CN106319044B - Biomarker and its application of a kind of nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction - Google Patents
Biomarker and its application of a kind of nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction Download PDFInfo
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Abstract
The invention discloses a kind of nasopharyngeal carcinoma auxiliary diagnosis, perhaps the biomarker of outcome prediction and its application are mainly used for preparing the real time fluorescent quantitative detection reagent of nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction.Real-Time Fluorescent Quantitative PCR Technique demonstrates expression of the long-chain non-coding RNA LINC01420 in nasopharyngeal carcinoma and chronic inflammation nasopharyngeal epithelium tissue, and LINC01420 expresses significant up-regulation in tissues of nasopharyngeal carcinoma.The present invention provides important reference frame for the early detection of nasopharyngeal carcinoma, early treatment.
Description
Technical field
The invention belongs to oncomolecularbiology fields, and in particular to a kind of nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction are used
Biomarker long-chain non-coding RNA LINC01420, and detect the reagent of the rna expression amount to be used to prepare nasopharyngeal carcinoma auxiliary
Help the purposes of 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.
Nasopharyngeal carcinoma is common high-incidence head-neck malignant tumor, is easy to happen metastasic cervical lymph nodes, and prognosis is poor.Research
The occurrence and development for showing this tumour are polygenes participation, multi-step, multistage complex process, and LncRNA is in nasopharyngeal carcinoma
Important role may be also played during occurrence and development.Recently, we found that lncRNA LINC01420 (NCBI
Accession number:NR_015367) up-regulation is expressed in tissues of nasopharyngeal carcinoma, it can also for the detection preparation of the lncRNA
With the auxiliary diagnosis for nasopharyngeal carcinoma.We further increase sample, have the nasopharyngeal carcinoma paraffin of Clinical Follow-up data to deposit at 110
In shelves sample, the expression of LINC01420 is had detected by the method for in situ hybridization (in situ hybridization),
It was found that LINC01420 expresses up-regulation in tissues of nasopharyngeal carcinoma, the Nasopharyngeal Carcinoma Patients of height expression LINC01420 compare low expression
The Nasopharyngeal Carcinoma Patients poor prognosis of LINC01420, therefore the detection preparation for being directed to the lncRNA can be used for the prognosis of nasopharyngeal carcinoma
Judgement.
We are transfected into human nasopharyngeal epithelioma 1, in vitro by designing and synthesizing targeting LINC01420siRNA sequence
Culture systems confirm that the expression of targeting interference LINC01420 can obviously inhibit invasion and the transfer ability of nasopharyngeal carcinoma cell.
Summary of the invention
For the above-mentioned prior art, the object of the present invention is to provide a kind of nasopharyngeal carcinoma auxiliary diagnosis or outcome predictions
The reagent of the rna expression amount is for making in biomarker long-chain non-coding RNA LINC01420, and detection tissues of nasopharyngeal carcinoma
The purposes of standby nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction preparation.
The biomarker of a kind of nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction is long-chain non-coding RNA
LINC01420, the sequence of long-chain non-coding RNA LINC01420 is as shown in SEQ NO:1.
Detect the application of the reagent of long-chain non-coding RNA LINC01420 expression quantity in tissues of nasopharyngeal carcinoma, the reagent
It is used to prepare nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction preparation；The sequence such as SEQ of long-chain non-coding RNA LINC01420
Shown in NO:1.
The nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction preparation be according to lncRNA LINC01420 in nasopharyngeal carcinoma
Expression come auxiliary diagnosis and outcome prediction, the significant up-regulation of lncRNA LINC01420 expression of Nasopharyngeal Carcinoma Patients.
The gender of the nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction preparation combination patient is used for nasopharyngeal carcinoma auxiliary diagnosis
Or outcome prediction.
The nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction preparation is real time fluorescent quantitative detection reagent.
Include drawing for real time fluorescent quantitative detection LINC01420 expression in the real time fluorescent quantitative detection reagent
Object sequence:
Forward primer: 5'-CACTCTACCCTCCGCACC-3',
Reverse primer: 5'-AGGAAGTGAAATCGTGCTGA-3'.
Include the GAPDH forward primer for control in the real time fluorescent quantitative detection reagent:
5 '-ACCACAGTCCATGCCATCAC-3 ' and reverse primer: 5 '-TCCACCACCCTGTTGCTGTA-3 '.
