Reverse primer: 5 '-TCCACCACCCTGTTGCTGTA-3 '.
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