CN119040329B

CN119040329B - A siRNA targeting glioblastoma and its application

Info

Publication number: CN119040329B
Application number: CN202411527574.4A
Authority: CN
Inventors: 张辰宇; 李菁; 陈梦寒; 方静雯
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2024-10-29
Filing date: 2024-10-29
Publication date: 2025-03-18
Anticipated expiration: 2044-10-29
Also published as: CN119040329A

Abstract

The invention relates to siRNA targeting glioblastoma and application thereof, belonging to the technical field of biology. The invention provides an siRNA targeting glioblastoma, wherein the antisense strand of the siRNA can be specifically combined with target nucleic acid to trigger degradation of the target nucleic acid, the target nucleic acid comprises a pathogenic gene of the glioblastoma, and the pathogenic gene of the glioblastoma comprises a gene encoding ribonucleotide reductase subunit M2 and/or a gene encoding heat shock protein 47. Studies show that the gene encoding ribonucleotide reductase subunit M2 and the gene encoding heat shock protein 47 have obvious correlation with the survival of glioblastoma patients, the two genes and the gene can be used as therapeutic targets of glioblastoma, siRNA capable of effectively knocking down the expression of the two genes in glioblastoma patients has great application prospects in preparing medicaments for preventing and/or treating glioblastoma.

Description

SiRNA targeting glioblastoma and application thereof

Technical Field

The invention relates to siRNA targeting glioblastoma and application thereof, belonging to the technical field of biology.

Background

Glioma (Glioma) is one of the most common brain tumors, accounting for 30% of central nervous system brain tumors. Gliomas are classified according to the World Health Organization (WHO) classification standard into grade I-IV, with the most malignant grade IV glioma, also known as glioblastoma (glioblastoma multiforme, GBM). Glioblastomas are frequently developed in brain parenchyma and are diffusely dispersed, are the most common and most invasive types of glioblastomas in clinic, have a median survival time of only 12-18 months in patients, and have a survival rate of less than 5% in 5 years.

Currently, standard treatments for glioblastoma mainly include three types of surgical resection, radiation Therapy (RT) and chemotherapy, where the only first line drug used for chemotherapy is temozolomide (Temozolomide, TMZ). However, about 55% of glioblastoma patients are found to be resistant to temozolomide primary during clinical treatment, and another part of glioblastoma patients inevitably acquire a temozolomide resistant phenotype during the treatment of temozolomide, so that the treatment effect is poor. Thus, there is an urgent need for more effective therapeutic agents against glioblastoma.

With the progress of the new generation sequencing technology, people have more in depth knowledge of the molecular basis and the genome overview of glioblastoma, and many researchers have begun to try to target glioblastoma. However, due to the existence of the blood brain barrier and the high heterogeneity of glioblastomas, glioblastoma targeted therapeutic drugs in many clinical research phases have poor therapeutic effects, and have not made significant progress in prolonging the overall survival of patients and improving prognosis. Therefore, there is an urgent need to break through central administration efficiency and tumor heterogeneity, thereby obtaining glioblastoma targeted therapeutic drugs with better therapeutic effects.

RNA interference (RNA INTERFERENCE, RNAI) is a small interfering RNA (SMALL INTERFERING RNA, SIRNA) -mediated, specific gene silencing phenomenon that is mediated by specific enzymes and is commonly found in organisms in recent years. The siRNA is a double-stranded RNA molecule composed of 20-25 nucleotides, and in an RNAi path, the siRNA interferes with gene expression by hybridization with a complementary mRNA molecule, and the interference triggers mRNA degradation so as to inhibit gene expression of a specific gene. RNAi can silence all genes theoretically, so that siRNA can be used as a targeted therapeutic drug to specifically regulate and control the expression of genes related to diseases.

If the expression of glioblastoma related genes in organisms can be effectively knocked down through siRNA, the targeting therapy of glioblastoma is very helpful. In addition, since the siRNA has specificity and effectiveness in knocking down the related genes of diseases, the development of glioblastoma targeting therapeutic drugs based on RNAi is expected to break through central administration efficiency and tumor heterogeneity. Naked sirnas are extremely susceptible to nuclease degradation, which makes the success of RNAi therapies largely dependent on the siRNA vector and delivery method. Conventional siRNA delivery systems, such as lipid nanoparticles, cationic polymers, viruses, and the like, all suffer from the problems of easy biodegradation, low biocompatibility, insufficient cycling stability, weak targeting ability, and the like. Therefore, on the basis of developing siRNA capable of effectively knocking down expression of glioblastoma related genes in organisms, the targeting therapy of glioblastoma is also critical for a delivery system which can effectively deliver the siRNA into a nerve center, can maintain stability and bioactivity of the siRNA in organisms for a long time and has high biocompatibility.

Disclosure of Invention

In order to solve the above problems, the present invention provides an siRNA targeting glioblastoma, the siRNA comprising a sense strand and an antisense strand, the sense strand of the siRNA being capable of being at least partially reverse-complementary to the antisense strand to form a double-stranded region, the antisense strand of the siRNA being capable of being specifically bound by base-complementary pairing to a target nucleic acid to trigger degradation of the target nucleic acid, the target nucleic acid comprising a pathogenic gene of glioblastoma, the pathogenic gene of glioblastoma comprising a gene encoding ribonucleotide reductase subunit M2 (RRM 2 gene) and/or a gene encoding heat shock protein 47 (HSP 47) (Serpinh gene).

In one embodiment of the invention, the target nucleic acid comprises mRNA encoding ribonucleotide reductase subunit M2 and/or mRNA encoding heat shock protein 47.

In one embodiment of the invention, the pathogenic genes of glioblastomas further include genes encoding epidermal growth factor receptor (EGFR gene), miR-214 and/or genes encoding vascular endothelial growth factor receptor (VEGFR gene).