The present invention demonstrates lncRNA LINC01420 in nasopharyngeal carcinoma (26) and slow by Real-Time Fluorescent Quantitative PCR Technique
Property inflammation nasopharyngeal epithelium tissue (10) in expression, the significant up-regulation (P=0.002) of LINC01420 expression.But also it sends out
The gender of existing expression and patient of the LINC01420 in nasopharyngeal carcinoma (NPC) is closely related, and LINC01420 expresses high patient male
Property it is on the high side, develop a kind of nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction real time fluorescent quantitative detection reagent accordingly, be nasopharynx
The early detection of cancer, early treatment provide important reference frame.
Detailed description of the invention
Fig. 1 is that fluorescence quantitative PCR detection finds that LINC01420 raises situation in nasopharyngeal carcinoma；
Real-Time Fluorescent Quantitative PCR Technique verifies lncRNA LINC01420 in nasopharyngeal carcinoma (26) and chronic inflammation nasopharynx
Expression in skin tissue (10), the significant up-regulation (P=0.002) of LINC01420 expression.
Fig. 2 is that in situ hybridization detects expression of the LINC01420 in nasopharyngeal carcinoma and normal nasopharyngeal epithelium；
LINC01420 expression in normal nasopharyngeal epithelium (NPE) is lower, only has 14 in 42 normal nasopharyngeal epitheliums
(33.3%) low expression that LINC01420 is detected in can't detect in remaining 28；And in 110 tissues of nasopharyngeal carcinoma
In have the high expression that LINC01420 is detected in 60 nasopharyngeal carcinoma (54.5%), detected in remaining 50 tissues of nasopharyngeal carcinoma
To the low expression of LINC01420；P=0.02.Expression of the LINC01420 in nasopharyngeal carcinoma (NPC) and Patients on Recurrence and distant place turn
Move closely related, LINC01420 expresses high patient and is easier to recur, and LINC01420 expresses the energy of high tumour DISTANT METASTASES IN
Power is stronger.The gender of expression and patient of the LINC01420 in nasopharyngeal carcinoma (NPC) is closely related, and LINC01420 expresses high trouble
Person male is on the high side.
Fig. 3 is that LINC01420 high expresses patient's poor prognosis, more easy to recur and transfer in tissues of nasopharyngeal carcinoma；
The high expression of LINC01420 is related to the poor prognosis of Nasopharyngeal Carcinoma Patients in nasopharyngeal carcinoma, i.e. LINC01420 high expression
The total life span (Over-all survival, OS) of patient will be significantly lower than LINC01420 low expression or the trouble that do not express
Person.
Fig. 4 is the interference siRNA sequence that LINC01420 is imported 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 has detected the expression of LINC01420 in nasopharyngeal carcinoma cell, and the expression of LINC01420 is significantly inhibited.Yin
Property control (NC) be Scramble interfere siRNA sequence.
Fig. 5 is cell invasion energy after importing the siRNA inhibition LINC01420 expression of LINC01420 in nasopharyngeal carcinoma cell
Power reduces；
Cell-penetrating (transwell) in Nasopharyngeal Carcinoma Cell Line 5-8F it is experimentally confirmed that be transferred to targeting LINC01420's
SiRNA, after the expression for inhibiting LINC01420, the nasopharyngeal carcinoma cell number that can pass through matrix glue film is substantially reduced, and shows that cell is invaded
Attack ability reduction.
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 discovery LINC01420 are raised in nasopharyngeal carcinoma
1. materials and methods:
Collect 10 chronic nasopharyngeal epithelium tissues and 26 tissues of nasopharyngeal carcinoma, extracted total RNA, 1 μ g RNA through reverse transcription at
After cDNA, real-time fluorescence quantitative PCR is carried out.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.
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
LINC01420 does not express or expresses very low, and the high expression P=in tissues of nasopharyngeal carcinoma in normal control tissue
0.002 (Fig. 1)
Embodiment 2, expression of the in situ hybridization detection discovery LINC01420 in nasopharyngeal carcinoma 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 LINC01420, 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 LINC01420 is expressed.
Oligonucleotide probe in situ hybridization detection LINC01420 expression:
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.
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.
