Application of si-SH3RF3-AS1 and CPT combined delivery system in the preparation of glioma drugs

Abstract

The present invention belongs to the field of biomedicine technology and relates to the application of a si-SH3RF3-AS1 and CPT combined delivery system in the preparation of glioma drugs. The present invention also proposes that the substance that inhibits the expression of SH3RF3-AS1 is a nanoparticle containing siRNA and CPT, the particle size of the nanoparticle is 200.9±4.371nm, the siRNA concentration in the nanoparticle is 50nM, and the CPT concentration is 20μM. The present invention proposes siRNA targeting SH3RF3-AS1 and a nanodrug containing the small interfering RNA. It has been verified that the nanodrug has a significant inhibitory effect on the proliferation of U251 and G1 cells, and the nanodrug synthesis process is controllable, with stable and reliable quality, providing new possibilities for the treatment of gliomas.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to the application of a si-SH3RF3-AS1 and CPT combined delivery system in the preparation of glioma drugs. Background Art

[0002] Gliomas are the most common primary tumors of the central nervous system, with both morbidity and mortality rates increasing annually. High-grade gliomas are highly malignant, lacking effective early diagnosis and treatment options, resulting in an extremely poor prognosis. Surgical resection of the tumor is often incomplete, leading to loss of neurological function, and resistance to chemotherapy drugs poses a serious clinical challenge. Currently, there is an urgent need to resolve the contradiction between the poor prognosis of gliomas and the limitations of treatment methods. Finding new therapeutic targets and treatments for gliomas is imperative. Summary of the Invention

[0003] In response to the problems existing in traditional brain glioma treatment, the present invention proposes a novel si-SH3RF3-AS1 and camptothecin (CPT) combined delivery system for the preparation of glioma drugs.

[0004] In order to achieve the above object, the present invention is implemented by adopting the following technical solutions:

[0005] The present invention proposes the use of a substance with SH3RF3-AS1 as a target, that is, a substance that reduces or inhibits the expression of SH3RF3-AS1, in the preparation of a drug for treating glioma, as well as the use of a substance that reduces or inhibits the expression of SH3RF3-AS1 in a preparation for enhancing the drug sensitivity of glioma patients, and the use of a substance that reduces or inhibits the expression of SH3RF3-AS1 in a preparation for improving the prognosis of patients; the sequence of SH3RF3-AS1 is shown in SEQ ID NO: 1 in the sequence listing.

[0006] The present invention also proposes the use of a substance for detecting the expression level of SH3RF3-AS1 in a drug or diagnostic kit for glioma diagnosis or prognosis prediction.

[0007] The present invention verifies that substances that inhibit the expression of SH3RF3-AS1 can play a role in inhibiting the proliferation and / or DNA replication of U251 cells and primary glioma cells G1.

[0008] The present invention proposes that the substance for inhibiting the expression of SH3RF3-AS1 is siRNA-SH3RF3-AS1 or a vector containing siRNA-SH3RF3-AS1, and the sequence of siRNA-SH3RF3-AS1 is

[0009] si-RNA-1 forward: GGGCUAACAUCAGAAGAGUTT,

[0010] si-RNA-1 reverse: ACUCUUCUGAUGUUAGCCCTT;

[0011] si-RNA-2 forward: CCCAACCUGUGAUAAUUAUTT,

[0012] si-RNA-2 reverse: AUAAUUAUCACAGGUUGGGTT.

[0013] The present invention also proposes that the substance that inhibits SH3RF3-AS1 expression is a nanoparticle containing siRNA-SH3RF3-AS1. As a preferred embodiment, the particle size of the nanoparticle is 200.9±4.371 nm, the siRNA concentration in the nanoparticle is 50 nM, and the CPT concentration is 20 μM.

[0014] Preferably, the steps for preparing the nano drug particles are as follows:

[0015] (1) Dissolve N-butylethanolamine and triethylamine in acetonitrile, mix well and cool to 0°C, add methacryloyl chloride dropwise, stir at low temperature for reaction and filter, extract the filtrate with dichloromethane and evaporate under reduced pressure to obtain monomer DBAMA.

[0016] 2-Hydroxyethyl disulfide and triethanolamine were dissolved in tetrahydrofuran, stirred evenly and cooled to 0°C. Methacryloyl chloride was then slowly added. After stirring at low temperature for reaction, the mixture was stirred at room temperature overnight. After filtration, the filtrate was extracted with ethyl acetate and evaporated under reduced pressure to obtain 2-[(2-hydroxyethyl)disulfide]ethyl-2-methyl-2-acrylate.

[0017] CPT, 4-dimethylaminopyridine and triphosgene were dissolved in dichloromethane and stirred to obtain solution A. 2-[(2-hydroxyethyl)dithio]ethyl-2-methyl-2-acrylate was dissolved in dichloromethane to obtain solution B. Solution B was slowly poured into solution A and stirred overnight. The mixture was concentrated under reduced pressure and passed through a silica gel column chromatography to obtain a yellow product OH-2S-CPT.

[0018] (2) 4-Cyano-4-[[(dodecylthio)thiomethyl]thio]pentanoic acid, 4-dimethylaminopyridine and N,N'-dicyclohexylcarbodiimide were dissolved in dichloromethane and stirred at room temperature to obtain a mixture C. The dichloromethane solution containing methoxypolyethylene glycol was added dropwise to the mixture C and stirred at room temperature for 36 h. After filtration, the filtrate was concentrated and purified by three rounds of precipitation in cold ether. The precipitate was collected and dried under vacuum overnight to obtain PEG-DCT as a light yellow powder.

