CN117721204A - ceRNA regulatory mechanism of circ0104727 and its application in glioma - Google Patents

Abstract

The invention belongs to the field of biomedicine, and specifically relates to the ceRNA regulatory mechanism of circ0104727 and its application in glioma. The circ0104727 described in the present invention, as a ceRNA, regulates the occurrence of glioma by competitively binding to miRNA-21-5p and eliminating the inhibitory effect of miRNA-21-5p on TIMP3/SMAD7. More specifically, the present invention also provides the application of circ0104727 in the diagnosis, grading, prognosis, and treatment of glioma.

Description

ceRNA regulatory mechanism of circ0104727 and its application in glioma

Technical field

The invention belongs to the field of biomedicine, and specifically relates to the ceRNA regulatory mechanism of circ0104727 and its application in glioma.

Background technique

As a star molecule studied in recent years, circular RNA (circRNA) can regulate the expression of downstream target genes by adsorbing miRNAs through sponges. This mechanism is called the competitive endogenous RNA (ceRNA) theory. The competitive endogenous ribonucleic acid (ceRNA) hypothesis is a new model of post-transcriptional regulation of genes, namely protein-coding messenger RNA (mRNA), long non-coding ribonucleic acid (lncRNA), pseudogene transcripts, and circular ribonucleic acid. (circRNA) and others can participate in the expression regulation of their target genes by binding to microRNA (miRNA). ceRNA is a supplement to the regulatory functions of traditional microRNA (miRNA) and messenger RNA (mRNA). It has been found that a variety of RNA molecules can form a ceRNA regulatory network through interaction to perform biological functions of post-transcriptional regulation. It plays an important role in maintaining normal physiological status and regulating the occurrence and development of diseases. The ceRNA network provides important scientific research value for a comprehensive understanding of the heterogeneity and molecular mechanisms of glioma.

Gliomas are the most common primary tumors of the brain and spinal cord and remain incurable. Different genetic and non-genetic effects determine tumor biology and clinical course. In 2007, the World Health Organization (WHO) classified central nervous system tumors into four WHO levels: I to IV. Among them, glioblastoma multiforme (GBM) is the most heterogeneous and most malignant type of brain glioma. Even if the patient actively cooperates with treatment, the median Survival period is currently only about 15 months.

Contents of the invention

In view of the technical problems currently faced, the present invention screened differentially expressed circRNAs through glioblastoma multiforme (GBM) gene chip and determined that circSH3GL3 (hsa_circ_0104727), which is significantly low-expressed in glioma, was used as the entry point. Based on the preliminary determination of its tumor suppressor gene role, through bioinformatics analysis combined with RNA pull down, protein profiling analysis, and miRNA and mRNA sequencing, a new mechanism by which CircSH3GL3 competitively binds to miR-21-5p to exert ceRNA effects was finally proposed. CircSH3GL3 Eliminate the inhibitory effect of miR-21-5p on the downstream target genes TIMP3 and SMAD7, inhibit the PI3K/AKT and wnt/β-catenin signaling pathways respectively through the functional activation of TIMP3 and SMAD7, and play an important anti-tumor role in glioma.

Based on the above results, a new strategy for the combined treatment of glioma based on circRNA and signaling pathway molecular inhibitors can be established, providing a theoretical basis and experimental basis for the development and exploration of tumor drug targets.

Specifically, the present invention provides the following technical solutions:

In a first aspect, the present invention provides the application of hsa_circ_0104727 in regulating the occurrence of glioma.

More specifically, the hsa_circ_0104727 acts as a ceRNA to regulate the occurrence of glioma by competitively binding to miRNA-21-5p.

The term "ceRNA" as used in the present invention, also known as competing endogenous RNA, refers to protein-coding messenger RNA (mRNA), long-chain non-coding ribonucleic acid (lncRNA), pseudogene transcripts, cyclic ribose Nucleic acids (circRNA) can participate in the expression regulation of their target genes by binding to microRNA (miRNA).

Preferably, the gliomas include WHO grades 1-4, where WHO grades 1-2 can be called low-grade gliomas, such as oligodendrogliomas and astrocytoma; WHO grades 3-4 can be called low-grade gliomas. High-grade gliomas, such as anaplastic astrocytoma and glioblastoma.

On the other hand, the present invention provides the application of the miRNA-circRNA-TIMP3 or miRNA-circRNA-SMAD7 regulatory module in regulating the occurrence of glioma, or,

The present invention provides the application of the miRNA-circRNA-TIMP3 or miRNA-circRNA-SMAD7 regulatory module in the preparation of products for regulating the occurrence of glioma,

The circRNA is hsa_circ_0104727, and the miRNA is miR-21-5p.

Preferably, the gliomas include WHO grades 1-4, where WHO grades 1-2 can be called low-grade gliomas, such as oligodendrogliomas and astrocytoma; WHO grades 3-4 can be called low-grade gliomas. High-grade gliomas, such as anaplastic astrocytoma and glioblastoma.

