Application of DNALI1 gene inhibitor in preparation of brain glioma drugs

Abstract

The application belongs to the technical field of biological medicine, and discloses application of a DNALI1 gene inhibitor in preparation of a brain glioma medicine. The DNALI1 gene inhibitor is siRNA for inhibiting expression of the DNALI1 gene. The siRNA provided in the application can efficiently silence expression of DNALI1 of brain glioma cells, inhibit cell proliferation and invasion, and has a prevention and treatment effect on brain glioma.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of a DNALI1 gene inhibitor in the preparation of drugs for glioma. Background Technology

[0002] Gliomas are the most common primary brain tumors, accounting for approximately 50% of all brain tumors. They arise from the malignant transformation of glial cells in the brain and spinal cord. Despite advancements in surgical treatment, radiation therapy, and chemotherapy, treatment options for gliomas remain limited. Due to their invasive growth, complete surgical resection is difficult, and gliomas readily develop resistance to chemotherapy drugs, resulting in a high recurrence rate and low 5-year survival rate. Therefore, there is an urgent need to find new and effective treatment strategies for gliomas.

[0003] With the continuous advancement of tumor molecular biology research, gene-targeted molecular therapies can now be used to treat malignant tumors, a field with enormous development potential. Unlike traditional chemotherapy, molecularly targeted therapy can inhibit cancer cell growth by acting on specific molecular targets. This treatment method has significant advantages such as high specificity, significant efficacy, and few adverse reactions. The development of gliomas is closely related to changes in genes or their expression levels, a complex process involving multiple genes, each of which has the potential to become a therapeutic target. However, to date, there are no effective targeted therapies against specific genes in gliomas available for clinical use. Therefore, finding biological target molecules applicable to the treatment of gliomas is a crucial and urgent problem that needs to be solved.

[0004] The protein encoded by the DNALI1 gene is an important component of axonal dynamism and plays a crucial role in animal growth and development. Studies have indicated that DNALI1 may be a prognostic biomarker for childhood asthma. Furthermore, DNALI1 has been found to be highly expressed in high-grade serous ovarian cancer, potentially possessing diagnostic value. However, the possible role of the DNALI1 gene in the diagnosis and treatment of gliomas has not yet been reported. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the technical problem to be solved by this invention is to provide the application of DNALI1 gene inhibitors in the preparation of drugs for glioma. Studies have shown that the expression of the DNALI1 gene in tumor tissues of patients with gliomas of all grades is significantly higher than that in normal tissues. Moreover, high expression of the DNALI1 gene is associated with poor prognosis in glioma patients. Inhibiting the expression of the DNALI1 gene in glioma cells can effectively inhibit the proliferation and invasion of tumor cells.

[0006] This invention provides the application of DNALI1 gene inhibitors in the preparation of glioma drugs, which include one of the following: drugs that inhibit glioma cell proliferation, drugs that inhibit glioma cell invasion, and drugs for the prevention and treatment of glioma.

[0007] Based on the inhibitory effect of DNALI1 gene inhibitors on the proliferation and invasion of human glioma cells, this invention also provides the application of DNALI1 gene inhibitors in the preparation of drugs for the prevention and treatment of human glioma.

[0008] In this invention, the DNALI1 gene inhibitor refers to a gene silencing technology targeting DNALI1, including adenovirus-mediated shRNA interference technology or lentivirus-mediated CRISPR CAS9 technology. Further, the DNALI1 inhibitor is DNALI1 siRNA, DNALI1 shRNA, the encoding DNA of shRNA, or DNALI1 sgRNA. Specifically, DNALI1 siRNA is a small interfering RNA targeting DNALI1, which can cause mRNA degradation, thereby silencing the DNALI1 gene. DNALI1 shRNA is a short hairpin RNA targeting DNALI1, which can be cloned into an expression vector and express a short interfering RNA, thereby achieving DNALI1 gene silencing. DNALI1 sgRNA is a guide RNA targeting the DNALI1 gene. sgRNA is an important component of the CRISPR gene knockout system and can bind to the CAS9 protein, guiding the CAS9 enzyme to target genomic DNA for cleavage of the DNALI1 gene.

