CN106511368A - Application of siRNA of long non-coding RNA LINC00152 and inhibiting preparation - Google Patents

Abstract

The invention discloses application of siRNA of long non-coding RNA LINC00152 and an inhibiting preparation. The siRNA sequence of the target LINC00152 is designed and synthesized, the siRNA is transfected into an oral cancer cell line, and thus it is proven that the expression of the target interfering LINC00152 can obviously inhibit the invasion and transfer ability of oral cancer cells. A new potential approach is provided for treatment of oral cancers.

Description

The application of the siRNA of long-chain non-coding RNA LINC00152 and inhibitory preparation

Technical field

The invention belongs to oncomolecularbiology field, 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 susceptible to metastasic cervical lymph nodes, Prognosis is poor.Research shows, the generation development of this tumour is polygenes participation, multi-step, multistage complex process, wherein The inactivation of tumor suppressor gene has played the effect of key with the activation of oncogene.

Long-chain non-coding RNAs (long non-coding RNAs, lncRNAs) be a class length more than 200nt, lack Special complete ORFs, without or seldom have the RNAs molecules of encoding histone function.It has recently been demonstrated that lncRNAs By epigenetic, transcription and the extensive regulatory gene of post-transcriptional level expression, they take part in biology growing, development, The regulation and control of the important vital movement such as aging and death.Increasing research shows the unconventionality expression or afunction of lncRNAs Develop with the generation of tumour closely related.Some lncRNAs molecules have great importance in terms of the diagnosis and treatment of tumour, And new molecular labeling can be judged as tumor prognosis.At present, the mankind lncRNAs genes cloned are more than 40,000 It is individual, 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 correlation of level and clinical case data.We are from after extracting RNA in mouth neoplasm and corresponding normal control tissue By reverse transcription, real-time quantitative PCR, the expression of LINC00152 is have detected, as a result show LINC00152 in mouth neoplasm Up-regulated in tissue.We subsequently again using the method for in situ hybridization, are further demonstrated in archive paraffin sample LINC00152 expresses significantly rise in mouth neoplasm, therefore the detection preparation for LINC00152 can be used for mouth neoplasm Auxiliary diagnosis, carry out survivorship curve analysis with reference to clinical return visit data and find that LINC00152 expresses high its life span of patient It is shorter than the lncRNA and expresses low patient, therefore the detection preparation for the lncRNA can be used for the prognosis of mouth neoplasm and sentence It is disconnected.

We target the siRNA sequence of LINC00152 by designing and synthesizing, and are transfected in cancer cell of oral cavity strain, in body Outer culture systems confirm that the expression of targeting interference LINC00152 can substantially suppress invasion and attack and the transfer ability of Stomatocyte.

The content of the invention

For above-mentioned prior art, it is an object of the invention to provide the siRNA's of long-chain non-coding RNA LINC00152 should With and inhibitory preparation.

The application of the siRNA of long-chain non-coding RNA LINC00152 of the present invention, using long-chain non-coding RNA The siRNA of LINC00152 prepares the reagent for suppressing 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 or two in following two：

siRNA-1：5 '-CAGGGAAUCUUUCAGCUGGAUUCCG-3 ',

siRNA-2：5′-UCUAUGUGUCUUAAUCCCUUGUCCU-3′.

A kind of suppression oral cavity cancerous invasion and the preparation for shifting, including the siRNA sequences of long-chain non-coding RNA LINC00152 Row.

One or two in two below specially：

siRNA-1：5 '-CAGGGAAUCUUUCAGCUGGAUUCCG-3 ',

siRNA-2：5′-UCUAUGUGUCUUAAUCCCUUGUCCU-3′

The described preparation for suppressing oral cavity cancerous invasion and transfer, also including negative control Scramble sequences：5’- GACACGCGACUUGUACCAC-3’。

The invention discloses the application of the siRNA of long-chain non-coding RNA LINC00152 and inhibitory preparation.By design simultaneously The siRNA sequence of synthesis targeting LINC00152, is transfected in cancer cell of oral cavity strain, confirms that targeting is dry in vitro in culture systems The expression for disturbing LINC00152 can substantially suppress invasion and attack and the transfer ability of cancer cell of oral cavity.The present invention is carried for the treatment of carcinoma of mouth 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) is than significantly raising in normal control (N) sample；

