Application of SOX18 as target spot in glioblastoma radiotherapy drug

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of SOX18 as a target spot in glioblastoma radiotherapy drugs. According to the invention, SOX18 is taken as a target spot, and RAD54L expression is down-regulated by specifically knocking out the SOX18 gene, so that DNA damage repair is inhibited to sensitize GBM radiotherapy; in a tumor model, deficiency of SOX18 significantly inhibits DNA damage repair of tumors and significantly sensibilizes radiotherapy, thereby effectively inhibiting development of glioblastoma.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of SOX18 as a target in radiotherapy drugs for glioblastoma. Background Technology

[0002] Glioblastoma (GBM) is the most aggressive and deadliest primary brain tumor in adults, with an extremely poor prognosis. Although radiotherapy and chemotherapy are the most effective non-surgical treatments for GBM patients, treatment resistance is common. GBM mediates radiotherapy resistance through highly efficient DNA damage repair, allowing it to survive and proliferate under genotoxic stress.

[0003] Radiotherapy has been proven to significantly prolong the overall survival of GBM patients. Theoretically, radiotherapy can kill all GBM cells; however, heterogeneity in radiation response among tumor cells leads to differences in efficacy. Radioresistance can cause GBM recurrence after radiotherapy, severely impacting its effectiveness. Its molecular mechanisms involve three core processes: enhanced DNA damage repair, cell cycle arrest, and apoptosis escape. Among these, homologous recombination repair (HRR) plays a crucial role in DNA double-strand break repair, and upregulation of HRR function is a typical characteristic of GBM and other malignant tumors. Therefore, finding new therapeutic strategies and drug targets targeting HRR is of significant clinical importance. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention aims to provide an application of SOX18 as a target in glioblastoma radiotherapy. By specifically knocking out the SOX18 gene and downregulating RAD54L expression, DNA damage repair is inhibited, thereby enhancing the sensitization of GBM radiotherapy. In tumor models, SOX18 deficiency significantly inhibits tumor DNA damage repair and significantly enhances radiotherapy sensitization, thereby effectively inhibiting glioblastoma progression.

[0005] To achieve the above objectives, the technical solution of the present invention is implemented as follows: Previous studies explored the molecular mechanisms of malignant progression of glioma and found that: (1) GBM upregulates transcription factor SOX18 to activate homologous recombination repair, enhances DNA repair capacity and confers radioresistance; SOX18 transcriptionally regulates the core subunit RAD54L of HRR, directly activating homologous recombination repair and enhancing DNA damage repair capacity. (2) Radiotherapy produces a large amount of ROS (reactive oxygen species) in tumor cells, and ROS can upregulate the total O-GlcNAc glycosylation level in cells; when DNA damage occurs, O-GlcNAc glycosylation modification stabilizes SOX18 protein and promotes its nuclear translocation, and transcriptionally regulates RAD54L to promote efficient HRR.

[0006] Therefore, the first objective of this invention is to provide the use of an inhibitor targeting SOX18 in the preparation of a radiotherapy drug for treating glioblastoma.

[0007] A second objective of this invention is to provide the application of an inhibitor targeting SOX18 in the preparation of a radiosensitizing drug for the treatment of glioblastoma.

[0008] Furthermore, the inhibitor is a drug that silences SOX18 expression.

[0009] Furthermore, the inhibitor is an shRNA designed based on SOX18.

[0010] The nucleotide sequence of the shRNA is shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. The shRNA is stably integrated into the genome via a vector (such as lentivirus) and continuously generates siRNA.

[0011] A third objective of this invention is to provide an inhibitor targeting SOX18, wherein the inhibitor is shRNA, and the nucleotide sequence of the shRNA is shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0012] A fourth objective of this invention is to provide a targeted drug for treating glioblastoma, said drug comprising the above-mentioned inhibitor or a drug comprising silencing RAD54L.

[0013] Among them, the SOX18 gene sequence is a publicly available gene sequence (NCBI Reference Sequence: NM_018419); the RAD54L gene sequence is a publicly available gene sequence (NCBI Reference Sequence: NM_003579.4).