In one embodiment of the invention, the target nucleic acid comprises mRNA encoding an EGF receptor, miR-214 and/or mRNA encoding a VEGF receptor.

In one embodiment of the present invention, when the pathogenic gene of glioblastoma is a gene encoding ribonucleotide reductase subunit M2, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence shown in SEQ ID No.4, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence shown in SEQ ID No. 9;

When the pathogenic gene of glioblastoma is a gene encoding heat shock protein 47, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.5, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 10.

In one embodiment of the present invention, when the pathogenic gene of glioblastoma is a gene encoding ribonucleotide reductase subunit M2, the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID No.4, and the nucleotide sequence of the antisense strand is shown as SEQ ID No. 9;

When the pathogenic gene of glioblastoma is a gene encoding heat shock protein 47, the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.5, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 10.

In one embodiment of the present invention, when the pathogenic gene of glioblastoma is a gene encoding an epidermal growth factor receptor, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence shown in SEQ ID No.1, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence shown in SEQ ID No. 6;

When the pathogenic gene of glioblastoma is miR-214, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.2, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 7;

when the pathogenic gene of glioblastoma is a gene encoding vascular endothelial growth factor receptor, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO.3, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 8.

In one embodiment of the present invention, when the pathogenic gene of glioblastoma is a gene encoding an epidermal growth factor receptor, the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 6;

When the pathogenic gene of glioblastoma is miR-214, the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.2, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 7;

When the pathogenic gene of glioblastoma is a gene for encoding vascular endothelial growth factor receptor, the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.3, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 8.

The invention also provides a recombinant nucleic acid molecule, which comprises shRNA, wherein the shRNA comprises a sense strand of the siRNA and an antisense strand of the siRNA, and the sense strand and the antisense strand are separated by a stem-loop sequence to form a hairpin structure.

In one embodiment of the present invention, when the pathogenic gene of glioblastoma is a gene encoding ribonucleotide reductase subunit M2, the shRNA comprises a nucleic acid molecule having the nucleotide sequence shown in SEQ ID No. 14;

when the pathogenic gene of glioblastoma is a gene encoding heat shock protein 47, the shRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 15.

In one embodiment of the present invention, when the pathogenic gene of glioblastoma is a gene encoding ribonucleotide reductase subunit M2, the nucleotide sequence of the shRNA is shown as SEQ ID No. 14;

When the pathogenic gene of glioblastoma is a gene encoding heat shock protein 47, the nucleotide sequence of the shRNA is shown as SEQ ID NO. 15.

In one embodiment of the present invention, when the pathogenic gene of glioblastoma is a gene encoding an epidermal growth factor receptor, the shRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID No. 11;

When the pathogenic gene of glioblastoma is miR-214, the shRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 12;

When the pathogenic gene of glioblastoma is a gene encoding vascular endothelial growth factor receptor, the shRNA comprises a nucleic acid molecule with a nucleotide sequence shown as SEQ ID NO. 13.

In one embodiment of the present invention, when the pathogenic gene of glioblastoma is a gene encoding an epidermal growth factor receptor, the nucleotide sequence of the shRNA is shown as SEQ ID No. 11;

when the pathogenic gene of glioblastoma is miR-214, the nucleotide sequence of the shRNA is shown as SEQ ID NO. 12;

When the pathogenic gene of glioblastoma is a gene for encoding vascular endothelial growth factor receptor, the nucleotide sequence of the shRNA is shown as SEQ ID NO. 13.

In one embodiment of the invention, the recombinant nucleic acid molecule further comprises a targeting nucleic acid molecule encoding a neural targeting peptide, a promoter and/or a functional nucleic acid molecule encoding a lysosomal associated membrane protein.

In one embodiment of the invention, the neural targeting peptide comprises a rabies glycoprotein peptide fragment (RVG), glioma targeting peptide (Angiopep-2), apolipoprotein (APOE) and/or Cell Penetrating Peptide (CPP) capable of penetrating the blood brain barrier.

In one embodiment of the invention, the promoters include a cytomegalovirus Promoter (CMV Promoter), a lentiviral Promoter (EF 1 Promoter ), and/or a polymerase III Promoter (U6 Promoter).

In one embodiment of the invention, the promoter is a cytomegalovirus promoter, and the nucleotide sequence of the cytomegalovirus promoter is shown in SEQ ID NO. 16.

In one embodiment of the invention, the lysosomal associated membrane proteins include lysosomal associated membrane protein 2B (LAMP 2B), transmembrane protein CD63, transmembrane protein CD9 and/or transmembrane protein CD81.

In one embodiment of the invention, the targeting nucleic acid molecule encodes rabies virus glycoprotein peptide fragment (RVG), the functional nucleic acid molecule encodes a lysosomal associated membrane protein 2B, the targeting nucleic acid molecule is inserted between the functional nucleic acid molecules to form a chimeric fragment, and the nucleotide sequence of the chimeric fragment is shown as SEQ ID NO. 17.

In one embodiment of the invention, the recombinant nucleic acid molecule comprises a cytomegalovirus promoter, a chimeric fragment and an shRNA connecting fragment which are sequentially connected in series, wherein the shRNA connecting fragment comprises shRNA corresponding to siRNA targeting mRNA encoding an epidermal growth factor receptor, shRNA corresponding to siRNA targeting mRNA encoding a vascular endothelial growth factor receptor, shRNA corresponding to siRNA targeting mRNA encoding ribonucleotide reductase subunit M2, shRNA corresponding to siRNA targeting mRNA encoding heat shock protein 47 and shRNA corresponding to siRNA targeting miR-214 which are sequentially connected in series.

In one embodiment of the invention, the nucleotide sequence of the shRNA junction fragment is shown as SEQ ID NO. 18.

