Preparation method of micro RNA nano microsphere and application of micro RNA nano microsphere in anti-tumor aspects

A nano-microsphere and tiny technology, applied in the field of tumor molecular biology, can solve the problems of difficult to confirm the effect, poor stability, difficult to apply to tumor patients, etc., to achieve the effect of inhibiting metastasis, solving preservation problems, and enhancing stability.

Inactive Publication Date: 2015-03-25
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are some problems in the application of miRNA mimics and AMOs, including 1) poor stability, miRNA mimics or miRNA-AMOs cannot be stored for a long time, and exogenous small molecule RNA is quickly degraded in serum, although chemically modified Stability can be increased, but is also quickly cleared from the body
2) Cannot directly enter cells to exert biological functions
miR-125b is a microRNA with carcinogenic effect. Previous studies have confirmed that miR-125b antisense molecule (miR-125b antisense) has a good effect on inhibiting tumor growth. However, miRNA research still has difficulties in transfection, poor stability, and biological Difficult to prove the role in the body and other problems, difficult to apply in tumor patients and other problems

Method used

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  • Preparation method of micro RNA nano microsphere and application of micro RNA nano microsphere in anti-tumor aspects
  • Preparation method of micro RNA nano microsphere and application of micro RNA nano microsphere in anti-tumor aspects
  • Preparation method of micro RNA nano microsphere and application of micro RNA nano microsphere in anti-tumor aspects

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1. Preparation scheme of tiny nano-microspheres

[0025] Dissolve miR-125b-AS (synthetic miR-125b-AS sample purchased from Shanghai Gemma Biopharmaceutical Company) in 10 times the volume of distilled water to prepare an RNA aqueous solution, and add the RNA aqueous solution to the protective oil layer (soybean oil at a volume ratio of 3:2 ), place the ultrasonic probe at the water-oil interface (≈150 W / cm 2 ), the reaction temperature was set at 20 °C, and the ultrasonic time was 3 Min. A 20kDa ultrafiltration membrane is used to separate the nano-microspheres. After the nano-microspheres are separated, their size and particle size distribution are analyzed by DLS and SME methods. The 125b-AS-RNs nanoparticles were fixed with glutaraldehyde and analyzed by scanning electron microscopy. The results of the implementation are shown in the appendix figure 1 Shown, scale bar: 500nm. The results show that the method can be used to prepare nano-RNA microspheres w...

Embodiment 2

[0026] Example 2. In vitro stability detection of microRNA nanospheres

[0027] In order to test the in vitro stability of microRNA nanospheres, the purchased miR-125b-AS samples synthesized by Shanghai Gemma Biopharmaceutical Company and the nanosphere mi125b-AS-RNs samples prepared by the present invention were respectively heated at 25°C. The UV absorbance of RNA was monitored at 260 nanometers with a UV spectrometer for 0-7 days. The results showed that after miR-125b-AS was placed for 7 days, the absorbance decreased significantly, but the absorbance of miR-125b-AS decreased significantly. There was no change, indicating that the stability of microRNA was significantly increased after being prepared as nanospheres.

[0028] Further, we used formaldehyde-denatured PAGE gel electrophoresis to detect the integrity of RNA to evaluate the storage time of microRNA nanospheres at room temperature. The implementation results are attached figure 2 , the results show that micr...

Embodiment 3

[0029] Example 3. Effect of miR-125b antisense nucleotide nanoparticles on growth and apoptosis of malignant glioma cells

[0030] cells by 10 3Each well was inoculated in a 96-well plate, cultured for 24 hours, and 87 cells were transfected with negative control miRNA (NC-AS) and miR-125b antisense strand (miR-125b-AS, marked as 125b-AS in the figure) , the transfection reagent lipofectamine 2000 from Invitrogen Company was used; the transfection reagent lipofectamine 2000 from Invitrogen Company was used; the nanospheres of miR-125b-AS (125b-AS-RNs) were directly transfected without the transfection reagent lipofectamin2000, and finally The concentration is 100nM / L. After 72 hours of culture, the cells were collected and 24 hours after transfection, the cells were collected to detect cell apoptosis by TUNEL assay and flow cytometry AV / FITC double staining. The results showed that both miR-125b-AS and its nanospheres could induce glial Tumor cell apoptosis, indicating tha...

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Abstract

The invention relates to a preparation method of a micro RNA nano microsphere and an application of the micro RNA nano microsphere in anti-tumor aspects. The nano microsphere comprises a nano-carrier and an antisense strand miR-125b-AS of micro RNA miR-125b which is wrapped or adsorbed by the carrier and is synthesized in vitro. According to the preparation method, miR-125b-AS is prepared into the nano microsphere, the stability of the nano microsphere can be significantly increased, and the nano microsphere cannot be degraded after being stored for a week at 25 DEG C; and detected by an MTT process, the nano microsphere can be transfected into a human neuroglioma cell U87 cell without using a transfection reagent, and the proliferation of tumor cells can be significantly inhibited. Invasion and migration experiments show that the transfer ability of the tumor cells can be significantly inhibited by the nano microsphere; influences of the nano microsphere to tumor cell cycles and cell apoptosis can be detected by using flow cytometry and a TUNEL process; and meanwhile, RT-PCR and Western-blot detection results show that changes of corresponding target genes and protein expression can be caused by the nano microsphere.

Description

technical field [0001] The invention belongs to the field of tumor molecular biology, and in particular relates to the preparation of a microRNA nano-microsphere and its application in anti-tumor. Background technique [0002] Non-coding small RNA (microRNA, miRNA) participates in the regulation of biological programs such as tumor cell proliferation, differentiation, apoptosis, migration, angiogenesis, and protein cleavage by regulating the expression of oncogenes or tumor suppressor genes. In addition to regulating gene expression, miRNAs themselves can also act as oncogenes or tumor suppressor genes and become targets for tumor therapy. By introducing tumor suppressor miRNAs, the tumor can be controlled or induced to develop in a normal direction, or even reversed to normal; at the same time, the introduction of miRNA antisense oligonucleotides complementary to miRNAs with oncogene characteristics can reduce the expression of miRNAs in tumors, which can inhibit The expre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K9/14A61P35/00
Inventor 林秀坤吴宁许焕丽刘晓卉
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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