CN104922695B - A kind of preparation and its application of long-chain non-coding RNA nanoparticle - Google Patents
A kind of preparation and its application of long-chain non-coding RNA nanoparticle Download PDFInfo
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- CN104922695B CN104922695B CN201410814933.4A CN201410814933A CN104922695B CN 104922695 B CN104922695 B CN 104922695B CN 201410814933 A CN201410814933 A CN 201410814933A CN 104922695 B CN104922695 B CN 104922695B
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Abstract
The present invention relates to a kind of preparation and its application of long-chain non-coding RNA nanoparticle, it is characterised in that：The HN lncRNA for wrapping up or adsorbing including nano-gene carrier and by genophore, the NH lncRNA genes are located at HN gene non-coding heads of district 236nt.Macromolecular RNA is directly prepared into stable nanoparticle by the present invention, and lncRNA molecules are prepared as the enhancing of nanoparticle rear stability, is preserved one week under the conditions of 25 DEG C and non-degradable, and this particle can not enter into the cell by any transfection reagent；The nanoparticle transfection efficiency of the present invention is high, moreover it is possible to plays a role in vivo.The present invention not only solves the problems, such as RNA preservation, and the research for RNA biological function opens new approach.The microballoon can enter cell with direct transfection, and increase the clonality of cell.In vivo study finds that long-chain RNA microballoons can dramatically increase the one-tenth knurl ability of mouse.
Description
Technical field
The invention belongs to oncomolecularbiology field, more particularly to a kind of system of long-chain non-coding RNA nanoparticle
The standby and its application in tumor research.
Background technology
Long-chain non-coding RNA（Long non-coding RNA, lncRNA）Be a kind of transcript length more than 200nt, no
The RNA of encoding proteins.Itself not encoding proteins of long-chain non-coding RNA, but adjusted in the form of RNA in epigenetics
The expression of controlling gene in a variety of aspects such as control, transcriptional control, post-transcriptional control, originally lncRNA is considered as genome
" noise " of transcription, it is the accessory substance of RNA polymerase transcription, without any biological function.Later research is found, some
LncRNA expression has tissue specificity and Space-time speciality, and many lncRNA have conservative secondary structure, prompt
LncRNA has important biological function.Further investigation revealed that many lncRNA have weight in the occurrence and development of tumour
Adjustment effect is wanted, it, which has, suppresses the biological action such as tumour growth or promotion metastases.Nano particle is as gene therapy
Carrier there are some significantly advantages, such as nucleic acid can be protected not degraded by nuclease, there is bioaffinity etc., especially
Suitable for being transfected inside gene.Therefore, nanometer technology is combined with lncRNA to provide superior technique for lncRNA research
Means.Long-chain RNA has extensive biological function, and NH-lncRNA genes are located at HN gene non-coding head of district 236nt, early stage
Research shows that NH-lncRNA has very high oncogenicity, can remarkably promote tumour growth.But the easy degraded of long-chain RNA presence,
The problems such as into cell difficulty, more it is difficult to operate compared to Short interfering RNA, also lacks research long-chain non-coding RNA molecule at present
Effective means.How to realize that lncRNA enters to play a role into the cell, be the technical bottleneck of current lncRNA researchs.
The present invention establishes a kind of nanometer technology, by a kind of non-coding long-chain RNA（HN-lncRNA）It is prepared as nanoparticle
Particle.Electrophoretic analysis shows that HN-lncRNA has very high stability, and room temperature is placed 7 days, structural integrity；Confirm long-chain simultaneously
RNA nanoparticles lncRNA-HN1-RNs can be directly entered cells play effect, and biology can be played in Mice Body
Function.The unique advantage of the technology of the present invention is, RNA molecule directly can be prepared as into RNA nanoparticles, it shows in vivo and in vitro
Show very high stability, there is good biological function.The invention provides a kind of long-chain RNA that can be used for treatment tumour to receive
Rice grain；The present invention not only solves the problems, such as RNA preservation simultaneously, and for long-chain RNA biological function research open up it is new
Approach.The present invention is also significant to development RNA medicines.