[0019] (3) The PEG-DCT obtained in step (2) and the DBAMA and 2,2'-azobis(2-methylpropionitrile) obtained in step (1) were dissolved in dioxane and placed in a Schlenk tube; after four freeze-pump-thaw cycles, the mixture was transferred to an oil bath and incubated with stirring for 48 h; the mixture was then cooled to room temperature, the product was dialyzed with deionized water, and freeze-dried to obtain PEG-PDBAMA powder.

[0020] (4) The PEG-PDBAMA powder obtained in step (3), the OH-2S-CPT obtained in step (1), and azobisisobutyronitrile were dissolved in a mixture of dioxane and dimethyl sulfoxide and placed in a Schlenk tube. After four freeze-pump-thaw cycles, the mixture was heated to 80°C to initiate polymerization and incubated with stirring for 48 hours. The mixture was cooled and purified by three rounds of precipitation in cold ether. A red powder was obtained after vacuum drying, which was designated as PEG-PDBAMA-PCPT.

[0021] (5) The PEG-PDBAMA-PCPT powder prepared in step (4) was dispersed in dimethyl sulfoxide to obtain dispersion C; siRNA-SH3RF3-AS1 was dissolved in dimethyl sulfoxide to obtain dispersion D; dispersion C and dispersion D were simultaneously added dropwise to deionized water and ultrasonically treated; dimethyl sulfoxide was removed by dialyzing in deionized water overnight to obtain PEG-PDBAMA-PCPT / siRNA-SH3RF3-AS1.

[0022] (6) U251 cells were washed three times in PBS, dispersed in a hypotonic buffer solution, and frozen at -80°C. After thawing, the resulting precipitate was collected by centrifugation and washed with PBS to obtain U251 cell membranes, which were suspended in PBS and mixed with PEG-PDBAMA-PCPT / siRNA-SH3RF3-AS1. The membranes were passed through a polycarbonate membrane using a liposome extruder to obtain PEG-PDBAMA-PCPT / siRNA-SH3RF3-AS1@U251, which was then freeze-dried to obtain nanomedicines.

[0023] Compared with the prior art, the advantages and positive effects of the present invention are:

[0024] The present invention proposes siRNA-SH3RF3-AS1 with SH3RF3-AS1 as the target and a nanodrug containing the small interfering RNA. It has been verified that the nanodrug has a significant inhibitory effect on the proliferation of U251 and G1 cells. The nanodrug synthesis process is controllable and the quality is stable and reliable. It provides new possibilities for the treatment of gliomas and provides a new drug for inhibiting proliferation for the treatment of gliomas. It can also enhance the therapeutic effect of chemotherapy drugs such as CPT in combination with other drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 MeRIP-seq results for glioma tissues. A: Distribution of m6A modifications at the start codon, CDS, and stop codons of mRNAs and lncRNAs in normal and glioma tissues. B: Distribution of m6A modifications in normal and glioma tissues. C: Highly enriched and conserved consensus RRACH sequences in normal and glioma tissues. D: Genes differentially modified with m6A between normal and glioma tissues (|log2FC| > 2); red indicates hypermethylation, while blue indicates demethylation. E: GO enrichment analysis results: Gene functions differentially modified with m6A between glioma and normal tissues. F: KEGG enrichment analysis: Gene pathways differentially modified with m6A between glioma and normal tissues. G: Four-quadrant plot showing the difference in m6A modification and RNA expression between normal and glioma tissues (SH3RF3-AS1: demethylated / upregulated). H: Visualization of m6A modification in SH3RF3-AS1.

[0026] Figure 2Regulation of the ALKBH5 / SH3RF3-AS1 / SH3RF3 axis in glioma. A: m6A modification levels of SH3RF3-AS1 in glioma and normal tissues were determined by m6A-RIP-qPCR. B: U251 cells were transfected with Flag-pcDNA3.1-ALKBH5 (Flag-ALKBH5) or Flag-pcDNA 3.1, and SH3RF3-AS1 expression was analyzed by Flag-RIP-qPCR. C: SH3RF3-AS1 expression was analyzed by RT-qPCR in U251 cells transfected with pcDNA3.1-ALKBH5 (over-ALKBH5) or pcDNA3.1 (over-control) and primary glioma G1 cells. D: U251 cells expressing ALKBH5 (over-ALKBH5) or control were treated with actinomycin D for 90 minutes, and SH3RF3-AS1 expression was measured by RT-qPCR. E: SH3RF3-AS1 subcellular localization and expression in U87 and U251 cells using the SH3RF3-AS1 probe (red) (200×). F: SH3RF3 protein and mRNA expression, as well as Rac1 protein expression, were detected in U251 cells transfected with pcDNA3.1-SH3RF3-AS1 (SH3RF3-AS1 overexpression) or siRNA-SH3RF3-AS1 (siRNA-1, siRNA-2). siRNA-SH3RF3-AS1 transfection significantly reduced SH3RF3 protein expression. G: SH3RF3-AS1-overexpressing / control U251 cells were treated with actinomycin D for 4 hours, and SH3RF3 mRNA expression was measured by RT-qPCR. Student's t-test was used. * P <0.05,*** P <0.001.

[0027] Figure 3The regulatory role of the ALKBH5 / SH3RF3-AS1 / SH3RF3 axis in glioma. A and B: Effects of overexpression of ALKBH5 and SH3RF3-AS1, and knockdown of SH3RF3-AS1 on the proliferation of U251 and primary glioma G1 cells. C: Effects of ALKBH5 overexpression, SH3RF3-AS1 overexpression or SH3RF3-AS1 knockdown on the cell cycle of U251 cells and primary glioma G1 cells. Representative images of primary glioma G1 cells are shown on the left, and the percentages of each cell cycle stage of U251 cells and primary glioma G1 cells are shown on the right. D: CCK-8 method was used to determine the effects of ALKBH5 overexpression, SH3RF3-AS1 overexpression or SH3RF3-AS1 knockdown on the IC50 value of CPT-11 in primary glioma G1 cells. Using t-test, * P <0.05, ** P <0.01 and *** P <0.001.