Preferably, the circRNA eliminates the inhibitory effect of miRNA on TIMP3/SMAD7.

More preferably, the TIMP3 inhibits the PI3K/AKT signaling pathway.

More preferably, the SMAD7 inhibits the Wnt/β-catenin signaling pathway.

Preferably, the PI3K/AKT signaling pathway and Wnt/β-catenin signaling pathway are important targets for glioma development.

On the other hand, the present invention provides the application of a reagent for detecting the expression level of hsa_circ_0104727 in the preparation of products with any of the following functions:

1) Diagnosis of glioblastoma,

2) Grading glioma,

3) Predict the prognosis of glioma patients.

Preferably, the glioblastoma is one type of glioma.

Preferably, the grading identifies the glioma patient as either a low-grade glioma or a high-grade glioma.

Preferably, the gliomas include WHO grades 1-4, where WHO grades 1-2 can be called low-grade gliomas, such as oligodendrogliomas and astrocytoma; WHO grades 3-4 can be called low-grade gliomas. High-grade gliomas, such as anaplastic astrocytoma and glioblastoma.

Preferably, the reagents include reagents used when detecting hsa_circ_0104727 expression in the following methods: detection methods based on PCR principles, Southern hybridization methods, Northern hybridization methods, dot hybridization methods, fluorescence in situ hybridization methods, DNA microarray methods, ASO method, high-throughput sequencing platform method.

Specifically, the detection method based on the PCR principle includes variable temperature amplification and constant temperature amplification.

Specifically, the reagent may be a primer pair, probe or antisense nucleotide that specifically binds to hsa_circ_0104727.

Further, the reagent further includes a detectable label.

Further, the detectable label includes radioactive isotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, and bioluminescent moieties.

On the other hand, the present invention provides a composition for treating glioma, which composition contains any one or more of the following:

1) hsa_circ_0104727 and/or hsa_circ_0104727 precursor,

2) Polynucleotide, which can be transcribed to form hsa_circ_0104727,

3) Delivery carrier containing 1) or 2),

4) Reagents that promote hsa_circ_0104727 expression,

5) Molecular pathway inhibitors, including the PI3K/AKT signaling pathway and the Wnt/β-catenin signaling pathway.

Preferably, the composition contains a molecular pathway inhibitor.

Preferably, the delivery carrier includes exosomes, viral vectors, lipid nanoparticles (LNP), polymer nanocarriers, inorganic nanocarriers or protein carriers.

Preferably, the viral vector includes lentiviral vector, adenoviral vector, and adeno-associated virus vector.

Preferably, pharmaceutically acceptable solid/liquid excipients are also included in the composition.

Preferably, the pharmaceutically acceptable solid/liquid excipients include binders, fillers, lubricants and disintegrants.

The dosage form and administration mode of the composition of the present invention are not particularly limited. Representative modes of administration include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular, or subcutaneous) injection, and topical administration.

In a specific embodiment, solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or compatibilizers, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) Humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) retarder, such as paraffin; (f) Absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, hard Calcium fatty acid, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents. Solid dosage forms such as tablets, dragees, capsules, pills and granules may be prepared using coatings and shell materials such as enteric casings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxy substances. If necessary, the active compounds can also be in microencapsulated form with one or more of the above-mentioned excipients.

In a specific embodiment, liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, specifically, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances. Besides these inert diluents, the compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents. For example, the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these substances.

In one specific embodiment, compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and for reconstitution into sterile injectable Sterile powder for solutions or dispersions. Suitable aqueous or non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

In a specific embodiment, dosage forms for topical administration include ointments, powders, patches, sprays and inhalants. It consists of mixing the active ingredient under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required.

Preferably, the dosage form of the composition includes capsules, tablets, pills, powders, granules, emulsions, solutions, suspensions, syrups or tinctures.

On the other hand, the present invention provides the use of the above composition in preparing medicine for treating glioma.

Preferably, the glioma includes WHO grade 1-4 glioma.

Preferably, the glioma includes oligodendroglioma, astrocytoma, anaplastic astrocytoma or glioblastoma.

Preferably, the glioma is glioblastoma.

On the other hand, the present invention provides a method for diagnosing glioblastoma, classifying glioma, or predicting the prognosis of a patient with glioma, the method comprising detecting the expression of hsa_circ_0104727 in a sample from a subject quantity.

Preferably, the sample includes: peripheral blood, tissue, blood, serum, plasma, urine, saliva, semen, milk, cerebrospinal fluid, tears, sputum, mucus, lymph, cytosol, ascites, pleural effusion, amniotic fluid , bladder wash fluid and bronchoalveolar lavage fluid.

Preferably, the sample is tissue.

On the other hand, the present invention provides a method for inhibiting the proliferation and invasion of glioma cells, which method includes increasing the expression of hsa_circ_0104727 in target cells.

Preferably, the method is non-therapeutic.

Preferably, the method is performed in vitro.

Preferably, the target cells are glioma cells, including glioblastoma cells.

