A dsRNA targeting enhancer II in the promoter II region of gdnf gene and its application

A promoter and gene technology, applied in dsRNA and its application fields, can solve problems such as ambiguity

Active Publication Date: 2020-10-23
XUZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, how to implement the above strategies is still unclear

Method used

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  • A dsRNA targeting enhancer II in the promoter II region of gdnf gene and its application
  • A dsRNA targeting enhancer II in the promoter II region of gdnf gene and its application
  • A dsRNA targeting enhancer II in the promoter II region of gdnf gene and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] 1.1 Cell culture

[0027] The human U251 astroblastoma cell line was purchased from ATCC cell bank and was identified based on its cell morphology and short tandem repeat sequence. Human primary astrocytes (human astrocytes, HA) were purchased from ScienCell. U251 and HA cells were cultured in modified Eagle's medium containing 10% fetal bovine serum (Gibco, USA) and astrocyte medium containing 2% fetal bovine serum and astrocyte growth supplement factor ( AM, ScienCell), add penicillin 100U / ml, streptomycin 100U / ml, place at 37℃, 5% CO 2 Cultivate in a saturated humidity incubator.

[0028] 1.2 Synthesis and transfection of dsRNA targeting different cis-acting elements in the II region of the GDNF gene promoter

[0029] Design and chemically synthesize 6 21-nucleotide dsRNAs targeting enhancer II or silencer II in the promoter II region of GDNF gene (GenBank accession number: AF053749) and control NC-dsRNA (Table 1). BLAST analysis ruled out the possibility that these dsRN...

Embodiment 2

[0048] 1.1 Cell viability detection

[0049] Divide U251 cells and HA cells into 2×10 3 The cell concentration per ml was seeded in a 96-well plate with 100 μl per well. When the cells grow to 50% confluency, they are infected with Lenti-S, Lenti-E and Lenti-NC lentivirus. After 72 hours of infection, the cells are processed according to the instructions of the CCK8 kit (Dojindo Molecular Technologies) and read by the Biotek microplate reader Absorbance at 450nm.

[0050] 1.2 Cell cycle detection

[0051] Divide U251 cells and HA cells into (5-8)×10 5 The density is inoculated at 25cm 2 Culture flask. After 72 hours of infection with Lenti-S, Lenti-E and Lenti-NC lentivirus, the cells were digested with 0.25% trypsin, washed twice with PBS, fixed in pre-chilled 70% (v / v) ethanol overnight, and in Stain with 100μg / ml propidium iodide (PI) for 1h at 4℃. Flow cytometry (FACScan) was used to detect the DNA content in the cells.

[0052] 2. Results

[0053] In order to clarify the effec...

Embodiment 3

[0055] 1.1 Scratch test

[0056] U251 cells and HA cells in the logarithmic growth phase were infected with lentivirus targeting the GDNF gene promoter II region. After 72 hours, they were seeded in a 12-well plate and continued to be cultured until the confluence was greater than 95%. Use a 10 μl sterile pipette tip to horizontally streak the cells in each well. After rinsing with PBS, they were replaced with fresh serum-free DMEM medium to continue the culture. Micrographs were taken at 0, 24, and 48h after the scratch, and the width of the scratch was measured with Image-Pro Plus 6.0 software.

[0057] 1.2 Transwell detection

[0058] U251 cells and HA cells in the logarithmic growth phase were infected with lentivirus targeting the GDNF gene promoter II region. After 72 hours, they were inoculated in the upper chamber of transwell containing fresh serum-free DMEM medium, and the lower chamber was added with 10% FBS in DMEM medium, after 24h incubation, take out the transwell ch...

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Abstract

The invention discloses dsRNA targeting enhancer II in the promoter II region of GDNF gene and application thereof. A dsRNA targeting enhancer II in the promoter II region of GDNF gene, the forward sequence is shown in SEQ ID NO.1, and the reverse sequence is shown in SEQ ID NO.2. The dsRNA of the present invention can be used in the preparation of drugs for inhibiting astroglioblastoma and the preparation of drugs for inhibiting the migration and invasion of astroglioblastoma. The dsRNA can specifically significantly reduce the transcription level of the GDNF gene in human U251 astroglioblastoma cells, and the lenti-E packaged according to its sequence can also significantly reduce the cell proliferation, migration and invasion capabilities of U251 cells, However, it has no effect on HA of human normal primary astrocytes, indicating that the dsRNA has a certain clinical application value in the treatment of glioblastoma.

Description

Technical field [0001] The invention belongs to the field of biomedicine, and relates to a dsRNA targeting enhancer II in the promoter II region of GDNF gene and its application. Background technique [0002] Glioblastoma (GBM) is the most common primary tumor of the central nervous system[1,2]. It is mostly invasive growth, easy to relapse and difficult to cure. The median survival time of the patient is only 14-16 months [ 3,4]. At present, the pathogenesis of GBM is not fully understood. Recent studies have found that the occurrence and development of GBM is related to the abnormal expression of many cytokines. Among them, glial cell-line derived neurotrophic factor (GDNF) is considered to be related to GBM, especially astrocytes. One of the most closely related cytokines of GBM derived from cells (Astrocyte, AST) [5-7]. In adult brain tissue, GDNF is mainly secreted by astrocytes [8,9], and is widely and lowly expressed in different brain regions [10,11]. GDNF secreted by...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/867A61K31/713A61P35/00A61P35/04
CPCA61P35/00A61P35/04C12N15/111C12N15/1136C12N15/86C12N2310/14C12N2320/32C12N2740/15043C12N2800/107C12N2310/531
Inventor 张宝乐高殿帅韩笑刘捷高擎
Owner XUZHOU MEDICAL UNIV
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