[0028] Figure 4 The role of the ALKBH5 / SH3RF3-AS1 / SH3RF3 axis in the prognosis of glioma patients. A: ALKBH5, SH3RF3-AS1, and SH3RF3 expression levels in patients with different WHO grades. B: The relationship between ALKBH5, SH3RF3-AS1, and SH3RF3 and 5-year overall survival and progression-free survival in glioma patients (Kaplan-Meier survival analysis). C: Glioma tissues with high ALKBH5 expression have a higher percentage of SH3RF3-AS1-positive cells. High ALKBH5 expression: greater than the upper quartile. D: Representative immunofluorescence histochemistry images of 148 glioma specimens. Pink: ALKBH5, white: SH3RF3, red: SH3RF3-AS1, blue: cell nuclei. The expression of SH3RF3-AS1 was analyzed on tissue microarrays using RNAScope® technology. The expression of ALKBH5 and SH3RF3 proteins in glioma tissues was detected using anti-ALKBH5 and anti-SH3RF3 antibodies. All values ​​are expressed as the percentage of positive cells. The Mann-Whitney U test was used. P <0.05,** P <0.01 and *** P <0.001.

[0029] Figure 5 Nano PCPT / siRNA@U251 inhibits glioma growth. A: Characteristics of PEG-PDBAMA-CPT / siRNA@U251: particle size distribution and morphology. Scale bar, 200 nm. B: CPT and SH3RF3-AS1 siRNA nanodrug group (Nano PCPT / siRNA-1+2@U251) than the control group Nano PCPT @U251 had a more significant proliferation inhibitory effect (79.47% vs. 85.47%, P =0.04). C: The combined administration of CPT and si-SH3RF3-AS1 has the greatest inhibitory effect on the survival of U251 cells. U251 cells were treated with Nano-siRNA@U251 and Nano PCPT The survival rates of the cells treated with @U251 were 94.43%±2.06% and 79.18%±4.19%, respectively, while those treated with Nano PCPT The survival rate of siRNA@U251 treatment was 74.94% ± 2.84%. The same pattern was also found in G1 cells. D and E: Tumors in the brain of nude mice were evaluated using in vivo imaging. Nano PCPT / siRNA@U251 inhibits in situ glial formation in nude mice. F:Nano PCPT Tumors in siRNA@U251 cells showed swelling and loosening of the tumors, with clearer borders. G: Expression of SH3RF3 (red) and Rac1 (green) in tumors. SH3RF3-AS1 siRNA inhibited the expression of SH3RF3 and Rac1. * P <0.05, ** P <0.01 and *** P <0.001. Data are expressed as mean ± standard deviation. DETAILED DESCRIPTION

[0030] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention is further described below in conjunction with specific embodiments. It should be noted that, in the absence of conflict, the embodiments of the present application and the features therein can be combined with each other.

[0031] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways than those described herein. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0032] Example 1

[0033] 1. Validation of SH3RF3-AS1 as a glioma-specific lncRNA target

[0034] MeRIP-seq sequencing was performed on three pairs of glioma and normal tissues. MeRIP-seq and data analysis were performed by Shanghai Gene Medical Technology Co., Ltd. This study was approved by the Research Ethics Committee of the Second Hospital of Shandong University (KYLL-2021(KJ)P-003), and patient samples were obtained after obtaining informed consent. MeRIP-seq sequencing identified 24,324 mA peaks in normal tissues and 23,351 mA peaks in glioma tissues. mA modification primarily occurs near the coding regions (CDSs) and 3' untranslated regions (UTRs) of mRNA transcripts in both normal and tumor tissues. In normal tissues, mA peaks were most abundant near CDSs (34.7%) and 3' UTRs (37.3%), followed by stop codons (19.8%). In tumor tissues, m6A peaks were most abundant near CDSs (37.7%) and 3'UTRs (34.5%), followed by stop codons (20.2%) ( Figure 1 A and 1B). The highly enriched and conserved consensus RRACH sequence is shown in Figure 1 C. Among the m6A modified peaks, protein-coding transcripts accounted for 93.94%, lncRNA transcripts accounted for 1.41%, pseudogenes accounted for 0.58%, and other types of transcripts or unmarked transcripts accounted for 4.07%. Most genes showed upregulation of m6A levels ( Figure 1 D). GO analysis showed that differentially m6A-modified genes play a role in transcription and transcriptional regulation ( Figure 1 E), KEGG analysis showed that differentially m6A-modified genes were enriched in cancer proteoglycan, endoplasmic reticulum protein processing and cancer signaling pathways ( Figure 1 F). Correlation analysis was performed on differentially m6A-modified genes and differentially expressed genes in glioma. The top 10 genes with the highest |log2FC| scores were as follows: Figure 1 G. SH3RF3-AS1 is the only glioma-specific lncRNA target, and SH3RF3-AS1 has only one significant m6A peak ( Figure 1 H).

[0035] 2. Regulation of the ALKBH5 / SH3RF3-AS1 / SH3RF3 axis in glioma

[0036] Total RNA was extracted from glioma or normal tissues according to the instructions of the EZNA Total RNA Extraction Kit I (Omega Bio-tek, USA). The RNA content and quality were measured at 260 nm using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). MeRIP experiments were performed according to the instructions of the m6A Methylated RNA Immunoprecipitation Kit (Merck Millipore, USA). After RNA purification, the RNA was purified using PrimeScript PCR. TM RT-qPCR was performed using the RT kit (Takara, China) and the SYBR Green Pro Taq HS qPCR (Rox) kit (Accurate Biotechnology, China) according to the manufacturer's instructions. Each experiment was repeated at least three times. Endogenous β-actin was used as a control, and 2 -ΔΔCt Methods The genes to be tested were relative quantified. The primer sequences for RT-qPCR amplification are shown in Table 1.