[0014] Furthermore, the targeted drug also includes pharmaceutically acceptable excipients.

[0015] Furthermore, the pharmaceutically acceptable excipient is any one or more of the following: excipients, stabilizers, diluents, binders, preservatives, lubricants, and antioxidants.

[0016] Furthermore, the pharmaceutically acceptable excipient is selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

[0017] Furthermore, the dosage forms of the targeted drug include, but are not limited to: tablets, liquids, capsules, powders, suppositories, granules, pills, sprays, or liniments.

[0018] Furthermore, the administration method of the targeted drug is selected from oral, intravenous, local, intradermal, or subcutaneous injection.

[0019] Advantages compared to existing technologies: This invention, through database bioinformatics analysis, determined that there are significant differences in the expression of SOX18 in glioblastoma tissue and adjacent non-tumor tissue, indicating that SOX18 is crucial for the proliferation and in vivo growth of glioblastoma. Furthermore, through SOX181 knockdown experiments, RAD54L overexpression experiments, and in vivo experiments, it was found that SOX18 knockdown can induce G2 phase arrest in GBM cells, while RAD54L overexpression reverses the inhibitory effect of SOX18 knockdown on GBM cells. Silencing RAD54L can reverse SOX18-driven radioresistance and restore in vivo radiosensitivity.

[0020] This invention demonstrates that SOX18 knockdown inhibits glioblastoma growth both in vivo and in vitro, promotes cell cycle arrest in the G2 / M phase, and inhibits DNA damage repair. Targeting SOX18-RAD54L in combination with radiotherapy effectively inhibits glioblastoma growth and prolongs survival in mice in vivo. Therefore, using SOX18 as a drug target can be used to develop drugs for the treatment of glioblastoma. Attached Figure Description

[0021] Figure 1 The study aimed to investigate the correlation between high SOX18 expression and GBM progression. (A) SOX18 expression levels in different grades of gliomas based on the TCGA database; (B) the correlation between SOX18 expression and patient prognosis using Kaplan-Meier analysis based on TCGA database data; (C) SOX18 expression in GBM tissues and adjacent non-tumor tissues detected by Western blot; (D) SOX18 expression in GBM tissues and corresponding adjacent non-tumor tissues assessed by immunohistochemistry (IHC); (E) Western blot analysis of SOX18 expression in different GBM cell lines and normal human astrocytes (NHA); and (F) SOX18 expression in stably transfected cell lines detected by Western blot.

[0022] Figure 2 SOX18 promotes GBM proliferation in vitro and in vivo; (A) shows the proliferation capacity of GBM cell lines as determined by the EdU assay, and (B) shows the results of hematoxylin-eosin (H&E) staining of mouse brain sections.

[0023] Figure 3 To clarify the regulatory mechanism of SOX18 on GBM through proteomics; (A) is a data-independent acquisition (DIA) proteomics analysis performed in U251 cells with SOX18 knockdown; (B) is a volcano plot showing the up / down regulation of proteins after SOX18 knockdown in U251 cells; (C) is a KEGG pathway enrichment analysis of differentially expressed proteomics genes; and (D) is a gene ontology (GO) functional enrichment analysis of differentially expressed proteomics genes.

[0024] Figure 4 Inhibition of SOX18 can promote GBM cell cycle arrest; (A) shows the protein-protein interaction (PPI) network of the top 50 differentially expressed genes (red represents upregulated proteins, green represents downregulated proteins), and (BC) shows cell cycle analysis performed by flow cytometry and Western blot.

[0025] Figure 5 SOX18 targets RAD54L transcription; (A) Chromatin immunoprecipitation sequencing (ChIP-Seq) analysis using the Illumina NovaSeq 6000 platform, (B) SOX18 binding sites in the U251 cell genome identified by ChIP-seq, (C) Venn diagram showing the intersection of downregulated genes identified by proteomics and SOX18 binding genes in promoter regions identified by ChIP-seq, and (D) Western blot analysis of CDK1 and Cyclin B2 protein expression.