[0009] The drug provided by this invention also includes cationic liposomes. In this invention, cationic liposome Lipofectamine 3000 is used as the transfection reagent.

[0010] In some embodiments, the DNALI1 gene inhibitor is a siRNA targeting DNALI1, consisting of complementary sense and antisense strands, wherein the sense strand is a nucleotide sequence as shown in SEQ ID NO.1 and the antisense strand is a nucleotide sequence as shown in SEQ ID NO.2; or, the sense strand is a nucleotide sequence as shown in SEQ ID NO.3 and the antisense strand is a nucleotide sequence as shown in SEQ ID NO.4; or, the sense strand is a nucleotide sequence as shown in SEQ ID NO.5 and the antisense strand is a nucleotide sequence as shown in SEQ ID NO.6.

[0011] Furthermore, the DNALI1 gene inhibitor used is a siRNA targeting DNALI1, consisting of complementary sense and antisense strands.

[0012] Furthermore, the sense strand of the DNALI1 siRNA is the nucleotide sequence shown in SEQ ID NO.1, and the antisense strand of the DNALI1 siRNA is the nucleotide sequence shown in SEQ ID NO.2.

[0013] Furthermore, the sense strand of DNALI1 siRNA has the nucleotide sequence shown in SEQ ID NO.3, and the antisense strand of DNALI1 siRNA...

[0014] The nucleotide sequence is as shown in SEQ ID NO.4.

[0015] Furthermore, the sense strand of DNALI1 siRNA has the nucleotide sequence shown in SEQ ID NO.5, and the antisense strand of DNALI1 siRNA has the nucleotide sequence shown in SEQ ID NO.6.

[0016] Furthermore, the glioma cells are human glioma U251 cells.

[0017] The pharmaceutical product of this invention also includes other therapeutic agents selected from other drugs used to treat gliomas. The dosage form of the drug is an injection solution or a powder for injection.

[0018] Advantages and beneficial effects of the present invention:

[0019] This invention is the first to discover that the expression of the DNALI1 gene in glioma patient tumor tissue is significantly higher than in normal tissue. Furthermore, high expression of the DNALI1 gene is associated with poor prognosis in glioma patients. Inhibiting the expression of the DNALI1 gene in glioma cells can effectively suppress cell proliferation and invasion. Therefore, DNALI1 can serve as an important molecular target for the prevention and treatment of glioma, potentially providing a powerful means for targeted therapy of glioma. Attached Figure Description

[0020] Figure 1 This shows the difference in DNALI1 gene expression between tumor tissue and normal brain tissue from patients with different grades of glioma in the TCGA database;

[0021] Figure 2 This study demonstrates the impact of DNALI1 gene expression on the survival rate of patients with glioma.

[0022] Figure 3 This study demonstrates the effects of different siRNAs on DNALI1 gene expression in human glioma cells.

[0023] Figure 4This study demonstrates the effect of siRNA knockdown of DNALI1 gene expression on glioma cell proliferation.

[0024] Figure 5 This study demonstrates the effect of siRNA knockdown of DNALI1 gene expression on glioma cell invasion. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or as recommended by the reagent manufacturer.

[0026] Example 1: Differences in DNALI1 gene expression between normal brain tissue and glioma tissue in the TCGA database.