Fig. 2 in situ hybridizations detect expression of the LINC00152 in mouth neoplasm and normal oral mucosa；

Left figure is organized for normal oral mucosa, and right figure is that mouth neoplasm organizes (multiplication factor：200 ×), LINC00152 exists In mouth neoplasm, expression is higher；

Fig. 3 is the correlation of the expression with patient's prognosis of LINC00152 in mouth neoplasm；

Patient's prognosis of LINC00152 low expressions expression higher than LINC00152 or expresser is not good, i.e. LINC00152 is 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；In figure, si152 is the cancer cell of oral cavity of the interference siRNA sequence for importing LINC00152, and NC is cloudy Property control；

After the siRNA mixtures of targeting LINC00152 are imported in cancer cell of oral cavity system Tca8113, real time fluorescent quantitative PCR method have detected the expression of LINC00152 in cancer cell of oral cavity, and the expression of LINC001520 is significantly pressed down System.Negative control (NC) disturbs siRNA sequence for Scramble.

Fig. 5 is cell migration energy after the siRNA of the importing LINC00152 in cancer cell of oral cavity suppresses LINC00152 expression Power is reduced；In figure, si152 is the cancer cell of oral cavity of the interference siRNA sequence for importing LINC00152, and NC is negative control；

Cell scratch experiment confirms, proceeds to the siRNA of targeting LINC00152, suppress in cancer cell of oral cavity system Tca8113 After the expression of LINC00152, cut healing rate slows down, and shows that cell is reduced to the ability of cut central authorities migration.

Fig. 6 is cell invasion energy after the siRNA of the importing LINC00152 in cancer cell of oral cavity suppresses LINC00152 expression Power is reduced；In figure, si152 is the cancer cell of oral cavity of the interference siRNA sequence for importing LINC00152, and NC is negative control；

Cell-penetrating (transwell) is it is experimentally confirmed that proceed to targeting LINC00152 in cancer cell of oral cavity system Tca8113 SiRNA, after suppressing the expression of LINC00152, can substantially reduce through the cancer cell of oral cavity number of matrix glued membrane, show cell Invasive ability is reduced.

Specific embodiment

The present invention is further illustrated below in conjunction with specific embodiment, and the unrestricted present invention.

Embodiment 1, the detection of real time fluorescent quantitative method confirm LINC00152 up-regulateds in mouth neoplasm

1. materials and methods：

14 normal oral mucosas and 15 mouth neoplasms organize extracted total RNA, after 2 μ g RNA are Jing reverse transcription into cDNA, Carry out real-time fluorescence quantitative PCR.

Forward primer：5 '-TTGATGGCTTGAACATTTGG-3 ',

Reverse primer：5’-TCGTGATTTTCGGTGTCTGT-3’.

For the internal reference house-keeping gene GAPDH Specific PCR primers of control：

Forward primer 5 '-ACCACAGTCCATGCCATCAC-3 '

Reverse primer 5 '-TCCACCACCCTGTTGCTGTA-3 '.

Real-time fluorescence quantitative PCR reaction system

Real-time fluorescence quantitative PCR reactions steps

Reaction confirms the amplification curve and melting curve of real-time fluorescence quantitative PCR, the expression intensity root of each gene after terminating After according to CT values (threshold cycle values), reference gene (GAPDH) markization, calculated using group t-test inspections P values.

2. result

LncRNA LINC00152 express relatively low in normal control tissue, and in mouth neoplasm tissue have higher expression 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 oligonucleotide probes for being used for mouth neoplasm and normal control tissue in situ hybridization

Root Ju LINC00152 gene sequencings are designed 3 best oligonucleotides of the optimal specificity of Crossbreeding parameters and are visited Pin, in addition, with house-keeping gene GAPDH as positive control.