Specifically, in the specific embodiments of the present invention, overexpression experiments were conducted on the LN229 glioblastoma cell line and U251 glioma cell line that lowly express hsa_circ_0104727 as examples, proving that the proliferation and invasion capabilities of cells are inhibited after overexpression of hsa_circ_0104727. In addition, a knockout experiment was conducted using the U87 (also known as ATCC HTB-14) glioblastoma cell line that highly expresses hsa_circ_0104727 as an example, and the opposite results were obtained.

In another aspect, the present invention provides a method of treating glioma, the method comprising administering the aforementioned composition or hsa_circ_0104727 to a patient.

Specifically, the administration mode of the administration is not particularly limited. Representative modes of administration include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular, or subcutaneous) injection, and topical administration.

Specifically, the glioma includes glioblastoma cells.

On the other hand, the present invention provides a system, which has a judgment device that can give any of the following results based on the expression amount of hsa_circ_0104727:

1) Whether the subject suffers from glioblastoma,

2) Subject’s glioma classification,

3) The prognosis of glioma patients.

The prognostic indicators of the present invention include objective response rate (ORR), overall survival rate (Overall survival rate, OS), progression-free survival (PFS), disease progression time ( time to progress (TTP), disease-free survival (DFS), time to treatment failure (TTF), response rate (RR), complete response (CR), partial response (PR).

Description of the drawings

Figure 1 is a cluster heat map of circRNA expression profile detection between clinical GBM specimens and corresponding paired adjacent normal tissues.

Figure 2 shows the qRT-PCR detection results of circRNA0071539, circRNA0104727 and circRNA0104722.

Figure 3 is a comparison of the expression levels of circSH3GL3 under different conditions.

Figure 4 is the electrophoresis result of amplifying linear genes SH3GL3 and circSH3GL3.

Figure 5 is a diagram of Sanger sequencing results.

Figure 6 is a diagram showing the results of RNase enzyme digestion of linear mRNA and circSH3GL3.

Figure 7 is a diagram showing the subcellular localization results of circSH3GL3.

Figure 8 is a graph showing the detection results of overexpression and knockdown efficiency.

Figure 9 is a graph showing the results of the CCK8 experiment.

Figure 10 is the result diagram of the Edu experiment.

Figure 11 is the result of the Transwell experiment.

Figure 12 is a clustering heat map obtained by mRNA sequencing enrichment.

Figure 13 is a plasmid vector map of the luciferase reporter gene plasmid vector pmirGLO.

Figure 14 is a graph of luciferase activity detection results.

Figure 15 is a diagram showing the results of RNA pull down experiment of biomarked miR-21-5p.

Figure 16 is a graph showing experimental results of proliferation analysis of LN229 and U87 cells transfected with miR21-5pmimic alone and co-transfected with circSH3GL3.

Figure 17 is a flow chart of differentially expressed genes and screening obtained by comparing U87-circSH3GL3 cells with U87-vector cells.

Figure 18 is a graph of qRT-PCR results of TIMP3, SMAD7, FMN1 and CREB5 mRNA.

Figure 19 is a schematic diagram of the miR-21-5p seed sequence matching TIMP3 and SMAD7 3'UTR.

Figure 20 is a picture of the results of Western blot experiment.

Figure 21 is a graph showing the results of luciferase reporter gene detection.

Figure 22 is a graph showing the results of Western blot detection of PI3K, p-PI3K, AKT1, P-AKT, and caspase3 protein expression levels.

Figure 23 is a graph showing the results of the TOP/FOP luciferase reporter plasmid experiment.

Figure 24 is a graph showing the results of immunofluorescence detection of the relative expression of β-catenin in the nucleus and cytoplasm of different treatment groups.

Figure 25 is a graph showing the protein expression levels of TCF4, c-myc, cyclinD1, and MMP2.

Figure 26 is a graph showing the results of the change in vivo experiment.

Figure 27 is a graph showing the results of detecting the expression levels of TIMP3, SMAD7, p-AKT, c-Myc, and cyclinD1.

Figure 28 is a picture of the results of immunohistochemical staining.

Figure 29 is a schematic diagram showing how CircSH3GL3 functions as ceRNA.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto. Any person familiar with the technical field can, within the technical scope disclosed in the present invention, use the technical solutions of the present invention and its Equivalent substitutions or changes of the inventive concept shall be included in the protection scope of the present invention.

Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

Experimental Materials

RNase, circRNA reverse transcription kit ( II First Strand cDNA SynthesisKit), quantitative PCR detection kit (/> qPCR/> Green Master Mix), circ_0104727 FISH probe and its circRNAs primers, and RNA lmmunoprecipitation kit were purchased from Guangzhou Gisai Biotechnology Co., Ltd. Actinomycin D was purchased from Sigma Company in the United States. RNA nucleocytoplasmic isolation and extraction kit was purchased from Ambion Company. CircSH3GL3 siRNA and well-verified overexpression lentivirus were constructed by Guangzhou Gisai Biotechnology Co., Ltd. and identified by our laboratory. The circ0104727 dual-luciferase reporter plasmid and its mutant plasmid that binds to miR-21-5p were also constructed and synthesized by Shanghai Jima Gene Co., Ltd. Immunohistochemistry detection kit and gDNA extraction kit were purchased from Beijing Solebao Biotechnology Co., Ltd.

