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

Abstract

The invention belongs to the technical field of biological medicine, and relates to an application of a si-SH3RF3-AS1 and CPT combined delivery system in preparation of glioma drugs. The invention also provides a substance for inhibiting the expression of the SH3RF3-AS1, namely nano-drug particles containing siRNA and CPT, the particle size of the nano-drug particles is 200.9 + / -4.371 nm, the concentration of the siRNA in the nano-drug particles is 50 nM, and the concentration of the CPT in the nano-drug particles is 20 [mu] M. The siRNA taking SH3RF3-AS1 as a target spot and the nano-drug containing the siRNA are provided, and verification shows that the nano-drug has a remarkable inhibition effect on proliferation of U251 and G1 cells, the synthesis process of the nano-drug is controllable, the quality is stable and reliable, and a new possibility is provided for treatment of glioma.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to an 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, and their morbidity and mortality rates are increasing year by year. High-grade gliomas are highly malignant, lack effective early diagnosis and treatment methods, and have extremely poor prognosis for patients. Surgical resection of tumors is often incomplete and leads to loss of neurological function. Problems such as resistance to chemotherapy drugs also seriously plague clinical practice. At present, there is an urgent need to resolve the contradiction between the poor prognosis of gliomas and the limitations of treatment methods, and it is urgent to find new targets and treatment methods for gliomas. Summary of the invention

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

[0004] In order to achieve the above object, the present invention is implemented by adopting the following technical solutions: The present invention proposes the use of a substance with SH3RF3-AS1 as a target, i.e., 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 SH3RF3-AS1 sequence is shown in the sequence listing SEQ ID NO: 1.

[0005] 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.

[0006] The present invention verifies that the substance that inhibits 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.

[0007] 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 si-RNA-1 forward: GGGCUAACAUCAGAAGAGUTT, si-RNA-1 reverse: ACUCUUCUGAUGUUAGCCCTT; si-RNA-2 forward: CCCAACCUGUGAUAAUUAUTT, si-RNA-2 reverse: AUAAUUAUCACAGGUUGGGTT.

[0008] The present invention also proposes that the substance for inhibiting the expression of SH3RF3-AS1 is a nano drug particle containing siRNA-SH3RF3-AS1. As a preferred embodiment, the particle size of the nano drug particle is 200.9±4.371 nm, the siRNA concentration in the nano drug is 50 nM, and the CPT concentration is 20 μM.

[0009] Preferably, 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 for reaction and filter, extract the filtrate with dichloromethane and evaporate under reduced pressure to obtain monomer DBAMA.

[0010] Dissolve 2-hydroxyethyl disulfide and triethanolamine in tetrahydrofuran, stir evenly and cool to 0°C, then slowly add methacryloyl chloride, stir at low temperature for reaction, and stir at room temperature overnight; after filtering, extract the filtrate with ethyl acetate, evaporate under reduced pressure to obtain 2-[(2-hydroxyethyl)disulfide]ethyl-2-methyl-2-acrylate.

[0011] 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 solution was concentrated under reduced pressure and passed through a silica gel column to obtain a yellow product OH-2S-CPT.

[0012] (2) Dissolve 4-cyano-4-[[(dodecylthio)thiomethyl]thio]pentanoic acid, 4-dimethylaminopyridine and N,N'-dicyclohexylcarbodiimide in dichloromethane and stir evenly at room temperature to obtain a mixed solution C. Add the dichloromethane solution containing methoxypolyethylene glycol dropwise into the mixed solution C and stir at room temperature for 36 hours. After filtering, concentrate the filtrate and purify it by three rounds of precipitation in cold ether. Collect the precipitate and dry it under vacuum overnight to obtain PEG-DCT light yellow powder.

[0013] (3) Dissolve the PEG-DCT obtained in step (2), the DBAMA obtained in step (1) and 2,2'-azobis(2-methylpropionitrile) in dioxane and place in a Schlenk tube; after four freeze-pump-thaw cycles, transfer to an oil bath and incubate with stirring for 48 hours; then cool to room temperature, dialyze the product with deionized water, and freeze-dry to obtain PEG-PDBAMA powder.