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 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 LINC01420 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 one of above-mentioned standard is pressed respectively by two pathology experts
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；②1
Divide and 2 points of persons are finally calculated as 1 point (+), 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 medium sun
Property 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 the 2.1LINC01420 in nasopharyngeal carcinoma is significantly increased than the expression in normal control tissue
LINC01420 (60/110) high expression in 54.5% tissues of nasopharyngeal carcinoma, and only 33.3%, (42 are chronic
14 in inflammation epithelial tissue sample) normal nasopharyngeal epithelial tissue in have expression (Fig. 2A), have between the two apparent
Statistical difference (P=0.02, Fig. 2 B).Expression and Patients on Recurrence and DISTANT METASTASES IN of the LINC01420 in nasopharyngeal carcinoma (NPC) are close
Cut phase is closed, and LINC01420 expresses high patient and is easier to recur, and the ability of LINC01420 DISTANT METASTASES IN is stronger (Fig. 2 C).
The gender of expression and patient of the LINC01420 in nasopharyngeal carcinoma (NPC) is closely related, and LINC01420 expresses high patient male
(Fig. 2 D) on the high side.
The highly expressed Nasopharyngeal Carcinoma Patients prognosis of 2.2LINC01420 is poor
We have carried out Effect of follow-up visit by telephone to 110 Nasopharyngeal Carcinoma Patients, have inquired their start time, treatment feelings in detail
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 to nasopharynx
The survival analysis that the expression of LINC01420 and the life span of patient and state carry out in cancerous tissue, finds LINC01420 high table
The patient (Fig. 3) that the life span total up to patient is significantly shorter than LINC01420 low expression or does not express.Illustrating LINC01420 is
One molecular labeling relevant to nasopharyngeal carcinoma prognosis, lncRNA expression is high, patient's poor prognosis.
Embodiment 3, siRNA interfere the expression of LINC01420
1. MATERIALS METHODS
1.1 reagents and 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
It is as follows to select optimal 3 corresponding target sequences for the siRNA best target for looking for the lncRNA:
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 cell culture and transfection
Nasopharyngeal Carcinoma Cell Line 5-8F is purchased from Central South University's cell centre, and RPMI 1640 used in cell culture trains base and tire ox
Trypsase used in serum and vitellophag is U.S.'s Gibco Products.
The good Nasopharyngeal Carcinoma Cell Line 5-8F of growth conditions is pressed 2 × 105A 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, which grows to 50-70% density, can start the transfection of siRNA；Transfection
Process is as follows:
The Hipefect that 3 μ l are added in sterile EP tube is mixed in 100 μ l serum free mediums stands 5min；
Three siRNA are mixed and are added in 100 μ l serum free mediums；Then with the above-mentioned 100 μ l comprising Hipefect
Serum free medium mildly mixes, and is stored at room temperature 30 minutes, and siRNA and liposome is made to form complex；
It is washed cell 3 times with D-Hank's liquid；
800 μ l serum free mediums (antibiotic-free) will be added in said mixture, be added in 6 orifice plates after mild mixing
1 hole；
6 orifice plates are placed in CO2In incubator, 37 DEG C are cultivated 6 hours, and supernatant is then abandoned, and complete medium is added and continues to train
It supports 48 hours.
1.5 real-time quantitative PCRs detect the effect of siRNA interference lncRNA expression:
Nasopharyngeal carcinoma cell extracted total RNA after siRNA is transfected, 2 μ g RNA are carried out glimmering in real time after reverse transcription is at cDNA
Fluorescent Quantitative PCR.LINC01420 primer are as follows: 5'-CACTCTACCCTCCGCACC-3', reverse primer:
5'-AGGAAGTGAAATCGTGCTGA-3'。
GAPDH primer for control is 5 '-ACCACAGTCCATGCCATCAC-3 ' and 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
After siRNA transfects nasopharyngeal carcinoma cell 5-8F, the expression of LINC01420 in nasopharyngeal carcinoma cell can be significantly lowered
(Fig. 4).
Embodiment 4, LINC01420siRNA inhibit the expression of LINC01420 and the invasion of nasopharyngeal carcinoma in nasopharyngeal carcinoma cell to turn
It moves
1. MATERIALS METHODS
1.1 cell culture and transfection
The good nasopharyngeal carcinoma cell 5-8F of growth conditions is pressed 2 × 105A cells/well is inoculated in 6 orifice plates, by 6 orifice plates
37 DEG C are placed in, 5%CO2In incubator, cell to be cultivated, which grows to 50-70% density, can start LINC01420siRNA turns
Dye.The experiment of 1.2 cell-penetratings
((Transwell) experiment is to verify the experimental method of tumor cell invasion ability to cell-penetrating.Transwell is small
Room (8 μm of aperture) and matrigel (Matrigel) are purchased from U.S. company BD, 4% paraformaldehyde fixer, crystal violet dye liquor
(0.1%g/ml) is purchased from Sigma company.Matrigel is diluted by 1:8, is coated on the upper chamber of the cell Transwell bottom film
Face, setting 37 DEG C makes Matrigel aggregate into gel in 30 minutes.Matrigel film water is carried out by BD company specification using preceding.
