CN115813945A

CN115813945A - Application of DHRSX inhibitor in preparation of glioma drug

Info

Publication number: CN115813945A
Application number: CN202211301541.9A
Authority: CN
Inventors: 陆亚鹏; 朱俐; 王丹; 王雪婷
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2022-10-24
Filing date: 2022-10-24
Publication date: 2023-03-21
Anticipated expiration: 2042-10-24
Also published as: CN115813945B

Abstract

The invention belongs to the technical field of biology, and discloses application of a DHRSX inhibitor in preparation of a glioma drug. The DHRSX gene shows specific high expression in the brain glioma, and the overall survival time of the brain glioma patients with the high DHRSX expression is short, and the prognosis is poor. The invention discovers that the expression of the DHRSX of the glioma cells can be efficiently silenced by the siRNA, so that the proliferation of the glioma cells is inhibited, the apoptosis of the glioma cells is promoted, and the prevention and treatment effects of the DHRSX inhibitor on the glioma are proved.

Description

Application of DHRSX inhibitor in preparation of glioma drug

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an application of a DHRSX inhibitor in preparation of a glioma drug.

Background

Glioma is the most fatal type of human brain malignant tumor, has high malignancy degree, rich blood vessels in tumor tissues, rapid growth of glioma, easy invasion and relapse, poor prognosis, and the highest morbidity and mortality rate in intracranial primary tumors. While brain gliomas have achieved some success with surgical treatments, radiation therapy and chemotherapy, treatment of brain gliomas to date remains a clinical oncology challenge. Glioma grows infiltratively, has no obvious boundary with surrounding normal brain tissues, is difficult to completely remove by operation, and is easy to generate drug resistance to chemotherapeutic drugs, so the recurrence rate is still high, and the 5-year survival rate of patients is low. Therefore, it is imperative to find new effective strategies to go out of this dilemma.

With the development of research on tumor pharmacology and molecular biology, molecular targeted therapy has become a new method for treating malignant tumors except for surgery, radiotherapy and chemotherapy. Different from traditional chemotherapy, the targeted therapy prevents the growth of cancer cells by interfering with specific target molecules required by the generation of cancer and the growth of tumors, and has the advantages of strong specificity, obvious curative effect, small adverse reaction and the like. Although the prior art finds that the occurrence and development of brain glioma have important relationship with the change of expression level of various genes. However, no particularly effective target gene can be applied to the prevention and treatment of glioma at present. Therefore, the search for effective biological target molecules for treating brain glioma is a key issue to be solved, which is crucial to the development of new effective prevention strategies.

DHRSX is a protein-coding Gene (Gene ID: 207063), a member of the reductase family, a newly discovered human Gene, and its function is poorly understood. It has been found that the DHRSX gene can play an important regulatory role in starvation-induced autophagy. Another study on breast cancer found that the DHRSX gene was associated with brain metastases in breast cancer. However, so far, the possible effect of the DHRSX gene in the prevention and treatment of brain glioma has not been reported.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to solve the technical problem of providing the application of the DHRSX inhibitor in preparing a brain glioma medicament. Research shows that the DHRSX gene has obviously higher expression in the tumor tissue of a brain glioma patient than that in the normal tissue. Moreover, high expression of the DHRSX gene is associated with poor prognosis in patients with brain glioma. The inhibition of the expression of DHRSX gene in glioma cells can effectively inhibit cell proliferation and promote apoptosis.

The invention provides an application of a DHRSX inhibitor in preparing a glioma drug, wherein the glioma drug comprises one of a drug for inhibiting the proliferation of glioma cells, a drug for promoting the apoptosis of the glioma cells and a drug for preventing and treating glioma.

Based on the inhibition effect of the DHRSX inhibitor on the proliferation of the human glioma cells and the promotion effect of the DHRSX inhibitor on the apoptosis of the human glioma cells, the invention also provides the application of the DHRSX inhibitor in the preparation of the medicine for preventing and treating the human glioma.

In the invention, the DHRSX inhibitor is DHRSX siRNA, DHRSX shRNA, shRNA coding DNA or DHRSX sgRNA through various DHRSX-targeting gene silencing technologies, including adenovirus-mediated shRNA interference technology or lentivirus-mediated Crispr CAS9 technology. The DHRSX siRNA is small interfering RNA targeting DHRSX, and can cause mRNA degradation and silence DHRSX gene. The DHRSX shRNA is short hairpin RNA targeting DHRSX, can be cloned to an expression vector and expresses short interfering RNA, thereby realizing the silencing of the DHRSX gene. The DHRSX sgRNA is a guide RNA for targeting a DHRSX gene, and the sgRNA is an important component based on a Crispr gene knockout system, can be combined with cas9 protein, and guides cas9 enzyme targeting genome DNA to shear the DHRSX gene.