H4 and NHA cell lines were purchased from Beijing Beina Chuanglian Biotechnology Research Institute, and U251, U87, LN229, A172, and T98G were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Medical Sciences. The above cell lines were cultured in 1640 or DMEM containing 10% fetal bovine serum (Gibco), 100 U/ml penicillin and 0.1 mg/ml streptomycin (Sangon biotech) at 37°C in a humidified atmosphere of 5% _CO2 . All cell lines tested negative for mycoplasma. Cells were transfected using Lipofectamine3000 (Invitrogen), and cells were collected for detection after 48-72 hours.

Example 1. Identification of circRNAG expression profiles in GBM tissue and adjacent normal brain tissue

In order to explore the role of circRNA in the occurrence and development of GBM, we used microarray chips to analyze ribosomal RNA-deleted total RNA from 3 clinical GBM specimens and corresponding paired adjacent normal tissues and constructed a circRNA database.

Three cases of fresh primary glioblastoma tissue and adjacent normal brain tissue specimens were collected from GBM patients treated with neurosurgery. During the operation, quick frozen section was used to determine the "normal boundary" of the resected tumor and then the corresponding "normal brain tissue" was taken. The size of the tissue in each case is about 0.5cm×0.5cm×0.5cm. It is washed with physiological saline and placed in a labeled cryopreservation tube. Quickly put it into a liquid nitrogen tank and transfer it to the laboratory for freezing at -80°C. All specimens were diagnosed as primary glioblastoma after surgery, and the adjacent histopathology confirmed that no cancer cells were involved. All patients did not receive radiotherapy or chemotherapy before surgery. All patients obtained consent from themselves or their family members and signed informed consent forms.

Three paired GBM specimens were tested for circRNA expression profiles using the Arraystar Human circRNAArray chip of Beijing Boao Biotechnology Co., Ltd. A total of 1812 abnormally expressed circRNAs were identified in GBM tissues, of which 711 were up-regulated and 1101 were down-regulated (Figure 1A) (fold change>5, p<0.05). Among these differentially expressed circRNAs, most were less than 1000 nucleotides in length, with different circRNA length distributions (Figure 1B), and the genomic origins of these significantly expressed circRNAs were also different. According to the circBase database source and the standard that the original detection signal value is greater than 200, qRT-PCR verified the 20 most significantly different circRNAs, including the top 10 significantly up-regulated and the top 10 most significantly down-regulated circRNAs. The clustering heatmap in Figure 1C shows 20 up-regulated and down-regulated circRNAs. Through qRT-PCR and sanger sequence verification, only circRNA0071539, circRNA0104727 and circRNA0104722 were consistent with the expression tendency of GBM chip analysis (Figure 2).

Based on expression abundance, we focused on the most significantly downregulated circRNA0104727. At the same time, we found that 4 of the 10 down-regulated circRNAs came from the linear gene SH3GL3, which according to bioinformatics analysis is related to the progression of GBM. Therefore, we selected circRNA0104727 (referred to as circSH3GL3 in the rest of the present invention) for further functional and molecular mechanism studies.

To further verify the downregulation of circSH3GL3, we examined 52 cases of gliomas of different grades, including 15 low-grade (WHOI-II) and 37 high-grade (WHO III-IV).

Fifty-two clinical cases of glioma specimens with different pathological tissue types were obtained from the biomedical specimen bank of Tianjin Institute of Neurosurgery, including 9 cases of oligodendrogliomas (WHO grade II) and 6 cases of astrocytomas (WHO grade). 2 cases of grade I and 4 cases of WHO grade II), 5 cases of anaplastic oligodendrogliomas (WHO grade III), 3 cases of anaplastic astrocytoma (WHO grade III), and 29 cases of primary glioblastoma Cytoma (WHO grade IV), all patients obtained consent from themselves or their families and signed informed consent forms.

The results show that compared with low-grade gliomas and normal controls, the expression of circSH3GL3 in GBM is significantly down-regulated and negatively correlated with WHO grade, and high expression of circSH3GL3 indicates good prognosis. In addition, Real-time PCR detected the expression of circSH3GL3 in glioma cell lines and found that, except for U251 cells, circSH3GL3 expression was significantly down-regulated in GBM cell lines compared with NHA cell lines (Figure 3).

Therefore, we further selected U251 cells for circSH3GL3 loss of function experiments, and LN229 and U87 cells for functional recovery experiments.

Example 2. Characteristics of circSH3GL3

1. Stability of circSH3GL3

We evaluated the exon structure of circSH3GL3, which is derived from exons 5 to 11 of the SH3GL3 gene on chromosome 15. We designed divergent primers across the circularization site to identify and specifically detect circSH3GL3 in U251 cell cDNA and genomic gDNA through PCR amplification and agarose gel electrophoresis.

