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Antisense oligonucleotide of targeted circular RNA circRHOBTB3 and application of antisense oligonucleotide in colorectal cancer treatment

An antisense oligonucleotide and targeting technology, which is applied in the design and application of ASO drugs, to achieve the effect of reducing the ability of metastasis in vivo, the ability of proliferation and migration and invasion in vitro

Pending Publication Date: 2022-01-28
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no research on antisense oligonucleotide (ASO) products targeting circRHOBTB3 and its application in inhibiting the occurrence and development of colorectal cancer

Method used

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  • Antisense oligonucleotide of targeted circular RNA circRHOBTB3 and application of antisense oligonucleotide in colorectal cancer treatment
  • Antisense oligonucleotide of targeted circular RNA circRHOBTB3 and application of antisense oligonucleotide in colorectal cancer treatment
  • Antisense oligonucleotide of targeted circular RNA circRHOBTB3 and application of antisense oligonucleotide in colorectal cancer treatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1A

[0025] Example 1 Inhibition of the Proliferative Ability of Colorectal Cancer Cells by ASO-cir and ASO-exo

[0026] 1. Materials

[0027] Colorectal cancer cell lines SW480, HT116, and RKO were provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. RPMI1640, DMEM, 0.05% Trypsin, and fetal bovine serum were purchased from Gibco; CCK8 reagent was purchased from Boster; NEOfect was purchased from Beijing Mayin Technology Co., Ltd. Other medicines were domestic analytically pure.

[0028] 2. Method

[0029] 2.1 Taking SW480 and ASO-cir as examples, count 5*10^5 respectively and spread them on two wells of a six-well plate.

[0030] 2.2 On the second day, configure the transfection system as follows: 100 microliters of DMEM, 2 microliters of NEOfect transfection reagent, and 2 microliters of 20 μMASO-cir.

[0031] 2.3 One day later, lay a 96-well plate for the CCK8 proliferation experiment.

[0032] 1) Digest the cells of the SW480-control gro...

Embodiment 2

[0039] Example 2 Inhibition of ASO-cir and ASO-exo on migration and invasion of colorectal cancer cells

[0040] 1. Materials

[0041] Colorectal cancer cell lines SW480, HT116, and RKO were provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. RPMI1640, DMEM, 0.05% Trypsin, and fetal bovine serum were purchased from Gibco Company; CCK8 reagent was purchased from Boster Company; Transwell double-layer plate was purchased from COSTAR Company; Matrigel glue was purchased from BD Company; crystal violet was purchased from Beyond Company. Other medicines were domestic analytically pure.

[0042] 2. Method

[0043] 2.1 Taking SW480 and ASO-cir as examples, count 5*10^5 respectively and spread them on two wells of a six-well plate.

[0044] 2.2 On the second day, configure the transfection system as follows: 100 microliters of DMEM, 2 microliters of NEOfect transfection reagent, and 2 microliters of 20 μMASO-cir.

[0045] 2.3 One day later, spr...

Embodiment 3

[0051] Example 3 Inhibition of ASO-cir and ASO-exo on the Metastatic Ability of Colorectal Cancer Cell Line Spleen Orthotopic Liver Metastasis Model

[0052] 1. Materials

[0053] The colorectal cancer cell line HCT116 was provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. 6-week-old male Nude mice were purchased from Shanghai Slack. Other medicines were domestic analytically pure.

[0054] 2. Method

[0055] 2.1 Suspend HCT116 cells, count 7*10^6 cells per milliliter, in PBS.

[0056] 2.2 Anesthetize the mouse, expose the spleen, inject 100 microliters of HCT116 cells into the mouse spleen through an insulin needle, and suture the skin and muscle layer.

[0057] 1) About 30 days later, inject 20nmol ASO-control or a mixture of 10nmol ASO-cir and 10nmol ASO-exo through the tail vein, once every 3 days, a total of 6 injections.

[0058] 2) About 60 days later, the mice were euthanized, and the liver and spleen were dissected to obtain ...

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Abstract

The circRHOBTB3 targeting ASO drug is designed and synthesized, and the level of circRHOBTB3 in cells can be effectively improved; after the ASO is used for treating colorectal cancer cells and an animal tumor metastasis model, proliferation, migration and invasion of the colorectal cancer cells in vitro and metastasis of the colorectal cancer cells in vivo are effectively inhibited, and a way is provided for treatment of malignant tumors.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to the design and application of ASO drugs. Specifically, the present invention designed and synthesized two antisense oligonucleotides ASO for the circularization and secretion regulatory element sequences of human circRHOBTB3, which can improve its intracellular expression by promoting circRHOBTB3 and inhibiting the exosome sorting of circRHOBTB3, The ASO can inhibit the growth and metastasis of colorectal cancer cells in vitro and in vivo. Background technique [0002] Circular RNA (circRNA) is one of the important members of non-coding RNA; different from linear long-chain non-coding RNA (lncRNA), circRNA is mainly produced by the precursor RNA transcribed by RNA polymerase II through reverse splicing and does not have 5 The 'end cap and 3' end poly(A) tail structure, but covalently bonded to form a circular RNA molecule with a closed loop structure. . circRNA is involved in the...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K31/7088A61P35/00A61P35/04A61P1/00
CPCC12N15/1135A61K31/7088A61P35/00A61P35/04A61P1/00C12N2310/11C12N2310/315C12N2310/321C12N2320/32
Inventor 张红河陈超一来茂德
Owner ZHEJIANG UNIV
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