The content of the invention
The invention provides a kind of preparation method and application of long-chain RNA nano particles,
The present invention is achieved by the following technical solutions：
A kind of long-chain non-coding RNA nanoparticle, it is characterised in that：Including nano-gene carrier and by genophore bag
The HN-lncRNA for the external synthesis wrapped up in or adsorbed.
Preferably, the NH-lncRNA is lncRNAHN1, and it is located at HN gene non-coding heads of district 236nt, its RNA widow
Nucleotides sequence is classified as：
。
Preferably, the nano-carrier is nano particle prepared by vegetable oil or dodecane, and the vegetable oil includes soybean
Oil, olive oil or the non-tempered oil of other edibles.
Preferably, the preparation method of described long-chain non-coding RNA nanoparticle, it is characterised in that step is as follows：Will
LncRNAHN1, which is dissolved in the aqua sterilisa of no RNase, is prepared as the RNA aqueous solution, and concentration is 200ng ~ 500ng/ml；lncRNA HN1
On the aqueous solution, vegetable oil is added, the volume ratio of vegetable oil and the lncRNA HN1 aqueous solution is 3：2, by above-mentioned vegetable oil with
LncRNA HN1 are dissolved in the distilled water of 10 times of volumes, ultrasonic probe are placed in into water oil level boundary, reaction temperature is set as 4
DEG C, ultrasound intensity is 150 W/cm2, ultrasonic time is 3 minutes；Then removed using 20 kDa Ultra filtration membrane and be not bound with
LncRNA, produce.
Present invention also offers described long-chain non-coding RNA nanoparticle answering in Antioncogene medicine
With.
Relative to prior art, the invention has the advantages that：
1）Some the long-chain non-coding RNA molecules having now been found that and the generation of tumour are closely related, NH- of the present invention
LncRNA is located at HN gene non-coding head of district 236nt, and its overexpression causes normal fibroblast NIH3T3 malignants.
2）Macromolecular RNA is directly prepared into stable nanoparticle by the present invention, and lncRNA molecules are prepared as nanoparticle
Rear stability strengthens, in room temperature（25℃）Under the conditions of preserve one week and non-degradable, this particle can not try by any transfection
Agent enters into the cell；The nanoparticle transfection efficiency of the present invention is high, moreover it is possible to plays a role in vivo.The present invention not only solves
RNA preservation problem, and the research for RNA biological function opens new approach.
3) preparation technology of RNA microballoons of the present invention has the characteristics that：Soybean oil is selected as lapping, soybean oil
Ratio with the lncRNA aqueous solution is 3：2（Volume ratio）, above-mentioned soybean oil and lncRNA are added in the distilled water of 10 times of volumes,
It is ultrasonically treated 3 minutes, then removes the lncRNA being not bound with using 20 kDa milipore filter.
3）The present invention establishes a kind of nanometer technology, by a kind of non-coding long-chain RNA（HN-lncRNA）It is micro- to be prepared as nanometer
Ball particle.RNA microballoons prepared by analysis shows have very high stability, and cell can be entered with direct transfection, and increase thin
The clonality of born of the same parents.In vivo study finds that long-chain RNA microballoons can dramatically increase the one-tenth knurl ability of mouse.Illustrate long-chain
RNA can be directly entered performance biological function in organism after being prepared as nanoparticle.
Brief description of the drawings
The present invention is further described below in conjunction with the accompanying drawings.
Accompanying drawing 1 is the electron-microscope scanning figure and grain size distribution of lncRNA-HN1-RNs nanoparticles of the present invention；In figure（a）
The lncRNA-HN1 nanoparticles of FITC marks are in its size of fluorescence microscopy Microscopic observation and particle diameter distribution；（b）ESEM
LncRNA-HN1-RNs sizes and particle diameter distribution；Scale：20 nm.