[0014] (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 recorded as PEG-PDBAMA-PCPT.

[0015] (5) The PEG-PDBAMA-PCPT powder prepared in step (4) is dispersed in dimethyl sulfoxide to obtain dispersion C; siRNA-SH3RF3-AS1 is dissolved in dimethyl sulfoxide to obtain dispersion D; dispersion C and dispersion D are simultaneously added dropwise into deionized water and subjected to ultrasonic treatment; dimethyl sulfoxide is removed by dialyzing in deionized water overnight to obtain PEG-PDBAMA-PCPT / siRNA-SH3RF3-AS1.

[0016] (6) U251 cells were washed three times in PBS, dispersed in a hypotonic buffer solution, and frozen at -80°C. After thawing, the 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 were then freeze-dried to obtain nanomedicines.

[0017] Compared with the prior art, the advantages and positive effects of the present invention are: The present invention proposes siRNA-SH3RF3-AS1 with SH3RF3-AS1 as a target and a nano drug containing the small interfering RNA. It has been verified that the nano drug has a significant inhibitory effect on the proliferation of U251 and G1 cells, and the synthesis process of the nano drug is controllable and the quality is stable and reliable, which provides new possibilities for the treatment of gliomas, provides new drugs for inhibiting proliferation for the treatment of gliomas, and enhances the therapeutic effect of chemotherapeutic drugs such as CPT in combination. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The results of MeRIP-seq in brain glioma tissues, where A: In normal tissues and glioma tissues, m6A modification is distributed on the start codon, CDS and stop codon of mRNA and lncRNA. B: Distribution map of m6A modification in normal tissues and glioma tissues. C: Highly enriched and conserved common RRACH sequences in normal tissues and glioma tissues. D: Genes with differential m6a modification in normal tissues and glioma tissues (|log2FC|>2); red indicates hypermethylation, while blue indicates demethylation. E: GO enrichment analysis results: Gene functions with differential m6A modification between glioma tissues and normal tissues. F: KEGG enrichment analysis: Gene pathways with differential m6A modification between glioma tissues and normal tissues. G: Four-quadrant diagram of differences in m6A modification and RNA expression in normal tissues and glioma tissues (SH3RF3-AS1: demethylation / upregulated expression). H: Visualization of mA modification in SH3RF3-AS1.

[0019] Figure 2Regulation of the ALKBH5 / SH3RF3-AS1 / SH3RF3 axis in glioma. A: m6A-RIP-qPCR was used to determine the m6A modification level of SH3RF3-AS1 in glioma and normal tissues. B: U251 cells were transfected with Flag-pcDNA3.1-ALKBH5 (Flag-ALKBH5) or Flag-pcDNA 3.1, and the expression of SH3RF3-AS1 was analyzed by Flag-RIP-qPCR. C: The expression of SH3RF3-AS1 in U251 cells transfected with pcDNA3.1-ALKBH5 (over-ALKBH5) or pcDNA3.1 (over-control) and glioma primary G1 cells was analyzed by RT-qPCR. D: ALKBH5 overexpression / control U251 cells were treated with actinomycin D for 90 minutes, and the expression of SH3RF3-AS1 was measured by RT-qPCR. E: SH3RF3-AS1 subcellular localization and expression detection in U87 and U251 cells using SH3RF3-AS1 probe (red) (200×). F: SH3RF3 protein and mRNA expression and Rac1 protein expression (anti-Rac1 antibody: Merck, clone 23A8, Germany, anti-SH3RF3 antibody: Abcam, ab184977, USA) 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 the expression of SH3RF3 protein. G: SH3RF3-AS1 overexpression / 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.

[0020] 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 stage of the cell cycle 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.