[0037] The m6A modification of SH3RF3-AS1 in glioma tissues and normal tissues is shown in Figure 3. Figure 2 A, m6A modification site in glioma tissue: chr2:109128916 is significantly demethylated. Bioinformatics analysis of the Cancer Genome Atlas (TCGA) database found that ALKBH5 mRNA expression in high-grade gliomas is correlated with SH3RF3-AS1 expression ( r =-0.18, P =0.03), but the other 12 common m6A regulators were not correlated with the expression of SH3RF3-AS1. The human glioma cell line U251 (provided by the Department of Neurosurgery, Qilu Hospital, Shandong University) was cultured at 37°C and 5% CO2 in DMEM medium containing 15% fetal bovine serum (FBS). Cell RNA was extracted as above, and RNA was enriched with Flag (ALKBH5) antibody according to the instructions of the RNA immunoprecipitation kit (Merck Millipore, USA). RT-qPCR was then performed as above to detect the expression of SH3RF3-AS1. The results showed that ALKBH5 was significantly enriched in SH3RF3-AS1 ( P =0.001)( Figure 2B). Fresh brain tumor tissue was cut into 1-2 mm pieces and dispersed in culture medium. The culture medium was changed every 2-3 days. After the cells crawled out of the tissue block, they were passaged and frozen. Primary glioma cells were identified by Giemsa staining and glial fibrillary acidic protein (GFAP) immunohistochemical staining. ALKBH5 overexpression plasmid was constructed using pcDNA3.1 (+) vector. U251 cells and primary glioma G1 cells overexpressing ALKBH5 were constructed by transient transfection. RT-qPCR detection showed that the expression of SH3RF3-AS1 was about 20-30% higher when ALKBH5 was overexpressed than that of control cells ( Figure 2 C). Cells were seeded in 6-cm culture dishes and incubated with 5 µg / mL actinomycin D (Aladdin, Shanghai, China) for 90 minutes. Total RNA was extracted from cells at 0 and 90 minutes and analyzed by RT-qPCR. This showed that ALKBH5 overexpression increased the stability of SH3RF3-AS1 ( P =0.02)( Figure 2 D). SH3RF3-AS1 probe was purchased from Advanced Cell Diagnostics (ACD) and the subcellular localization of SH3RF3-AS1 in U87 and U251 cells was evaluated according to the instructions of the RNA fluorescence in situ hybridization (FISH) kit (Jima, Shanghai, China). Figure 2 E), the results showed that SH3RF3-AS1 is distributed in the nucleus and cytoplasm. Based on the bioinformatics analysis of the TCGA database, the potential target genes of SH3RF3-AS1 include 6 trans-acting genes and 1 cis-acting gene. In high-grade gliomas, the expression of SH3RF3-AS1 is positively correlated with the expression of SH3RF3 mRNA ( r =0.73, P <0.001). SH3RF3-AS1-targeting siRNAs (si-SH3RF3-AS1-1 and si-SH3RF 3-AS1-2) and a negative control siRNA (NC) were synthesized by Shanghai Gene Gene Co., Ltd. (siRNA sequences are shown in Table 1). SH3RF3-AS1 overexpression vectors were constructed. SH3RF3-AS1 siRNA transfection significantly inhibited SH3RF3 expression in U251 cells. SH3RF3-AS1 overexpression led to upregulation of SH3RF3 mRNA ( Figure 2 F). SH3RF3-AS1 overexpression increased the stability of SH3RF3 mRNA after 4 hours of actinomycin D treatment ( P =0.04)( Figure 2 G).

[0038] Table 1 Primer sequences and small interfering sequences for RT-qPCR amplification

[0039]

[0040] 3. ALKBH5 / SH3RF3-AS1 axis promotes glioma cell proliferation and CPT-11 resistance

[0041] Cell proliferation was detected using CCK-8 (Elabscence, China) reagent and 5-ethynyl-2'-deoxyuridine (EdU) (Elabscence, China) reagent. CCK-8 assay for cell proliferation: After cell treatment, cells were incubated in a medium containing 10% CCK-8 reagent at 37°C for 1.5 hours, and the absorbance was measured at 450 nm. EdU assay for cell DNA replication: After cell treatment, U251 cells were incubated with EdU for 2 hours / G1 cells were incubated with EdU for 12 hours. Unlabeled cells were used as controls. Cells were collected and stained according to the manufacturer's instructions and detected by flow cytometry. In U251 cells and primary glioma G1 cells, overexpression of ALKBH5 and SH3RF3-AS1 upregulated cell proliferation and DNA replication, while knockdown of SH3RF3-AS1 downregulated cell proliferation and DNA replication ( Figure 3 A). We stained the cells with propidium iodide and analyzed the cell cycle by flow cytometry. In U251 cells and primary glioma G1 cells, overexpression of ALKBH5 or SH3RF3-AS1 increased the percentage of cells in the S phase, while the percentage of cells in the G0 / G1 phase was significantly decreased ( Figure 3 B). In addition, SH3RF3-AS1 knockdown increased the percentage of G0 / G1 phase cells and decreased the proportion of S phase cells in U251 cells and primary glioma G1 cells ( Figure 3 C). Taken together, these results suggest that ALKBH5 / SH3RF3-AS1 upregulates glioma cell proliferation. Cells were seeded in 96-well plates at a density of 5000 cells / well. After overnight culture, the medium was replaced with irinotecan (CPT-11; Qilu Pharmaceutical, China) at varying concentrations, and the cells were cultured for another 48 hours. Cell viability was determined using CCK-8 assay, and the IC50 concentration of CPT-11 was calculated. The IC50 value of CPT-11 was reduced in primary glioma G1 cells with SH3RF3-AS1 knockdown ( Figure 3 D), while SH3RF3-AS1 overexpression resulted in an increased IC50 value of CPT-11. Taken together, these data suggest that SH3RF3-AS1 knockdown enhances the therapeutic sensitivity of glioma to CPT-11.