[0026] Figure 6 SOX18 promotes DNA damage repair through RAD54L; (A) is a schematic diagram of potential SOX18 binding sites in the promoter region of the RAD54L gene; (B) DNA gel electrophoresis shows the enrichment of PCR products in the ChIP experiment; (C) luciferase reporter gene assay shows the regulatory effect of SOX18 on the RAD54L promoter; (D) Western blot analysis shows the expression level of RAD54L; and (E) immunofluorescence assay shows the expression of ph2A.X protein.

[0027] Figure 7 To investigate the inhibitory effect of RAD54L overexpression on SOX18 knockdown on glioblastoma progression; (A) Western blot analysis of changes in the expression levels of CDK1, Cyclin B1 and ph2A.X proteins; (B) flow cytometry analysis of changes in cell cycle after RAD54L overexpression; and (C) EdU assay analysis of cell proliferation capacity.

[0028] Figure 8To construct a nude mouse intracranial xenograft model with SOX18 knockdown and RAD54L overexpression; (A) is a schematic diagram of the establishment of the nude mouse intracranial xenograft model, and (B) is an assessment of the fluorescence intensity and size of intracranial tumors in each group of nude mice by in vivo fluorescence imaging.

[0029] Figure 9 To enhance the in vivo radiotherapy effect of GBM by targeting the SOX18-RAD54L axis; (A) is the curve of total fluorescence intensity changing over time, reflecting the dynamics of tumor burden; (B) is the survival analysis of tumor-bearing mice; (C) is the curve of body weight change at different time points; (D) is a typical H&E stained section showing the difference in tumor size.

[0030] Figure 10 Immunofluorescence was used to detect the expression of related proteins in tissues; green represents the immunofluorescence of SOX18 in the tumor, red represents the immunofluorescence of ph2A.X, yellow represents the immunofluorescence of RAD54L, and co-localization immunofluorescence. Detailed Implementation

[0031] The above-described technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. However, the present invention is not limited to these embodiments, nor are these embodiments limited in any way.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the formulations involved in the following examples are all commercially available products that can be purchased from the market.

[0033] The U87MG cell line is a malignant glioma cell line, while the U251 cell line is derived from human brain astrocytoma. The SOX18 and RAD54L sequences are from the National Center for Biotechnology Information (https: / / www.ncbi.nlm.nih.gov / gene).

[0034] The relevant experimental methods are as follows: 1. Flow cytometry for detecting cell cycle arrest Cells were seeded in 6-well plates and cultured for 24 hours until adherence was achieved and cell confluence reached approximately 50%. Cells were then washed with PBS, digested with trypsin, and the enzyme reaction was terminated by adding complete medium containing FBS. The cell pellet was collected after centrifugation, resuspended in pre-chilled 70% ethanol, and fixed at -20°C for at least 4 hours. The fixed cells were centrifuged again, washed twice with PBS, and resuspended in 0.5 mL of PI / RNase staining buffer (BD, catalog number 550825). After incubation at room temperature in the dark for 15 minutes, cell cycle distribution was analyzed using a CytoFLEX flow cytometer (Beckman Coulter), and data were processed using FlowJo software (version 10.0).

[0035] 2. Western blot detection of gene knockdown levels Blank control U251 cells and U251 cells infected with knockdown lentivirus were added to culture dishes in RIPA solution mixed with 1% PMSF. Lysis buffer was collected, and cells were disrupted using an ultrasonic homogenizer. The lysate was centrifuged at 14000 rpm for 15 min in a refrigerated centrifuge to obtain protein. Cellular protein was quantified using the BCA method. Proteins were denatured by boiling with 5×SDS buffer. Equal amounts of protein were loaded onto PVDF membranes and immobilized by electrophoresis and transfer. Antigenic epitopes were blocked in skim milk, and antibodies were incubated with corresponding primary antibody dilutions. After rinsing, secondary antibodies were incubated, and the membranes were developed in ECL chemiluminescence buffer to detect the expression of internal control and target proteins.