The oligonucleotide probe of LINC00152 expression is detected in situ hybridization：

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 kit and in situ hybridization detection reagent

Dig Oligonucleitide Tailing Kit(2^ndGeneration) kit (Roche companies), anti-ly High octyl- horseradish peroxidase complex detection kit (Anti-Digoxigenin-POD, Fab fragments, Roche Company).Strengthen the TSA signal amplifying system (TSA of detection of expression signal in situ^TMBiotin System, NEL700 kit, 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), the frog essence DNA (denatured of denaturation 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, blocks 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 (pH7.2～ 7.4, NaCl 137mmol/L, KCl 2.7mmol/L, Na₂HPO₄4.3mmol/L, KH₂PO₄1.4mmol/L)；3% methyl alcohol-bis- The oxygen aqueous solution (80% methyl alcohol and the configuration of 30% hydrogen peroxide)；0.01mol/L citrate buffers (citrate buffer, CB, PH6.0 ± 0.1,9ml 0.1M citric acid solutions and 41ml 0.1M sodium citrate solutions add provisional configuration in 450ml distilled water 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 cover glass (480 × 240mm²) customize 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 are prepared), haematoxylin, Yihong, neutral mounting natural gum, cover glass, Slide.

The mark of 1.4 oligonucleotide probes

Oligonucleotide probe mark 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)

Mix, be slightly centrifuged.37 DEG C of water-bath 30min, plus 2ul EDTA (0.2M, pH 8.0) stopped reactions.

Purify after 1.5 oligonucleotide probes mark

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

A) 100% cold ethanol (- 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 ice-cold 70% (V/V) ethanol of 50 μ l.

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

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

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

1.6 histotomies hybridize pre-treatment

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

B) dimethylbenzene is dewaxed 3 × 5min successively.

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

D) 300 μ l pepsin K (10 μ g/ml) are added dropwise in section, 37 DEG C digest 20min.

E) to cut into slices 1min, stopped reaction are washed into PBS (0.1M PBS+2mg/ml glutamic acid).

F) cut into slices into 0.2N HCL, 20-30min is reacted in 37 DEG C, increase the permeability of tissue.

G) section 4% paraformaldehyde (0.1M PBS dissolvings) fixes 10min, room temperature afterwards.

H) in order to increase tissue positive intensity for hybridization, acetylation process is carried out to section.Cut into slices 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 consumption of prehybridization solution is often flat 12.5 μ l of square cm chips area, correspondingly sized Parafilm cover section, prehybridization 2 hours in 37 DEG C of wet box.(prehybridization solution Composition includes：2XSSC, 10%Dextran sulphate, 1X Denhardt ' s solution, 50mM Phosphate The μ g/ml poly A of Buffer (PH 7.0), 50mM DTT, 250 μ l, 100,5 μ g/ml poly dA, 250 μ g/ml yeast T-RNA, 500 μ g/ml ssDNA, 47%Deionized formamide).

B) Parafilm is removed, gets rid of prehybridization solution, section is placed in 5min in 2 × SSC.

C) hybridization reaction：37 DEG C of hybridized overnights (18-20h).Each section adds 250 μ l hybridization solutions and is covered with Parafilm Lid.Corresponding probe is added just to become hybridization solution in prehybridization solution.Hybridization solution is prepared in prehybridization, is placed 37 DEG C of incubations, is made Probe is completely dissolved in hybridization solution, and this experiment is mixed with a plurality of oligonucleotide probe, is matched somebody with somebody 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 Develop the color during detection reaction during positive quantitatively probe the naked probe mark reaction theory spy compared with 30 bases of 100pmol Pin yield carries out the concentration that COMPREHENSIVE CALCULATING goes out label probe for two kinds of standards of 900ng.

D) post-hybridization washing, section immersion 2 × SSC, 10min, throws off Parafilm.Shake on shaking table successively and wash, 2 × SSC (0.5%SDS), 2 × 15min → 0.25 × SSC (0.5%SDS), 2 × 15min.

Color developing detection reaction after 1.8 hybridization

A) compound is combined with purpose RNA using Anti-Digoxigenin-POD detection digoxigenin-probes；TSA amplifies system System strengthens the positive signal of in situ hybridization reaction solution reaction, DAB colour developings.

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

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

D) do not wash, suck unnecessary blocking agent, 1:Anti-Digoxigenin-POD (the TBS+0.1%Triton of 100 dilutions X-100+1% blocking agents), room temperature 4 hours.

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

F) signal is added dropwise in section and amplifies reagent Biotinyl Tyamid, 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 storage liquid), room temperature 10 minutes.

G) TNT is washed, 3 × 5min.

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

I) TNT is washed, 3 × 5min.

J) distilled water washing, 1 × 1min.

K) DAB colour developings, control chromogenic reaction under microscope.

L) haematoxylin is redyed,

M) alcohol step dehydration, chip drying.