The specific PCR primers are F: 5'-GTGAAGACGACAGCTATTTA-3' (SEQ ID NO. 1); R: 5'-GACACAGTGTTCAGCATTCC-3' (SEQ ID NO. 2). The amplified fragment size of hsa_circ_0104727 is 192 bp.

The electrophoresis results show that the heterogeneous primer can only amplify in cDNA and cannot amplify in gDNA, while its linear gene SH3GL3 can amplify in both cDNA and gDNA (Figure 4). Sanger was further performed on the PCR product amplified from cDNA. Sequencing, the specific cyclization site is shown in Figure 5. These results confirmed that circSH3GL3 is a closed circular RNA formed by back-splicing at the post-transcriptional level and can be specifically amplified by Divergent primers.

After RNase digestion of U251 cell RNA, quantitative PCR was used to detect the expression of circSH3GL3 and its corresponding linear RNA molecule SH3GL3. The results showed that compared with linear mRNA, circSH3GL3 was indeed more resistant to digestion by RNaseR enzyme, and the expression level of SH3GL3 in the RNaseR(+) group was significantly reduced compared with the RNaseR(-) group (Figure 6A). U251 cells were further treated with actinomycin D to indirectly verify the stability of circSH3GL3. The qRT-PCR results showed that after treatment with actinomycin D for 24 hours, circSH3GL3 was significantly degraded compared with the linear RNA molecule SH3GL3 (Figure 6B). The results confirmed that circSH3GL3 is more stable than linear SH3GL3 molecules.

2. Subcellular localization of circSH3GL3

The subcellular localization of circRNA molecules is closely related to their different biological functions. circRNAs mainly located in the cytoplasm mainly function through the competitive ceRNA mechanism, while circRNAs located in the nucleus mainly regulate the transcription and splicing of downstream target genes by binding to RNA-binding proteins.

We first used RNA nucleocytoplasmic separation experiments to extract nuclear and cytoplasmic RNA from U251 cells respectively. After reverse transcribing equal volumes of RNA, we detected the distribution of circSH3GL3 in the cells by quantitative PCR. The distribution of GAPDH and U6 mRNA in the nucleus and cytoplasm were used as controls. The results showed that circSH3GL3 was mainly located in the cytoplasm of glioma cells (Figure 7A).

We then applied fluorescence in situ hybridization (FISH) method to further clarify the cellular localization of circTOP2A. FISH in situ hybridization probe sequence: hsa_circ_0104727 (5’, 3’ CY3 Labeled): TTCACTAAATAGCTGTCGTCTTCA (SEQ ID NO. 3). The single-stranded DNA probe that specifically corresponds to the circSH3GL3 cleavage site carries Cy3-labeled red fluorescence. The results observed under laser confocal microscopy are consistent with the results of the RNA nucleocytoplasmic separation experiment. circSH3GL3 is mainly located in the cytoplasm (Figure 7B).

Two methods were used to clarify the cytoplasmic localization of circSH3GL3 and lay a theoretical foundation for its competitive binding to miRNA.

Example 3. High expression of circSH3GL3 significantly inhibits glioma cell proliferation and invasion

The role of circSH3GL3 in glioma progression was evaluated through gain-of-function and loss-of-function assays.

Based on the expression of circSHGL3 in glioma cell lines, the circSH3GL3 (circSH3GL3 wild-type expression plasmid lentivirus) was overexpressed in LN229 and U87 cells. The sequencing identification, recombinant lentiviral vector packaging, and dripping have been completed at Guangzhou Gisai Biotechnology Co., Ltd. Degree determination, expression verification and other work, contract number: GS200413-CZQ034), and knock down the expression of circSH3GL3 in U251 cells.

qRT-PCR verified the overexpression and knockdown efficiency of circSH3GL3 and showed that transfection had no effect on the parental SH3GL3 gene (Figure 8A-B).

CCK8, Edu and Transwell experiments showed that compared with the vector group, circSH3GL3 overexpression could significantly inhibit the proliferation and invasion of tumor cells. The si-circSH3GL3-mediated inhibition of circSH3GL3 can significantly promote cell growth and invasion (Figure 9-11).

Example 4. CircSH3GL3 competitively binds to miR-21-5p

The above results indicate that circSH3GL3 is mainly located in the cytoplasm, and circSH3GL3 may play an anti-tumor gene role in GBM through competitive binding with miRNA via a ceRNA mechanism.

In order to identify the downstream miRNA of circSH3GL3, we performed a biotinylated circSH3GL3 probe RNA pull down experiment and performed miRNA high-throughput sequencing on the enriched RNA. The sequence results showed that 20 miRNAs were enriched. According to the enrichment and Fold change>2, qRT-PCR further verified 7 miRNAs, including has-miR-1-3p, has-miR-1246, has-miR-615-3p, has-miR-21-5p , hsa-miR-125b-1-3p, hsa-miR-4787-3p and hsa-miR-191-3p. Among them, the specific enrichment amounts of miR-1-3p, miR-21-5p and miR-615-3p were significantly higher than those in the NC group, as shown in Figure 12A.

