CN115820647A

CN115820647A - ARG2 gene-targeted siRNA and application thereof in improving drug resistance of breast cancer

Info

Publication number: CN115820647A
Application number: CN202211687761.XA
Authority: CN
Inventors: 朱英华; 陆小梅; 王佳颐
Original assignee: Dongguan Eighth People's Hospital (dongguan Children's Hospital)
Current assignee: Dongguan Eighth People's Hospital (dongguan Children's Hospital)
Priority date: 2022-12-27
Filing date: 2022-12-27
Publication date: 2023-03-21

Abstract

The invention relates to the technical field of biomedicine, in particular to siRNA targeting ARG2 gene and application thereof in improving drug resistance of breast cancer, the invention designs and synthesizes siRNA aiming at nucleotide sequence of ARG2 gene, after the siRNA is transferred into human breast cancer tamoxifen drug-resistant cells MCF7TR, the expression of ARG2 gene can be effectively inhibited, and the drug resistance of MCF7TR cells to endocrine therapy drug tamoxifen is obviously reduced, thereby achieving the purpose of inhibiting tumor. The siRNA molecule targeting the ARG2 gene can be used for preparing a medicament for treating tamoxifen drug-resistant breast cancer, and has wide application prospect.

Description

ARG2 gene-targeted siRNA and application thereof in improving drug resistance of breast cancer

Technical Field

The invention relates to the technical field of biomedicine, in particular to siRNA targeting ARG2 gene and application thereof in improving drug resistance of breast cancer.

Background

Tamoxifen is the principal endocrine treatment for patients with era positive breast cancer, and tamoxifen resistance is produced in approximately 40% of patients. Relapse and metastasis caused by tamoxifen resistance become a difficult problem to be solved urgently in clinical treatment of ER alpha positive breast cancer. A large number of researches reveal that the occurrence of breast cancer tamoxifen resistance is accompanied by the abnormal expression of a large number of genes, and the inhibition of the abnormally high expression genes in drug-resistant cells or the functions thereof is an important strategy for treating breast cancer resistance.

Small interfering RNA (siRNA) is a double-stranded RNA with the length of 20 to 25 nucleotides, and the siRNA is known to specifically inhibit the expression of related genes in a complementary pairing mode, so that the siRNA becomes an important tool for researching the gene function and drug targets.

Disclosure of Invention

In order to overcome the defects and shortcomings in the prior art, the invention aims to provide an ARG2 gene targeted siRNA and application thereof in improving drug resistance of breast cancer.

The purpose of the invention is realized by the following technical scheme: an ARG2 gene targeting siRNA, wherein the ARG2 gene targeting siRNA sequence is: si-ARG2-F:5'-CCUAUCGAGAAGGCAUGUATT-3', si-ARG2-R:5'-UACAUGCCUUCUCGAUAGGTT-3'.

Application of siRNA targeting ARG2 gene in preparation of medicine for improving drug resistance of patients to breast cancer.

Preferably, the resistance is to tamoxifen, an endocrine treatment drug.

Preferably, the breast cancer is era positive breast cancer.

Preferably, the siRNA targeting the ARG2 gene can effectively inhibit the expression of the ARG2 gene and obviously reduce the drug resistance of human breast cancer tamoxifen drug-resistant cells to an endocrine therapy drug tamoxifen.

Preferably, the sequence of the siRNA targeting the ARG2 gene is as follows: si-ARG2-F:5'-CCUAUCGAGAAGGCAUGUATT-3', si-ARG2-R:5'-UACAUGCCUUCUCGAUAGGTT-3'.

An application of siRNA targeting ARG2 gene and tamoxifen in preparing medicine for treating breast cancer.

Preferably, the breast cancer is ER α positive breast cancer.

The invention has the beneficial effects that: the invention designs and synthesizes siRNA aiming at the nucleotide sequence of ARG2 gene, after the siRNA is transferred into human breast cancer tamoxifen drug-resistant cells MCF7TR, the expression of the ARG2 gene can be effectively inhibited, and the drug resistance of MCF7TR cells to the endocrine therapy drug tamoxifen is obviously reduced, thereby achieving the purpose of inhibiting tumor. The siRNA molecule targeting the ARG2 gene can be used for preparing a medicament for treating tamoxifen drug-resistant breast cancer, and has a wide application prospect.

Drawings

FIG. 1 is a graph showing the measurement of the expression level of ARG2 in tamoxifen-sensitive cells and drug-resistant cells;

FIG. 2 is a graph of ARG2 expression levels versus prognosis for tamoxifen-treated breast cancer patients;

FIG. 3 is a graph of detection of siARG2 interference efficiency;

figure 4 is the effect of interfering siARG2 expression on proliferation of tamoxifen-resistant cells and on resistance to tamoxifen (. About.. About.p.. About.0.01).

Detailed Description

For the understanding of those skilled in the art, the present invention will be further described with reference to the following examples and accompanying fig. 1-4, which are not intended to limit the present invention.

Example A study of ARG2 expression levels in tamoxifen-resistant breast cancer cells

1. Establishment of tamoxifen drug-resistant breast cancer cell research model MCF7TR by using breast cancer cell line MCF7

2. Extraction of total RNA of cells, cDNA Synthesis

Total RNA of the cells is extracted by a column method Total RNA miniprep kit (EZB-TZ 1), and the steps are carried out according to the kit instructions.

cDNA synthesis was performed using 4 XEZscript Reverse Transcription Mix II (EZB-RT 2 GQ), according to the kit instructions.