The invention also provides a recombinant plasmid which expresses the siRNA, or carries the recombinant nucleic acid molecule.

In one embodiment of the invention, the recombinant plasmid vector comprises at least one of a viral vector or a non-viral vector, wherein the viral vector comprises at least one of a flavivirus vector, a retrovirus vector, a phage vector, an adenovirus vector, an adeno-associated virus vector, a vaccinia virus vector, a hybrid virus vector, a baculovirus vector, a herpes simplex virus vector or a lentivirus vector, and the non-viral vector comprises a plasmid vector.

In one embodiment of the invention, the plasmid vector comprises a pcDNA6.2 plasmid, a pLKO.1 plasmid, a PGEM-3zf plasmid, a PUC19 plasmid and/or a PUC57 plasmid.

In one embodiment of the invention, the recombinant plasmid is prepared by connecting the recombinant nucleic acid molecule with a linearized vector after enzyme digestion.

The invention also provides an engineering cell, wherein the genome of the engineering cell is integrated with the siRNA, or the genome of the engineering cell is integrated with the recombinant nucleic acid molecule, or the engineering cell carries the recombinant plasmid.

In one embodiment of the invention, the engineered cell comprises a liver cell.

In one embodiment of the invention, the engineered cell is an engineered body liver cell.

The invention also provides a small extracellular vesicle, wherein the small extracellular vesicle wraps the siRNA, or the small extracellular vesicle wraps the recombinant nucleic acid molecule, or the small extracellular vesicle wraps the recombinant plasmid, or the small extracellular vesicle is secreted by the engineering cell.

In one embodiment of the present invention, the small extracellular vesicles secreted by the engineered cells are internally packaged with siRNA expressed by the recombinant nucleic acid molecules in the engineered cells.

The invention also provides application of the siRNA or the recombinant nucleic acid molecule or the recombinant plasmid or the engineering cell or the small extracellular vesicle in preparing a medicament for preventing and/or treating brain tumor of a central nervous system.

In one embodiment of the invention, the composition of the medicament comprises an inhibitor comprising the siRNA, the recombinant nucleic acid molecule, the recombinant plasmid, the engineered cell and/or the small extracellular vesicle.

In one embodiment of the invention, the inhibitor is an engineered cell as described above.

In one embodiment of the invention, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

In one embodiment of the invention, the pharmaceutically acceptable excipients further comprise a solvent.

In one embodiment of the invention, the brain tumor of the central nervous system comprises glioma, and the glioma comprises glioblastoma.

The invention also provides a medicine for preventing and/or treating brain tumors of the central nervous system, wherein the components of the medicine comprise inhibitors, and the inhibitors comprise the siRNA, the recombinant nucleic acid molecules, the recombinant plasmids, the engineering cells and/or the small extracellular vesicles.

The technical scheme of the invention has the following advantages:

1. The invention provides an siRNA targeting glioblastoma, wherein the antisense strand of the siRNA can be subjected to base complementary pairing and target nucleic acid specific binding to trigger degradation of target nucleic acid, the target nucleic acid comprises a pathogenic gene of the glioblastoma, and the pathogenic gene of the glioblastoma comprises a gene (RRM 2 gene) encoding ribonucleotide reductase subunit M2 and/or a gene (Serpinh gene) encoding heat shock protein 47 (HSP 47). The differential gene enrichment analysis and the gene channel enrichment analysis of glioblastoma patients and healthy people show that the gene encoding the ribonucleotide reductase subunit M2 and the gene encoding the heat shock protein 47 have obvious correlation with the survival of glioblastoma patients, and the gene encoding the ribonucleotide reductase subunit M2 and the gene encoding the heat shock protein 47 can be used as therapeutic targets of glioblastoma, so that siRNA capable of effectively knocking down the expression of the two genes in glioblastoma patients has great application prospects in preparing medicaments for preventing and/or treating central nervous system brain tumors, especially glioblastoma.

Further, the pathogenic genes of glioblastoma also include genes encoding epidermal growth factor receptor (EGFR gene), miR-214 and/or genes encoding vascular endothelial growth factor receptor (VEGFR gene). The gene encoding the EGFR, the miR-214 and the gene encoding the VEGF receptor are obviously related to the survival of glioblastoma patients, and therefore, the gene encoding the EGFR, the miR-214 and the gene encoding the VEGF receptor can also be used as therapeutic targets of glioblastoma, and the siRNA capable of effectively knocking down the expression of the three genes in the glioblastoma patients has great application prospects in preparing medicines for preventing and/or treating central nervous system brain tumors, especially glioblastoma.

Further, when the pathogenic gene of glioblastoma is a gene encoding ribonucleotide reductase subunit M2, the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.4, the nucleotide sequence of the antisense strand is shown as SEQ ID NO.9, when the pathogenic gene of glioblastoma is a gene encoding heat shock protein 47, the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.5, the nucleotide sequence of the antisense strand is shown as SEQ ID NO.10, when the pathogenic gene of glioblastoma is a gene encoding epidermal growth factor receptor, the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.1, the nucleotide sequence of the antisense strand is shown as SEQ ID NO.6, when the pathogenic gene of glioblastoma is miR-214, the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.7, and when the pathogenic gene of glioblastoma is a gene encoding vascular growth factor receptor, the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 8. The siRNA can target the pathogenic gene of glioblastoma, the research shows that the siRNA has high interference efficiency, can remarkably lower the expression of the pathogenic gene of glioblastoma in organisms, and the research shows that the small extracellular vesicles wrapping the siRNA can remarkably inhibit the growth of glioblastoma in the brain of mice. Therefore, the siRNA has great application prospect in preparing medicaments for preventing and/or treating brain tumors of the central nervous system, especially glioblastoma.

