CN106511368B - The application of the siRNA of long-chain non-coding RNA LINC00152 and inhibitory preparation - Google Patents
The application of the siRNA of long-chain non-coding RNA LINC00152 and inhibitory preparation Download PDFInfo
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Abstract
The invention discloses the application of the siRNA of long-chain non-coding RNA LINC00152 and inhibitory preparations.It by designing and synthesizing the siRNA sequence of targeting LINC00152, is transfected into cancer cell of oral cavity strain, it was demonstrated that the expression of targeting interference LINC00152 can obviously inhibit invasion and the transfer ability of cancer cell of oral cavity.The present invention provides new potential approach for the treatment of carcinoma of mouth.
Description
Technical field
The invention belongs to oncomolecularbiology fields, and in particular to the siRNA's of long-chain non-coding RNA LINC00152
Application process.
Background technology
Oral and maxillofacial malignancy is common head-neck malignant tumor occurred frequently, 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 molecules 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 and control of the important vital movement such as aging and death.More and more research shows 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 new molecular labeling can be judged as tumor prognosis.Currently, the mankind lncRNAs genes cloned are more than 40,000
It is a, but the Unknown Function of overwhelming majority lncRNA.
We have detected expressions of the LINC00152 in clinical Ex vivo Tumor sample, and analyze LINC00152 tables
Up to the horizontal correlation with clinical case data.We are after extracting RNA in mouth neoplasm and corresponding normal control tissue
By reverse transcription, real-time quantitative PCR has detected the expression of LINC00152, as a result shows LINC00152 in mouth neoplasm
Up-regulated expression in tissue.We further demonstrate in archive paraffin sample then again using the method for in situ hybridization
LINC00152 expresses notable up-regulation in mouth neoplasm, therefore can be used for mouth neoplasm for the detection preparation of LINC00152
Auxiliary diagnosis carries out survivorship curve analysis in conjunction with clinical return visit data and finds that LINC00152 expresses its life span of high patient
The shorter than lncRNA expresses low patient, therefore the prognosis that can be used for for the detection preparation of the lncRNA mouth neoplasm is sentenced
It is disconnected.
We target the siRNA sequence of LINC00152 by designing and synthesizing, and are transfected into cancer cell of oral cavity strain, in body
Outer culture systems confirm that the expression of targeting interference LINC00152 can obviously inhibit invasion and the transfer ability of Stomatocyte.
Invention content
For the above-mentioned prior art, answering the object of the present invention is to provide the siRNA of long-chain non-coding RNA LINC00152
With and inhibitory preparation.
The application of the siRNA of long-chain non-coding RNA LINC00152 of the present invention, utilizes long-chain non-coding RNA
The siRNA of LINC00152 prepares the reagent for inhibiting oral cavity cancerous invasion and transfer, the sequence of long-chain non-coding RNA LINC00152
Row such as SEQ NO:Shown in 1.
The siRNA sequence of long-chain non-coding RNA LINC00152 is one or two in following two：
siRNA-1：5 '-CAGGGAAUCUUUCAGCUGGAUUCCG-3 ',
siRNA-2：5′-UCUAUGUGUCUUAAUCCCUUGUCCU-3′.
A kind of preparation inhibiting oral cavity cancerous invasion and transfer, includes the siRNA sequences of long-chain non-coding RNA LINC00152
Row.
One or two below specially in two：
siRNA-1：5 '-CAGGGAAUCUUUCAGCUGGAUUCCG-3 ',
siRNA-2：5′-UCUAUGUGUCUUAAUCCCUUGUCCU-3′
The preparation of inhibition the oral cavity cancerous invasion and transfer, further includes negative control Scramble sequences：5'-
GACACGCGACUUGUACCAC-3’。
The invention discloses the application of the siRNA of long-chain non-coding RNA LINC00152 and inhibitory preparations.By designing simultaneously
The siRNA sequence of synthesis targeting LINC00152, is transfected into cancer cell of oral cavity strain, confirms that targeting is dry in culture systems in vitro
Invasion and the transfer ability of cancer cell of oral cavity can obviously be inhibited by disturbing the expression of LINC00152.The present invention is that the treatment of carcinoma of mouth carries
New approach is supplied.