Accompanying drawing 2 is denaturing formaldehyde SDS- after lncRNA-HN1-RNs nanoparticles of the present invention are placed 0-7 days at room temperature
PAGE electrophoresis detection figures.
Accompanying drawing 3 is the result figure of lncRNA-HN1-RNs nanoparticles transfection efficiency of the present invention measure.
Accompanying drawing 4 is the functional evaluation result figure of lncRNA-HN1-RNs nanoparticles of the present invention in animal body, A in figure,
Using the HN-lnc-RNA expression vectors of structure as control, by HN-lnc-RNA nanoparticle rotaring copolymering NIH 3 T 3 cells, agar clone
Detect Cell clonality；The NIH-3T3 cells inoculation nude mice of B in figure, HN-lnc-RNA nanoparticle transfection, detection
HN-lnc-RNA nanoparticles influence on the one-tenth knurl ability of NIH-3T3 cells；Based on this evaluation HN-lnc-RNA nanoparticle
Transfection efficiency in inside and outside function and its organism.
Embodiment
With reference to embodiment, the present invention is further described.
Embodiment 1：HN-lnc-RNA nanoparticles prepare scheme
LncRNAHN1 is dissolved in the aqua sterilisa of no RNase and is prepared as the RNA aqueous solution, concentration is 200ng ~ 500ng/ml；
On the lncRNA HN1 aqueous solution, vegetable oil is added, the volume ratio of vegetable oil and the lncRNA HN1 aqueous solution is 3：2, by above-mentioned plant
Thing oil is dissolved in the distilled water of 10 times of volumes with lncRNA HN1, and ultrasonic probe is placed in into water oil level boundary, reaction temperature
It is set as 4 DEG C, ultrasound intensity is 150 W/cm2, ultrasonic time is 3 minutes；Then removed using 20 kDa Ultra filtration membrane
The lncRNA being not bound with, is produced.Its size and particle diameter distribution are analyzed through DLS and SME methods, sees accompanying drawing 1（b）It is shown.For
The size and form of lncRNA-HN1-RNs nano particles are detected, after lncRNA-HN1-RNs is fixed with 2.5% glutaraldehyde, is put
The observation analysis under Electronic Speculum, see accompanying drawing 1（a）It is shown.As a result show, nanoparticle particle diameter prepared by the present invention is all between 20-
Between 30nm, particle is in spherical, and single dispersing is uniformly distributed.
Embodiment 2：HN-lnc-RNA nanoparticles vitro stability detects
After long-chain non-coding RNA HN1 is prepared as into the preparation of lncRNA-HN1-RNs nanoparticles, in 25 DEG C of bars of room temperature
Placed 7 days under part, while be control sample with lncRNA-HN1, with denaturing formaldehyde PAGE detected through gel electrophoresis RNA integrality,
Evaluating RNA nanoparticles at ambient temperature can the holding time.As a result as shown in accompanying drawing 2, the results showed that, it is untreated
LncRNA-HN1 is degradable, but lncRNA-HN1-RNs nano particles are placed rear electrophoresis detection in 7 days and still showed well at room temperature
Integrality.
Embodiment 3：HN-lnc-RNA nanoparticles transfection efficiency determines
LncRNA-HN1 3 ' ends are marked with fluorescence probe Cy3, and nanoparticle is prepared as according to embodiment 1, will be made
Standby lncRNA-HN1-RNs-Cy3 nanoparticles, according to the final concentration and human melanoma cell that contained RNA is 100ng/ml
YU-SIT1 is incubated altogether, does not add the 37 DEG C of cultures in 5% CO2 cell culture incubator of transfection reagent lipofectamine 2000
24h, cell is evaluated into lncRNA-HN1-RNs nanometers as green cells ratio is detected under fluorescence microscope with this afterwards
The efficiency of particulate transfectional cell.As a result as shown in accompanying drawing 3, the results showed that, the transfection of lncRNA-HN1-RNs-Cy3 nano particles
Afterwards, fluorescence is presented in most cells, illustrates that long-chain RNA nanoparticles need not can enter melanoma by means of transfection reagent
Cell.