[0036] 3. Constructing a nude mouse orthotopic xenograft tumor model An orthotopic glioblastoma model was established using five-week-old male BALB / c nude mice (Jicui Yaokang). The procedure was briefly as follows: Genetically modified U87MG cells (SOX18 overexpression group, SOX18 overexpression combined with RAD54L knockdown group, and their corresponding control groups) were resuspended in pre-chilled PBS. Under isoflurane anesthesia, 3 × 10⁻⁶ cells were... 5 Cells (suspended in 6 μL PBS) were implanted intracranially into the right frontal lobe of mice, with the location coordinates 2 mm lateral and 1 mm posterior to the anterior fontanelle as a reference point. Tumor growth was monitored using bioluminescence imaging with the IVISLumina III in vivo imaging system. Starting 15 days after cell implantation, mice were given local radiotherapy (2 Gy) every 3 days. Mice exhibiting neurological symptoms or seizures were euthanized by cervical dislocation. At the experimental endpoint, brain tissue was collected, fixed in 4% paraformaldehyde, and then subjected to hematoxylin-eosin (H&E) staining and immunohistochemical (IHC) analysis.

[0037] The present invention will be further described in detail below with reference to specific embodiments: Example 1: Detection of the role of SOX18 in the progression of glioblastoma To investigate the expression of SOX18 in gliomas, clinical data and sequencing information obtained from the TCGA database were analyzed, and the results are as follows: Figure 1 and 2 As shown.

[0038] from Figure 1 The results show that the expression level of SOX18 is positively correlated with the grade of glioma, and is significantly elevated in glioblastoma (e.g., Figure 1 (As shown in A). Furthermore, patients with high SOX18 expression have a significantly worse prognosis (e.g., ...). Figure 1 (As shown in B in the figure). Similarly, Western blot and immunohistochemical analyses of GBM specimens showed that, where T represents tumor tissue, N represents adjacent normal tissue, and the numbers are paired numbers, the expression of SOX18 in GBM tissue was significantly higher than that in the paired adjacent normal tissue (e.g., T represents tumor tissue, N represents adjacent normal tissue, and the numbers are paired numbers). Figure 1 (As shown in C and D). Furthermore, compared to normal human fibroblasts (NHA), the expression level of SOX18 protein was significantly increased in all five glioblastoma cell lines (U87MG, U251, LN229, U118, T98) (as shown in C and D). Figure 1 As shown in E).

[0039] To further investigate the role of SOX18 in the biological function of GBM cells, stable cell lines with SOX18 overexpression and knockdown were established in U87MG and U251 cells. sh-NC served as the knockdown control group, sh-1 and sh-2 represented SOX18 knockdown, EV represented the overexpression control group, and OV represented SOX18 overexpression. Western blot results showed successful cell line construction. Figure 1 As shown in F, sh-NC, sh-1, and sh-2 have the nucleotide sequences shown in Table 1. EdU assay (5-ethynyl-2'-deoxyuridine assay) indicated that SOX18 knockdown significantly reduced cell proliferation (e.g., ...). Figure 2 (As shown in A). Furthermore, U251 cells were injected into nude mice to construct orthotopic xenografts, followed by hematoxylin and eosin (H&E) staining. The results showed that SOX18 knockdown significantly inhibited GBM growth (as shown in A). Figure 2 (As shown in B in the diagram).

[0040] In summary, the above results indicate that SOX18 is crucial for the proliferation and in vivo growth of GBM.

