CN113373177A - Method for improving curative effect of glioblastoma temozolomide - Google Patents
Method for improving curative effect of glioblastoma temozolomide Download PDFInfo
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- CN113373177A CN113373177A CN202110490438.2A CN202110490438A CN113373177A CN 113373177 A CN113373177 A CN 113373177A CN 202110490438 A CN202110490438 A CN 202110490438A CN 113373177 A CN113373177 A CN 113373177A
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- 238000000034 method Methods 0.000 title claims abstract description 12
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- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims abstract description 144
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Abstract
An interference plasmid for knocking down the expression of COPS5 protein in a glioblastoma cell line, application of the interference plasmid in serving as a medicine for solving the drug resistance problem of temozolomide in treatment, and application of a combined medicine formed by the interference plasmid for knocking down the expression of COPS5 protein and the temozolomide in serving as a glioblastoma chemotherapy medicine. The interference plasmid knockdown the expression of COPS5 protein, and regulates the iron death process of glioblastoma cells, specifically including increasing the expression of iron death promoting protein and reducing the expression of iron death inhibiting protein.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to an interference plasmid for knocking down the expression of COPS5 protein in a glioblastoma cell line, application of the interference plasmid as a medicine for solving the drug resistance problem of temozolomide in treatment, and application of a combined medicine formed by the interference plasmid for knocking down the expression of COPS5 protein and the temozolomide as a glioblastoma chemotherapy medicine.
Background
Gliomas are the most common primary intracranial tumors, with glioblastoma accounting for approximately 50% of all gliomas. Glioblastoma has the characteristics of high invasiveness, easy recurrence and high lethal disability rate, and the 5-year total survival rate of the glioblastoma is one of the worst tumors in all human cancers. Despite aggressive treatment, median survival is approximately 15 months.
Temozolomide is an oral alkylating agent type antitumor drug, has broad-spectrum antitumor activity, can permeate a blood brain barrier, has bioavailability close to 100 percent, and is the first choice chemotherapeutic drug for glioblastoma. However, temozolomide resistance is common and complicated in clinical practice, and the biggest problem limiting the effect is the resistance problem.
Therefore, how to effectively solve the drug resistance problem of temozolomide in glioblastoma, and improving the curative effect of temozolomide in glioblastoma has become a problem to be solved in clinical practice.
Therefore, it is necessary to overcome the deficiencies of the prior art by providing an interference plasmid for knocking down the expression of COPS5 protein in a glioblastoma cell line, an application of the interference plasmid in solving the drug resistance problem of temozolomide in treatment, and an application of a combined drug formed by the interference plasmid for knocking down the expression of COPS5 protein and the temozolomide in the aspect of being used as a glioblastoma chemotherapy drug.
Disclosure of Invention
The invention aims to avoid the defects of the prior art and provides an interference plasmid for knocking down the expression of COPS5 protein in a glioblastoma cell line, application of the interference plasmid as a medicine for solving the drug resistance problem of temozolomide in treatment, and application of a combined medicine formed by the interference plasmid for knocking down the expression of COPS5 protein and the temozolomide as a glioblastoma chemotherapy medicine.
The object of the invention is achieved by the following technical measures.
Provides an interference plasmid for knocking down the expression of COPS5 protein in a glioblastoma cell line, and the gene sequence is CCAGACTATTCCACTTAATTT.
Preferably, the interference plasmid for knocking down the expression of COPS5 protein in a glioblastoma cell line promotes iron death of glioblastoma cells, and solves the problem of drug resistance of temozolomide in treatment.
Preferably, the interfering plasmid for knocking down the expression of COPS5 protein in a glioblastoma cell line as described above regulates the iron death process of glioblastoma cells.
Preferably, the above-mentioned interfering plasmid for knocking down the expression of COPS5 protein in glioblastoma cell line has increased expression of iron death promoting protein and decreased expression of iron death suppressing protein.
Preferably, the above-mentioned interfering plasmid for knocking down the expression of COPS5 protein in glioblastoma cell line has increased expression of iron death promoting protein ACSL4 and decreased expression levels of SLC7A11 and GPX4, which are related proteins inhibiting iron death.