[0027] TCGA (The Cancer Genome Atlas) is a database jointly developed by the National Cancer Institute and the National Human Genome Institute in 2006. It collects clinical data, genomic variations, mRNA expression, miRNA expression, and methylation data for various human cancers. This data is a crucial source of information for cancer researchers. This study downloaded RNA-seq data of gliomas from the TCGA dataset (https: / / portal.gdc.com) and performed statistical analysis using R software v4.0.3. The expression distribution of the DNALI1 gene in different grades of glioma tissues and normal tissues is shown below. Figure 1 As shown in the figure, the horizontal axis represents different tissue samples, and the vertical axis represents the distribution of gene expression. A p-value less than 0.05 was considered statistically significant. The results indicate that DNALI1 expression was significantly higher in glioma tissues of different grades than in normal brain tissues.

[0028] Example 2: The effect of DNALI1 gene expression on the survival rate of glioma patients

[0029] The Chinese Glioma Genome Atlas (CGGA) database contains clinical and sequencing data from over 2,000 brain tumor samples from patients in China and is equipped with a web application for data storage and exploration. Currently, CGGA includes image-genomic data (274 samples), scRNA-seq data (6,148 cells, 73 regions from 14 patients), whole-exome sequencing data (286 samples), mRNA sequencing data (693 samples), mRNA microarray data (325 samples), mRNA microarray data (301 samples), DNA methylation data (159 samples), microRNA microarray data (198 samples), and matched clinical data. In this study, we analyzed the impact of DNALI1 gene expression on the survival of glioma patients using mRNA sequencing data mRNAseq-325 (CGGA325) and corresponding clinical data. First, based on the median DNALI1 expression level in glioma patients, patients were divided into high and low groups. Then, the Kaplan-Meier survival curves were plotted using an online tool in the CGGA database, and the results are as follows: Figure 2 The results suggest that high expression of DNALI1 is a high-risk factor for poor prognosis in patients with glioma.

[0030] Example 3: Knockdown of DNALI1 gene expression in human glioma U251 cells using siRNA

[0031] We purchased the human glioma U251 cell line from ATCC (Manassas, VA, USA). The cells were cultured in a humid environment at 37°C and 5% CO2 in Dulbecco-modified Eagle medium supplemented with 10% fetal bovine serum. Two × 10⁶ cells were cultured the day before siRNA transfection. 5 U251 cells were seeded in 6-well plates and cultured at 37°C with 5% CO2 for 24 h, and then transfected according to the Lipofectamine 3000 instructions.

[0032] The following are three sequences used to knock down DNALI1 gene expression:

[0033] siRNA1#

[0034] The justice chain is 5'-CCGAAGUAGUGAUGUUAAUUA-3' (SEQ ID NO.1)

[0035] The antisense chain is 5'-AUUAACAUCACUACUUCGGGG-3' (SEQ ID NO.2)

[0036] siRNA2#

[0037] The justice chain is 5'-AAGUAGUGAUGUUAAUUAACA-3' (SEQ ID NO.3)

[0038] The antisense chain is 5'-UUAAUUAACAUCACUACUUCG-3' (SEQ ID NO.4)

[0039] siRNA3#

[0040] The justice chain is 5'-AGGAGCAGUUAGACUUAAAGC-3' (SEQ ID NO.5)

[0041] The antisense chain is 5'-UUUAAGUCUAACUGCUCCUGG-3' (SEQ ID NO.6)

[0042] Forty-eight hours after transfection with siRNA, total protein was extracted, and the expression of the DNALI1 gene in different experimental groups was detected by Western blot. Cells were lysed using RIPA lysis buffer containing PMSF to extract total protein, and protein concentration was determined using the BCA method. Subsequently, proteins were separated by electrophoresis on a 10% SDS-PAGE gel. After electrophoresis, the proteins were electrotransferred to a PVDF membrane, blocked with TBST containing 5% skim milk powder for 2 hours, and then washed three times with TBST. The washed membrane was incubated overnight at 4°C with diluted DNALI1 antibody (Santa Cruz Biotechnology, 1:1000), followed by three washes with TBST; then incubated with secondary antibody m-IgG2b BP at room temperature for 1.5 hours, followed by three washes with TBST; finally, ECL was used for imaging. Experimental results are shown below. Figure 3 siRNA1#, siRNA2# and siRNA3# can all significantly downregulate the expression of DNALI1 protein in glioma cells.