[0042] 4. ALKBH5 / SH3RF3-AS1 / SH3RF3 overexpression indicates poor prognosis in glioma patients

[0043] Immunofluorescence was used to detect SH3RF3 and ALKBH5 expression on tissue microarrays from glioma patients. SH3RF3-AS1 expression was detected on tissue microarrays using the RNAscope kit (Advanced Cell Diagnostics, USA) according to the manufacturer's instructions. The SH3RF3-AS1 probe was purchased from Advanced Cell Diagnostics (ACD). The clinical characteristics of the glioma patients included in the tissue microarrays are shown in Table 2.

[0044] Table 2 Clinical characteristics of glioma patients

[0045]

[0046] According to Table 2, the expression of ALKBH5 protein in WHO grade III tissues was higher than that in WHO grade I and II tissues ( P <0.001, P =0.005). ALKBH5 protein expression in WHO IV grade tissue was also higher than that in grade I tissue ( P =0.009). The expression of ALKBH5 in high-grade glioma (grade III and IV) samples was significantly higher than that in low-grade glioma (grade I and II) samples ( P <0.001) ( Figure 4 A). Kaplan-Meier survival analysis showed that ALKBH5 expression levels in glioma samples were associated with progression-free survival (PFS) ( P =0.04, Figure 4 B), but not associated with overall survival (OS) ( P >0.05). In addition, ALKBH5 expression level was significantly correlated with WHO grade ( r =0.30, P <0.001) and SH3RF3-AS1 expression levels ( r =0.19, P =0.02). The percentage of SH3RF3-AS1 positive cells in tissues with low ALKBH5 expression was lower than that in tissues with high ALKBH5 expression ( P <0.05) ( Figure 4 C). The expression of SH3RF3-AS1 in high-grade glioma samples was significantly higher than that in low-grade glioma samples ( P =0.002). Figure 4 As shown in A, the expression of SH3RF3-AS1 in grade III glioma was significantly higher than that in grade I ( P <0.0001) or Grade II ( P =0.003), SH3RF3-AS1 levels were correlated with WHO grade ( r=0.23, P =0.006). Kaplan-Meier survival analysis showed that SH3RF3-AS1 expression levels above the median were associated with shorter 5-year overall survival and progression-free survival ( P =0.04, P =0.009; Figure 4 B). Typical images of immunofluorescence staining of ALKBH5, SH3RF3, and SH3RF3-AS1. Figure 4 D. In addition, the expression of SH3RF3 in high-grade glioma samples was significantly higher than that in low-grade glioma samples ( P =0.03). Figure 4 As shown in A, compared with grade I glioma tissue, the SH3RF3 level in grade III glioma tissue was significantly increased ( P =0.04), SH3RF3 levels were correlated with WHO grade ( r =0.25, P Multivariate Cox regression analysis including SH3RF3-AS1 expression, ALKBH5 expression, and all clinical variables of glioma patients (age, sex, Ki67, EGFR, and PDL1 expression) showed that high SH3RF3-AS1 expression and high WHO grade were independent factors affecting the 5-year overall survival of glioma (Table 3).

[0047] Table 3. Cox regression analysis of 5-year overall survival rate of glioma patients

[0048]

[0049] 5. Nano PCPT Synthesis and efficacy verification of siRNA@U251

[0050] This example provides a method for preparing Nano PCPT / siRNA@U251 preparation process to achieve targeted co-delivery of CPT and si-SH3RF3-AS1, thereby improving the bioavailability and efficacy of the drug / gene combination approach.

[0051] In the following steps, 4-cyano-4-[[(dodecylthio)thiomethyl]thio]pentanoic acid (DCT), N,N'-dicyclohexylcarbodiimide (DCC, CAS No. 538-75-0), methoxypolyethylene glycol (DP 3000-5000), and azobisisobutyronitrile (AIBN) were purchased from Sigma-Aldrich. 2-Hydroxyethyl disulfide (CAS No. 1892-29-1) was purchased from Macklin, and 4-dimethylaminopyridine (DMAP) was purchased from Heowns. Triphosgene, methacryloyl chloride, and CPT were purchased from Energy Chemical. All other reagents and procedures used in bioengineering or chemical engineering experiments were commercially available unless otherwise specified.

[0052] (1) Dissolve 3.46 g of N-butylethanolamine and 2.317 g of triethylamine in 50 mL of acetonitrile and mix well. Cool the mixture to 0°C and add 2.394 g of methacryloyl chloride dropwise. After 1-2 min of dropwise addition, place the mixture in ice water, maintain the temperature at 0°C, stir at 200 rpm for 2 hours, and then continue stirring at 200 rpm at room temperature for 24 hours. Filter the mixture, extract the resulting filtrate with 30 ml of dichloromethane (DCM), and evaporate under reduced pressure to obtain the monomer methacrylate n-butylaminoethanol (DBAMA). The NMR data of the product are: 1 H NMR (400 MHz, Chloroform-d) δ 6.10(s, 1H), 5.55 (t, J = 1.8 Hz, 1H), 4.20 (t, J = 6.2 Hz, 2H), 2.74 (t, J = 6.2Hz, 2H), 2.52-2.42 (m, 4H), 1.94 (s, 3H), 1.48-1.37 (m, 4H), 1.30 (dq, J =14.2, 7.0 Hz, 4H), 0.90 (t, J = 7.3 Hz, 6H).

