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CircRNA related to MTX resistance of tumor cells and application of circRNA to reversal of MTX resistance of tumor cells

A tumor cell, drug resistance technology, applied in the field of tumor biodiagnosis and treatment, to reduce DMs, improve efficiency, and reverse tumor drug resistance

Active Publication Date: 2019-04-09
HARBIN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is not yet known whether circular RNAs will affect changes in the form of gene amplification such as double minutes

Method used

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  • CircRNA related to MTX resistance of tumor cells and application of circRNA to reversal of MTX resistance of tumor cells
  • CircRNA related to MTX resistance of tumor cells and application of circRNA to reversal of MTX resistance of tumor cells
  • CircRNA related to MTX resistance of tumor cells and application of circRNA to reversal of MTX resistance of tumor cells

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Experimental program
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Effect test

Embodiment 1c

[0041] Example 1 Differential expression of circRNA7746 in MTX-sensitive and drug-resistant cells

[0042] 1. Screening differentially expressed circular RNAs in HT-29 MTX sensitive cells and HT-29 MTX resistant cells containing DMs

[0043] (1) Cell culture

[0044] The HT-29 MTX sensitive cell line was cultivated with DMEM high glucose medium containing 15% fetal bovine serum, and the final concentration of MTX was 10 -4 mol / L medium to culture HT-29 MTX drug-resistant cell line containing DMs. All cells were cultured in 5% CO 2 , 37 ℃ cell culture incubator.

[0045] Entrusted Beijing Nuohezhiyuan Company to conduct HT-29 MTX sensitive cells and HT-29 10 -4 mol / L MTX drug-resistant cell lines (each cell provided three independent biological replicates) were independently constructed for circular RNA, followed by next-generation sequencing. Identification of circular RNAs using the find_circ bioinformatics method. The relative expression of circular RNA in each sample ...

Embodiment 2

[0115] Example 2 Effect of interfering with circRNA7746 on cell drug resistance

[0116] 1. Establishment and verification of DMs-containing HT-29MTX drug-resistant cell interference circRNA7746 clone

[0117] (1) HT-29MTX drug-resistant cells containing DMs interfered with the establishment of circRNA7746 clones.

[0118] Firstly, the optimal purine concentration was screened in HT-29 MTX drug-resistant cells containing DMs. The HT-29 MTX drug-resistant cells containing DMs in the logarithmic growth phase were digested with trypsin for 5 minutes, and then resuspended with medium to form a single cell suspension. After cell counting, 10 5 Cells were inoculated into a 24-well plate at a density of 24 cells, shaken evenly in a cross method, and placed in an incubator for cultivation to adhere to the wall. After culturing for 24 hours, replace the fresh medium, and add different amounts of puromycin to each well in turn, so that the final concentrations of puromycin in the medi...

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Abstract

The invention discloses a circRNA related to MTX resistance of tumor cells and application of the circRNA to reversal of the MTX resistance of the tumor cells. An HT-29 MTX-sensitive and DMs-containing MTX-resistant cell line is selected as a study subject, the circRNA highly expressed in MTX-resistant cells is obtained through second-generation sequencing, validation and optimization and is namedas circRNA7746, and the expression quantity of the circRNA7746 is further determined to be positively correlated to the degree of the methotrexate (MTX) resistance of the tumor cells through establishment of an MTX treatment model of a tumor-bearing mouse and in combination with PCR and an agarose gel electrophoresis method. According to a virus transfection method, influence of the interfering circRNA7746 on the drug resistance of the cells, the amplification degree and the amplification form of a DHFR gene and micronuclear excretion is detected respectively, and the facts that inhibition ofthe circRNA7746 can promote DMs excretion and reverse the MTX resistance of the tumor cells are determined. Through proposal of the invention, a novel target is provided for tumor treatment and an effective technical means is provided for effectively combating the MTX resistance.

Description

technical field [0001] The present invention relates to a circRNA and its application, in particular to a circRNA related to the MTX drug resistance of tumor cells and its application in reversing the MTX drug resistance of tumor cells. The invention belongs to the field of tumor biological diagnosis technology and treatment technology. Background technique [0002] Methotrexate (MTX) is the most widely used folic acid antagonist in the treatment of malignant tumors, it can competitively bind to dihydrofolate reductase (Dihydrofolatereductase, DHFR), inhibit nucleotide metabolism, thereby blocking DNA synthesis , specifically kill proliferating cells. Although MTX is a commonly used drug in clinical tumor treatment, the generation of drug resistance in the body has become the bottleneck of MTX chemotherapy. Therefore, finding molecular markers related to MTX resistance has an important guiding role in clinical tumor chemotherapy, and overcoming MTX resistance Drugs are the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/519A61K31/7105A61P35/00
CPCA61P35/00A61K31/519A61K31/7105C12N15/1135C12N2310/531A61K2300/00
Inventor 孟祥宁蔡梦迪吕雅凡朱静关荣伟高巍马金法王萍王旭宋盈张慧姝韩玥
Owner HARBIN MEDICAL UNIVERSITY
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