The invention also provides application of the interference plasmid for knocking down the expression of COPS5 protein in a glioblastoma cell line as a medicine for solving the problem of drug resistance of temozolomide in treatment.
The invention also provides application of a combined medicament consisting of the interference plasmid for knocking down the expression of the COPS5 protein and temozolomide in the aspect of being used as a glioblastoma chemotherapy medicament, wherein the gene sequence of the interference plasmid is CCAGACTATTCCACTTAATTT.
Preferably, the drug resistance problem of temozolomide in treatment is solved by knocking down COPS5 protein expression through interference plasmids.
Preferably, the interference plasmid knocks down COPS5 protein expression, regulating the process of glioblastoma cell iron death.
Preferably, the expression of the iron death promoting protein is increased, and the expression of the iron death inhibiting protein is decreased.
Preferably, the expression of the iron death promoting protein ACSL4 is increased, and the expression levels of SLC7A11 and GPX4 which are related proteins for inhibiting iron death are reduced.
The interference plasmid of the invention, which realizes the expression of the knockdown COPS5 protein in a glioblastoma cell line, solves the drug resistance problem of the mozamide in treatment. The interference plasmid knockdown the expression of COPS5 protein, and regulates the iron death process of glioblastoma cells, specifically including increasing the expression of iron death promoting protein and reducing the expression of iron death inhibiting protein. The interference plasmid which knocks down the expression of COPS5 protein and the combination drug which is formed by combining temozolomide can be used as the drug for chemotherapy of glioblastoma.
Drawings
The invention is further illustrated by means of the attached drawings, the content of which is not in any way limiting.
FIG. 1 shows the expression of ferritin in human glioblastoma U87MG cells.
FIG. 2 is the expression of the iron death protein under the effect of Erastin, an iron death agonist, at various concentrations.
FIG. 3 shows the expression of the iron death protein under the action of the iron death agonist Erastin.
FIG. 4 shows the U87MG iron death in human glioblastoma U87MG cells at different concentrations of the iron death agonist Erastin.
FIG. 5 shows the U87MG iron death in human glioblastoma U87MG cells at different concentrations of the iron death agonist RSL 3.
FIG. 6 shows the iron death of human glioblastoma U87MG cells in human glioblastoma U87MG cells at different concentrations of iron promoter.
FIG. 7 shows U87MG iron death in human glioblastoma U87MG cells by the action of the different iron death agonists Erastin.
FIG. 8 shows the results of different treatment modalities of example 2 of the present invention for glioblastoma inhibition.
FIG. 9 shows the results of different treatment modalities of example 3 of the present invention for glioblastoma inhibition.
Detailed Description
The invention is further illustrated by the following examples.
Example 1.
An interference plasmid for knocking down the expression of COPS5 protein in a glioblastoma cell line has a gene sequence of CCAGACTATTCCACTTAATTT. COPS 5: photogenic factor 9 complex subunit 5 protein.
In the research, the photogenic factor 9 complex subunit 5 (COPS5) protein is expressed in glioblastoma and is involved in regulating the iron death process of glioblastoma cells so as to influence the drug resistance of temozolomide in treatment.
The COPS5 expression interference plasmid is specifically shRNA (short hairpin RNA) plasmid, two short inverted repeat sequences specially aiming at the COPS5 gene are cloned into the expression vector plasmid, and after entering cells through transfection, mRNA transcribed by COPS5 is degraded, so that the expression of the COPS5 gene is interfered, and the level of intracellular COPS5 protein is reduced. This interference plasmid was specifically designed for the COPS5 gene, with the gene sequence CCAGACTATTCCACTTAATTT.
In glioblastoma cell lines, knockdown of COPS5 protein regulates the process of glioblastoma cell iron death. The research shows that the expression of the COPS5 protein is knocked down by the interference plasmid, the expression of the iron death promoting protein is increased, and the expression of the iron death inhibiting protein is reduced.