Serum free medium and 1 × 10 is added on each cell Transwell upper layer5It is a to have transfected LINC01420siRNA
With the nasopharyngeal carcinoma cell of negative control, the culture medium for containing 20% fetal calf serum is added in the cell Transwell lower layer.Cell continues
After culture 36 hours, is fixed with 4% paraformaldehyde fixer, violet staining, dabs off the non-migrating cell in upper layer with cotton swab,
It is washed 3 times with PBS.The nasopharyngeal carcinoma cell across matrix glue film is observed under the microscope.
2. result
After 2.1 import LINC01420siRNA inhibition LINC01420 in nasopharyngeal carcinoma cell, cell invasion ability is reduced
Cell-penetrating (Transwell) in Nasopharyngeal Carcinoma Cell Line 5-8F it is experimentally confirmed that be transferred to targeting
LINC01420siRNA, after the expression for inhibiting LINC01420, the nasopharyngeal carcinoma cell number that can pass through matrix glue film is substantially reduced,
Show that cell invasion ability reduces (Fig. 5).
Claims (6)
1. detecting the application of the reagent of long-chain non-coding RNA LINC01420 expression quantity in tissues of nasopharyngeal carcinoma, which is characterized in that institute
The reagent stated is used to prepare nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction preparation；The sequence of long-chain non-coding RNA LINC01420
Column are as shown in SEQ NO:1.
2. application according to claim 1, which is characterized in that the nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction preparation
It is the expression according to long-chain non-coding RNA LINC01420 in nasopharyngeal carcinoma to carry out auxiliary diagnosis and outcome prediction, nose
The significant up-regulation of LINC01420 expression in pharynx cancer tissue.
3. application according to claim 1 or 2, which is characterized in that the nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction
The gender of preparation combination patient can be used for nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction.
4. application according to claim 1, which is characterized in that the nasopharyngeal carcinoma auxiliary diagnosis or outcome prediction preparation
For real time fluorescent quantitative detection reagent.
5. application according to claim 4, which is characterized in that comprising being used in the real time fluorescent quantitative detection reagent
Real time fluorescent quantitative detects the primer sequence of LINC01420 expression:
Forward primer: 5 '-CACTCTACCCTCCGCACC-3 ',
Reverse primer: 5 '-AGGAAGTGAAATCGTGCTGA-3 '.
6. application according to claim 4 or 5, which is characterized in that include in the real time fluorescent quantitative detection reagent
GAPDH forward primer for control: 5 '-ACCACAGTCCATGCCATCAC-3 ' and reverse primer: 5 '-
TCCACCACCCTGTTGCTGTA-3’。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104388543A (en) * | 2014-10-27 | 2015-03-04 | 中南大学 | In situ hybridization probe, reagent and application of long non-coding RNA LOC401317 |
| CN105233304A (en) * | 2015-10-30 | 2016-01-13 | 中南大学 | Application of long-chain non-coding RNA gene LOC553103 to preparation of nasopharyngeal darcinoma cell inhibitors |
-
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104388543A (en) * | 2014-10-27 | 2015-03-04 | 中南大学 | In situ hybridization probe, reagent and application of long non-coding RNA LOC401317 |
| CN105233304A (en) * | 2015-10-30 | 2016-01-13 | 中南大学 | Application of long-chain non-coding RNA gene LOC553103 to preparation of nasopharyngeal darcinoma cell inhibitors |
Non-Patent Citations (3)
| Title |
|---|
| BC062451.1;Anonymous;《GenBank》;20050509;第1-2页 * |
| Long non-coding RNA expression in primary human monocytes;Hoda Mirsafian et al;《Genomics》;20160108;第108卷(第1期);第37-45页 * |
| 白介素1受体II mRNA在小鼠子宫蜕膜中的转录;于永生等;《吉林农业大学学报》;20131231;第35卷(第1期);第72-75页 * |
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