[0041] Example 2: Study on SOX18 knockdown-induced GBM cell cycle arrest To investigate the molecular mechanism by which SOX18 affects the biological function of glioblastoma, proteomic analysis was performed on SOX18-knockdown U251 cells, and the results were compared with control cells. The experimental procedure and results are as follows: Figure 3 As shown in A in the figure. This analysis identified 1721 differentially expressed proteins, of which 974 proteins were downregulated and 747 proteins were upregulated. The fold change threshold used was greater than 1, and the significance level was P < 0.05 (e.g., ...). Figure 3 (As shown in B). Subsequent KEGG, GO, and PPI analyses of 974 downregulated proteins revealed that SOX18 knockdown significantly disrupted cell cycle and DNA repair-related pathways (e.g., ...). Figure 3 CD in Figure 4 (As shown in A in the diagram). Flow cytometry and Western blot analysis further demonstrated that SOX18 knockdown induced G2 phase arrest in GBM cell lines and significantly reduced the expression of G2 phase-related cell cycle proteins CDK1 and Cyclin B1 (as shown in the diagram). Figure 4 (As shown in BC). These results indicate that SOX18 knockdown can induce G2 phase arrest in GBM cells.

[0042] Example 3: SOX18 promotes DNA damage repair by transcriptionally regulating RAD54L. To further investigate the transcriptional dysregulation of downstream genes associated with SOX18-driven cell cycle arrest in glioblastoma, we used Illumina technology to sequence ChIP-Seq (chromatin immunoprecipitation sequencing) products to locate SOX18 binding sites across the entire genome. The procedure is as follows: Figure 5 As shown in A in the diagram. Subsequently, SOX18 binding sites were identified in U251 cells, most of which were located in intergenic regions, intronic regions, and promoter regions (such as...). Figure 5 (As shown in B). By integrating ChIP-seq and proteomics datasets, six potential targets that may be directly regulated by SOX18 were identified: EML1, MELK, XRCC2, NTMT1, P3H4, and RAD54L (as shown in B). Figure 5 (As shown in C). Three genes closely related to the cell cycle—MELK, XRCC2, and RAD54L—were selected, and related small interfering RNAs were constructed (related nucleotide sequences are shown in Table 1). The results showed that silencing RAD54L significantly inhibited the expression of cell cycle-related proteins (CDK1 and Cyclin B1) and the DNA damage marker ph2A.X (as shown in Table 1). Figure 5 (as shown in D in the diagram).

[0043] Table 1 RNA Sequences

[0044] Furthermore, online computational tools were used to identify potential binding regions of SOX18 and RAD54L. Chromatin immunoprecipitation-PCR (ChIP-PCR) analysis showed that SOX18 directly binds to region 3 of the RAD54L promoter (e.g., Figure 6 (As shown in AB). This binding was further confirmed by dual-luciferase reporter gene assay (RAD54L-WT represents the wild-type RAD54L promoter region, and RAD54L-MUT represents the RAD54L mutant region), which confirmed that SOX18 can directly regulate the expression of RAD54L (e.g., Figure 6 (As shown in C). Knockdown of SOX18 leads to a decrease in RAD54L protein levels (as shown in C). Figure 6 (As shown in D in the diagram). Previous studies have confirmed a strong association between RAD54L and homologous recombination repair. To determine whether SOX18 knockdown leads to DNA damage, further immunofluorescence assays were performed to assess the expression of the DNA damage marker ph2A.X. The results showed that SOX18 knockdown significantly increased the expression of ph2A.X (as shown in D in the diagram). Figure 6 (As shown in E in the figure). These results indicate that SOX18 can directly regulate RAD54L and induce homologous recombination repair.

[0045] Example 4: Study on periodic arrest induced by reversing SOX18 knockdown due to RAD54L overexpression To elucidate the regulatory role of SOX18 in glioblastoma DNA damage repair mediated by RAD54L, SOX18 knockdown experiments were performed in stable RAD54L-overexpressing cell lines. Western blot experiments showed that RAD54L overexpression significantly enhanced the expression levels of G2 phase-related proteins (CDK1, Cyclin B1) and DNA damage markers (ph2A.X). Figure 7 As shown in Figure A, where + indicates transfection with the plasmid and - indicates no transfection with the plasmid. Subsequent experiments showed that RAD54L overexpression effectively alleviated cell cycle arrest induced by SOX18 knockdown and significantly reduced the proportion of GBM cells in G2 phase. Figure 7 (As shown in B in the image). Further EdU detection was performed, with Hoechst representing nuclear staining, EdU representing cells in the proliferative phase, and Merge representing fused images, confirming that RAD54L overexpression significantly promotes cell proliferation. Figure 7 (As shown in C in the figure). These results collectively suggest that RAD54L overexpression may reverse the inhibitory effect of SOX18 knockdown on GBM cells.