[0053] To synthesize OH-2S-CPT, 2-[(2-hydroxyethyl)dithio]ethyl-2-methyl-2-acrylate was first synthesized: 2 g of 2-hydroxyethyl disulfide and 1.97 g of triethanolamine were dissolved in 60 mL of tetrahydrofuran (THF) and cooled to 0°C. 2.03 mg of methacryloyl chloride was slowly added to the mixture. After stirring at 0°C and 200 rpm for 2 hours, the mixture was brought to room temperature and stirred at 200 rpm overnight. The mixture was then filtered, and the filtrate was concentrated and extracted with 100 mL of ethyl acetate. Evaporation under reduced pressure yielded 2-[(2-hydroxyethyl)dithio]ethyl-2-methyl-2-acrylate as a light yellow oil.

[0054] 800 mg of CPT, 1122 mg of 4-dimethylaminopyridine, and 272.6 mg of triphosgene were dissolved in 3 mL of anhydrous dichloromethane (DCM) and stirred at 200 rpm for 0.5 hours at room temperature to obtain Solution A. 560.8 mg of the synthesized 2-[(2-hydroxyethyl)dithio]ethyl-2-methyl-2-propenoate was dissolved in 2 mL of DCM to obtain Solution B. Solution B was slowly poured into Solution A and stirred at 200 rpm overnight. Subsequently, the solvent was removed by concentration under reduced pressure, and the pale yellow pure product, OH-2S-CPT, was obtained by silica gel column chromatography. The NMR data of this product were as follows: 1 H NMR (400 MHz, DMSO-d6) δ8.70 (s, 1H), 8.21-8.09 (m, 2H), 7.86 (ddd, J=8.5, 6.8, 1.5 Hz, 1H 2 (m, 4H), 2.17 (dq, J=10.3, 7.1 Hz, 2H), 1.82 (d, J=1.2 Hz, 3H), 0.92 (t, J=7.4 Hz, 3H).

[0055] (2) 178.95 mg of 4-cyano-4-[[(dodecylthio)thiomethyl]thio]pentanoic acid (CAS No.: 870196-80-8), 54.16 mg of 4-dimethylaminopyridine (DMAP) and 91.47 mg of N,N'-dicyclohexylcarbodiimide were dissolved in 30 mL of anhydrous DCM and stirred at room temperature to mix evenly. 10 mL of anhydrous DCM solution containing 1.71 g of methoxypolyethylene glycol (DP 3000-5000) was added dropwise to the above mixture and stirred at room temperature for 36 hours at a stirring rate of 200 rpm. The filtrate was then filtered and concentrated by evaporation and purified by three rounds of precipitation in cold ether at -20°C. The precipitate was collected and dried under vacuum overnight to obtain PEG-DCT as a light yellow powder.

[0056] (3) PEG-PDBAMA was synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization. 540.48 mg of PEG-DCT obtained in step (2) was weighed and dissolved together with 1205 mg of DBAMA obtained in step (1) and 4.94 mg of 2,2'-azobis(2-methylpropionitrile) in 5 ml of dioxane and placed in a Schlenk tube. After four freeze-pump-thaw cycles, the reaction solution was transferred to an oil bath at 70°C and incubated at 100 rpm for 48 hours. The reaction solution was then cooled to room temperature, dialyzed against deionized water to purify the product, and freeze-dried at -4°C to obtain PEG-PDBAMA powder.

[0057] (4) Preparation of PEG-PDBAMA-PCPT using RAFT polymerization technique. 700 mg of PEG-PDBAMA powder obtained in step (3), 490 mg of OH-2S-CPT obtained in step (1), and 2.4 mg of azobisisobutyronitrile (AIBN) were weighed and dissolved in 50 ml of a mixture of dioxane and dimethyl sulfoxide (DMSO) with a volume ratio of 1:1 and placed in a Schlenk tube. After four freeze-pump-thaw cycles, the mixture was heated to 80 °C to initiate polymerization and incubated at 100 rpm for 48 h. The solution was then cooled to room temperature and purified by three rounds of precipitation in cold ether at -20 °C. A red powder was obtained after vacuum drying and was designated as PEG-PDBAMA-PCPT.

[0058] (5) Weigh 8.3 mg of the PEG-PDBAMA-PCPT powder prepared in step (4) and evenly disperse it in 0.5 mL of dimethyl sulfoxide. Subsequently, 4 OD of siRNA was dissolved in 0.5 mL of dimethyl sulfoxide. The two dimethyl sulfoxide mixtures were added dropwise to 4 mL of deionized water and sonicated for 30 minutes. The mixture was dialyzed in deionized water overnight to remove dimethyl sulfoxide to obtain PEG-PDBAMA-PCPT / siRNA; two siRNAs targeting SH3RF3-AS1 were prepared using the above method to obtain PEG-PDBAMA-PCPT / siRNA-1, PEG-PDBAMA-PCPT / siRNA-2, and PEG-PDBAMA-PCPT / siRNA-1+2 (two siRNAs were mixed at 2 OD each).