FIG. 1 shows the expression of ferritin in human glioblastoma U87MG cells. As can be seen from FIG. 1, in human glioblastoma U87MG cells, the expression of the iron death-promoting protein ACSL4 was increased after the interference of the expression level of COPS5 protein with the interference plasmid shCOPS5, compared with the control shScr, while the expression levels of two related iron death-inhibiting proteins SLC7A11 and GPX4 were decreased, and β -actin was an internal reference.
FIG. 2 shows the expression of the iron death protein under the action of Erastin, an iron death agonist, at various concentrations. FIG. 2 shows that under the action of Erastin (iron death is increased after Erastin is added) which is an iron death agonist with different concentrations, the expression of SLC7A11 which is a protein related to iron death is obviously increased. FIG. 3 shows the expression of the iron death protein under the action of the iron death agonist Erastin. FIG. 3 shows that the expression level of SLC7A11 of the ferroptosis protein is increased under the action of Erastin which is an iron death agonist. As can be seen from both FIGS. 2 and 3, the expression level of the ferroptosis inhibitory protein was significantly reduced after interfering with the expression of COPS5, as compared to the control group.
Applicants found that knocking down COPS5 protein expression in glioblastoma cell lines promotes glioblastoma iron death.
FIG. 4 shows the U87MG iron death in human glioblastoma U87MG cells at different concentrations of the iron death agonist Erastin. FIG. 4 shows that U87MG iron death is significantly increased (the lower the Relative Cell Viability, the more iron death) in human glioblastoma U87MG cells after interference of the expression level of COPS5 by the shCOPS5 plasmid compared to the control shScr under the action of different concentrations of the iron death agonist Erastin. FIG. 5 shows the U87MG iron death in human glioblastoma U87MG cells at different concentrations of the iron death agonist RSL 3. FIG. 5 shows similar results to FIG. 4, except that another iron death agonist, RSL3, was used.
FIG. 6 shows that under the action of Erastin, the iron death of U87MG cells of human glioblastoma multiforme is increased after the interference of plasmid shCOPS5 with the expression level of COPS5, and under the action of Erastin-Ferr-1, the iron death of U87MG cells is obviously reduced. FIG. 7 shows that U87MG iron death is significantly increased (higher lipid Reactive Oxygen Species (ROS) is higher, more iron is killed) after the interference of COPS5 expression level by shCOPS5 plasmid compared with the control group under the action of Erastin, an iron death agonist.
Therefore, the interference plasmid can knock down the expression of COPS5 protein in a glioblastoma cell line, regulate the iron death process of glioblastoma cells, promote the iron death of the glioblastoma cells and solve the problem of drug resistance of temozolomide in treatment
Example 2.
The use of the interfering plasmid for knocking down COPS5 protein expression in glioblastoma cell line of example 1 as a drug to solve the problem of drug resistance of temozolomide in therapy.
The knockdown of the expression of COPS5 in combination with temozolomide treatment has a better and significant effect in inhibiting the growth of glioblastoma in combination treatment mode compared with the independent knockdown of the expression of COPS5 and the independent application of temozolomide treatment. As shown in fig. 8, the combined use of COPS5 interference plasmid COPS5 and temozolomide in nude mice resulted in a smaller volume of subcutaneous tumors compared to the control group, the group using interference plasmid shCOPS5 alone and the group using temozolomide alone.
The interference plasmid for knocking down the expression of COPS5 protein in a glioblastoma cell line can realize more obvious tumor growth inhibition effect by promoting the death of glioblastoma cell iron and further reducing the drug resistance of temozolomide, and can be used as a drug for solving the drug resistance problem of the temozolomide in treatment.
Example 3.
Provides an application of a combined medicine consisting of interference plasmids for knocking down the expression of COPS5 protein and temozolomide in the aspect of being used as a glioblastoma chemotherapy medicine.
The knockdown COPS5 and temozolomide combined drug can achieve a more obvious tumor growth inhibition effect by promoting the iron death of glioblastoma cells and further reducing the drug resistance of temozolomide. Can be used as a new medicine for improving the curative effect of the glioblastoma temozolomide.