[0021] Figure 4 The role of ALKBH5 / SH3RF3-AS1 / SH3RF3 axis in the prognosis of glioma patients. A: Expression levels of ALKBH5, SH3RF3-AS1 and SH3RF3 in patients with different WHO grades. B: Relationship between ALKBH5, SH3RF3-AS1 and SH3RF3 and 5-year overall survival and progression-free survival of glioma patients (Kaplan-Meier survival analysis). C: The percentage of SH3RF3-AS1-positive cells is high in glioma tissues with high ALKBH5 expression. High expression of ALKBH5: greater than the upper quartile. D: Representative immunofluorescence histochemistry images in 148 glioma specimens. Pink: ALKBH5, white: SH3RF3, red: SH3RF3-AS1, blue: cell nucleus. The expression of SH3RF3-AS1 was analyzed on tissue microarray by RNAScope® technology. The expression of ALKBH5 and SH3RF3 proteins in glioma tissues was detected by 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.

[0022] Figure 5 For 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) compared with the control group Nano PCPT @U251 had a more significant proliferation inhibition effect (79.47% vs. 85.47%, P =0.04). C: The combined administration of CPT and si-SH3RF3-AS1 had the greatest inhibitory effect on the survival of U251 cells. 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 / siRNA@U251 treatment survival rate was 74.94%±2.84%. The same pattern was also found in G1 cells. D and E: Evaluation of tumors in the brain of nude mice using in vivo imaging. Nano PCPT / siRNA@U251 inhibits in situ gliogenesis in nude mice. F: Nano PCPT / Tumors in siRNA@U251 showed swelling and loose arrangement, and the tumors showed 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

[0023] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described below in conjunction with specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0024] 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 of the following disclosure.

[0025] Example 1 1. Validation of SH3RF3-AS1 as a glioma-specific lncRNA target 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 mainly occurs near the coding regions (CDSs) and 3' untranslated regions (UTRs) of mRNA transcripts in normal and tumor tissues. In normal tissues, mA peaks are 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'UTR (34.5%), followed by stop codons (20.2%) ( Figure 1 A and 1B). The highly enriched and conserved consensus RRACH sequences are 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).

[0026] 2. Regulation of the ALKBH5 / SH3RF3-AS1 / SH3RF3 axis in glioma Total RNA was extracted from glioma or normal tissues according to the instructions of EZNA Total RNA Extraction Kit I (Omega Bio-tek, USA). The content and quality of RNA were measured at 260 nm by NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). MeRIP experiments were performed according to the instructions of m6A methylated RNA immunoprecipitation kit (Merck Millipore, USA). After RNA purification, PrimeScript TM RT-qPCR was performed according to the instructions of the RT kit (Takara, China) and the SYBRGreen Pro Taq HS qPCR (Rox) kit (Accurate Biotechnology, China). Each experiment was repeated at least three times. Endogenous control β-actin was used as a control, and 2 -ΔΔCt Methods The genes to be tested were relatively quantified, and the primer sequences for RT-qPCR amplification are shown in Table 1.

[0027] The m6A modification of SH3RF3-AS1 in glioma tissues and normal tissues is shown in Figure 2 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 the mRNA expression of ALKBH5 in high-grade gliomas is correlated with the expression of SH3RF3-AS1 ( 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% CO 2 The cells were cultured in an environment containing 15% fetal bovine serum (FBS) in DMEM medium. Cell RNA was extracted as above, and RNA was enriched with Flag (ALKBH5) antibody according to the instructions of RNA immunoprecipitation kit (Merck Millipore, USA). RT-qPCR was performed to detect the expression of SH3RF3-AS1 as above. The results showed that ALKBH5 significantly enriched SH3RF3-AS1 ( P =0.001)( Figure 2B). Fresh brain tumor tissue was cut into 1-2 mm size 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 acid 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 found that the expression of SH3RF3-AS1 was about 20-30% higher than that of control cells when ALKBH5 was overexpressed ( Figure 2 C). The cells were seeded in 6-cm culture dishes and incubated with 5 µg / mL actinomycin D (Aladdin, Shanghai, China) for 90 min. Total RNA was extracted from cells at 0 and 90 min and RT-qPCR analysis 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 RNA fluorescence in situ hybridization (FISH) kit (Jima, Shanghai, China). Figure 2 E), the results showed that SH3RF3-AS1 was 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 was positively correlated with the expression of SH3RF3 mRNA ( r =0.73, P <0.001). siRNA targeting SH3RF3-AS1 (si-SH3RF3-AS1-1 and si-SH3RF 3-AS1-2) and negative control siRNA (NC) were synthesized by Shanghai Gene Gene Co., Ltd. The siRNA sequences are shown in Table 1, and the SH3RF3-AS1 overexpression vector was constructed. SH3RF3-AS1 siRNA transfection significantly inhibited the expression of SH3RF3 in U251 cells, and SH3RF3-AS1 overexpression led to upregulation of SH3RF3 mRNA ( Figure 2 F). After 4 h of actinomycin D treatment, SH3RF3-AS1 overexpression increased the stability of SH3RF3 mRNA ( P =0.04)( Figure 2 G).

