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Preparation and application of a long-chain non-coding RNA nanosphere

A long-chain non-coding, nano-microsphere technology, which is applied in the direction of non-effective components of oil/fat/wax, gene therapy, genetic material components, etc., can solve the lack of effective means for long-chain non-coding RNA molecules, and long-chain RNA is easy to degrade , Difficulty entering cells and other issues, achieving high transfection efficiency, enhanced stability, and solving preservation problems

Inactive Publication Date: 2018-03-23
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, long-chain RNA has problems such as easy degradation and difficulty entering cells. Compared with short-chain RNA, it is more difficult to manipulate. At present, there is still a lack of effective means to study long-chain non-coding RNA molecules.

Method used

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  • Preparation and application of a long-chain non-coding RNA nanosphere
  • Preparation and application of a long-chain non-coding RNA nanosphere
  • Preparation and application of a long-chain non-coding RNA nanosphere

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Preparation scheme of HN-lnc-RNA nanoparticles

[0024] Dissolve lncRNAHN1 in RNase-free sterilized water to prepare an RNA aqueous solution with a concentration of 200ng~500ng / ml; add vegetable oil to the lncRNA HN1 aqueous solution, the volume ratio of vegetable oil to lncRNA HN1 aqueous solution is 3:2, mix the above vegetable oil with lncRNA Dissolve HN1 in 10 times the volume of distilled water, place the ultrasonic probe at the boundary between water and oil, set the reaction temperature at 4°C, and the ultrasonic intensity at 150 W / cm 2 , and the ultrasonic time was 3 minutes; then, a 20 kDa ultrafiltration membrane was used to separate and remove unbound lncRNA. Its size and particle size distribution were analyzed by DLS and SME methods, see attached figure 1 (b) shown. In order to detect the size and shape of lncRNA-HN1-RNs nanoparticles, lncRNA-HN1-RNs were fixed with 2.5% glutaraldehyde and observed and analyzed under electron microscope, see at...

Embodiment 2

[0025] Example 2: In vitro stability testing of HN-lnc-RNA nanospheres

[0026] The long-chain non-coding RNA HN1 was prepared as lncRNA-HN1-RNs nanospheres, and then placed at room temperature at 25 °C for 7 days. At the same time, lncRNA-HN1 was used as a control sample, and the integrity of the RNA was detected by formaldehyde-denaturing PAGE gel electrophoresis. To evaluate the storage time of RNA nanospheres at room temperature. see attached results figure 2 As shown, the results showed that the untreated lncRNA-HN1 was completely degraded, but the lncRNA-HN1-RNs nanoparticles still showed good integrity after being placed at room temperature for 7 days by electrophoresis detection.

Embodiment 3

[0027] Example 3: Determination of transfection efficiency of HN-lnc-RNA nanospheres

[0028] The 3' end of lncRNA-HN1 was labeled with fluorescent probe Cy3, and prepared as nanospheres according to Example 1, and the prepared lncRNA-HN1-RNs-Cy3 nanospheres were prepared according to the final concentration of 100ng / ml RNA contained. Concentrations were co-incubated with human melanoma cells YU-SIT1 without adding the transfection reagent lipofectamine 2000 and cultured in a 5% CO2 incubator at 37°C for 24 hours, and then the cells were placed under a fluorescence microscope to detect the ratio of green fluorescent cells to evaluate lncRNA - Efficiency of transfecting cells with HN1-RNs nanoparticles. see attached results image 3 As shown, the results showed that after transfection of lncRNA-HN1-RNs-Cy3 nanoparticles, most cells showed fluorescence, indicating that long-chain RNA nanospheres could enter melanoma cells without the aid of transfection reagents.

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Abstract

The invention relates to preparation and application of a long non-coding RNA (ribonucleic acid) nanometer microsphere. The long non-coding RNA nanometer microsphere is characterized by comprising a nanometer gene vector and an HN (haemagglutinin neuraminidase)-lncRNA (long chain non-coding ribonucleic acid) which is wrapped or absorbed by the gene vector, wherein the HN-lncRNA gene is located at HN gene non-coding area length 236nt. A macro molecule RNA is directly prepared into a stable nanometer microsphere; after a lncRNA molecule is prepared into the nanometer microsphere, the stability is enhanced; after being preserved at 25 DEG C for a week, the nanometer microsphere is not degraded; the particle can enter a cell without assistance of transfection reagent. The transfection efficiency of the nanometer microsphere is high and the nanometer microsphere can also exert effects in a human boy. The problem on storage of the RNA is solved, and a new way for studying biological functions of the RNA is also developed. The microsphere can be directly transfected into the cell, so that the cloning formation ability of the cell is improved. In-vitro study shows that the tumorigenic ability of a mouse can be obviously increased by the long non-coding RNA nanometer microsphere.

Description

technical field [0001] The invention belongs to the field of tumor molecular biology, and in particular relates to the preparation of a long-chain non-coding RNA nanometer microsphere and its application in tumor research. Background technique [0002] Long non-coding RNA (long non-coding RNA, lncRNA) is a type of RNA whose transcript length exceeds 200nt and does not encode proteins. Long non-coding RNA itself does not encode protein, but regulates the expression level of genes in the form of RNA at various levels such as epigenetic regulation, transcriptional regulation, and post-transcriptional regulation. "Noise", which is a by-product of RNA polymerase transcription, does not have any biological function. Later studies found that the expression of some lncRNAs has tissue-specific and spatio-temporal specificity, and many lncRNAs have conserved secondary structures, suggesting that lncRNAs have important biological functions. Further studies have found that many lncRNA...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K31/7088A61K47/44A61P35/00
Inventor 林秀坤吴宁许焕丽刘晓卉
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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