As shown in fig. 9, in the experiment of subcutaneous tumor formation in nude mice, the volume and weight of subcutaneous tumor in nude mice were significantly reduced after combined treatment with COPS5 interference plasmid shCOPS5 and temozolomide, which was more significant under the action of Erastin, an iron death agonist, and disappeared under the action of Erastin-Fer-1, an iron death inhibitor.
Therefore, the combined medicine formed by the interference plasmid with reduced COPS5 protein expression and temozolomide can be used as the medicine for chemotherapy of glioblastoma.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Sequence listing
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<120> a method for improving the therapeutic effect of temozolomide in glioblastoma multiforme
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Claims (10)
1. An interfering plasmid for knock-down of expression of COPS5 protein in a glioblastoma cell line, characterized by: the gene sequence is CCAGACTATTCCACTTAATTT.
2. The interference plasmid of claim 1, for knocking down expression of COPS5 protein in a glioblastoma cell line, wherein: promoting the death of glioblastoma cell iron and overcoming the drug resistance problem of temozolomide in treatment.
3. The interference plasmid of claim 2, for knocking down expression of COPS5 protein in a glioblastoma cell line, wherein: regulate the process of iron death in glioblastoma cells.
4. The interference plasmid of claim 3, for knocking down expression of COPS5 protein in a glioblastoma cell line, wherein: the expression of the iron death promoting protein is increased, and the expression of the iron death inhibiting protein is reduced.
5. The interference plasmid of claim 4 for knocking down expression of COPS5 protein in a glioblastoma cell line, wherein: the expression of the iron death promoting protein ACSL4 is increased, and the expression levels of SLC7A11 and GPX4 which are related proteins for inhibiting iron death are reduced.
6. Use of the interfering plasmid according to any one of claims 1 to 5 for knock-down of expression of COPS5 protein in glioblastoma cell lines as a drug to solve the problem of resistance of temozolomide in therapy.
7. The application of the combined medicine consisting of the interference plasmid and temozolomide for knocking down the expression of the COPS5 protein in the aspect of being used as a glioblastoma chemotherapy medicine is characterized in that: the gene sequence of the interference plasmid is CCAGACTATTCCACTTAATTT.
8. The use of the combination of the interference plasmid for knocking down COPS5 protein expression and temozolomide according to claim 7 as a glioblastoma chemotherapy drug, wherein: the drug resistance problem of temozolomide in treatment is solved by knocking down COPS5 protein expression through interference plasmids.
9. The use of the combination of the interference plasmid for knocking down COPS5 protein expression and temozolomide according to claim 8 as a glioblastoma chemotherapy drug, wherein: the interference plasmid knocks down the expression of COPS5 protein, and regulates the iron death process of glioblastoma cells.
10. The use of the combination of the interference plasmid for knocking down COPS5 protein expression and temozolomide according to claim 8 as a glioblastoma chemotherapy drug, wherein: increase the expression of the iron death promoting protein and reduce the expression of the iron death inhibiting protein.
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| CN202110490438.2A CN113373177A (en) | 2021-05-06 | 2021-05-06 | Method for improving curative effect of glioblastoma temozolomide |
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| CN114515285A (en) * | 2022-03-11 | 2022-05-20 | 中国人民解放军陆军军医大学第一附属医院 | Application of iron death inducer RSL3 in preparation of drug for inhibiting glioblastoma |
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| US20050069918A1 (en) * | 2003-05-29 | 2005-03-31 | Francois Claret | JAB1 as a prognostic marker and a therapeutic target for human cancer |
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| US20050069918A1 (en) * | 2003-05-29 | 2005-03-31 | Francois Claret | JAB1 as a prognostic marker and a therapeutic target for human cancer |
| CN106047879A (en) * | 2016-08-18 | 2016-10-26 | 广州市锐博生物科技有限公司 | Oligonucleotide molecule used for inhibiting expression of mRNA of target gene and composition set thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114515285A (en) * | 2022-03-11 | 2022-05-20 | 中国人民解放军陆军军医大学第一附属医院 | Application of iron death inducer RSL3 in preparation of drug for inhibiting glioblastoma |
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