Description of the drawings
Fig. 1 is the expression water that LINC00152 in the in vitro sample of mouth neoplasm is detected using the method for real-time fluorescence quantitative PCR
It is flat；
Expression of the LINC00152 in mouth neoplasm (T) in normal control (N) sample than significantly raising；
Fig. 2 in situ hybridizations detect expression of the LINC00152 in mouth neoplasm and normal oral mucosa；
Left figure is normal oral mucosa tissue, and right figure is mouth neoplasm tissue (amplification factor：200 ×), LINC00152 exists
Expression is higher in mouth neoplasm；
Fig. 3 is the correlation of the expression and patient's prognosis of LINC00152 in mouth neoplasm；
Patient's prognosis of LINC00152 low expressions is expressed than LINC00152 high or expresser is not good, i.e. LINC00152 high
The patient of expression is faster dead.
Fig. 4 is the interference siRNA sequence that LINC00152 is imported in cancer cell of oral cavity, can significantly inhibit LINC00152 and exist
Expression in cancer cell of oral cavity；Si152 is the cancer cell of oral cavity for the interference siRNA sequence for importing LINC00152 in figure, and NC is the moon
Property control；
After the siRNA mixtures of targeting LINC00152 being imported in cancer cell of oral cavity system Tca8113, real time fluorescent quantitative
PCR method has detected the expression of LINC00152 in cancer cell of oral cavity, and the expression of LINC001520 is significantly pressed down
System.Negative control (NC) is that Scramble interferes siRNA sequence.
Fig. 5 is cell migration energy after importing the siRNA inhibition LINC00152 expression of LINC00152 in cancer cell of oral cavity
Power reduces；Si152 is the cancer cell of oral cavity for the interference siRNA sequence for importing LINC00152 in figure, and NC is negative control；
Cell scratch experiment confirms, the siRNA of targeting LINC00152 is transferred in cancer cell of oral cavity system Tca8113, inhibits
After the expression of LINC00152, cut healing rate slows down, and shows that the ability that cell is migrated to cut center reduces.
Fig. 6 is cell invasion energy after importing the siRNA inhibition LINC00152 expression of LINC00152 in cancer cell of oral cavity
Power reduces；Si152 is the cancer cell of oral cavity for the interference siRNA sequence for importing LINC00152 in figure, and NC is negative control；
Cell-penetrating (transwell) in cancer cell of oral cavity system Tca8113 it is experimentally confirmed that be transferred to targeting LINC00152
SiRNA, after the expression for inhibiting LINC00152, can pass through matrix glued membrane cancer cell of oral cavity number substantially reduce, show cell
Invasive ability reduces.
Specific implementation mode
It further illustrates the present invention, is not intended to limit the present invention below in conjunction with specific implementation mode.
Embodiment 1, the detection of real time fluorescent quantitative method confirm LINC00152 up-regulated expressions in mouth neoplasm
1. materials and methods：
14 normal oral mucosas and 15 mouth neoplasm tissue extracted total RNAs, 2 μ g RNA after reverse transcription is at cDNA,
Carry out real-time fluorescence quantitative PCR.
Forward primer：5 '-TTGATGGCTTGAACATTTGG-3 ',
Reverse primer：5'-TCGTGATTTTCGGTGTCTGT-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 values (threshold cycle values), reference gene (GAPDH) markization, is examined and calculated using group t-test
P values.
2. result
LncRNA LINC00152 express relatively low in normal control tissue, and have higher expression in mouth neoplasm tissue
P=0.043 (Fig. 1)
Embodiment 2, in situ hybridization detection find that expression of the LINC00152 in mouth neoplasm is 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 LINC00152 gene sequencings are designed 3 best oligonucleotides of the best specificity of Crossbreeding parameters and are visited
Needle, in addition, using house-keeping gene GAPDH as positive control.
Oligonucleotide probe in situ hybridization detection LINC00152 expression：
LINC00152 probes 1：5'-CTATGTCTTAATCCCTTGTCCTTCATTAAAAGC-3'；
LINC00152 probes 2：5'-CTTCATTGAACAGTTTGTATATTGGAAACTTGCC-3'；
LINC00152 probes 3：5'-GCTGCTTTTAAGTTTCAAATTG ACATTCCAGAC-3'.