In addition, combined with our previous miRNA chip analysis of 3 pairs of GBM and adjacent non-tumor tissues, miR-21-5p is one of the most up-regulated miRNAs and is abundant in GBM (Figure 12B). Therefore, we selected miR-21-5p for further verification. miR-21-5p contains a putative targeting site in the circSH3GL3 region, as shown in Figure 12C.

In order to verify the binding ability of miRNA to circSH3GL3, we constructed a circSH3GL3 luciferase reporter plasmid system. The target gene prediction website was used to predict the seed sequences of miR-21-5p that bind to the TIMP3 and SMAD7 3'UTR regions respectively, and the mutant (MT) and wild type (WT) of the specific binding regions were constructed into luciferase reporters. On the gene plasmid vector pmirGLO, the plasmid vector map is shown in Figure 13. The construction process was synthesized by Shanghai Jima Gene Co., Ltd., and the experimental methods were based on the articles published by this research group: Jikui Sun; Jinhuan Wang; Meng Li; Shengjie Li; HanyunLi; Yan Lu; Feng Li; Tao Xin; Feng Jin; circTOP2A functions as a ceRNA to promote glioma progression by upregulating RPN2, Cancer Science, 2023, 114(2): 490-503.

MiR-21-5p mimic and circSH3GL3 luciferase reporter gene were co-transfected into LN229 and U87 cells. The results confirmed that compared with the NC group, miR-21-5p could significantly reduce the luciferase activity of circSH3GL3-WT, while there was no difference in the luciferase activity of the circSH3GGL3-MUT group (Figure 14).

Then, biotin-labeled miR-21-5p pull down experiments confirmed that circSH3GL3 was significantly enriched in the miR-21-5p capture fraction compared with the negative control (Figure 15).

In addition, further EDU and CCK8 functional experiments confirmed that overexpression of miR-21-5p promoted cell proliferation, while overexpression of circSH3GL3 could reverse the proliferation-promoting effect mediated by miR-21-5p mimic (Figure 16).

These results indicate that circSH3GL3 can competitively bind to miR-21-5p and participate in the malignant progression of glioma.

Example 5. CircSH3GL3 targets and regulates TIMP3 and SMAD7 by competitively binding to miR-21-5p.

Current research shows that miR-21-5p, as a star molecule, plays an important oncogene role in the occurrence and development of glioma by inhibiting a variety of anti-cancer genes. Therefore, we hypothesized that circSH3GL3 inhibits tumor progression by protecting the direct target genes of miR-21-5p. RNA-seq analysis after increasing circSH3GL3 expression found that compared with U87-Vector cells, 293 differentially expressed genes (DEGs) were up-regulated (fold>1.5) and 254 DEGs were down-regulated (foldchange>0.5) in U87-circSH3GL3 cells. (Figure 17). Applying the upregulated degs together with the miRanda database to predict potential targets of miR-21-5p, we obtained 10 overlapping genes, including TIMP3, SMAD7, TRIM2, ABCA1, FMN1, RAB27B, SEMA5A, SAMD9, CYBRD1, CREB5 . Finally, TIMP3, SMAD7, FMN1 and CREB5 were selected for qRT-PCR verification. The detailed screening flow chart is shown in Figure 17.

The qRT-PCR results showed that up-regulation of circSH3GL3 could significantly increase the mRNA levels of TIMP3 and SMAD7, but had no significant effect on the mRNA levels of FMN1 and CREB5 (Figure 18).

Combined with the ENCORI online database, multiple target gene prediction software such as PITA, PicTar, microT, and targetscan suggested that TIMP3 and SMAD7 are direct targets of miR-21-5p. The seed sequence of miR-21-5p matching the 3'UTR of TIMP3 and PDCD4 genes is shown in Figure 19.

In order to determine whether circSH3GL3 can regulate the downstream genes TIMP3 and SMAD7, Westernblot testing was first performed. The results showed that the upregulation of circSH3GL3 could significantly increase the protein levels of TIMP3 and SMAD7, but miR-21-5p mimic partially reversed this effect (Figure 20A). Next, to further confirm whether TIMP3 and SMAD7 are direct targets of miR-21-5p, western blot and dual-luciferase reporter plasmid detection were performed. The results showed that in LN229 and U87 cells, the expression of TIMP3 and SMAD7 proteins was significantly up-regulated after knocking down the expression of miR-21-5p (20B).

