CircRNAs associated with mtx drug resistance of tumor cells and their application in reversing mtx drug resistance of tumor cells

A tumor cell and drug resistance technology, applied in the field of tumor biodiagnosis and treatment, to achieve the effect of reversing tumor drug resistance, reducing the degree of gene amplification and improving efficiency

Active Publication Date: 2022-04-15
HARBIN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • CircRNAs associated with mtx drug resistance of tumor cells and their application in reversing mtx drug resistance of tumor cells
  • CircRNAs associated with mtx drug resistance of tumor cells and their application in reversing mtx drug resistance of tumor cells
  • CircRNAs associated with mtx drug resistance of tumor cells and their application in reversing mtx drug 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 the drug resistance of tumor cells to MTX and its application in reversing the drug resistance of tumor cells to MTX. The present invention selects HT-29 MTX-sensitive and MTX-resistant cell lines containing DMs as research objects, and obtains a highly expressed circRNA in MTX-resistant cells through next-generation sequencing, verification and optimization, and named it circRNA7746. The mouse MTX treatment model was combined with PCR and agarose gel electrophoresis to further confirm that the expression level of circRNA7746 was positively correlated with the degree of resistance of tumor cells to methotrexate (MTX). The effects of interfering circRNA7746 on cell drug resistance, DHFR gene amplification degree and amplification form, and micronucleus efflux were detected by virus transfection method, and it was confirmed that inhibiting circRNA7746 can promote the efflux of DMs and reverse the resistance of tumor cells to MTX. medicine. The proposal of the present invention provides a new target for tumor treatment, and an effective technical means for effectively combating MTX drug 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 Patents(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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