Embodiment 4：LncRNA-HN1-RNs nanoparticles in vivo functionality detects
（1）Using lncRNA-HN1 as control, lncRNA-HN1-RNs nanoparticle direct transfection NIH3T3 cells are determined
Ability, detection cell clonal formation method measure is cloned using agar.Specific implementation is as follows：
The preparation of cell：LncRNA-HN1-RNs under conditions of transfection reagent is not added with, is transfected respectively with its nanoparticle
NIH3T3 cells, 24h after transfection, cell are used for soft agar and form colony growth experiment.The preparation of 0.6% bottom agar：Take 8 ml
10% (v/v) NBCS and DMEM high glucose mediums have been added in 50mL centrifuge tubes, has been placed in 42 DEG C of water-bath preheatings；Add
Enter 3% agarose of 2 mL, 60 DEG C of preheatings, it is rapid to mix；The laying of 0.6% bottom agar：By amount of 6 orifice plates per hole 1mL
Lay bottom agar.After room temperature solidification, that is, form 0.6% bottom agar in semi-solid；The preparation of 0.3% top-layer agar：6
Orifice plate needs the top-layer agars of 1mL 0.3% per hole, and in order to reduce error, every kind of cell line needs 3 multiple holes, therefore for every kind of
Cell line, prepare the top-layer agars of 4ml 0.3%.3.6 mL are taken to add 10% (v/v) NBCS and the high sugar cultures of DMEM
Based on 15mL centrifuge tubes, 42 DEG C of water-bath preheatings are placed in；3% agarose of 60 DEG C of preheatings of 0.4ml is added, rapid to mix, weight
It is new to be placed in 42 DEG C of water-bath insulations (be solidified in 0.3% top-layer agar short time at this temperature)；Digested with trypsin solution,
Cell is collected, 1,000g 5 min of centrifugation, supernatant is removed, cell suspension is configured to proper volume (small size is preferably) culture medium,
And counted with blood counting chamber；By 6 orifice plates two cell quantity gradients are done per 1000,5000, hole cell：Take respectively containing 4,
000th, 20, the cell suspension of 000 cell is rapid to mix in the top-layer agars of 4ml 0.3% of 42 DEG C of insulations, and 1 is added per hole
Ml (has laid 0.6% bottom agar).It is in semi-solid after room temperature solidification.
Cell is placed in 37 DEG C, 5% CO2Incubator, cultivate 7 days, and timing supplementing culture medium in the training period；After 7 days, fall
Micro- sem observation is put, and counts number of cell clones, 1ml crystal violet dye liquors, room temperature 15 minutes are added per hole；Abandon dye liquor, clear water drift
Wash and dry counting clone.Transmission scan instrument scan image.As a result show, the culture plate for transfecting lncRNA-HN1 does not clone shape
Into, but the culture plate for transfecting lncRNA-HN1-RNs every square centimeter has 50 clones.Illustrate that long-chain RNA is being prepared as nanometer
Cells play effect need not can be directly entered after grain by means of transfection reagent.