[0028] Table 1 Primer sequences and small interfering sequences for RT-qPCR amplification 3. ALKBH5 / SH3RF3-AS1 axis promotes glioma cell proliferation and CPT-11 resistance Cell proliferation was detected using CCK-8 (Elabscence, China) reagent and 5-ethynyl-2'-deoxyuridine (EdU) (Elabscence, China) reagent. CCK-8 detection of cell proliferation: After cell treatment, cells were incubated in medium containing 10% CCK-8 reagent at 37°C for 1.5 hours, and the absorbance was measured at 450 nm. EdU detection of 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, the percentage of cells in the S phase increased after overexpression of ALKBH5 or SH3RF3-AS1, while the percentage of cells in the G0 / G1 phase decreased significantly ( Figure 3 B). In addition, knockdown of SH3RF3-AS1 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 containing different concentration gradients of irinotecan (CPT-11; Qilu Pharmaceutical, China) was replaced and cultured for another 48 h. Cell viability was determined using CCK-8 reagent, 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 increase in the IC50 value of CPT-11. Taken together, these data indicate that SH3RF3-AS1 knockdown can enhance the therapeutic sensitivity of glioma to CPT-11.

[0029] 4. ALKBH5 / SH3RF3-AS1 / SH3RF3 overexpression indicates poor prognosis in patients with glioma Immunofluorescence was used to detect the expression of SH3RF3 and ALKBH5 on tissue microarrays of glioma patients. The expression of SH3RF3-AS1 on tissue microarrays was detected using the RNAscope kit (Advanced Cell Diagnostics, USA) according to the instructions. The SH3RF3-AS1 probe was purchased from Advanced Cell Diagnostics (ACD). The clinical characteristics of glioma patients on tissue microarrays are shown in Table 2.

[0030] Table 2 Clinical characteristics of glioma patients 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). The expression of ALKBH5 protein 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 level in glioma samples was associated with progression-free survival (PFS) ( P =0.04, Figure 4 B), but not associated with overall survival (OS) ( P >0.05). In addition, the expression level of ALKBH5 was significantly correlated with WHO grade ( r =0.30, P <0.001) and SH3RF3-AS1 expression level ( 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 gliomas was significantly higher than that in grade I ( P <0.0001) or Grade II ( P =0.003), SH3RF3-AS1 level was 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 level was correlated with WHO grade ( r =0.25, P =0.002). Multivariate Cox regression analysis incorporating SH3RF3-AS1 expression, ALKBH5 expression, and clinical variables of all glioma patients (age, gender, expression of Ki67, EGFR, and PDL1) showed that high expression of SH3RF3-AS1 and high WHO grade were independent factors affecting the 5-year overall survival of glioma (Table 3).

[0031] Table 3. Cox regression analysis of 5-year overall survival rate of glioma patients 5. Nano PCPT Synthesis and efficacy verification of siRNA@U251 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.

[0032] 4-Cyano-4-[[(dodecylthio)thiomethyl]thio]pentanoic acid (DCT), N,N'-dicyclohexylcarbodiimide (DCC, CAS No. 538-75-0), methoxypolyethylene glycol (degree of polymerization 3000-5000) and azobisisobutyronitrile (AIBN) in the following steps 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 conventional commercial reagents or conventional experimental operations in biological genetic engineering or chemical engineering experiments were used unless otherwise specified.