2. The invention also provides a recombinant nucleic acid molecule, which comprises shRNA, a targeting nucleic acid molecule, a promoter and/or a functional nucleic acid molecule, wherein the shRNA comprises a sense strand of siRNA targeting glioblastoma and an antisense strand of siRNA targeting glioblastoma, the sense strand and the antisense strand are separated by a stem-loop sequence to form a hairpin structure, the targeting nucleic acid molecule codes for a neural targeting peptide, and the functional nucleic acid molecule codes for a lysosomal associated membrane protein. The invention uses a synthetic biology method to connect nerve center targeting peptide capable of penetrating blood brain barrier, lysosome related membrane protein capable of forming small extracellular vesicles and shRNA containing siRNA in series to obtain a gene loop, and the gene loop is injected into a body through intravenous injection, so that the liver of the body can be used as a biological reaction base plate, and the small extracellular vesicles wrapping therapeutic siRNA can be generated by self-assembly in the body, thereby realizing the accurate delivery and multi-target joint treatment of glioblastoma RNAi drugs. Therefore, the recombinant nucleic acid molecule has great application prospect in preparing medicaments for preventing and/or treating brain tumors of the central nervous system, especially glioblastoma.

3. The invention provides a small extracellular vesicle, the small extracellular vesicles encapsulate siRNA targeting glioblastoma. The small extracellular vesicles (Small extracellular vesicles, sEV) are small membrane particles (particle size of 30-150 nm) secreted by cells, which are important communication means for cells to help the communication between cells by transporting nucleic acids and proteins between adjacent cells. Compared to traditional delivery systems, small extracellular vesicles, due to their natural nature, are able to evade phagocytosis, prolong the in vivo half-life of the agent, and reduce immunogenicity. The siRNA targeting glioblastoma through the small extracellular vesicles has the advantages that firstly, the siRNA targeting glioblastoma can be effectively delivered into a nerve center, the stability and the bioactivity of the siRNA targeting glioblastoma in a living body can be maintained for a long time, meanwhile, the siRNA targeting glioblastoma also has high biocompatibility, the siRNA targeting glioblastoma can be beneficial to inhibiting the expression of glioblastoma pathogenic genes in the living body, so that an anti-tumor effect is achieved, and secondly, the siRNA targeting glioblastoma can be delivered through the small extracellular vesicles, the problems of using extracellular vesicles from exogenous cells to deliver, such as complex technical operation, high cost, endotoxin and the like, can be effectively avoided, and thirdly, experiments prove that the small extracellular vesicles injected into a mouse can be used for remarkably inhibiting the growth of glioblastoma in the brain of the mouse, and the targeted glioblastoma can be remarkably treated. Therefore, the small extracellular vesicles have great application prospect in preparing medicines for preventing and/or treating brain tumors of the central nervous system, especially glioblastoma.

Drawings

FIG. 1 correlation of genes encoding deoxyribonucleotide reductase (RRM 2) with patient survival.

FIG. 2 correlation of the gene encoding heat shock protein 47 (HSP 47) with patient survival.

FIG. 3 interference efficiency of siRNA targeting VEGFR genes.

FIG. 4 interference efficiency of siRNA targeting RRM2 gene.

FIG. 5 interference efficiency of siRNA targeting Serpinh gene.

FIG. 6 is a diagram of the construction of five siRNA tandem multi-target gene loops.

FIG. 7 plasmid map of recombinant plasmid CMV-RVG-SiR ^{EGFR+VEGFR+RRM2+Serpinh1+anti-214}.

FIG. 8 is a flow chart of an experiment for in vitro functional verification of sEV-siRNA produced by self-assembly.

FIG. 9 influence of self-assembled sEV-siRNA on EGFR gene mRNA (self-assembled sEV-siRNA significantly reduced target gene mRNA expression compared to control).

FIG. 10 effect of self-assembled sEV-siRNA on VEGFR gene mRNA (self-assembled sEV-siRNA significantly reduced target gene mRNA expression compared to control).

FIG. 11 effect of self-assembled sEV-siRNA on RRM2 gene mRNA (self-assembled sEV-siRNA significantly reduced expression of target gene mRNA compared to control).

FIG. 12 effect of self-assembled sEV-siRNA on Serpinh gene mRNA (sEV-siRNA produced by self-assembly significantly reduced expression of target gene mRNA compared to control).

FIG. 13 effect of self-assembled sEV-siRNA on proteins (EGF receptor, VEGF, DNase and HSP 47) (Table of self-assembled sEV-siRNA significantly reduced target protein compared to control).

FIG. 14 results of in vivo images of mice from different groups of mice.

FIG. 15 therapeutic effect of gene loops in mice (tail vein injection of gene loops inhibits growth of intracranial tumors in situ xenograft mice).

Detailed Description

The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.

The following examples do not identify specific experimental procedures or conditions, which may be followed by procedures or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.

Experimental example 1 acquisition of therapeutic target of glioblastoma

The specific process is as follows:

According to the characteristics of high proliferation speed, high angiogenesis capacity and immune escape of glioblastoma, firstly, an Epidermal Growth Factor Receptor (EGFR), miR-214 and Vascular Endothelial Growth Factor (VEGFR) are selected as possible treatment targets of GBM, then, the special treatment targets of glioblastoma are screened by a TCGA database, and the specific screening process comprises the steps of downloading gene information of 10 healthy people and 528 glioblastoma patients from an Affymetrix HG-UG133A platform, finding out genes with obvious changes of the glioblastoma patients through differential gene enrichment analysis and gene passage enrichment analysis, and correlating the genes with survival of the patients, wherein the results are shown in figures 1-2. From fig. 1-2, it can be seen that the gene encoding ribonucleotide reductase subunit M2 and the gene encoding heat shock protein 47 have significant correlation with the survival of glioblastoma patients, and thus, new therapeutic targets of glioblastoma deoxyribonucleotide reductase (RRM 2) and heat shock protein 47 (HSP 47, encoded by Serpinh gene) were finally determined.