[0059] (6) U251 cells (from the Neurosurgery Laboratory of Qilu Hospital) were collected, washed three times in PBS buffer (pH 7.4, the same below), dispersed in 3 mL of hypotonic buffer solution, and frozen at -80°C. The concentrations of the substances in the hypotonic buffer solution were as follows: 20 mM Tris-HCl (pH = 7.5), 10 mM KCl, 2 mM MgCl2, and 1 mM PMSF. After thawing, the supernatant was collected by centrifugation at 2000 × g for 5 minutes, then at 10,000 × g for 30 minutes, and then at 100,000 × g for 1 hour. The resulting precipitate was collected and washed once with PBS to obtain U251 cell membranes, which were suspended in PBS buffer and mixed with freshly prepared PEG-PDBAMA-PCPT / siRNA-1+2 at a volume ratio of 1:1. The resulting solution was then physically passed through a 400 nm polycarbonate membrane 10 times using a liposome extruder to obtain PEG-PDBAMA-PCPT / siRNA@U251 (i.e., PEG-PDBAMA-PCPT / siRNA-1+2, referred to as Nano PCPT / siRNA@U251), and then freeze-dried to obtain nanomedicine. An agarose gel blocking experiment was used to verify whether the PEG-PDBAMA-PCPT nanodrug (NP) prepared by the above method successfully carried siRNA. Compared with free siRNA, the siRNA can be completely compressed at a ratio of 20:1 (w / w). The size, ζ potential and morphology of PEG-PDBAMA-PCPT / siRNA@U251 particles were evaluated by dynamic light scattering (DLS) and transmission electron microscopy (TEM). After testing, the particle size of PEG-PDBAMA-PCPT / siRNA@U251 obtained in this example was 200.9±4.371 nm, and the particle size was uniform, with a polydispersity index (PDI) of 0.316±0.088 ( Figure 5 A). The siRNA concentration in the final prepared nanomedicine was fixed at 50 nM, and the CPT concentration was 20 μM.

[0060] In order to verify the effect of nanomedicine, PEG-PDBAMA-PCPT / siRNA-1+2 in the above steps were replaced with PEG-PDBAMA-PCPT (without RNA), PEG-PDBAMA-PCPT / siRNA-1, and PEG-PDBAMA-PCPT / siRNA-2 to obtain Nano PCPT @U251 (omit step (5), no siRNA), Nano PCPT / siRNA-1@U251 (containing the single si-SH3RF3-AS1-1 in Table 1), Nano PCPT / siRNA-2@U251 (containing the single si-SH3RF3-AS1-2 in Table 1) was set aside. The above substances were treated with U251 cells respectively, and the treatment process was as follows: First, Nano PCPT @U251 (omit steps (1) (4) (5), does not contain CPT and siRNA), Nano PCPT / siRNA-1@U251、Nano PCPT / siRNA-2@U251 and Nano PCPT / siRNA-1+2@U251, after dispersion, except for the control group, the siRNA concentration in each sample was 5μM and the CPT concentration was 2mM, as the mother solution. When used, dilute Nano with complete cell culture medium PCPT @U251、Nano PCPT / siRNA-1@U251、Nano PCPT / siRNA-2@U251 and Nano PCPTThe mother solution of / siRNA-1+2@U251 was used to make the final concentration of CPT 20μM and siRNA 50nM in addition to the control group. The same volume of complete culture medium with PBS buffer was added as a further control and treated U251 cells for 24 hours. CCK-8 assay was then performed to determine the inhibitory effect of different nanomedicines on tumor cells. The results showed that Nano PCPT / siRNA-1+2@U251 vs Nano PCPT / siRNA-1@U251 and Nano PCPT / siRNA-2@U251 has a more significant inhibitory effect ( Figure 5 B). For the convenience of description, Nano PCPT / siRNA-1+2@U251 is abbreviated as Nano PCPT / siRNA@U251. Then, the above method was used to treat G1 cells for 24 hours. The results showed that Nano PCPT / siRNA@U251 has the most significant inhibitory effect on the proliferation of U251 and G1 cells ( Figure 5 C).

[0061] In this example, BALB / c nude mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. and housed under SPF conditions. All animal experiments were conducted with the approval of the Ethics Committee of the Second Hospital of Shandong University (KYLL-2021 (KJ) A-0024). U251 cells were used to orthotopically in nude mice: 3×10 fluorescently labeled 5 U251 human glioma cells (U251 luc) were implanted intracranially in BALB / c nude mice (male, 4 weeks old). The growth of orthotopic U251 luc tumors was monitored by bioluminescence imaging (BLI) system. The day of glioma cell implantation was designated as day 0. At week 1 and week 4, images were acquired using IVIS Lumina II in vitro imaging system (PerkinElmer, USA) 5 minutes after intraperitoneal injection of 150 mg / kg luciferin. Nude mice carrying U251 luc cells were randomly divided into six groups (n=7 / group). Starting from day 7, mice received PBS buffer, Nano@U251 (omitted steps (1) (4) (5), without CPT and siRNA), free CPT ... PCPT @U251 (omit step (5), no siRNA), Nano-siRNA@U251 (omit steps (1) and (4), no CPT) or Nano PCPT200 μL of / siRNA@U251 drug (diluted with PBS buffer, except for the PBS buffer control group, the mice were given 5 mg / kg of CPT, 0.5 mg / kg of siRNA, and 5 mg / kg of free CPT) and injected intraperitoneally every three days. In addition, mice were weighed before administration, and the efficacy was evaluated by BLI. U251 luciferase cells were injected intracranially into nude mice, Nano@U251, free CPT, Nano PCPT @U251, Nano-siRNA@U251 and Nano PCPT siRNA@U251 was administered to mice starting 7 days after cell injection. In vivo imaging of nude mice showed that 7 days after modeling, all six groups of nude mice showed bioluminescence in vivo, with no significant difference in luminescence intensity. 28 days after modeling, the bioluminescence intensity of all six groups of mice increased ( Figure 5 D and 5E), Nano PCPT @U251 or Nano-siRNA@U251 inhibited tumor growth compared with either monotherapy. PCPT / siRNA@U251 significantly inhibited tumor growth. H&E staining showed that the tumor tissues of PBS and Nano@U251 groups were significantly PCPT The cell density of the siRNA@U251 and Nano-siRNA@U251 groups was higher, and the Nano PCPT @U251, Nano-siRNA@U251 and Nano PCPT The boundary between tumor and normal tissue in the siRNA@U251 group was clearer ( Figure 5 F). The weight of mice in the free CPT group decreased dramatically. In addition, the expression of SH3RF3 and Rac1 in the Nano-siRNA@U251 group was lower than that in the Nano@U251 group ( Figure 5 G).