N) the special mounting glue of dropwise addition histotomy, the cover glass cover plate of dimension, what dicing-tape transfer system was equipped with Crosslinking section 1min under uviol lamp.

1.9 result judges and standard

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

Carried out with two kinds of standards of cell number of the intensity and positive expression of the detection rna expression position positive signal respectively again Comprehensive grading, criterion is：(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 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 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.

It is in order to reduce the subjective factor of appraisal result as far as possible, respective by one of above-mentioned standard respectively by two pathology experts Judged and scored, then both are scored multiplication, as a result for：1. 0 point of person is finally calculated as 0 point, it is believed that radiolucent table reaches；2. 1 point 1 point is calculated as finally 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 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 are carried out to experimental result using SPSS13.0 statistical softwares, is compared two-by-two and is used χ²Test or Fisher Exact test, correlation analysis adopt Spearmen correlation methods；P ＜ 0.05 are that difference is statistically significant. Survivorship curve analysis is using Kaplan-Meier method and log-rank test；Multi-variables analysis adopts 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 is not expressed in normal control tissue or is expressed very low (Fig. 2 is left), and swollen in most of oral cavity Higher (Fig. 2 is right) is expressed in tumor tissue, between the two with obvious significant difference (P<0.05).

2) Kaplan-Meier survival analysises

We have carried out Effect of follow-up visit by telephone to all 182 mouth neoplasm patients, inquired their start time in detail, have controlled Treatment situation, whether there is recurrence, whether there is, and registering life span and state, and it is right The survival analysis that the expression of LINC00152 is carried out with the life span of patient and state in mouth neoplasm tissue, finds It is short (Fig. 3) that the survival of patients time of the high expression of LINC00152 expresses patient that is low or not expressing compared with LINC00152.Explanation LINC00152 is a molecular labeling related to mouth neoplasm prognosis, and the lncRNA expresses low, patient's good prognosis.

Embodiment 3, siRNA disturb the expression of LINC00152

1. MATERIALS METHODS

1.1 reagents and kit

TRIZOL^TMReagent(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 The siRNA best targets of the lncRNA are looked for, the corresponding target sequence in optimal 2 is selected as follows：

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, the training bases of RPMI 1640 and tire used by cell culture Cow's serum, and the trypsase used by vitellophag is U.S.'s Gibco Products.

Growth conditions good cancer cell of oral cavity system Tca8113 is pressed into 2 × 10⁵Individual cells/well is inoculated in 6 orifice plates, by 6 Orifice plate is placed in 37 DEG C, 5%CO₂In incubator, treat that cultured cells starts the transfection of siRNA by growing to 50-70% density；Turn Dye process is as follows：

In aseptic EP pipes, add the Hipefect of 3 μ l that standing 5min is mixed in 100 μ l serum free mediums；

2 siRNA mixing are added in 100 μ l serum free mediums；Then with the above-mentioned 100 μ l comprising Hipefect without Blood serum medium is gently mixed, and is stored at room temperature 30 minutes, makes siRNA form complex with liposome；

With D-Hank's liquid washed cell 3 times；

800 μ l serum free mediums (antibiotic-free) will be added in said mixture, in 6 orifice plates being added after gentle mixing 1 hole；

6 orifice plates are placed in into CO₂In incubator, 37 DEG C are cultivated 6 hours, then abandon supernatant, add complete medium to continue training Support 48 hours.

The effect of 1.5 real-time quantitative PCRs detection siRNA interference lncRNA expression：

Cancer cell of oral cavity extracted total RNA after siRNA is transfected, after 2 μ g RNA are Jing reverse transcription into cDNA, is carried out glimmering in real time Fluorescent Quantitative PCR.

LINC00152 forward primers：5 '-TTGATGGCTTGAACATTTGG-3 ',

LINC00152 reverse primers：5’-TCGTGATTTTCGGTGTCTGT-3’.

For the GAPDH of control

Forward primer is 5 '-ACCACAGTCCATGCCATCAC-3 '

Reverse primer 5 '-TCCACCACCCTGTTGCTGTA-3 '.

Real-time fluorescence quantitative PCR reaction system

Real-time fluorescence quantitative PCR reactions steps

Reaction confirms the amplification curve and melting curve of real-time fluorescence quantitative PCR, the expression intensity root of each gene after terminating After according to CT values (threshold cycle values), reference gene (GAPDH) markization, calculated using group t-test inspections P values.

