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Inhibitor for knocking down circXPO1 and application of inhibitor in preparation of glioma treatment medicine

A technology for glioma and glioma cells, which can be used in anti-tumor drugs, drug combinations, medical preparations containing active ingredients, etc.

Active Publication Date: 2022-07-22
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Current studies have reported that the expression of circXPO1 is related to the occurrence of osteosarcoma, lung adenocarcinoma, and prostate cancer, but there is no report on its effect on malignant glioma

Method used

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  • Inhibitor for knocking down circXPO1 and application of inhibitor in preparation of glioma treatment medicine
  • Inhibitor for knocking down circXPO1 and application of inhibitor in preparation of glioma treatment medicine
  • Inhibitor for knocking down circXPO1 and application of inhibitor in preparation of glioma treatment medicine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Sanger sequencing and RNase R treatment confirmed the formation of circular RNA

[0026] 1. Experimental method

[0027] 1. RT-PCR to obtain circXPO1

[0028] Total RNA of GBM cell lines (ie U87-MG, U251 cells) was extracted and reverse transcribed using Trizol reagent (ThermoFisher Scientific) for RT-PCR analysis. Primers are shown in (Table 1).

[0029] RT-PCR system:

[0030] In order to prove that circXPO1 forms circular RNA rather than linear, the RT-PCR product was recovered and sent to the company for sanger sequencing, and the sequencing results were compared with DNASTAR software. The results show that circXPO1 is indeed formed by the head-to-tail circularization of exons 2, 3, and 4 of the parent gene XPO1 ( figure 1 A, B, C).

[0031] 2. To confirm the circular structure of circXPO1, its stability was assessed by treatment with exonuclease (RNase R). The results showed that circXPO1 was resistant to RNase R ( figure 1 D), while XPO1 was di...

Embodiment 2

[0034] Example 2: Expression of circXPO1 in normal and glioma tissues and GBM cell lines

[0035] 1. Experimental method:

[0036] 1. Fluorescence quantitative PCR to detect the expression of circXPO1 in normal and glioma tissues and GBM cell lines

[0037]Total RNA was extracted from glioma tissue and GBM cell lines (i.e. U87-MG, U251 cells), and Trizol reagent (Thermo Fisher Scientific) was used for quantitative PCR (qRT-PCR) analysis. The amplification reaction was completed by the cfx96 real-time PCR detection system (Bio-Rad Company, Hercules, CA, USA) using SYBR Green fluorescence quantitative PCR kit according to the operating specifications. For circXPO1 and XPO1 primers (Table 1), use The expression of related genes was calculated by the method. All qRT-PCR experiments were repeated in triplicate and all data used GAPDH as a control.

[0038] qRT-PCR system:

[0039] 2. Experimental results

[0040] The expression of circXPO1 in normal brain tissue, low-grade...

Embodiment 3

[0041] Example 3: Inhibition of circXPO1 expression using shRNA in U87-MG and U251 cells

[0042] 1. Experimental method

[0043] 1. Construction of scramble, circXPO1-sh plasmid (GFP co-expression): the most commonly used interference plasmid construction method is used, through endonuclease (Biolabs), enzyme digestion shRNA sequence and P GreenPuro vector (Sangon), using T4 Ligase (Biyuntian) ligates to form a plasmid.

[0044] The method for obtaining the virus liquid: (1) Take two 1.5mL centrifuge tubes, add 30 μL DMEM and 30 μL Fectin (ThermoFisher). (2) Take another 2 1.5mL centrifuge tubes, add 60μg DMEM, 0.75μg pspax2 packaging plasmid, 0.25μg pmd2.G packaging plasmid, add 1μg scramble, circXPO1-sh interference plasmid respectively, and then mix with the solution in step (1) centrifuge tube, After standing for 20 minutes, the cells were added to the 293T cell liquid cultured in DMEM medium containing 10% fetal bovine serum, respectively. After 24 and 48 hours, the su...

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Abstract

The invention discloses an inhibitor for knocking down circXPO1 and application of the inhibitor in preparation of a medicine for treating glioma. At present, the research reports that the expression of circXPO1 is related to osteosarcoma, lung adenocarcinoma and prostate cancer, and the related effect of circXPO1 on malignant glioma is not reported. According to the invention, a pair of shRNAs is designed according to the splicing site of circXPO1, and the shRNAs can significantly inhibit the expression level of circXPO1 and can inhibit the activity, proliferation and migration of glioma.

Description

technical field [0001] The invention relates to the technical field of molecular diagnosis, in particular to an inhibitor for knocking down circXPO1 and its application in the preparation of a drug for treating glioma. Background technique [0002] Human glioblastoma (Glioma) is the most common primary malignant tumor in the central nervous system, accounting for 80% of all malignant primary brain and central nervous system tumors. According to histological morphology and molecular features, the World Health Organization (WHO) reclassified glioblastomas into types I-IV in 2016, among which types I and II are often referred to as low-grade glioblastomas. glioma, LGG), types III and IV include glioblastoma (GBM), anaplastic astrocytoma, and anaplastic oligodendroglioma. Type III and IV gliomas are often regarded as research object. The incidence of low-grade gliomas is relatively low, accounting for only 15% of the total number of gliomas. Clinical treatment methods such as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/713A61P35/00A61P35/04A23L33/13
CPCA61K31/713A61P35/00A61P35/04A23L33/13A23V2002/00A23V2200/308
Inventor 谭舟王学辉邱猛生
Owner HANGZHOU NORMAL UNIVERSITY
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