（2）Nude mice is inoculated with the NIH-3T3 cells of lncRNA-HN1-RNs nanoparticles transfection, detects lncRNA-HN1-
RNs nanoparticles influence on the one-tenth knurl ability of NIH-3T3 cells.Based on external work inside this evaluation HN-lnc-RNA nanoparticle
Transfection efficiency in energy and its organism.Specific embodiment is as follows：
Human desmocyte NIH3T3 cells are cultivated 24 hours in 37 DEG C, are transfected respectively in the case where being not added with transfection reagent
LncRNA-NH1 and lncRNA-HN1-RNs, transfect lncRNA-HN1-RNAd cell（1 x 106）It is thin with normal NIH-3T3
Born of the same parents and the NIH-3T3 cells for transfecting lncRNA-HN1, the nude mice armpit of 6 week old is seeded in by hypodermic mode respectively
Under, every kind of cell is inoculated with 6 nude mices, and normal diet and rearing conditions are given after inoculation, and mouse was put to death after 6 weeks, calculates nude mice
Form the ratio of tumour.The external tumorigenesis ability of lncRNA-HN1-RNs nanoparticles is evaluated with this.It the results are shown in Table 1,
As shown in Table 1, the average knurl weight of tumour for transfecting lncRNA-HN1 is 0.2 g, but transfects lncRNA-HN1-RNs and put down
Equal knurl weight is 2.3 g.After illustrating that lncRNA is prepared as nanoparticle, tumorigenesis ability significantly improves, and can play in vivo
Function.
Claims (3)
- A kind of 1. long-chain non-coding RNA nanoparticle, it is characterised in that：Wrapped up including nano-gene carrier and by genophore Or the HN-lncRNA of the external synthesis of absorption；It is located at HN gene non-coding head of district 236nt to the NH-lncRNA, and its long-chain is non- The oligonucleotide sequence of coding RNA is：35565dest_path_image001The nano-carrier is nano particle prepared by vegetable oil, and the vegetable oil includes soybean oil, olive oil or other edible With anharmonic oil.
- 2. the preparation method of long-chain non-coding RNA nanoparticle as claimed in claim 1, it is characterised in that step is as follows：Will HN-lncRNA, which is dissolved in the aqua sterilisa of no RNase, is prepared as the RNA aqueous solution, and concentration is 200ng ~ 500ng/ml；In HN- On the lncRNA aqueous solution, vegetable oil is added, the volume ratio of vegetable oil and the HN-lncRNA aqueous solution is 3：2, by above-mentioned vegetable oil with HN-lncRNA is dissolved in the distilled water of 10 times of volumes, ultrasonic probe is placed in into water oil level boundary, reaction temperature is set as 4 DEG C, ultrasound intensity is 150 W/cm2, ultrasonic time is 3 minutes；Then removed using 20 kDa Ultra filtration membrane and be not bound with HN-lncRNA, produce.
- 3. application of the long-chain non-coding RNA nanoparticle as claimed in claim 1 in antineoplastic is prepared.
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| CN112587663B (en) * | 2020-12-29 | 2022-01-07 | 浙江大学 | Application of long-chain non-coding RNA-lncIVRL in prevention and treatment of influenza A virus infection |
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| CN101633923A (en) * | 2009-07-10 | 2010-01-27 | 四川大学 | Long non-coding RNA sequence relevant to human melanoma cells and application thereof |
| US20100233706A1 (en) * | 2009-03-11 | 2010-09-16 | The Rockefeller University | Methods to fix and detect nucleic acids |
| CN103501821A (en) * | 2011-03-08 | 2014-01-08 | 艾克塞斯制药公司 | Targeted nanocarrier systems for delivery of actives across biological membranes |
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Patent Citations (3)
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|---|---|---|---|---|
| US20100233706A1 (en) * | 2009-03-11 | 2010-09-16 | The Rockefeller University | Methods to fix and detect nucleic acids |
| CN101633923A (en) * | 2009-07-10 | 2010-01-27 | 四川大学 | Long non-coding RNA sequence relevant to human melanoma cells and application thereof |
| CN103501821A (en) * | 2011-03-08 | 2014-01-08 | 艾克塞斯制药公司 | Targeted nanocarrier systems for delivery of actives across biological membranes |
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| Physicochemical Investigations on Solid Lipid Nanoparticles and on Oil-Loaded Solid Lipid Nanoparticles: A Nuclear Magnetic Resonance and Electron Spin Resonance Study;Katja Jores等;《Pharmaceutical Research》;20030830;第20卷(第8期);1274-1283 * |
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