The invention also provides a cationic liposome in the medicine. In the present invention, cationic liposome Lipofectamine 3000 was used as a transfection reagent.

In some embodiments, the DHRSX inhibitor is siRNA against DHRSX, consisting of a complementary sense strand and antisense strand, wherein the sense strand is the nucleotide sequence shown in SEQ ID No.1 and the antisense strand is the nucleotide sequence shown in SEQ ID No. 2; or, the sense strand is the nucleotide sequence shown as SEQ ID NO.3, and the antisense strand is the nucleotide sequence shown as SEQ ID NO. 4.

Further, the DHRSX inhibitor is siRNA aiming at DHRSX, and consists of a complementary sense strand and an antisense strand.

Further, the sense strand of the siRNA of DHRSX is the nucleotide sequence shown in SEQ ID NO.1, and the antisense strand of the siRNA of DHRSX is the nucleotide sequence shown in SEQ ID NO. 2.

Further, the sense strand of the siRNA of DHRSX is the nucleotide sequence shown in SEQ ID NO.3, and the antisense strand of the siRNA of DHRSX is the nucleotide sequence shown in SEQ ID NO. 4.

Further, the brain glioma cell is a human glioma U251 cell.

The pharmaceutical of the invention further comprises an additional therapeutic agent selected from other agents useful in the treatment of brain gliomas. The medicament is in the form of injection liquid or powder injection for injection.

The invention has the advantages and beneficial effects that:

the invention discovers for the first time that the expression of the DHRSX gene in the tumor tissue of a patient with glioma is obviously higher than that of the normal tissue. Moreover, high expression of the DHRSX gene is associated with a poor prognosis for patients with brain glioma. The inhibition of the expression of DHRSX gene in glioma cells can effectively inhibit cell proliferation and promote apoptosis. Therefore, DHRSX can be used as an important molecular target for treating brain glioma, and a powerful means is possibly provided for the targeted treatment of the brain glioma.

Drawings

FIG. 1 shows the difference in expression of DHRSX gene in normal tissues of brain glioma patients and brain glioma tissues in TCGA database;

FIG. 2 shows the effect of DHRSX gene expression on survival in patients with brain glioma;

FIG. 3 effect of different siRNAs on DHRSX gene expression in human glioma cells;

FIG. 4 shows the effect of silencing the expression of DHRSX gene on glioma cell proliferation;

FIG. 5 shows the effect of silencing the expression of DHRSX gene on glioma cell apoptosis.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, for which specific conditions are not indicated in the examples, are generally carried out under conventional conditions or under conditions recommended by the reagent manufacturers.

Example 1 differential expression of DHRSX Gene in Normal brain tissue and glioma tissue of patients with brain glioma in TCGA database

TCGA (The Cancer Genome atlas) is a database jointly developed in 2006 by National Cancer Institute (NCI, national Institute of Cancer) and National Human Genome Research Institute (NHGRI, national Institute of Human Genome), and it is a very important data source for Cancer researchers, including clinical data of various Human cancers, and also including data of genomic variation, mRNA expression, miRNA expression, methylation, and The like. The study obtained RNAseq data and corresponding clinical information for brain gliomas from the TCGA dataset (https:// portal.gdc.com). Statistical analysis was performed using R software v4.0.3. P values <0.05 were considered statistically significant. The expression distribution of the DHRSX gene in glioma tissues and normal tissues is shown in figure 1, wherein the abscissa represents samples of different groups, the ordinate represents the expression distribution of the gene, and different colors represent different groups. Two groups of samples were tested for significance by wilcox. The results show that DHRSX expression in brain glioma tissues is significantly higher than in normal tissues (p < 0.0001).

Example 2 Effect of DHRSX Gene expression on survival of brain glioma patients

The overall survival rate (OS) between the high-and low-expression groups of the DHRSX gene in TCGA-enrolled patient data sets of brain gliomas was analyzed using the GEPIA website (http:// GEPIA. Cancer-pku. Cn). The parameters are set as follows: group cutoff, median; confidence interval, 95%; log rank P value, <0.05. FIG. 2 shows the effect of DHRSX gene expression on OS in brain glioma patients, HR represents the risk factor for the high versus low expression group samples, where HR >1 represents a risk factor for the gene (higher expression, worse prognosis), and HR <1 represents a protective factor (higher expression, better prognosis). The results suggest that high expression of DHRSX is a high risk factor for poor prognosis in patients with brain glioma.

Example 3 silencing of DHRSX Gene expression in human glioma cells Using siRNA

Human glioma U251 cell line was purchased from ATCC (Manassas, VA, USA). Place the cells at 37 ℃ 5% CO ₂ In a humidified environment, using 10% fetal bovine serum supplemented in Dulbecco's modified Eagle's medium. 2X 10 day before siRNA transfection ⁵ U251 cell seeding ofIn 6-well plate, 37 ℃,5% ₂ After 24h incubation under conditions, transfection was performed according to the Lipofectamine 3000 instructions.