Experimental example 2 acquisition of siRNA targeting glioblastoma

The specific process is as follows:

1. Design of

SiRNA targeting EGFR gene, miR-214, VEGFR gene, RRM2 gene and Serpinh gene (see Table 1 for details, complementary DNA sequences of sense strand and antisense strand of siRNA in Table 1) were designed with mRNA encoding Epidermal Growth Factor Receptor (EGFR), miR-214, mRNA encoding Vascular Endothelial Growth Factor Receptor (VEGFR), mRNA encoding ribonucleotide reductase subunit M2 (RRM 2) and mRNA encoding heat shock protein 47 (HSP 47, encoded by Serpinh gene) as target genes, respectively, as follows:

The nucleotide sequence of the sense strand is shown as SEQ ID NO.1, the nucleotide sequence of the antisense strand is shown as SEQ ID NO.6, and the EGFR-siRNA is named as EGFR-siRNA;

the nucleotide sequence of the sense strand is shown as SEQ ID NO.2, the nucleotide sequence of the antisense strand is shown as SEQ ID NO.7, and the miR-214-siRNA is named;

siRNA1 of targeted VEGFR gene, the nucleotide sequence of sense strand is shown as SEQ ID NO.19, the nucleotide sequence of antisense strand is shown as SEQ ID NO.25, and the sequence is named VEGFR-siRNA1;

siRNA2 of targeted VEGFR gene, the nucleotide sequence of sense strand is shown as SEQ ID NO.3, the nucleotide sequence of antisense strand is shown as SEQ ID NO.8, and the sequence is named VEGFR-siRNA2;

The nucleotide sequence of the sense strand is shown as SEQ ID NO.20, the nucleotide sequence of the antisense strand is shown as SEQ ID NO.26, and the siRNA3 of the targeted VEGFR gene is named as VEGFR-siRNA3;

siRNA1 of target RRM2 gene, the nucleotide sequence of sense strand is shown as SEQ ID NO.21, the nucleotide sequence of antisense strand is shown as SEQ ID NO.27, and it is named RRM2-siRNA1;

siRNA2 of target RRM2 gene, the nucleotide sequence of sense strand is shown as SEQ ID NO.4, the nucleotide sequence of antisense strand is shown as SEQ ID NO.9, and it is named RRM2-siRNA2;

the nucleotide sequence of the sense strand is shown as SEQ ID NO.22, the nucleotide sequence of the antisense strand is shown as SEQ ID NO.28, and the target RRM2 gene is named RRM2-siRNA3;

siRNA1 of target Serpinh1 gene, the nucleotide sequence of sense strand is shown as SEQ ID NO.23, the nucleotide sequence of antisense strand is shown as SEQ ID NO.29, and it is named Serpinh-siRNA 1;

siRNA2 of target Serpinh1 gene, the nucleotide sequence of sense strand is shown as SEQ ID NO.24, the nucleotide sequence of antisense strand is shown as SEQ ID NO.30, and it is named Serpinh-siRNA 2;

siRNA3 of target Serpinh gene, the nucleotide sequence of sense strand is shown in SEQ ID NO.5, the nucleotide sequence of antisense strand is shown in SEQ ID NO.10, and the sequence is named Serpinh-siRNA 3;

2. Screening

Mixing 5. Mu.L of transfection reagent lipo2000 (purchased from Siemens Fed. Co.) with 100. Mu.L of Opti-MEM (purchased from Siemens Fed. Co.) and standing for 5 min to obtain a mixture A, diluting different siRNAs with 100. Mu.L of Opti-MEM to a concentration of 100 pmol and standing for 5 min to obtain a mixture B, mixing the mixture A and the mixture B and standing for 20 min to obtain a transfection solution containing different siRNAs;

U87 cells (purchased from a cell bank of China academy of sciences) were inoculated into 6-well plates each of which was added 2mL DMEM medium (purchased from Siemens Fedder) containing 10% (v/v) fetal bovine serum at an inoculum size of 1X 10 ⁵/well, cultured in a cell incubator at 37℃for 24 h in 5% (v/v) CO ₂, after 24 h, a blank control group, a negative control group (siNC), an EGFR-siRNA test group, a miR-214-siRNA test group, a VEGFR-siRNA1 test group, a VEGFR-siRNA2 test group, a VEGFR-siRNA3 test group, an RRM2-siRNA1 test group, an RRM2-siRNA2 test group, an RRM2-siRNA3 test group, a Serpinh-siRNA 1 test group, a Serpinh-siRNA 2 test group, a Serpinh 1-3 test group were set, and 4 multiple wells were set in each of the 6-well plates;

After the setting, respectively adding transfection solutions containing different siRNAs into wells of an experimental group (the EGFR-siRNA experimental group is given with the transfection solution containing siRNA targeting EGFR gene, the miR-214-siRNA experimental group is given with the transfection solution containing siRNA targeting miR-214 gene, the VEGFR-siRNA1 experimental group is given with the transfection solution containing siRNA1 targeting VEGFR gene, so as to push the same), adding the transfection solution containing siNC into wells of a negative control group (the nucleotide sequence of the sense strand of siNC is shown as SEQ ID NO.31, the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 32), adding the transfection solution containing no siRNA into a blank control group, culturing 6 h in a 5% (v/v) CO ₂ and 37 ℃ cell culture box, and after 6 h is transfected, adding 2 mL new 10% (v/v) DMEM culture medium containing 10% (v/v) bovine serum into each well of a 6-well plate, and continuing to transfer cells of ₂ and h ℃ in a 37 ℃ cell culture box;