[0046] Example 5: In vivo radiotherapy efficacy of targeted SOX18-RAD54L axis-sensitized GBM Given the crucial role of SOX18-mediated RAD54L transcriptional regulation in promoting GBM radiotherapy resistance, an in vivo experiment was conducted to investigate whether RAD54L knockdown could eliminate SOX18-driven treatment resistance. The experimental procedure is as follows: Figure 8 As shown in A, NC is the transfection control group, IR is the radiotherapy group, OE is the SOX18 overexpression group, and OE+sh is the SOX18 overexpression and RAD54L knockdown group.

[0047] Experiments showed that SOX18 overexpression conferred radioprotective effects on GBM xenograft models, while inhibition of RAD54L significantly weakened the SOX18-mediated radioprotective effect (e.g., Figure 8 B in Figure 9 (As shown in A). Furthermore, RAD54L deletion alleviated weight loss and prolonged survival in tumor-bearing mice overexpressing SOX18 (e.g., ...). Figure 9 (As shown in BC). Hematoxylin-eosin (H&E) staining further validated the above results by displaying consistent histopathological features (e.g., BC). Figure 9 (As shown in D in the figure). Simultaneous experiments revealed that RAD54L knockdown significantly upregulated the expression level of the DNA damage marker ph2A.X in SOX18-overexpressing GBM tissues (e.g., as shown in D in the figure). Figure 10 (As shown). These results confirm that silencing RAD54L can reverse SOX18-driven radioresistance and restore in vivo radiosensitivity.

[0048] In summary, this invention targets SOX18 and can influence glioblastoma proliferation by controlling its expression level. Knocking down or silencing SOX18 can induce G2 phase arrest in GBM cells and slow down the growth of glioblastoma tissue. SOX18 is also a key target affecting the radiotherapy efficacy of glioblastoma. Overexpression of RAD54L in glioblastoma tissue after radiotherapy may reverse the inhibitory effect of SOX18 knockdown on GBM cells, while silencing RAD54L can reverse SOX18-driven radioresistance and restore in vivo radiosensitivity. Therefore, targeting the SOX18-RAD54L axis can enhance the in vivo radiotherapy efficacy of GBM cells, providing new evidence for the role of SOX18 in enhancing radiosensitivity in glioblastoma radiotherapy.

[0049] Finally, it should be emphasized that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of SOX18-targeting inhibitors in the preparation of radiotherapy drugs for the treatment of glioblastoma.

2. Application of SOX18-targeting inhibitors in the preparation of radiosensitizing drugs for the treatment of glioblastoma.

3. The application according to claim 1 or 2, characterized in that, The inhibitor is a drug that silences SOX18 expression.

4. The application according to claim 1 or 2, characterized in that, The inhibitor is an shRNA designed based on SOX18.

5. The application according to claim 4, characterized in that, The nucleotide sequence of the shRNA is shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:

3.

6. An inhibitor targeting SOX18, characterized in that, The inhibitor is shRNA, and the nucleotide sequence of the shRNA is shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:

3.

7. A targeted drug for treating glioblastoma, characterized in that, The drug comprises the inhibitor of claim 6 or a drug comprising silencing RAD54L.

8. The targeted drug according to claim 7, characterized in that, The targeted drug also includes pharmaceutically acceptable excipients.

9. The targeted drug according to claim 7, characterized in that, The pharmaceutically acceptable excipients are any one or more of the following: excipients, stabilizers, diluents, binders, preservatives, lubricants, and antioxidants.

10. The targeted drug according to claim 7, characterized in that, The pharmaceutically acceptable excipients are selected from at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, magnesium stearate, and mineral oil.

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