[0062] The above description is merely a preferred embodiment of the present invention and does not constitute any other form of limitation to the present invention. Any person skilled in the art may utilize the technical contents disclosed above to change or modify them into equivalent embodiments with equivalent changes for application in other fields. However, any simple modification, equivalent change, and modification of the above embodiments made in accordance with the technical essence of the present invention without departing from the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. Use of a substance that inhibits or reduces the expression of SH3RF3-AS1 in the preparation of a drug for treating glioma or improving the prognosis of glioma, wherein the sequence of SH3RF3-AS1 is shown in SEQ ID NO: 1 in the sequence listing; the substance that inhibits the expression of SH3RF3-AS1 is siRNA-SH3RF3-AS1 or a vector containing siRNA-SH3RF3-AS1, and the sequence of siRNA-SH3RF3-AS1 is si-RNA-1 forward: GGGCUAACAUCAGAAGAGUTT, si-RNA-1 reverse: ACUCUUCUGAUGUUAGCCCTT; si-RNA-2 forward: CCCAACCUGUGAUAAUUAUTT, si-RNA-2 reverse: AUAAUUAUCACAGGUUGGGTT.

2. The application according to claim 1, characterized in that The substance for inhibiting the expression of SH3RF3-AS1 is nano drug particles containing siRNA-SH3RF3-AS1.

3. The application according to claim 2, characterized in that: The steps for preparing the nano drug particles are as follows: (1) Dissolve N-butylethanolamine and triethylamine in acetonitrile, mix well and cool to 0°C, add methacryloyl chloride dropwise, stir at low temperature to react and filter, extract the filtrate with dichloromethane and evaporate under reduced pressure to obtain monomer DBAMA; Dissolve 2-hydroxyethyl disulfide and triethanolamine in tetrahydrofuran, stir evenly, and cool to 0°C. Then slowly add methacryloyl chloride, stir at low temperature to react, and then stir at room temperature overnight. After filtering, the filtrate is extracted with ethyl acetate and evaporated under reduced pressure to obtain 2-[(2-hydroxyethyl)dithio]ethyl-2-methyl-2-acrylate. Camptothecin, 4-dimethylaminopyridine, and triphosgene were dissolved in dichloromethane and stirred to obtain solution A. 2-[(2-hydroxyethyl)dithio]ethyl-2-methyl-2-acrylate was dissolved in dichloromethane to obtain solution B. Solution B was slowly poured into solution A and stirred overnight. The solution was concentrated under reduced pressure and passed through a silica gel column chromatography to obtain a yellow product, OH-2S-CPT. (2) 4-cyano-4-[[(dodecylthio)thiomethyl]thio]pentanoic acid, 4-dimethylaminopyridine and N,N'-dicyclohexylcarbodiimide were dissolved in dichloromethane and stirred at room temperature to obtain a mixed solution C. The dichloromethane solution containing methoxypolyethylene glycol was added dropwise to the mixed solution C and stirred at room temperature for 36 hours. After filtration, the filtrate was concentrated and purified by precipitation in cold ether for three rounds. The precipitate was collected and dried under vacuum overnight to obtain PEG-DCT light yellow powder; (3) The PEG-DCT obtained in step (2), the DBAMA obtained in step (1), and 2,2'-azobis(2-methylpropionitrile) were dissolved in dioxane and placed in a Schlenk tube; after four freeze-pump-thaw cycles, the tube was transferred to an oil bath and incubated with stirring for 48 h; the tube was then cooled to room temperature, the product was dialyzed with deionized water, and freeze-dried to obtain PEG-PDBAMA powder; (4) The PEG-PDBAMA powder obtained in step (3), the OH-2S-CPT obtained in step (1), and azobisisobutyronitrile were dissolved in a mixture of dioxane and dimethyl sulfoxide and placed in a Schlenk tube. After four freeze-pump-thaw cycles, the mixture was heated to 80°C to initiate polymerization and incubated with stirring for 48 hours. The mixture was cooled and purified by three rounds of precipitation in cold ether. A red powder was obtained after vacuum drying, which was designated as PEG-PDBAMA-PCPT. (5) The PEG-PDBAMA-PCPT powder prepared in step (4) was dispersed in dimethyl sulfoxide to obtain dispersion C; siRNA-SH3RF3-AS1 was dissolved in dimethyl sulfoxide to obtain dispersion D; dispersion C and dispersion D were simultaneously added dropwise to deionized water and ultrasonically treated; the dimethyl sulfoxide was removed by dialyzing in deionized water overnight to obtain PEG-PDBAMA-PCPT / siRNA-SH3RF3-AS1; (6) U251 cells were washed three times in PBS, dispersed in a hypotonic buffer solution, and frozen at -80°C. After thawing, the resulting precipitate was collected by centrifugation and washed with PBS to obtain U251 cell membranes, which were suspended in PBS and mixed with PEG-PDBAMA-PCPT / siRNA-SH3RF3-AS1. The membranes were passed through a polycarbonate membrane using a liposome extruder to obtain PEG-PDBAMA-PCPT / siRNA-SH3RF3-AS1@U251, which was then freeze-dried to obtain nanomedicines.

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