after transfection of 24 h, cell lysate (from Sigma Co.) was added to 6-well plates at an addition of 500. Mu.L per well, chloroform was added to 6-well plates at an addition of 100. Mu.L per well, vortexed vigorously, placed on ice for 5 min, and centrifuged at 12000 rpm, 4℃for 10 min, after centrifugation, the supernatant was taken, isopropanol was added in an equal volume to the supernatant, placed at room temperature (25 ℃) for 16 h, centrifuged at 12000 rpm, 4℃for 10 min, after centrifugation, the pellet was taken, resuspended to the original volume (i.e., equal volume to the supernatant) using 75% (v/v) ethanol, centrifuged at 12000 rpm, 4 ℃) for 10 min, after centrifugation, mRNA was obtained, the mRNA was obtained by centrifugation, the mRNA obtained by extraction was used as the sample to be tested, 18S was used as the internal standard gene, the expression level of the target gene in the cells after interference of different siRNAs was detected by qPCR (the measurement of C _T was obtained by qPCR, the relative level of mRNA was normalized to 18S by qPCR) Determining the expression level of a target gene by the method) so as to verify the interference efficiency of the designed siRNA, wherein the verification result is shown in figures 3-5;

the specific steps of qPCR are as follows:

reverse transcription, namely adding the reagent into a 200 mu L enzyme-removing centrifuge tube according to the reaction system of the table 2, and placing the mixture into a PCR instrument after vortex mixing to obtain cDNA (specific reverse transcription reaction program is shown in the table 2);

qPCR the reagents were added to 200. Mu.L enzyme-removed centrifuge tubes according to the reaction system of Table 3, vortexed, centrifuged and then added to 96-well plates (specific qPCR reaction procedure is shown in Table 3, primers used for qPCR are shown in Table 4).

As can be seen from fig. 3, in the siRNA targeting VEGFR gene, the interference efficiency of siRNA2 was optimal, and expression of VEGFR MRNA was significantly knocked down.

As can be seen from fig. 4, in the siRNA targeting RRM2 gene, the interference efficiency of siRNA2 was optimal, and the expression of RRM2 mRNA was significantly knocked down.

As can be seen from fig. 5, in siRNA targeting Serpinh1 gene, the interference efficiency of siRNA3 was optimal, and expression of Serpinh mRNA was significantly knocked down.

Table 1 siRNA and its sequence

TABLE 2 reverse transcription reaction System and reaction procedure

Table 3 qPCR reaction System and reaction procedure

Table 4 qPCR primers and sequences thereof

Experimental example 3 influence of self-assembled sEV-siRNA on target protein and mRNA in vitro

The specific process is as follows:

1. construction of recombinant plasmid CMV-RVG-siR ^{EGFR+VEGFR+RRM2+Serpinh1+anti-214}

Designing shRNA (the nucleotide sequence of the shRNA is shown as SEQ ID NO. 11) according to siRNA targeting EGFR gene, the nucleotide sequence of the sense strand of which is shown as SEQ ID NO.1, the nucleotide sequence of the antisense strand of which is shown as SEQ ID NO.6, and naming the shRNA as Hsa-siEGFR;

according to siRNA targeting miR-214, wherein the nucleotide sequence of the sense strand is shown as SEQ ID NO.2, the nucleotide sequence of the antisense strand is shown as SEQ ID NO.7, shRNA (the nucleotide sequence of the shRNA is shown as SEQ ID NO. 12) is designed and named as Anti-miR-214;

Designing shRNA (the nucleotide sequence of the shRNA is shown as SEQ ID NO. 13) according to siRNA targeting VEGFR genes, wherein the nucleotide sequence of the sense strand is shown as SEQ ID NO.3, the nucleotide sequence of the antisense strand is shown as SEQ ID NO.8, and the shRNA is named as mmu-siVEGFR;

Designing shRNA (the nucleotide sequence of the shRNA is shown as SEQ ID NO. 14) according to siRNA targeting RRM2 genes, wherein the nucleotide sequence of the sense strand is shown as SEQ ID NO.4, the nucleotide sequence of the antisense strand is shown as SEQ ID NO.9, and the shRNA is named Hsa-siRRM2;

Designing shRNA (the nucleotide sequence of the shRNA is shown as SEQ ID NO. 15) according to siRNA targeting Serpinh gene, the nucleotide sequence of which is shown as SEQ ID NO.5, the nucleotide sequence of which is shown as SEQ ID NO.10, of the sense strand, and the shRNA is named Hsa-SISERPINH1;

The chimeric fragment of a gene encoding rabies glycoprotein peptide (RVG), a gene encoding lysosomal associated membrane protein 2B (LAMP 2B), hsa-siEGFR, mma-siVEGFR, hsa-siRRM2, hsa-SISERPINH1 and Anti-miR-214 are sequentially connected in series to obtain a multi-target gene loop (the construction map of five siRNA tandem multi-target gene loops is shown in FIG. 6, the nucleotide sequence of the cytomegalovirus Promoter is shown in SEQ ID NO.16, the nucleotide sequence of the chimeric fragment is shown in SEQ ID NO.17, and the nucleotide sequence of the multi-target gene loop is shown in SEQ ID NO. 43).