Positive control probe (detection house-keeping gene GAPDH)：
GAPDH probes 1：5'-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3'；
GAPDH probes 2：5'-GTCAGAGGAGACCACCTGGTGCTCAGTGTA-3'；
GAPDH probes 3：5'-CAGTAGAGGCAGGG ATGATGTTCTGGAGAG-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(2ndGeneration) kit (Roche companies), anti-ly
High octyl- horseradish peroxidase complex detection kit (Anti-Digoxigenin-POD, Fab fragments, Roche
Company).TSA signal amplifying systems (the TSA of enhancing detection of expression signal in situTMBiotin System, NEL700 kits,
PerkinElmer companies).DAB staining kits (Beijing Zhong Shan companies).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 sperm DNA, ssDNA), yeast transfer RNA (yeast t-RNA, tRNA), DTT, 50 ×
Denhardts ' s solution, PBS buffer, 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
Absolute ethyl 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-bis-
Oxygen aqueous solution (80% methanol and the configuration of 30% hydrogen peroxide)；0.01mol/L citrate buffers (citrate buffer, CB,
Provisional configuration in 450ml distilled water is added in pH6.0 ± 0.1,9ml 0.1M citric acid solutions and 41ml 0.1M sodium citrate solutions
Correction work liquid pH value again afterwards)；0.1% trypsase；Haematoxylin；1% hydrochloride alcohol (70% alcohol of 1ml concentrated hydrochloric acids+99ml
Configuration)；The special mounting glue of micro-array tissue (PTS Cure Mount II)；Special coverslip (480 × 240mm2) customization Zheng Yu
State glass apparatus factory.Leica low melting points (58 DEG C) paraffin, domestic beeswax, absolute alcohol, dimethylbenzene, 10% neutral paraformaldehyde
(0.01mol/L, pH7.4, DEPC distilled water and PBS buffer solution are prepared), haematoxylin, Yihong, neutral mounting natural gum, coverslip,
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 (control:control oligonucleutide 5μ
l+ddH2O 4μl)
Mixing slightly centrifuges.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) 100% cold ethyl alcohol (- 20 DEG C) of+2.5 μ l 4M LiCL+75 μ l of probe reaction mixture (22 μ l)
B) -70 DEG C of precipitations 60min, or-20 DEG C of 2h.
C) 13.000 × g, 4 DEG C of centrifugation 15min.
D) supernatant is abandoned, is washed with 70% ice-cold (V/V) ethyl alcohol of 50 μ l.
E) 4 DEG C of 13.000xg centrifuge 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 dewaxes 3 × 5min successively.
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 washings → DEPC-PBS washs 2 × 5min.
D) 300 μ l pepsins K (10 μ g/ml) are added dropwise on slice, 37 DEG C digest 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, reacts 20-30min in 37 DEG C, increase the permeability of tissue.
G) slice fixes 10min, room temperature afterwards with 4% paraformaldehyde (0.1M PBS dissolvings).
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 triethanolamines), room temperature 10min.
I) 1M PBS wash 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
12.5 μ l of square cm chips area, parafilm covering of corresponding size are 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 basis：The concentration of each probe by its with
Colour developing is compared and the label reaction theory spy of the naked probe of 30 bases of 100pmol when detection reaction when the positive quantifies probe
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.It is washed successively 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) Anti-Digoxigenin-POD detection digoxigenin-probes and purpose RNA combination compounds are used；TSA amplifications system
The positive signal of system enhancing in situ hybridization reaction solution reaction, DAB colour developings.
B) slice is gone in TNT buffer solutions, 3 × 5min.
C) TNB is added dropwise and blocks buffer solution, 300 μ l/TMAs, room temperature, 30min.
D) it does not wash, sucks extra blocking agent, 1:100 diluted Anti-Digoxigenin-POD (TBS+0.1%Triton
X-100+1% blocking agents), room temperature 4 hours.
E) TNT Buffer (0.1M Tris-HCl, PH7.5,0.15M NaCL, 0.05%Tween 20) wash 3 × 5min.
F) signal amplification reagent Biotinyl Tyamid are added dropwise on slice, 300 μ l/TMAs, (BiotinylTyramid stores
Liquid storage：Biotinyl Tyramid are dissolved in 0.2ml DMSO, Biotinyl Tyramid working solutions：1 × dilution, 1:50 is dilute
Release Biotinyl Tyramid storages liquid), room temperature 10 minutes.
G) TNT is washed, 3 × 5min.
H) SA-HRP (strepto- avidin-horseradish peroxidase), 300 μ l/TMAs, room temperature 30min are added dropwise in slice.
I) TNT is washed, 3 × 5min.
J) distilled water washing, 1 × 1min.
K) DAB develops the color, and chromogenic reaction is controlled under microscope.
