Applications of circRRM2B gene as new target in screening Vemurafenib-resistant melanoma treatment drugs

A fenib-resistant, melanoma technology, applied in the field of bioengineering, can solve the problem of no biological function of circRRM2B, and achieve the effect of large toxic side effects and high price

Active Publication Date: 2020-05-29
SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the research on circRNA is in its infancy, there is no research report on the biological function of circRRM2B

Method used

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  • Applications of circRRM2B gene as new target in screening Vemurafenib-resistant melanoma treatment drugs
  • Applications of circRRM2B gene as new target in screening Vemurafenib-resistant melanoma treatment drugs
  • Applications of circRRM2B gene as new target in screening Vemurafenib-resistant melanoma treatment drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Co-cultivation of exosomes derived from drug-resistant strains and sensitive strains can increase the resistance of sensitive strains to vemurafenib.

[0018] 1-1) Multiple strains of melanoma cells were cultured, IC50 values ​​were measured after vemurafenib treatment, and sensitive strains (A375S) were selected. The corresponding drug-resistant strain (A375R) was established by using the drug concentration increasing method.

[0019]1-2) Cultivate drug-resistant and sensitive strains, collect the cell supernatant, centrifuge at 110,000×g for 2 hours, remove the supernatant, and resuspend the pellet in PBS. Centrifuge at 110,000×g for 70 minutes at 4°C. The pellet was resuspended in PBS, centrifuged at 110,000×g for 70 minutes at 4°C (twice), and the obtained pellet was resuspended in PBS and stored at -80°C. The morphology of exosomes was observed by transmission electron microscopy. Exosome marker proteins CD63 and CD81 were detected by WB.

[0020] 1-3...

Embodiment 2

[0022] Example 2 The circRNA chip screened out the 10 most differentially expressed circRNAs in the exosomes of sensitive strains and drug-resistant strains.

[0023] 2-1) Digest the total RNA in exosomes with RNase R enzyme to remove linear RNA and enrich circular RNA. Then, the labeled cRNA was hybridized to Arraystar Human circRNA Array V2 (8x15K, Arraystar) by random primer method, the chip was washed after incubation at 65°C for 17 hours, and the array was scanned with Agilent G2505C scanner.

[0024] 2-2) The collected array images were analyzed using Agilent Feature Extraction software (version 11.0.1.1). Quantile normalization and subsequent data processing were performed using the R software limma package. Statistically significant differentially expressed circRNAs were identified between the two groups by volcano plot screening. Differentially expressed circRNAs between two samples were identified by the Fold Change filtering method. A hierarchical clustering appr...

Embodiment 3

[0027] Example 3 qRT-PCR verification found that the expression of circRRM2B was significantly increased in drug-resistant strains and their exosomes. The expression of circRRM2B in sensitive strains was significantly increased after the exosomes secreted by drug-resistant strains were co-cultured with sensitive strains.

[0028] 3-1) Extract RNA with TRIZOL, measure RNA purity and concentration, reverse transcribe RNA into cDNA, and configure Realtime PCR reaction systems for all cDNA samples. qRT-PCR was used to detect the expression of circRRM2B in drug-resistant strains, drug-resistant strain exosomes, sensitive strains, and sensitive strain exosomes.

[0029] 3-2) Exosomes derived from sensitive strains and drug-resistant strains were co-cultured with sensitive strains for 48 hours, respectively, and the expression of circRRM2B in cells was detected by qRT-PCR.

[0030] Depend on image 3 It can be seen that the expression of circRRM2B was significantly increased in dru...

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Abstract

The invention provides applications of a circRRM2B gene as a new target in screening Vemurafenib-resistant melanoma treatment drugs. The drugs take the circRRM2B gene as a target and inhibit or silence the expression of the circRRM2B gene. Applications of reagents for inhibiting or silencing the expression of the circRRM2B gene in preparing drugs for inhibiting melanoma are also provided. Throughchip screening, the circRRM2B is found being related to the Vemurafenib resistance of the melanoma and is the new target to screen the Vemurafenib-resistant melanoma treatment drugs, so that the circRRM2B gene has positive significance on screening new drugs. Meanwhile, the discovery of the new target provides a new thought for the treatment of drug-resistant melanoma.

Description

technical field [0001] The invention belongs to the field of bioengineering and relates to melanoma, and specifically uses the circRRM2B gene as a target in screening drugs for inhibiting melanoma. Background technique [0002] 1.1 Vemurafenib resistance is a difficult problem in the treatment of melanoma [0003] Melanoma is a malignant tumor derived from melanocytes, with poor curative effect and high mortality rate of traditional treatment. About 40-60% of melanoma patients carry BRAF V600E Mutation, the mutation of this site can activate the MEK-ERK pathway and promote the continuous division and proliferation of tumor cells. for BRAF V600E The mutation-targeted drug Vemurafenib (Vemurafenib PLX4032) can significantly prolong the progression-free survival and overall survival of patients. However, it has been clinically found that almost all patients will develop drug resistance within 1 year of vemurafenib treatment. Major acquired resistance mechanisms include: ME...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886A61K45/00A61P35/00
CPCC12Q1/6886A61K45/00A61P35/00C12Q2600/158C12Q2600/178C12Q2600/136
Inventor 程琼
Owner SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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