Synthesizing a multi-target gene loop, carrying out double enzyme digestion (enzyme digestion system see Table 5,37 ℃ for enzyme digestion reaction 30 min and then 85 ℃ for inactivation 5 min) on the multi-target gene loop and a PCDNA.2 plasmid vector (purchased from Shanghai Ji Ma Co., ltd.) by BamHI and XhoI (purchased from Simer Fei Co.), carrying out electrophoresis gel running on the product after the enzyme digestion reaction is finished, and recovering a target fragment gel to obtain a vector and an insert fragment subjected to double enzyme digestion; the recombinant plasmid with recombinant nucleotide sequence of SEQ ID 35-R35 (shown in SEQ ID NO. 35) was obtained by streaking a double digested vector and insert with T4 DNA ligase and T4 DNA ligase buffer (purchased from Takara Corp.) (the ligation system was shown in Table 6,16 ℃ C. Incubated at 14 h) to obtain ligation product, transforming E.coli competent cells DH 5. Alpha. (purchased from Optimago family) to obtain transformation product, culturing the transformation product in LB agar plate medium (formula: 5g/L yeast, 10g/L peptone, 10g/L sodium chloride and 18g/L agar powder) containing 50. Mu.g/mL ampicillin (Amp) at 37 ℃ C., culturing at 12 h, picking single colony, inoculating single colony to LB liquid medium (formula: 5g/L yeast, 10g/L peptone and 10g/L sodium chloride) containing 50. Mu.g/mL spectinomycin at 37 ℃ C., culturing at h to obtain plasmid liquid, extracting and purifying recombinant plasmid in the liquid, sequencing to obtain recombinant plasmid with recombinant nucleotide sequence of SEQ ID 35-R35 (shown in SEQ ID NO. 35).

2. Influence of self-assembled sEV-siRNA on target protein and mRNA in vitro

According to the experimental procedure of FIG. 8, control empty plasmid and recombinant plasmid CMV-RVG-SiR ^EGFR ^{+VEGFR+RRM2+Serpinh1+anti-214} were tail-vein injected into 6 week-old BALB/C mice (from Proteus) twice daily in a dose of 10 mg/kg (200. Mu.L PBS buffer), and CO-injected four times daily, plasma from the mice was obtained by taking blood from the eyeballs after the last injection for 6 hours, exosomes (sEV-siRNA produced by self-assembly of the exosomes, i.e., a multi-target gene loop, in the mice) were extracted from the plasma by ultracentrifugation, U87 cells (from Shanghai cell Bioinstitute) were inoculated to a 6-well plate (from Gifumeo) supplemented with 10% (v/v) fetal bovine serum (Novain) at an inoculum size of 7.5X10 ⁶ cells/well), 12-35 were incubated in a 5% (v/v) CO ₂, 37℃cell incubator, h were incubated in a 5% (v/v) cell incubator, and CO-expressed by CO-protein (5. Mu.v) in a target gene (5. Mu.L) was CO-37% protein was CO-expressed in the following PCR experiment using the following protocol, and the following PCR protocol was carried out at a total protein level of 5.37 to a target protein of the 5% (5.v) and a 5.mu.L, 50.L, and a target protein was added to a PCR protocol of the target protein (4.9.37) in the PCR protocol, and a sample from the experiment was added to the PCR protocol (experiment).

RNA extraction, namely adding cell lysate (purchased from Takara company) into a 6-well plate with the addition amount of 1mL per well, placing the cell lysate on ice for 45min, and shaking vigorously every 15min to obtain a cell lysate; centrifuging the cell lysate at 12000 Xg and 4 ℃ for 10 min to remove cell residues, and taking supernatant A after centrifugation; adding 200 mu L of chloroform into the supernatant A, shaking vigorously, standing on ice for layering (consuming 5 min) to obtain an extraction product; centrifuging the extracted product at 12000 Xg and 4 ℃ for 15min, taking supernatant B after centrifugation, adding 600 mu L of isopropanol into the supernatant B and uniformly mixing, standing for 3 h in a refrigerator at-20 ℃ to obtain crude extracted RNA, centrifuging the crude extracted RNA at 12000 Xg and 4 ℃ for 20 min, tilting the centrifuge tube after centrifugation is finished, pouring the supernatant out of the centrifuge tube to obtain a centrifuge tube filled with white RNA sediment, preparing 75% (v/v) ethanol with RNase-free water, pouring 1mL of 75% ethanol into the centrifuge tube filled with white RNA sediment, firstly upside down washing the sediment, centrifuging for 10 min at 12000 Xg and 4 ℃, tilting the centrifuge tube after centrifugation is finished, pouring out the supernatant out of the centrifuge tube, centrifuging for a short time to obtain washed RNA sediment, sucking redundant liquid in the centrifuge tube filled with washed RNA sediment by a pipette gun, reversely buckling, airing the sediment to obtain dry RNA sediment, adding 30 mu L of RNase-free water into the centrifuge tube filled with dry RNA sediment to dissolve the obtained RNA sediment, measuring the concentration of the RNA sediment to be the concentration of the sample to be ng mu L of the RNA sediment, centrifuging the obtained by measuring the concentration of the RNA sediment before the concentration is measured to be the RNA sediment is measured, the concentration is required to be the concentration of the RNA is measured to be the RNA solution is measured by using the sample and the solution is adjusted to be the concentration of 62 mu L, obtaining a sample to be tested;

Adding the reverse transcription reaction system into a centrifuge tube, mixing uniformly by vortex, and placing into a PCR instrument for reverse transcription reaction (reverse transcription reaction program: 42 ℃,2 min, 37 ℃,15min, 85 ℃, 5s, 4 ℃ and preservation) to obtain cDNA solution;

qPCR detection, quantitative detection is carried out by using 18S as an internal reference and double-stranded DNA dye (SYBR Geen), a qPCR reaction system is prepared according to the table 8, the qPCR reaction system is added into a microtube, vortex mixing is carried out, and then the mixture is placed into an LC480 fluorescent quantitative PCR instrument for carrying out PCR reaction (the PCR reaction program is 95 ℃ for 5 minutes, 95 ℃ for 30 seconds for 40 cycles, 6 ℃ for 30 seconds and 72 ℃ for 1 minute, and primers used for qPCR are shown in the table 4).