L) haematoxylin is redyed,
M) alcohol step is dehydrated, chip drying.
N) the dropwise addition special mounting glue of histotomy, the coverslip cover plate of dimension, what dicing-tape transfer system was equipped with
Crosslinking slice 1min under ultraviolet lamp.
1.9 results judge 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：Positioned 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, criterion are：(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 the light brown back of the body
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：A. no positive cell expression, 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 possible, one of above-mentioned standard is pressed respectively respectively by two pathology experts
Judged and scored, then the two is scored and is multiplied, result is：1. 0 point of person is finally calculated as 0 point, it is believed that feminine gender expression；2. 1 point
It is finally calculated as 1 point with 2 points of persons, it is believed that weakly positive is expressed；3. 3 points and 4 points of persons are finally calculated as 2 points, it is believed that moderate positive is expressed；④
6, which assign to 9 points of persons, is finally calculated as 3 points, it is believed that strong positive is expressed.
1.10 analyses and statistical software
Statistical analysis is carried out to experimental result using SPSS13.0 statistical softwares, compares use χ two-by-two2Test or Fisher
Exact test, correlation analysis use Spearmen correlation methods；P < 0.05 are 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 are that difference is statistically significant.
2 results
1) expression of the lncRNA LINC00152 in mouth neoplasm and normal control tissue
LINC00152 does not express or expresses very low (Fig. 2 is left) in normal control tissue, and swollen in most of oral cavity
Higher (Fig. 2 is right) is expressed in tumor tissue, and there is apparent significant difference (P< between the two;0.05).
2) Kaplan-Meier survival analysis
We have carried out Effect of follow-up visit by telephone to all 182 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 LINC00152 and the life span of patient and state carry out in mouth neoplasm tissue, finds
It is short (Fig. 3) that the survival of patients time of LINC00152 high expression compared with LINC00152 expresses patient that is low or not expressing.Explanation
LINC00152 is one and the relevant molecular labeling of mouth neoplasm prognosis, which expresses low, patient's good prognosis.
Embodiment 3, siRNA interfere the expression of LINC00152
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 softwares of LINC00152 sequence inputting Invitrogen companies, seek
It is as follows to select the corresponding target sequence of best 2 for the siRNA best targets for looking for the lncRNA：
siRNA-1：5 '-CAGGGAAUCUUUCAGCUGGAUUCCG-3 ',
siRNA-2：5 '-UCUAUGUGUCUUAAUCCCUUGUCCU-3 ',
Negative control Scramble sequences：5'-GACACGCGACUUGUACCAC-3'.
1.3 cell culture and transfection
Cancer cell of oral cavity system Tca8113 is purchased from Central South University's cell centre, and RPMI 1640 trains base and tire used in cell culture
Trypsase used in cow's serum and vitellophag is U.S.'s Gibco Products.
The good cancer cell of oral cavity system Tca8113 of growth conditions is pressed 2 × 105A cells/well is inoculated in 6 orifice plates, by 6
Orifice plate is placed in 37 DEG C, 5%CO2In incubator, cell growth to be cultivated can start the transfection of siRNA to 50-70% density；Turn
Dye process is as follows：
The Hipefect of the 3 μ l mixing in 100 μ l serum free mediums is added in sterile EP tube and stands 5min；
2 siRNA are mixed and are added in 100 μ l serum free mediums；Then with the above-mentioned 100 μ l comprising Hipefect without
The mild mixing of blood serum medium is stored at room temperature 30 minutes, and siRNA is made to form complex with liposome；
Use D-Hank'S liquid washs cell 3 times；
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：
Cancer cell of oral cavity 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.
LINC00152 forward primers：5 '-TTGATGGCTTGAACATTTGG-3 ',
LINC00152 reverse primers：5'-TCGTGATTTTCGGTGTCTGT-3'.
GAPDH for control
Forward primer is 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 values (threshold cycle values), reference gene (GAPDH) markization, is examined and calculated using group t-test
P values.
2. result
After siRNA transfects cancer cell of oral cavity Tca8113, the expression water of LINC00152 in cancer cell of oral cavity can be significantly lowered
Flat (Fig. 4).
Embodiment 4, LINC00152siRNA inhibit the expression of LINC00152 and the invasion of carcinoma of mouth in cancer cell of oral cavity to move
It moves
1. MATERIALS METHODS
1.1 cell culture and transfection
The good cancer cell of oral cavity Tca8113 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 growth to be cultivated to 50-70% density can start LINC00152siRNA and turn
Dye.