3. Fluorescent quantitative PCR detection of ARG2 expression

The fluorescent quantitative PCR adopts 2 xEZ Color SYBR Green qPCR Master Mix (CQ 20), the steps are carried out according to the kit instruction, the experimental result is shown in figure 1, and the ARG2 expression is obviously increased in tamoxifen drug-resistant breast cancer cells.

Example study of correlation of ARG2 expression with prognosis of breast cancer patients

The correlation of ARG2 expression with prognosis in breast cancer patients treated with tamoxifen was analyzed using the tumor database Kaplan-Meier Plotter, and as shown in FIG. 2, it was found that the higher the level of ARG2 expression, the lower the disease-free survival rate of breast cancer patients.

Example study on interference efficiency of interfering ARG2 with siRNA in MCF7TR cells resistant to triple breast cancer

The cultured MCF7TR cells are transfected by a dose of 20nM, total RNA of the cells is extracted after 48 hours, the total RNA is reversely transcribed into cDNA, and the expression level of ARG2 is detected by fluorescence quantitative PCR, and the experimental result is shown in FIG. 3, and the siRNA has the interference efficiency of about 90 percent on the ARG 2. Wherein, the siRNA sequence of the target ARG2 gene is as follows: si-ARG 2-F5'-CCUAUCGAGAAGGCAUGUATT-3', si-ARG2-R:5'-UACAUGCCUUCUCGAUAGGTT-3'; the control sequence was: 5' -si-NC-F:5'-UUCUCCGAACGUGUCACGUTT-3', si-NC-R:5'-ACGUGACACGUUCGGAGAATT-3'.

Example Effect of interfering ARG2 expression with siRNA in four Breast cancer resistant cells MCF7TR on tamoxifen resistance

After 48 hours of siRNA transfection, 1uM tamoxifen is added into a culture solution, the cell survival rate is detected by a CCK-8 kit after 48 hours, and the experimental result is shown in figure 4.

The invention designs and synthesizes siRNA aiming at the nucleotide sequence of ARG2 gene, after the siRNA is transferred into human breast cancer tamoxifen drug-resistant cells MCF7TR, the expression of the ARG2 gene can be effectively inhibited, and the drug resistance of MCF7TR cells to the endocrine therapy drug tamoxifen is obviously reduced, thereby achieving the purpose of inhibiting tumor. The siRNA molecule targeting the ARG2 gene can be used for preparing a medicament for treating tamoxifen drug-resistant breast cancer, and has wide application prospect.

The above-described embodiments are preferred implementations of the present invention, and the present invention may be implemented in other ways without departing from the spirit of the present invention.

Claims

1. An siRNA targeting the ARG2 gene, characterized by: the siRNA sequence of the targeting ARG2 gene is as follows: si-ARG2-F:5'-CCUAUCGAGAAGGCAUGUATT-3', si-ARG2-R:5'-UACAUGCCUUCUCGAUAGGTT-3'.

2. Application of siRNA targeting ARG2 gene in preparation of medicine for improving drug resistance of patients to breast cancer.

3. The use of an ARG2 gene targeting siRNA of claim 2 in the manufacture of a medicament for improving the resistance of a patient to breast cancer, wherein said siRNA comprises: the resistance is due to the endocrine therapy drug tamoxifen.

4. The use of an ARG2 gene targeting siRNA of claim 2 in the manufacture of a medicament for improving the resistance of a patient to breast cancer, wherein said siRNA comprises: the breast cancer is ER alpha positive breast cancer.

5. The use of an ARG2 gene targeting siRNA of claim 2 in the manufacture of a medicament for improving the resistance of a patient to breast cancer, wherein said siRNA comprises: the siRNA targeting the ARG2 gene can efficiently inhibit the expression of the ARG2 gene and obviously reduce the drug resistance of the tamoxifen for endocrine therapy of human breast cancer drug-resistant cells.

6. The use of an ARG2 gene targeting siRNA of claim 2 in the manufacture of a medicament for improving the resistance of a patient to breast cancer, wherein said siRNA comprises: the siRNA sequence of the targeting ARG2 gene is as follows: si-ARG2-F:5'-CCUAUCGAGAAGGCAUGUATT-3', si-ARG2-R:5'-UACAUGCCUUCUCGAUAGGTT-3'.

7. An application of siRNA targeting ARG2 gene and tamoxifen in preparing medicine for treating breast cancer.

8. The use of the ARG2 gene targeting siRNA and tamoxifen in the preparation of a medicament for treating breast cancer according to claim 7 wherein: the breast cancer is ER alpha positive breast cancer.

9. The use of the ARG2 gene targeting siRNA and tamoxifen in the preparation of a medicament for treating breast cancer according to claim 7 wherein: the siRNA targeting the ARG2 gene can effectively inhibit the expression of the ARG2 gene and obviously reduce the drug resistance of human breast cancer tamoxifen drug-resistant cells to an endocrine therapy drug tamoxifen.

10. The use of the ARG2 gene targeting siRNA and tamoxifen in the preparation of a medicament for treating breast cancer according to claim 7 wherein: the siRNA sequence of the targeting ARG2 gene is as follows: si-ARG2-F:5'-CCUAUCGAGAAGGCAUGUATT-3', si-ARG2-R:5'-UACAUGCCUUCUCGAUAGGTT-3'.