[0033] (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 for 24 hours at room temperature. Filter the mixture, extract the filtrate with 30 ml of dichloromethane (DCM), and evaporate under reduced pressure to obtain the monomer methacrylate n-butyl-N-butylaminoethanol (DBAMA). The product NMR data 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).

[0034] 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 moved to room temperature and stirred at 200 rpm overnight. The mixture was then filtered, and the resulting filtrate was concentrated and extracted with 100 mL of ethyl acetate, and evaporated under reduced pressure to obtain a light yellow oily liquid to obtain 2-[(2-hydroxyethyl)dithio]ethyl-2-methyl-2-acrylate.

[0035] Then 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 room temperature at 200 rpm for 0.5 hours to obtain solution A. 560.8 mg of the synthesized 2-[(2-hydroxyethyl)dithio]ethyl-2-methyl-2-acrylate was dissolved in 2 mL of DCM to obtain solution B, which was slowly poured into solution A and stirred at 200 rpm overnight. Subsequently, the solvent was removed by concentration under reduced pressure, and a light yellow pure product OH-2S-CPT was obtained by silica gel column chromatography. After testing, the nuclear magnetic resonance data of the product were: 1H 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).

[0036] (2) Dissolve 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 in 30 mL of anhydrous DCM and stir to mix at room temperature. Add 10 mL of anhydrous DCM solution containing 1.71 g of methoxypolyethylene glycol (DP 3000-5000) dropwise to the above mixture and stir at room temperature for 36 hours at a stirring rate of 200 rpm. Then filter and evaporate the filtrate, and purify it by precipitation in cold ether at -20°C for three rounds. Collect the precipitate and dry it under vacuum overnight to obtain PEG-DCT light yellow powder.

[0037] (3) Synthesize PEG-PDBAMA using reversible addition-fragmentation chain transfer (RAFT) polymerization technology. Weigh 540.48 mg of PEG-DCT obtained in step (2) and dissolve it 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 place it 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 transferred to room temperature for cooling, and the product was purified by dialyzing with deionized water, and freeze-dried at -4°C to obtain PEG-PDBAMA powder.

[0038] (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 hours. 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 recorded as PEG-PDBAMA-PCPT.

[0039] (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. Then, dissolve 4 OD of siRNA in 0.5 mL of dimethyl sulfoxide. Add the above two dimethyl sulfoxide mixtures dropwise into 4 mL of deionized water and ultrasonicate for 30 minutes. The mixture is dialyzed in deionized water overnight to remove dimethyl sulfoxide to obtain PEG-PDBAMA-PCPT / siRNA; two siRNAs of SH3RF3-AS1 are prepared by the above method to obtain PEG-PDBAMA-PCPT / siRNA-1, PEG-PDBAMA-PCPT / siRNA-2 and PEG-PDBAMA-PCPT / siRNA-1+2 (two siRNAs are mixed at 2 OD each).

[0040] (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 MgCl 2 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 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 physically passed through a 400nm 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 the nanodrug. 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.

[0041] In order to verify the effect of nanomedicine, PEG-PDBAMA-PCPT / siRNA-1+2 in the above steps was 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 a single si-SH3RF3-AS1-1 in Table 1), Nano PCPT / siRNA-2@U251 (containing a single si-SH3RF3-AS1-2 in Table 1) for later use. The above substances were used to treat U251 cells. The treatment process was as follows: First, Nanoparticles were dispersed in PBS buffer. 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, the siRNA concentration in each sample except the control group 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 PCPT / siRNA-1+2@U251 mother solution, so that the sample, except for the control group, the final concentration of CPT was 20μM, the final concentration of siRNA was 50nM, and the same volume of PBS buffer complete culture medium was added as a further control, and U251 cells were treated for 24 hours. Then CCK-8 assay was 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).