1.2 cell scratch experiments
Cell scratch experiment is the experimental method for verifying tumor cell migration ability.LINC00152siRNA interference is transfected
The cancer cell of oral cavity of sequence or control sequence (scramble) is inoculated in 6 orifice plates, when cell density reaches 90%, uses 200ul
Pipet draws a straight line (cut) in each 6 orifice plates, then in the Each point in time such as 0,24,48,64 hour (depending on different thin
Depending on born of the same parents' transfer ability) cut healing state is observed under the microscope, it takes pictures, and calculate each group cell migration rates.
1.3 cell-penetratings are tested
((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 companies.Matrigel is pressed 1:8 dilutions, are coated on the upper chamber of the cells Transwell bottom film
Face, setting 37 DEG C makes Matrigel aggregate into gel in 30 minutes.Using preceding matrigel film water is carried out by BD companies specification.
Serum free medium and 1 × 10 is added on each cells Transwell upper layer5It is a to have transfected LINC00152siRNA
With the cancer cell of oral cavity of negative control, in the cells Transwell, the culture medium containing 20% fetal calf serum is added in lower layer.Cell continues
After culture 36 hours, is fixed with 4% paraformaldehyde fixer, violet staining, the non-migrating cell in upper layer is dabbed off with cotton swab,
It is washed 3 times with PBS.The cancer cell of oral cavity across matrix glued membrane is observed under the microscope.
2. result
After 2.1 interference sequences for importing LINC00152 in cancer cell of oral cavity inhibit LINC00152 expression, cell migration
Ability reduces
Cell scratch experiment confirms, the interference sequence of targeting LINC00152 is transferred in cancer cell of oral cavity system Tca8113,
After the expression for inhibiting LINC00152, the speed that cancer cell of oral cavity is migrated from cut both sides toward cut center slows down, cut healing
Time lengthening shows that cell movement transfer ability reduces (Fig. 5).
After 2.1 import LINC00152siRNA inhibition LINC00152 in cancer cell of oral cavity, cell invasion ability reduces
Cell-penetrating (Transwell) in cancer cell of oral cavity system Tca8113 it is experimentally confirmed that be transferred to targeting
LINC00152siRNA after the expression for inhibiting LINC00152, can pass through the cancer cell of oral cavity number of matrix glued membrane to substantially reduce,
Show that cell invasion ability reduces (Fig. 6).
SEQUENCE LISTING
<110>Central South University
<120>The application of the siRNA of long-chain non-coding RNA LINC00152 and inhibitory preparation
<130>Nothing
<160> 14
<170> PatentIn version 3.3
<210> 1
<211> 828
<212> RNA
<213>LINC00152 RNA sequences
<400> 1
gugcgccuuu uuuuuuuuuu ccuucuuagu cguguguaca ucauugggaa uggagggaaa 60
uaaaugacug gauggucgcu gcuuuuuaag uuucaaauug acauuccaga caagcggugc 120
cugagcccgu gccugucuuc agaucuucac agcacaguuc cugggaaggu ggagccacca 180
gccucuccuu guccuggagg cuggaagugc aaaaggaagg ugucggcaag aucguuuuuu 240
ucugagagcu cucuccuugg cuugcagaug gcagccugcu ccuggcacag ucuuuucucu 300
acucaugccc aaaguuacgg aggacccagc aaccaucucc ugcagccccu ggaaaccucu 360
ugacucuucu gugauguccc cagugaucca gcagcccugg ccuucuuuug auggcuugaa 420
cauuuggucu ucauugaaca guuuguauau uggaaacuug ccagccucca uccacauucc 480
aaccuccguc ugcaucccuc gaauaacugg gagaugaaac aggaagcucu augacacacu 540
ugaucgaaua ugacagacac cgaaaaucac gacucagccc ccuccagcac cucuaccugu 600
ugcccgccga ucacagccgg aaugcagcug aaagauuccc uggggccugg uuccaaccgc 660
ccacugugga cucugaggcc ucugcauuug cggguggucu gccugugaua uuuuggucau 720