In addition, luciferase reporter gene assay confirmed that down-regulation of miR-21-5p resulted in a significant increase in luciferase activity in the pmirGLO-TIMP3-WT and pmirGLO-SMAD7-WT groups, while pmirGLO-TIMP3-MT and pmirGLO-SMAD7-MT There was no change in luciferase activity in the group (Figure 21), suggesting that miR-21-5p regulates the expression of TIMP3 and SMAD7 by directly binding to the 3'UTR of TIMP3 and SMAD7. Finally, luciferase reporter gene detection showed that compared with the mutated luciferase reporter gene, the luciferase activity of TIMP3 and SMAD7 wild-type was significantly increased when stably upregulating circSH3GL3 expression (Figure 21), once again confirming that circSH3GL3 has an effect on miR The regulatory role of -21-5p.

Example 6. CircSH3GL3 regulates PI3K/AKT and Wnt/β-catenin signaling pathways through TIMP3 and SMAD7 respectively

Based on the mRNA sequence of U87 cells with previous circSH3GL3 overexpression, bioinformatics analysis showed enrichment of multiple signaling pathways, including the PI3K/AKT and Wnt/β-catenin signaling pathways, which are the two main targets for GBM drug design. Further searching the literature in the Pubmed database, more and more evidence shows that TIMP3 can significantly inhibit the PI3K/AKT signaling pathway, and SMAD7 is an inhibitory gene of the Wnt/β-catenin signaling pathway. A study on glioma reported that exosome-mediated MIF further activates the PI3K/AKT signaling pathway by downregulating TIMP3, thereby promoting TMZ resistance in glioma.

Therefore, we next explored whether circSH3GL3 regulates the PI3K/AKT and Wnt/β-catenin signaling pathways by targeting TIMP3 and SMAD7, respectively. Western blot analysis showed that circSH3GL3 overexpression could significantly inhibit the expression of p-PI3K, P-AKT and P-AKT, without affecting the total expression levels of PI3K and AKT (Figure 22). In LN229 and U87 cells that stably overexpressed circSH3GL3, siRNA TIMP3-mediated inhibition can partially reverse the circSH3GL3-mediated inhibition of the PI3K/AKT pathway, as shown in Figure 22.

TOP/FOP luciferase reporter material experiment was used (for specific experimental methods, please refer to the articles published by this research group: MaQ, Li B, Wang C, Mo L, Zhang X, Tang F, Wang Q, Yan X, Yao X et al: RPN2 is targeted by miR-181c and mediates glioma progression and temozolomide sensitivity via the wnt/beta-catenin signaling pathway. Cell death&disease 2020,11(10):890.), western blot and immunofluorescence detection analysis of β-catenin circSH3GL3 targets SMAD7 mediation Lead the role of Wnt/β-catenin pathway. The TOP/FOP luciferase reporter plasmid experiment confirmed that lentivirus-mediated circSH3GL3 can significantly reduce the luciferase activity of the TOP plasmid group. In the U251 and LN229 cell lines that stably knock down circTOP2A expression, knocking down SMAD7 expression at the same time can reverse the decrease in luciferase activity mediated by circSH3GL3 to a certain extent (Figure 23).

The results of Western blot experiments on important factors downstream of the wnt signal transduction pathway confirmed that circSH3GL3 can significantly inhibit the expression of TCF4, c-myc, cyclinD1 and MMP2. Similarly, after knocking down the expression of SMAD7 at the same time, the expression of TCF4, c-myc, cyclinD1 The expression level was significantly up-regulated (Figure 24). In addition, immunofluorescence analysis was used to detect the expression changes of β-catenin, a core factor of the wnt pathway, in different treatment groups. The results showed that after overexpression of circSH3GL3, the level of β-catenin in the nucleus was significantly reduced. Knockdown of SMAD7 also reversed the β-catenin mediated by circSH3GL3. -catenin transcriptional activity (Fig. 25).

Example 7. Effect of circSH3GL3 on the growth of glioma in vivo

In order to study the effect of circSH3GL3 on glioma growth, 5 × 10 ⁵ luciferase-labeled U87 cells stably overexpressing circSH3GL3 (Lv-circSH3GL3) or Lv-miR-21-5p were stereotaxically injected to construct nude mouse skulls. Internal model. Tracking tumor progression via in vivo bioluminescence. The experiment was divided into three groups: Lv-NC, Lv-circSH3GL3, Lv-circSH3GL3+LV-miR-21-5p group.

The volume of xenograft tumors formed by Lv-circSH3GL3 U87 cells was significantly smaller than that of the control group (Lv-NC) (Figure 26A), and Kaplan-Meier analysis results showed that the survival period of the Lv-circSH3GL3 group was significantly prolonged (Figure 26B).

Western blot results showed that the protein levels of TIMP3 and SMAD7 were significantly increased in the LV-circSH3GL3 group, and the expressions of p-AKT, c-Myc, and cyclinD1 were significantly decreased (Figure 27). Immunohistochemical staining showed that the expression of TIMP3 and SMAD7 increased in the Lv-circSH3GL3 group, which was consistent with the western blot results (Figure 28). However, the results of the Lv-circSH3GL3+LV-miR-21-5p group suggest that the restored expression of miR-21-5p reverses the up-regulation of TIMP3 and SMAD7 by circSH3GL3 and the inhibitory effect of circSH3GL3 on the PI3K/AKT and wnt/β-catenin signaling pathways to a certain extent ( Figure 29), the results in vivo and in vitro are consistent.