[0043] Example 4: Effect of knockdown of DNALI1 gene expression on glioma cell proliferation

[0044] To investigate the effect of downregulating DNALI1 gene expression on glioma cell proliferation, we used the MTT assay to detect cell proliferation. Human glioma U251 cells were cultured in Dulbecco-modified Eagle medium containing 10% fetal bovine serum at 37°C and 5% CO2. U251 cells were divided into a control group and different DNALI1 siRNA knockdown groups. Glioma cells were transfected with the three siRNAs described in Example 3 for 48 hours and seeded at an initial density of 3000 cells / well in 96-well plates. After 48 hours, 5 mg / mL MTT was added to each well (20 μL per well). After 4 hours of incubation, 150 μL of DMSO was added to dissolve the crystals for 20 min, and the experimental data were recorded at 570 nm using a microplate reader. Results are shown below. Figure 4 Cell proliferation in the DNALI1 siRNA group was significantly lower than that in the control group (P<0.001), demonstrating that downregulating DNALI1 gene expression can effectively inhibit glioma cell proliferation.

[0045] Example 5: Effect of knocking down DNALI1 gene expression on glioma cell invasion

[0046] The effect of knocking down DNALI1 gene expression on the invasion of U251 glioma cells was investigated using a Transwell assay. The Transwell assay utilizes the serum concentration difference between the upper and lower chambers to induce cell migration to the lower chamber. During migration, cells pass through the Matrigel. After fixation and staining, the cells are photographed under a microscope, and their invasive ability is determined by the number of cells in the field of view. Before the experiment, Transwell chambers were placed in 24-well plates, and Matrigel was diluted with basal medium at a ratio of 1:6. 60 μL of Matrigel was added to each well, and the plates were incubated at 37°C for 4 hours. After incubation, the plates were dried on a sterile surface. Before use, the Transwell chambers containing Matrigel were incubated for 1 hour, and then cells were seeded. DNALI1 gene expression was knocked down using siRNA3# as described in Example 3. U251 glioma cells were transfected with siRNA3# and cultured for 48 hours. The culture medium was then replaced with medium containing 1% serum, and the cells were pre-starved for 6 hours. The cells were then digested with 0.25% trypsin to adjust the cell concentration to 5 × 10⁻⁶. 4Cells / mL: 200 μL was added to the upper chamber of a Transwell chamber, and 600 μL of complete culture medium containing 10% fetal bovine serum was added to the lower chamber. The chambers were incubated at 37°C and 5% CO2 for 24 h. After 24 h, the culture medium in the chambers was discarded, and the cells were fixed with pre-cooled methanol for 1 h. The cells were then washed with pre-cooled 1×PBS, stained with crystal violet for 30 min, and photographed using an inverted microscope. The number of cells reflected the invasiveness of the tumor cells. Results are as follows: Figure 5 As shown, compared with the control group, the invasion rate of cells in the DNALI1siRNA group was significantly reduced (P<0.001), suggesting that the DNALI1 gene is a key gene affecting glioma cell invasion.

[0047] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all changes falling within the meaning and scope of the equivalents of the claims are intended to be included within the scope of the invention. Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of a DNALI1 gene inhibitor in the preparation of a glioma drug, wherein the glioma drug is a drug for the prevention and treatment of glioma; The DNALI1 gene inhibitor is one of siRNA1#, siRNA2# and siRNA3#; The sense strand of siRNA1# is the sequence shown in SEQ ID NO.1, and the antisense strand is the sequence shown in SEQ ID NO.2; The sense strand of siRNA2# is the sequence shown in SEQ ID NO.3, and the antisense strand is the sequence shown in SEQ ID NO.4; The sense strand of siRNA3# is the sequence shown in SEQ ID NO.5, and the antisense strand is the sequence shown in SEQ ID NO.6.

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