gggcuggucu ggucgguuuc ccauuugucu ggccagucuc uaugugucuu aaucccuugu 780
ccuucauuaa aagcaaaacu aaagaaaaaa aaaaaaaaaa aaaaaaaa 828
<210> 2
<211> 20
<212> DNA
<213>Real time fluorescent quantitative detects the forward primer of LINC00152 expression
<400> 2
ttgatggctt gaacatttgg 20
<210> 3
<211> 20
<212> DNA
<213>Real time fluorescent quantitative detects the reverse primer of LINC00152 expression
<400> 3
tcgtgatttt cggtgtctgt 20
<210> 4
<211> 20
<212> DNA
<213>Reference gene GAPDH specific PCR forward primers
<400> 4
accacagtcc atgccatcac 20
<210> 5
<211> 20
<212> DNA
<213>Reference gene GAPDH specific PCR reverse primers
<400> 5
tccaccaccc tgttgctgta 20
<210> 6
<211> 33
<212> DNA
<213>In situ hybridization detects the oligonucleotide probe 1 of LINC00152 expression
<400> 6
ctatgtctta atcccttgtc cttcattaaa agc 33
<210> 7
<211> 34
<212> DNA
<213>In situ hybridization detects the oligonucleotide probe 2 of LINC00152 expression
<400> 7
cttcattgaa cagtttgtat attggaaact tgcc 34
<210> 8
<211> 33
<212> DNA
<213>In situ hybridization detects the oligonucleotide probe 3 of LINC00152 expression
<400> 8
gctgctttta agtttcaaat tgacattcca gac 33
<210> 9
<211> 30
<212> DNA
<213>GAPDH probes 1
<400> 9
ccactttacc agagttaaaa gcagccctgg 30
<210> 10
<211> 30
<212> DNA
<213>GAPDH probes 2
<400> 10
gtcagaggag accacctggt gctcagtgta 30
<210> 11
<211> 30
<212> DNA
<213>GAPDH probes 3
<400> 11
cagtagaggc agggatgatg ttctggagag 30
<210> 12
<211> 25
<212> RNA
<213> siRNA-1
<400> 12
cagggaaucu uucagcugga uuccg 25
<210> 13
<211> 25
<212> RNA
<213> siRNA-2
<400> 13
ucuauguguc uuaaucccuu guccu 25
<210> 14
<211> 19
<212> RNA
<213>Negative control Scramble sequences
<400> 14
gacacgcgac uuguaccac 19
Claims (2)
1. the application of the siRNA of long-chain non-coding RNA LINC00152, which is characterized in that utilize long-chain non-coding RNA
The siRNA of LINC00152 prepares the reagent for inhibiting oral cavity cancerous invasion and transfer, the sequence of long-chain non-coding RNA LINC00152
Row such as SEQ NO:Shown in 1.
2. the application of the siRNA of long-chain non-coding RNA LINC00152 according to claim 1, which is characterized in that long-chain
The siRNA sequence of non-coding RNA LINC00152 is one or two in following two：
siRNA-1：5 '-CAGGGAAUCUUUCAGCUGGAUUCCG-3 ',
siRNA-2：5′-UCUAUGUGUCUUAAUCCCUUGUCCU-3′.
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|---|---|---|---|---|
| CN103966311A (en) * | 2014-03-04 | 2014-08-06 | 宁波大学 | Method for detecting lncRNA (long noncoding RNA) in plasma |
| CN105018498A (en) * | 2015-05-12 | 2015-11-04 | 中南大学 | Application method of lnc RNA (long-chain non-coding ribonucleic acid) AFAP1-AS1 |
| CN105200152A (en) * | 2015-10-30 | 2015-12-30 | 中南大学 | Application of long-chain non-coding RNA (ribonucleic acid) gene LOC553103 in preparation of nasopharynx cancer prognosis preparation |
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| CN103966311A (en) * | 2014-03-04 | 2014-08-06 | 宁波大学 | Method for detecting lncRNA (long noncoding RNA) in plasma |
| CN105018498A (en) * | 2015-05-12 | 2015-11-04 | 中南大学 | Application method of lnc RNA (long-chain non-coding ribonucleic acid) AFAP1-AS1 |
| CN105200152A (en) * | 2015-10-30 | 2015-12-30 | 中南大学 | Application of long-chain non-coding RNA (ribonucleic acid) gene LOC553103 in preparation of nasopharynx cancer prognosis preparation |
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| Upregulation of long non-coding RNA LINC00152 by SP1 contributes to gallbladder cancer cell growth and tumor metastasis via PI3K/AKT pathway;Qiang Cai et al;《AM J Transl Res》;20161030;第8卷(第10期);第4068-4081页 * |
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