[0042] In this example, BALB / c nude mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. and raised under SPF conditions. All animal experiments were approved by the Ethics Committee of the Second Hospital of Shandong University (KYLL-2021 (KJ) A-0024). U251 cells were used to form tumors in nude mice: 3×10 fluorescently labeled 5 U251 human glioma cells (U251 luc) were implanted intracranially into BALB / c nude mice (male, 4 weeks old). The growth of orthotopic U251 luc tumors was monitored by a bioluminescence imaging (BLI) system. The date of glioma cell implantation was designated as day 0, and images were acquired using an IVIS Lumina II ex vivo imaging system (PerkinElmer, USA) 5 minutes after intraperitoneal injection of 150 mg / kg luciferin at week 1 and week 4. 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 PCPT / siRNA@U251 drug 200μL (diluted with PBS buffer, except for the PBS buffer control group, the mice were given 5mg / Kg CPT, 0.5mg / Kg siRNA, and 5mg / Kg free CPT), intraperitoneally injected once every three days. In addition, the mice were weighed before administration, and the efficacy was evaluated based on 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 given 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 luminescence in vivo, and there was 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 Nano-siRNA@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 in the PBS and Nano@U251 groups were PCPT / siRNA@U251 and Nano-siRNA@U251 groups had higher cell densities, and 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).

[0043] The above description is only a preferred embodiment of the present invention and does not limit the present invention in other forms. Any technician familiar with the profession may use the technical content disclosed above to change or modify it into an equivalent embodiment with equivalent changes and apply it to other fields. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the protection scope 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 or prognosing glioma, the sequence of SH3RF3-AS1 being shown in the sequence listing as SEQ ID NO:

1.

2. Use of substances that inhibit or reduce the expression of SH3RF3-AS1 in the preparation of preparations that enhance the sensitivity of patients to glioma drug treatment.

3. Use of a substance for detecting the expression level of SH3RF3-AS1 in the preparation of a reagent or kit for glioma diagnosis and / or prognosis prediction.

4. The use according to claim 1 or 2, characterized in that: 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.

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

6. The use according to claim 5, characterized in that: The steps of 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 for reaction 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 for reaction, and stir at room temperature overnight; filter and extract the filtrate with ethyl acetate, evaporate under reduced pressure to obtain 2-[(2-hydroxyethyl) disulfide] 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, concentrated under reduced pressure, and passed through a silica gel column to obtain a yellow product OH-2S-CPT; (2) Dissolve 4-cyano-4-[[(dodecylthio)thiomethyl]thio]pentanoic acid, 4-dimethylaminopyridine and N,N'-dicyclohexylcarbodiimide in dichloromethane and stir evenly at room temperature to obtain a mixed solution C. Add the dichloromethane solution containing methoxypolyethylene glycol dropwise into the mixed solution C and stir at room temperature for 36 hours. After filtering, concentrate the filtrate and purify it 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) dissolving the PEG-DCT obtained in step (2), the DBAMA obtained in step (1) and 2,2'-azobis(2-methylpropionitrile) in dioxane and placing them in a Schlenk tube; after four freeze-pump-thaw cycles, transferring them to an oil bath and incubating them with stirring for 48 hours; then cooling them to room temperature, dialyzing the purified product with deionized water, and freeze-drying them to obtain PEG-PDBAMA powder; (4) dissolving the PEG-PDBAMA powder obtained in step (3), the OH-2S-CPT obtained in step (1) and azobisisobutyronitrile in a mixture of dioxane and dimethyl sulfoxide, and placing the mixture 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. After vacuum drying, a red powder was obtained, which was recorded as PEG-PDBAMA-PCPT. (5) The PEG-PDBAMA-PCPT powder prepared in step (4) is dispersed in dimethyl sulfoxide to obtain dispersion C; siRNA-SH3RF3-AS1 is dissolved in dimethyl sulfoxide to obtain dispersion D; dispersion C and dispersion D are simultaneously added dropwise to deionized water and subjected to ultrasonic treatment; dimethyl sulfoxide is 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 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 were then freeze-dried to obtain nanomedicines.

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