From the results of fig. 9 to 13, it was found that the expression levels of EGFR, VEGFR, RRM, serpinh1 protein and mRNA in U87 cells could be significantly reduced by injecting recombinant plasmid CMV-RVG-siR ^{EGFR+VEGFR+RRM2+Serpinh1+anti-214} into mice as compared with control empty plasmid. The results show that the exosomes extracted from the mouse plasma have higher interference efficiency on the target genes.

Table 5 enzyme digestion System

Table 6 connection system

TABLE 7 reverse transcription reaction system

Table 8 qPCR reaction System

Experimental example 4 therapeutic Effect of Gene Loop in mice

The specific process is as follows:

U87-luc cells (purchased from Shanghai cell Biotechnology institute) were implanted intracranially (from Jiuzhikang) in nude mice (from bregma) at an injection level of 1X 10 ⁶ (5. Mu.L of high sugar medium) at the following coordinates relative to bregma: post 0.5mm, lateral 2.5mm and intra-brain 3.5mm to construct glioblastoma xenograft models, and after 7 days of U87 cell implantation, tumor size was examined using in vivo imaging to ensure successful formation of glioblastoma in the brain. The nude mice carrying glioblastoma are randomly divided into 3 groups, wherein the 3 groups are respectively a blank group (n=10), a control group (n=10) and a treatment group (n=10), the treatment group mice are injected with recombinant plasmid CMV-RVG-SiR ^{EGFR+VEGFR+RRM2+Serpinh1+anti-214} (solvent is 200 mu L PBS buffer) according to tail vein of 5 mg/kg, the control group mice are injected with equivalent doses of control empty plasmid (solvent is 200 mu L PBS buffer) according to tail vein, the blank group mice are injected with equivalent volumes of PBS buffer for one time in two days, the injection is carried out for 14 times in total, during the plasmid injection, live body imaging of the mice is shot every seven days, and the tumor size is detected, and the detection results are shown in figures 14-15.

From the results of fig. 14-15, in vivo imaging of the animals on day 28 after injection of the recombinant plasmid CMV-RVG-siR ^{EGFR+VEGFR+RRM2+Serpinh1+anti-214} via the tail vein showed significantly lower fluorescence intensity in the intracranial tumor in the treated mice than in the control (about 48% decrease) compared to the control empty plasmid. The result shows that the gene loop can effectively inhibit the deterioration of the intracranial tumor of the mice.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims

1. The siRNA targeting glioblastoma is characterized in that an antisense strand of the siRNA can be specifically combined with target nucleic acid through base complementation pairing to trigger degradation of the target nucleic acid, the target nucleic acid is a gene encoding heat shock protein 47, the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.5, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 10.

2. A recombinant nucleic acid molecule is characterized in that the recombinant nucleic acid molecule comprises an shRNA connecting fragment, wherein the shRNA connecting fragment comprises shRNA corresponding to siRNA targeting mRNA encoding an epidermal growth factor receptor, shRNA corresponding to siRNA targeting mRNA encoding a vascular endothelial growth factor receptor, shRNA corresponding to siRNA targeting mRNA encoding ribonucleotide reductase subunit M2, shRNA corresponding to siRNA targeting mRNA encoding heat shock protein 47 and shRNA corresponding to siRNA targeting miR-214;

the shRNA corresponding to an siRNA targeting mRNA encoding heat shock protein 47 comprises the sense strand of the siRNA of claim 1 and the antisense strand of the siRNA of claim 1;

The shRNA corresponding to the siRNA targeting mRNA encoding ribonucleotide reductase subunit M2 comprises a sense strand of the siRNA with a nucleotide sequence shown as SEQ ID NO.4 and an antisense strand of the siRNA with a nucleotide sequence shown as SEQ ID NO. 9;

The shRNA corresponding to the siRNA targeting the mRNA encoding the epidermal growth factor receptor comprises a sense strand of the siRNA with a nucleotide sequence shown as SEQ ID NO.1 and an antisense strand of the siRNA with a nucleotide sequence shown as SEQ ID NO. 6;

The shRNA corresponding to the siRNA targeting miR-214 comprises a sense strand of the siRNA with a nucleotide sequence shown as SEQ ID NO.2 and an antisense strand of the siRNA with a nucleotide sequence shown as SEQ ID NO. 7;

The shRNA corresponding to the siRNA targeting the mRNA encoding the vascular endothelial growth factor receptor comprises a sense strand of the siRNA with a nucleotide sequence shown as SEQ ID NO.3 and an antisense strand of the siRNA with a nucleotide sequence shown as SEQ ID NO. 8;

the sense strand and the antisense strand are separated by a stem loop sequence to form a hairpin structure.

3. A recombinant plasmid, wherein the siRNA of claim 1 is expressed by the recombinant plasmid, or the recombinant plasmid carries the recombinant nucleic acid molecule of claim 2.

4. An engineered cell having the siRNA of claim 1 integrated into its genome, or the recombinant nucleic acid molecule of claim 2 integrated into its genome, or the engineered cell carrying the recombinant plasmid of claim 3.

5. A small extracellular vesicle, wherein the small extracellular vesicle is packaged with the siRNA of claim 1, or the small extracellular vesicle is packaged with the recombinant nucleic acid molecule of claim 2, or the small extracellular vesicle is packaged with the recombinant plasmid of claim 3.

6. Use of the siRNA of claim 1 or the recombinant nucleic acid molecule of claim 2 or the recombinant plasmid of claim 3 or the engineered cell of claim 4 or the small extracellular vesicle of claim 5 in the manufacture of a medicament for the prevention and/or treatment of glioblastoma.

7. A medicament for the prevention and/or treatment of glioblastoma characterized in that the composition of the medicament comprises an inhibitor comprising the siRNA of claim 1 or the recombinant nucleic acid molecule of claim 2 or the recombinant plasmid of claim 3 or the engineered cell of claim 4 or the small extracellular vesicle of claim 5.