2. result

After siRNA transfection 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, the expression of LINC00152 and the invasion and attack of carcinoma of mouth in LINC00152siRNA suppression cancer cell of oral cavity are moved Move

1. MATERIALS METHODS

1.1 cell culture and transfection

Growth conditions good cancer cell of oral cavity Tca8113 is pressed into 2 × 10⁵Individual cells/well is inoculated in 6 orifice plates, by 6 holes Plate is placed in 37 DEG C, 5%CO₂In incubator, treat that cultured cells starts LINC00152siRNA by growing to 50-70% density and turns Dye.

1.2 cell scratch experiments

Cell scratch experiment is the experimental technique 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 in each 6 orifice plate (cut), subsequently in the Each point in time such as 0,24,48,64 hours (depending on different thin Depending on born of the same parents' transfer ability) cut healing state is examined under a microscope, take pictures, and calculate each group cell migration rates.

1.3 cell-penetratings are tested

((Transwell) experiment is the experimental technique for verifying tumor cell invasion ability to cell-penetrating.Transwell is little Room (8 μm of aperture) and matrigel (Matrigel) purchased from U.S. company BD, 4% paraformaldehyde fixer, crystal violet dye liquor (0.1%g/ml) purchased from Sigma companies.Matrigel is pressed into 1:8 dilutions, are coated on the upper room of Transwell cell bottom films Face, putting 37 DEG C makes Matrigel aggregate into gel in 30 minutes.Matrigel film water is carried out by BD companies specification using front.

Serum free medium and 1 × 10 is added on each Transwell cells upper strata⁵It is individual to have transfected LINC00152siRNA With the cancer cell of oral cavity of negative control, the culture medium containing 20% hyclone is added in Transwell cells lower floor.Cell continues After culture 36 hours, fixed with 4% paraformaldehyde fixer, violet staining, the non-migrating cell in upper strata dabbed off with cotton swab, 3 times are washed with PBS.Examine under a microscope the cancer cell of oral cavity through matrix glued membrane.

2. result

After 2.1 interference sequences that LINC00152 is imported in cancer cell of oral cavity suppress LINC00152 expression, cell migration Ability is reduced

The confirmation of cell scratch experiment, proceeds to the interference sequence of targeting LINC00152 in cancer cell of oral cavity system Tca8113, After suppressing the expression of LINC00152, cancer cell of oral cavity slows down from cut both sides toward the speed of cut central authorities migration, cut healing Time lengthening, shows that cell movement transfer ability reduces (Fig. 5).

After 2.1 import LINC00152siRNA suppression LINC00152 in cancer cell of oral cavity, cell invasion ability is reduced

Cell-penetrating (Transwell) is it is experimentally confirmed that proceed to targeting in cancer cell of oral cavity system Tca8113 LINC00152siRNA, after suppressing the expression of 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

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<213>LINC00152 RNA sequences

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<213>The reverse primer of real time fluorescent quantitative detection LINC00152 expression

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<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 (5)

1. the application of the siRNA of long-chain non-coding RNA LINC00152, it is characterised in that using long-chain non-coding RNA The siRNA of LINC00152 prepares the reagent for suppressing 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, it is characterised in that long-chain The siRNA sequence of non-coding RNA LINC00152 is or two in following two：

siRNA-1：5 '-CAGGGAAUCUUUCAGCUGGAUUCCG-3 ',

siRNA-2：5′-UCUAUGUGUCUUAAUCCCUUGUCCU-3′.

3. a kind of suppression oral cavity cancerous invasion with transfer preparation, it is characterised in that including long-chain non-coding RNA LINC00152's SiRNA sequence.

4. the preparation of suppression oral cavity according to claim 3 cancerous invasion and transfer, it is characterised in that specially following two In one or two：

siRNA-1：5 '-CAGGGAAUCUUUCAGCUGGAUUCCG-3 ',

siRNA-2：5′-UCUAUGUGUCUUAAUCCCUUGUCCU-3′.

5. suppression oral cavity cancerous invasion according to claim 3 or 4 and the preparation of transfer, it is characterised in that also including feminine gender Control Scramble sequences：5’-GACACGCGACUUGUACCAC-3’.