The three siRNA sequences used to silence DHRSX gene expression are as follows:

siRNA1#

the sense strand is 5'-GGAUGGAGUUCAAACUAAUUG-3' (SEQ ID NO. 1)

The antisense strand is 5'-AUUAGUUUGAACUCCAUCCUG-3' (SEQ ID NO. 2)

siRNA2#

The sense strand is 5'-CUUUGGUGUUAGAGAGAUAGA-3' (SEQ ID NO. 3)

The antisense strand is 5'-UAUCUCUCUAACACCAAAGGA-3' (SEQ ID NO. 4)

siRNA3#

The sense strand is 5'-GGCAGUUUGUGCAGAAGUUCA-3'

The antisense strand is 5'-AACUUCUGCACAAACUGCCGG-3'

After siRNA transfection for 48h, cells are washed for 2 times by precooled PBS, total protein is extracted after human RIPA lysate is added, and the expression of DHRSX genes of different experimental groups is detected by Western blot technology. The total protein was subjected to SDS PAGE. And cutting a PVDF membrane with a proper size after the electrophoresis is finished, and electrically transferring the protein to the PVDF membrane. After the membrane transfer was completed, the membrane was placed in a box and then an appropriate amount of 5% skim milk was added, and the box was closed on a shaker for 1h, and then the membrane was washed 3 times with TBST. Washed membranes were incubated overnight with diluted DHRSX antibody (Invitrogen, 1; then, incubating for 1.5h at room temperature by using a fluorescent secondary antibody, and washing for 3 times by using TBST; and finally, developing by using fluorescence. The experimental results are shown in FIG. 3, and siRNA1# and siRNA2# both can obviously reduce the expression of DHRSX protein.

Example 4 Effect of silencing DHRSX Gene expression on glioma cell proliferation

The effect of DHRSX gene on U251 glioma cell proliferation was investigated by trypan blue staining. The cells were divided into control group and DHRSX siRNA group. After 48h of transfection of glioma cells with siRNA # 1 described in example 3, the cells were harvested and seeded in 6-well plates. At various time points (0, 12, 24, 48 h), pancreatin and single cell suspensions were prepared, followed by trypan blue staining to a final concentration of 0.2% and viable cell counts were counted. The results are shown in fig. 4, the proliferation rate of the DHRSX gene silencing group cells is significantly lower than that of the control group cells, and the difference is particularly significant after 24h (P < 0.001), which suggests that inhibiting the expression of the DHRSX gene can effectively inhibit the proliferation of the human glioma cells.

Example 5 Effect of silencing expression of DHRSX Gene on glioma cell apoptosis

Placing human glioma U251 cells at 37 ℃,5% CO ₂ In a humidified environment, using 10% fetal bovine serum supplemented in Dulbecco's modified Eagle's medium. The expression of DHRSX gene was knocked down using siRNA #2 as described in example 3. And (3) inoculating the human glioma U251 cells after siRNA1#24h transfection into a 6-well plate, and culturing for 72h. Apoptosis was determined by annexin V-FITC/PI staining. Cells were stained in 100. Mu.L of binding buffer containing 5. Mu.L of annexin V-FITC and 10. Mu.L of propidium iodide (20 ng/mL), then incubated in dark at room temperature for 10-15 minutes and apoptosis was detected by flow cytometry. As shown in FIG. 5, the percentage of apoptosis was significantly increased in the DHRSX gene-silenced cells as compared with the control cells (P)<0.01). These results indicate that DHRSX has an anti-apoptotic effect in glioma cells. Inhibition of DHRSX gene expression promotes glioma cell apoptosis.

While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. An application of DHRSX inhibitor in preparing medicine for treating brain glioma is disclosed, wherein the medicine for treating brain glioma includes one of medicine for inhibiting proliferation of brain glioma cell, medicine for promoting apoptosis of brain glioma cell and medicine for preventing and treating brain glioma.

2. The use according to claim 1, wherein the DHRSX inhibitor is DHRSX siRNA.

3. The use of claim 2, wherein the sense strand of the DHRSX siRNA is the nucleotide sequence shown in SEQ ID No.1, and the antisense strand of the DHRSX siRNA is the nucleotide sequence shown in SEQ ID No. 2.

4. The use of claim 2, wherein the sense strand of the DHRSX siRNA is a nucleotide sequence shown as SEQ ID No.3, and the antisense strand of the DHRSX siRNA is a nucleotide sequence shown as SEQ ID No. 4.

5. The use of any one of claims 1 to 4, wherein the brain glioma cells are human glioma U251 cells.