The above content is a further detailed description of the present invention in combination with specific preferred embodiments, and it cannot be concluded that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all of them should be regarded as belonging to the protection scope of the present invention.

Claims (10)

1. Application of hsa_circ_0104727 in regulation of glioma occurrence;

   more specifically, the hsa_circ_0104727 as ceRNA modulates glioma occurrence by competitive binding to miRNA-21-5 p;

   preferably, the glioma comprises an oligodendroglioma, an astrocytoma, a modified astrocytoma or a glioblastoma.
2. Use of a miRNA-circRNA-TIMP3 or miRNA-circRNA-SMAD7 regulatory module for modulating glioma occurrence, wherein the circRNA is hsa_circ_0104727 and the miRNA is miR-21-5p;

   preferably, the glioma comprises an oligodendroglioma, an astrocytoma, a modified astrocytoma or a glioblastoma;

   preferably, the circRNA module in the regulation module eliminates the inhibition effect of the miRNA module on the TIMP3 module or SMAD7 module, thereby regulating the occurrence of glioma;

   more preferably, the TIMP3 inhibits the PI3K/AKT signaling pathway;

   more preferably, the SMAD7 inhibits the Wnt/β -catenin signaling pathway.
3. 3. Use of a reagent for detecting hsa_circ_0104727 expression level in the preparation of a product having any one of the following functions:

   1) Diagnosing glioblastoma, and diagnosing the glioblastoma,

   2) The glioma is classified into a grade and a grade,

   3) Predicting prognosis of glioma patients;

   preferably, the glioblastoma is one of gliomas;

   preferably, the grading is to identify the glioma patient as a low grade glioma or a high grade glioma;

   preferably, the glioma comprises a WHO 1-4 grade, in particular, WHO 1-2 grade is a low grade glioma and WHO 3-4 is a high grade glioma;

   preferably, the glioma comprises an oligodendroglioma, an astrocytoma, a modified astrocytoma or a glioblastoma.
4. 4. The use according to claim 3, wherein the reagent comprises a reagent used in the following method for detecting hsa_circ_0104727 expression: detection method based on PCR principle, southern hybridization method, northern hybridization method, dot hybridization method, fluorescence in situ hybridization method, DNA microarray method, ASO method, high throughput sequencing platform method;

   specifically, the detection method based on the PCR principle comprises variable temperature amplification and constant temperature amplification;

   specifically, the reagent is a primer pair, probe or antisense nucleotide that specifically binds to hsa_circ_ 0104727;

   further, the reagent further comprises a detectable label;

   further, the detectable label includes a radioisotope, a nucleotide chromophore, an enzyme, a substrate, a fluorescent molecule, a chemiluminescent moiety, a magnetic particle, a bioluminescent moiety.
5. 5. A composition for treating glioma, said composition comprising any one or more of the following:

   1) hsa_circ_0104727 and/or hsa_circ_0104727 precursors,

   2) A polynucleotide which is transcribed to form hsa_circ_0104727,

   3) A delivery vehicle comprising 1) or 2) of,

   4) An agent for promoting hsa_circ_0104727 expression,

   5) A molecular pathway inhibitor, the molecular pathway comprising a PI3K/AKT signaling pathway and a Wnt/β -catenin signaling pathway;

   preferably, the composition contains a molecular pathway inhibitor;

   preferably, a pharmaceutically acceptable solid/liquid excipient is also included in the composition;

   preferably, the pharmaceutically acceptable solid/liquid excipients include binders, fillers, lubricants and disintegrants.
6. 6. The composition of claim 5, wherein the delivery vehicle comprises an exosome, a viral vector, a lipid nanoparticle, a polymeric nanocarrier, an inorganic nanocarrier, or a protein carrier;

   preferably, the viral vector comprises a lentiviral vector, an adenoviral vector, an adeno-associated viral vector.
7. Use of hsa_circ_0104727 or a composition according to claim 5 for the manufacture of a medicament for the treatment of glioma;

   preferably, the glioma comprises a WHO 1-4 grade glioma;

   preferably, the glioma comprises an oligodendroglioma, an astrocytoma, a modified astrocytoma or a glioblastoma;

   preferably, the glioma is glioblastoma.
8. 8. A method of inhibiting glioma cell proliferation and invasion, the method comprising increasing the expression level of hsa_circ_0104727 in a target cell;

   preferably, the method is non-therapeutic;

   preferably, the method is performed in vitro.
9. 9. A system having a judging means for giving any one of the following results by the expression level of hsa_circ_ 0104727:

   1) Whether the subject has glioblastoma,

   2) Glioma typing of the subject,

   3) Prognosis of glioma patients.
10. 10. The system of claim 9, further comprising one or more of a detection device, an input device, an output device, and a communication device.